Secretory pathway function in Saccharomyces cerevisiae.

Secretory Pathway Function in Saccharomyces cerevisiae ANN E. CLEVES and VYTAS A . BANKAITIS Department of Microbiology, University of Illinois, Urbana, Illinois, USA

I. Introduction . . . . . . . . . . . . . . . 11. The yeast secretory pathway . . . . . . . . . . . . A. Elucidation of form . . . . . . . . . . . . . . 111. Protein transport from the cytoplasm into the endoplasmic reticulum A. The paradigms: mammalian andprokaryoticmodels . . . . . B. Genetic analyses . . . . . . . . . . . . . C. In vitro systems . . . . . . . . . . . . . IV. Protein trafficking from the endoplasmic reticulum to the Golgi complex . . A. The mammalian paradigm . . . . . . . . . . . B. Reconstructionoftheyeastprocessinvitro . . . . . . . C. Molecular analysis of genes whose products stimulate transport from the endoplasmic reticulum to the Golgi complex . . . . . . D. The retention problem. . . . . . . . . . . . V. TheGolgicomplexasasecretoryorganelle . . . . . . . . A. Functional compartmentalization of the yeast Golgi complex . . . B. Involvementof aphospholipid-transferprotein . . . . . . C. Theretentionproblem: theroleofclathrin . . . . . . . D. Coupling of Golgi-complex and actin-cytoskeleton functions . . . . . VI . Fusion of Golgi complex-derived vesicles with the plasma membrane A. Involvement of a GTP-binding protein . . . . . . . . B. Other gene products that potentiate GTP-binding protein function . VII. Summary . . . . . . . . . . . . . . . . VIII. Acknowledgements . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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I. Introduction

The eukaryotic secretory pathway plays an essential role in maintaining the cellular requirements for biochemical compartmentation, and it represents a major aspect of intracellular protein traffic within the eukaryotic cell. It is ADVANCES IN MICROBIALPHYSIOLOGY, VOL. 33 ISBN &l2-0277324

Copyright 0 1992. by Academic Press Limited All rightsofreproduction inany form reserved

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defined by a set of biochemically and morphologically distinct membraneenclosed organelles that house activities associated with catalysis of protein transport, and sorting of proteins from the cytoplasm to various intracellular organelles and the cell surface. Deservedly, the study of intracellular protein transport now commands a great deal of scientific effort and represents a major discipline in cell biology. The basic form of the eukaryotic secretory pathway was elucidated in a series of studies performed by Palade and his coworkers (Palade, 1975). Proteins are synthesized on cytoplasmic ribosomes, inserted into the lumen of the endoplasmic reticulum (ER), and subsequently transported to the Golgi complex (a distinct organelle). Delivery of proteins to the cell surface is the end result of fusion of Golgi complex-derived secretory vesicles, or secretory granules, to the plasma membrane. It is now appreciated that intercornpartmental protein traffic (and, in the case of the Golgi complex, intracompartmental protein traffic) is driven by the budding and specific fusion of transitional vesicles from donor compartments to specific acceptor organelles (Pfeffer and Rothman, 1987). An appreciation of the mechanisms by which these transport vesicles bud from donor organelles and specifically target to (and fuse with) acceptor membranes is a necessary component of an understanding of secretory pathway function. Superimposed upon these issues are considerations of maintenance of organelle identity in the face of a massive bulk flow of protein and lipid from the ER, movement of phospholipids between organelle membranes, protein sorting, and secretory polarity. The definitive demonstration of the basic form of the yeast secretory pathway, and its direct analogy to that of mammalian cells, was provided by the pioneering work of Schekman and his colleagues (Novick et al., 1980; Schekman, 1985). The tractability of Saccharamyces cerevisiae t o genetic, molecular, biochemical and cell biological analyses has made this organism an increasingly attractive experimental system with which to study various aspects of secretory pathway function. A number of fundamental insights concerning the molecular aspects of eukaryotic secretory pathway function have already been forthcoming from this system, with the promise of many more to follow. In this review, we will consider only those topics that are immediately relevant to biosynthetic protein transport through the yeast secretory pathway in Sacch. cerevisiae with some consideration of protein retention within organelles and secretory polarity. Detailed considerations of signal-sequence function, protein glycosylation, protein sorting from the secretory pathway to the vacuole, and aspects of vacuolar biogenesis are discussed in recent reviews (Ballou, 1982; Wickner and Lodish, 1985; Klionsky et al., 1990).

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11. The Yeast Secretory Pathway A . ELUCIDATION OF FORM

The basic scheme of the yeast secretory pathway is directly analagous to that of higher eukaryotes, namely E R + Golgi complex + vesicles + ultimate compartment (i.e. the plasma membrane or vacuole). Elucidation of this pathway involved the isolation and characterization of yeast mutants elaborating temperature-sensitive ( t s ) defects at specific stages of the secretory pathway. These defects exhibited the dual properties of being both recessive and thermoreversible traits. As such, these sec mutants identified gene products whose activity was required to stimulate secretory pathway function in yeast (Schekman, 1985). The isolation of such sec mutants was greatly facilitated by a density-enrichment strategy that took advantage of the fact that sec mutants became dense, with respect to wild-type cells, upon imposition of the secretory block (Novick and Schekman, 1979; Novick, et al., 1980). Two classes of secretion-defective mutants were obtained. Class A sec mutants exhibited intracellular accumulation of active invertase (a secretory protein) under the restrictive condition whereas class B sec mutants did not accumulate active invertase at 3TC, even though protein synthesis was not affected. The class B mutants defined two complementation groups, SEC.53 and SEC59, whose products were somehow involved in glycosylation of proteins that had been inserted into the E R lumen (FerroNovick et al., 1984; Feldman et al., 1987). Subsequent molecular and biochemical analyses have identified SEC.53 as the structural gene for phosphomannomutase, the enzyme that converts mannose 6-phosphate to mannose 1-phosphate (Kepes and Schekman, 1988). Thus, in sec53 mutants, production of the sugar donor (GDP-mannose) for synthesis of dolichol-linked oligosaccharides is blocked. The SEC.59 gene product encodes a hydrophobic 59 kDa polypeptide that also plays some role in transfer of mannose to the dolichol-linked oligosaccharide (Bernstein et al., 1989). Consequently, class B mutants were not remarkably informative with respect to mechanisms of secretory pathway function. Genetic analysis of class A mutants revealed that these fall into 23 complementation groups whose products are involved in secretory protein transport through defined stages of the secretory pathway (Fig. 1). The latter conclusion was reached by electron-microscopic visualization of the terminal phenotypes of such mutants (Novick et al., 1980). These experiments permitted classification of the mutants into several classes: (i) those that were blocked in protein traffic from the E R to the Golgi complex (these ER-blocked mutants defined nine complementation groups, SEC12, SEC13, SEC16, SECl7, SEC18, SEC20, SEC21, SEC22 and SEC23);

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FIG. 1 . Diagram illustrating the yeast secretory pathway. Yeast secretory glycoproteins are synthesized in the cytoplasm and are transported into the lumen of the endoplasmic reticulum (ER) where they undergo N-linked core glycosyl modification. After passage through the Golgi complex, where the remodelling of glycosyl chains is completed, glycoproteinsare delivered to their final destinations,either the cell surface or the vacuole. The involvement of 21 of the 23 original SEC gene products is indicated (Novick et al., 1980). (ii) those that were blocked in protein traffic through the Golgi complex (i.e.

sec7 and secl4 mutants); (iii) those mutants that were defective for fusion

of Golgi complex-derived secretory vesicles to the plasma membrane (10 complementation groups, S E C l , SEC2, SEC3, SEC4, SECS, SEC6, SEC8, SEC9, SEClO and SECI.5). Mutations in two complementation groups, SECI1 and SEC19, exhibited noteworthy terminal phenotypes. The secll mutants failed to exaggerate any organelle membranes whereas the secl9 mutants exaggerated all of them. That these class A mutants affected the function of the same pathway was confirmed by epistasis analyses where the appropriate sec double-mutant combinations were genetically constructed and, upon imposition of the 37°C block, visualized for terminal phenotype. Novick et al. (1981) found that the ER-blocked mutations were epistatic to the Golgi complex-block and vesicle-block lesions, and that Golgi-complex blocks were epistatic to the secretory vesicle blocks. This is consistent with the concept of a linear pathway (ER -+ Golgi complex + vesicles) as opposed to a concept of independent parallel pathways. Moreover, these epistasis analyses established the relative order of the first execution points of these SEC gene products on the pathway, and demonstrated the relative order of organelle involvement. This temporal order of organelle involvement on the secretory pathway was further

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supported by the studies of Esmon et af. (1981), who showed that assembly of oligosaccharide chains on secretory glycoproteins in yeast occurred in defined stages. Glycoproteins blocked at the E R possessed immature (i.e. core) oligosaccharide chains and Golgi complex-blocked glycoproteins had either more extensively (but nonetheless incompletely) modified oligosaccharide chains (i.e. the case of sec7), or fully modified sugar chains (i.e. s e c l l ) . Glycoproteins blocked at the secretory vesicle stage appeared uniformly to exhibit fully matured glycosyl chains (Esmon et al., 1981). Thus, class A sec mutants provided the means by which the general form of the yeast secretory pathway could be demonstrated. Furthermore, these mutants provided a convenient starting point for biochemical and genetic strategies aimed at determining the mechanisms that underlie stage-specific secretory protein transport. Much of this review is devoted to a discussion of what the second-generation experiments with these mutants have revealed with respect to protein-transport mechanisms. 111. Protein Transport from the Cytoplasm into the Endoplasmic Reticulum

The biochemical identity of intracellular organelles is in part a function of the unique sets of proteins that reside within each of them. At one level, the organelle membranes serve as impermeable barriers to diffusion of such resident proteins (although the problem of protein retention is still a very real one; see below). This barrier function for organelle membranes is not compatible with the first event that signifies entry of a polypeptide into the secretory pathway, that is, export of proteins from their cytoplasmic site of synthesis into the E R lumen. The problem of protein translocation across the first membrane is one that is faced by all cells, and it is one that has received a great deal of experimental scrutiny in both mammalian and prokaryotic systems. A. T H E PARADIGMS; MAMMALIAN AND PROKARYOTIC MODELS

Protein export from the cytoplasm can be considered to involve four conceptually distinct events: (a) the initial sorting event where proteins destined for export are recognized by a cytoplasmic machinery by virtue of N-terminal signal peptides that are elaborated by precursor forms of such secretory proteins, (b) delivery of the engaged secretory precursor polypeptides to the target membrane, (c) translocation of the polypeptides across the membrane (possibly through a proteinaceous pore), and (d) proteolytic processing, or maturation, of the translocated proteins (Blobel and Dobberstein, 1975).

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In mammalian systems, it is the signal recognition particle (SRP) that is involved in serving as a cytoplasmic adapter between nascent secretory polypeptide chains and the protein translocation apparatus of the ER membrane (Fig. 2; for a review, see Walter and Lingappa, 1986). The SRP is a ribonucleoprotein particle that sediments at 1Is in sucrose velocity gradients, has an affinity for ribosomes, and consists of a 300-nucleotide RNA component (termed 7SL RNA) that is complexed with six unique polypeptides of 9,14,19,54,68 and 72 kDa (Siege1 and Walter, 1985). The SRP experiences a dramatic increase in its binding affinity for those ribosomes, presenting an emerging nascent polypeptide chain that exhibits a signal sequence. Subsequent to binding of the signal peptide by the 54 kDa SRP subunit, the SRP-nascent chain-ribosome complex is targeted to the E R membrane. This targeting is realized on the basis of the direct interaction of the 68 kDa/72 kDa SRP subunits with a heterodimeric integral membrane protein of the E R membrane, the SRP receptor. Translocation

FIG. 2. Diagram illustrating the signal hypothesis. This diagram describes thc cotranslational export of a secretory protein from the cytoplasm into the endoplasmic reticulum (ER) lumen. The basic tenets of this scenario have been established by elegant studies in mammalian cell-free systems (Walter and Lingappa, 1986). Nascent polypeptide chains are bound by the signal-recognition particle (SRP) to form a soluble SRP-nascent chain-ribosome complex that is targeted to ER membranes by virtue of the interaction of the SRP with a heterodimeric membrancbound SRP receptor (docking protein). The signal peptide then engages the signalsequence receptor and the SRP is released into the cytosol for participation in another round of ER targeting. Translocation of the nascent polypeptide ensues in a manner that is not at all understood, and signal-peptidecleavage occurs either during or immediately after the translocation step.

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of the nascent secretory polypeptide is believed to ensue in a cotranslational, but otherwise undetermined, fashion. It is not known whether translocation proceeds through a proteinaceous pore or through the membrane bilayer itself. Finally, the signal sequences of secretory polypeptides are proteolytically removed by a signal peptidase, an enzymic activity associated with a complex composed of six non-identical polypeptide chains (Evans et af., 1986). In Escherichia coli, the basic features of the signal hypothesis for protein export are also fulfilled (Bankaitis et af., 1986a). Although there is still some question as to whether there exists an obvious direct analogue to the SRP in E. cofi, the cytoplasmic adapter function appears to be provided by a peripheral membrane protein, the secA gene product (abbreviated to SecAp; Oliver and Beckwith, 1982). This gene product appears to interact with both signal sequence and mature regions of precursor proteins, and exhibits an ATPase activity that is stimulated by acidic phospholipids (Lill et af., 1990). Maximal stimulation of the SecAp ATPase activity also requires the SecYp, an integral protein of the cytoplasmic membrane in E. cofi(Crooke etal., 1988). Thus, one can broadly consider the SecYp to act as the SecAp receptor. It is also clear that proteins can be exported from E. coli in either co- or post-translational modes in vivo (Randall, 1983) and that cytoplasmicfactors, termed chaperonins, are involved in the maintenance of polypeptides in a translocation-competent (i.e. less tightly folded) state, a consideration that is of particular importance to the latter mode of protein export (Collier et al., 1988). While the mechanistic details of the translocation step are entirely unclear, the signal peptidase in E. cofi has been shown to be a 36 kDa polypeptide that is essential for viability of the bacterium (Date, 1983). Conditional defects in signal peptidase function lead to the general accumulation of precursors for exported proteins that have, nonetheless, been translocated across the cytoplasmic membrane (Dalbey and Wickner, 1985). B. GENETIC ANALYSES

Unlike our current understanding of the scheme for protein translocation across the cytoplasmic membrane in E. cofi, which is built upon both powerful in vivo and in vitro evidence, the mamma,lian paradigm is based solely upon in vitro arguments, compelling and elegant though these be. Thus, Sacch. cerevisiae has provided a flexible experimental system that has been amenable to the application of both in vivo and in vitro approaches directed at the study of protein transport from the cytoplasm into the E R lumen. A classic genetic approach to the understanding of such protein transport in yeast required the isolation of mutants unable to export proteins

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into the E R lumen. The initial sec mutant hunt failed to produce such mutants. Fortunately, the insights acquired from genetic selections designed to generate the analogous export mutants in bacteria yielded proven strategies for the positive selection for yeast mutants defective in protein export from the cytoplasm into the E R lumen. 1. The HOL'

Selection

The strategy for isolating mutants of Sacch. cerevisiae that were defective in the cellular machinery that translocates secretory precursors from the cytoplasm into the E R lumen was described by Deshaies and Schekman (1987) and extended by Rothblatt et al. (1989). This selection was analogous to the one devised by Oliver and Beckwith (1982) that yielded the first secA" mutants of E. coli.Briefly, the normally cytoplasmic HIS4 gene product (the HIS4p) was fused to an N-terminal signal peptide provided by the prepro-afactor, the precursor of the secreted a-factor pheromone. This chimera targeted to the E R lumen and, in yeast strains that were otherwise mutant for HZS4, rendered cells incapable of growing on minimal media supplemented with histidinol (rather than histidine). This hol- phenotype presumably reflected the inability of histidinol to gain access into the ER lumen so that the HIS4p chimera (which retained HIS4p enzymic activity) could convert the histidinol to histidine (Deshaies and Schekman, 1987; Rothblatt et al., 1989). A positive selection for HOL+ mutants failing to translocate the HIS4p chimera into the E R lumen was thus generated. By selecting for HOL' derivatives, a number of ts mutants were recovered that exhibited conditional defects in protein translocation from the cytoplasm into the E R lumen.

2. Genes Involved in Protein Translocation Three genes (SEC61 , SEC62 and SEC63) were identified by the HOL" selection (Deshaies and Schekman, 1987; Rothblatt et al., 1989). Thermosensitive alleles of each of these three genes appeared to block protein translocation at the same point in the pathway, that is, prior to translocation into the E R lumen and signal-peptide cleavage. Such mutants were found to accumulate non-glycosylated precursor forms of three secretory proteins (prepro-a-factor, acid phosphatase and invertase) and a vacuolar protein (carboxypeptidase Y). It should be noted, however, that sec62 mutants were only partially defective for invertase transport. Subcellular fractionation experiments indicated that, while the precursor forms exhibited an association with intracellular membranes, these precursors had clearly not been translocated into the E R lumen. This was indicated by the susceptibility of

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the precursors to proteases that had access to the cytoplasmic surfaces, but not lumenal aspects, of intracellular membranes (Rothblatt et al., 1989). Finally, it was observed that the sec6Zfssec63" double-mutant combination resulted in loss of cell viability, whereas the sec6Zfssec62" and sec62" sec63" double-mutant combinations exhibited exaggerated ts growth and translocation defects. On the basis of such genetic interactions, Rothblatt et al. (1989) suggested that these three gene products act along the same functional pathway. Genomic clones of the SEC62 and SEC63 genes have been recovered and characterized. The nucleotide sequence of SEC62 suggested that the SEC62p was a 283-residue polypeptide, predicted to be of 32,381 Da and, for yeast proteins, an unusually basic isoelectric point of 10.7 (Deshaies and Schekman, 1989). O n the basis of the inferred SEC62p primary sequence, Deshaies and Schekman (1989) speculated that this polypeptide did not contain an N-terminal signal peptide that would serve to direct insertion of the SEC62p into E R membranes. Nevertheless, these workers suggested that the SEC62p was an integral membrane protein of the E R , one that spanned the membrane bilayer twice. These suggestions rested on the identification of two potential membrane-spanning domains in the inferred SEC62p primary sequence, and the fact that E R functions were affected (both in vivo and in vitro) under conditions of SEC62p dysfunction (Rothblatt et al., 1989; Deshaies and Schekman, 1989). The idea that the SEC62p is an integral membrane protein of the E R is an attractive one as it would be entirely consistent with the proposed role of the SEC62p as a component of the E R translocation machinery. The demonstration that this protein is exclusively localized in E R membranes, as would also be predicted, still awaits the appropriate fractionation and immunolocalization experiments. In any event, the SEC62p clearly plays a unique and essential cellular function in yeast as sec62 disruption mutations represented recessive, haploid-lethal events (Deshaies and Schekman, 1989). The SEC63 gene was similarly essential for vegetative growth of Sacch. cerevisiae, as reported by Sadler et al. (1989), who obtained genomic clones of SEC63. Interestingly, Sadler et al. (1989) were engaged in a molecular analysis of cellular components (such as the NPLZ gene product) that facilitated import of proteins from the cytoplasm into the yeast nucleus. Mutants conditionally defective in NPLZ gene function were unable to support efficient nuclear-protein transport under restrictive conditions, and the allelism of NPLZ and SEC63 was unambiguously established by genetic criteria. Sequence analysis of DNA has established that SEC63 potentially encodes a 663-residue polypeptide that exhibits three potential membranespanning regions (Sadler et al., 1989). The most interesting aspect of the inferred primary sequence was that a region of 72 amino-acid residues of the

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SEC63p, which accounted for most of the domain bounded by the second and third potential transmembrane segments, exhibited a 43% identity with the N-terminus of the DnaJ protein in E. coli, a member of the heat-shock family in this bacterium. At this point, it is unclear whether the SEC63p region of DnaJ homology is exposed to the yeast cytosol or whether it is exposed to the lumenal E R surface. In either event, this homology suggests that the SEC63p may be involved in maintaining the translocation competence of polypeptides (i.e. a chaperonin function) or may serve to nucleate formation of some sort of translocation complex. The dual function of the SEC63p in E R and nuclear transport still needs to be reconciled, however. One possibility is that the SEC63p serves as a common translocator component for both E R and nuclear membranes with compartment specificity being determined by some other component(s). Alternatively, defects in the SEC63p may cause general dysfunction of the E R and nuclear membranes, which are contiguous, thereby affecting E R and nuclear transport processes indirectly. It is of interest to note that sec6Z and sec62 mutations failed to cause nuclear-transport defects (Sadler et al., 1989). Localization of the SEC63p in an intracellular compartment should help resolve the question of whether the primary function of this gene product is most directly relevant to E R processes, nuclear processes, or both.

3. The Cytosolic Factors Involved in Translocation The HOL’ selection, while yielding mutants that are likely to be defective for the translocation step in protein transport into the E R lumen, failed to generate mutants defective in the earliest steps of this process, namely the recognition and membrane-targeting events that precede the translocation step (see Section 1II.A). The mammalian and prokaryotic paradigms for this process, however, laid some of the groundwork for fruitful “wreck and check” approaches. These strategies have provided key insights into the role for chaperonins in intracellular protein transport, and have revealed promising avenues for further investigation with respect to SRP-like activities in yeast. Deshaies et al. (1988) investigated whether the yeast 70 kDa heat-shock protein (HSP70) homologues found in Sacch. cerevisiae served as molecular chaperones for polypeptides destined for insertion into the E R lumen. The SSA subgroup of the nine HSP70 homologues in Sacch. cerevisiae consists of four genes (SSAZ-SSAQ) whose products execute interchangeable functions in vivo (Werner-Washburne et al., 1987). Although these genes are functionally redundant (and therefore invisible to classical loss-of-function mutant screens), ssal ssa2 double-mutant strains are ts for growth whereas ssal ma2 ssal triple mutants are inviable. Thus, Deshaies et al. (1988)

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engineered galactose-inducible expression of SSAl in the ssal ssa2 ssal triple mutant background and asked whether depletion of SSAlp (induced by glucose challenge of such cells) had any specific consequence for protein transport. Depletion of the SSAlp in vivo resulted in the accumulation of precursor forms of the soluble vacuolar proteinase carboxypeptidase Y (CPY) and the secretory prepro-a-factor. These precursors were blocked in transport at a point prior to their insertion into the E R lumen. The evidence came from their accessibility to exogenously added proteases in cell-free lysates, and their comigration in sodium dodecyl sulphate-polyacrylamidegel electrophoresis (SDS-PAGE) systems with the unmodified forms of the corresponding preproteins obtained from sec61 and sec62 mutants. This precursor accumulation was considered to be specific for two reasons. First, cells ceasing to grow due to glucose-dependent depletion of at least two other essential gene products failed to show a secretory defect. Second, the SSAlp by itself was capable of restoring translocation activity to SSAlpdeficient extracts in vitro (Deshaies et al., 1988; Chirico et al., 1988). These data clearly showed an involvement of the SSA .gene products in protein transport. That this involvement was not limited to E R transport was dramatically demonstrated by the concomitant in vivo defects in transport of the mitochondrial F,ATPase p-subunit into the mitochondrial matrix under SSA-deficient conditions (Deshaies et al., 1988). Given the central role that the SRP occupies in the mammalian scheme for ER protein transport, some effort has been directed at identifying genes whose products could potentially be involved in SRP-like activities in yeast. The two components that have received the most attention to date include the 7SL RNA component of the SRP and the 54 kDa SRP subunit that is thought to be the direct mediator of signal-peptide recognition. Ribes et al. (1988), as well as Brennwald et al. (1988) and Poritz et a f . (1988), described the identification of a 7SL RNA in Schizosaccharomyces pombe, reported the cloning of the structural gene, and found significant structural and primary sequence homologies between the Schiz. pornbe and human 7SL RNA species. Both Ribes et al. (1988) and Brennwald et al. (1988) employed gene-disruption experiments to show that 7SL RNA was essential for vegetative growth of Schiz. pombe. That the 7SL RNA in Schiz. pombe might assemble into an SRP-like particle in vivo was suggested by several biochemical experiments. First , the 7SL RNA was recovered from post-ribosomal supernatants in an 11sparticle. Second, the 7SL RNA from this yeast bound to canine SRP proteins under stringent conditions. Third, the canine SRP proteins of 19 kDa and the 68 kDa/72 kDa heterodimer footprinted to homologous regions of both the mammalian and Schiz. pombe RNAs (Poritz et al., 1988). Interestingly, although the dimorphic yeast Yarrowia lipolytica also contains a 7SL RNA that showed essentially

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the same biochemical properties as that from Schiz. pombe (Poritz et al., 1988), Sacch. cereuisiae was devoid of the 7SL species (Brennwald et al., 1988).Thus, the Schiz. pombe system appears to be the eukaryotic model of choice for dissecting 7SL function in uiuo. The current challenge is to determine whether or not 7SL function is relevant to secretory function in Schiz. pombe. To investigate further the role of SRP polypeptide subunits in ER transport, cDNA clones for the mammalian 54 kDa SRP subunit (i.e. the signal peptide-binding subunit SRP54) were identified and characterized (Bernstein et al., 1989;Romisch et al., 1989). The inferred primary sequence of the SRP54 revealed two domains of interest; an N-terminal domain that contained a potential GTP-binding motif and a C-terminal rnethionine-rich domain that was suggested to form a "methionine-bristle" structure that could serve as a signal peptide-binding groove. Comparisons of the SRP54 primary sequence with available protein data bases revealed several homologies of interest, but the most relevant homology for the purposes of this review was that observed between SRP54 and a 48 kDa protein from E. coli that associates with a 4.5s RNA, in an SRP-like particle, and has been named FFH (fifty-four hornologue) (Bernstein et al., 1989; Romisch et al., 1989; Ribes et al., 1990). Both SRP54 and FFH were significantly homologous over their entire length, and FFH exhibited the putative GTPbinding and methionine-bristle sequence motifs. This similarity suggested the presence of SRP-like subunit polypeptides in organisms that did not produce 7SL RNAs. Hann et al. (1989) took advantage of the highly conserved regions in the GTP-binding domains of mammalian SRP54 (SRP54mam)and FFH to design PCR oligonucleotides for amplification of genomic sequences in both Sacch. cereuisiae and Schir. pombe. While initial attempts to amplify DNA from Sacch. cereuisiae were equivocal, the reactions of Schiz. pombe were productive and ultimately yielded an SRP54 homologue (SRP54"P) of this yeast. Nucleotide-sequence analysis indicated that SRP54'P (the designation given to SRP54 from Schiz. pombe) was composed of 522 amino-acid residues of 57 kDa and a predicted isoelectric point of 9.9 (Hann et al., 1989). Moreover, comparison of the inferred SRP54""" and SRP54sp primary sequences revealed additional homologies that ultimately yielded a productive strategy for identification and cloning of SRP54", the Sacch. cerevisiae SRP54 homologue. This homologue was predicted to be a basic protein (pZ9.5) of 541 residues of 60 kDa. The yeast and mammalian SRP54 species were notably homologous over their entire primary sequence, and both yeast SRP54 homologues exhibited recognizable GTP-binding and methionine-bristle domains. That at least SRP54" played an essential in uiuo function was indicated by the demonstration that disruption of the

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SRP54" structural gene is a haploid-lethal event in Sacch. cerevisiae. It is anticipated that the same conclusion will be reached for SRP54'P. Further delineation of SRP54 function in vivo awaits construction of the appropriate conditional mutants defective in SRP54"" and SRP54'P function, and the test of whether such defects lead to dysfunction in transport of proteins into the ER lumen. However, it is tempting to believe it likely that such will be the case. Hann and Walter (1990) found that SRP54'P was associated in vivo with 7SL RNA in Schiz. pombe, in direct analogy to the mammalian SRP, whereas SRP.54" associated in vivo with RNA in Sacch. cerevisiae of some 600 bp in size and sedimented as a 16s ribonucleoprotein particle. 4. Signal-Peptide Processing

Transport of a secretory protein into the E R lumen is marked by cleavage of the N-terminal signal peptide by a highly specific endoprotease, the signal peptidase. As already noted, vertebrae signal peptidases are multisubunit complexes. Mammalian signal peptidase consists of six unique polypeptide subunits whereas the signal peptidase from the hen oviduct consists of two unique polypeptide subunits (Evans et al., 1986; Baker and Lively, 1987). The signal peptidase in E. coli is an ectopic 36 kDa protein of the cytoplasmic membrane (Wolfe et al., 1983). The signal peptidase is an important component of the transport process as the rate of protein export from the E R is greatly hastened by signal peptide cleavage, even though translocation into the E R lumen does not depend upon the processing event. Finally, the processing event displays remarkable fidelity and conserved specificity. Prokaryotic and eukaryotic signal peptidases are able to process correctly and efficiently precursors of either prokaryotic or eukaryotic origin in vitro (Bankaitis et al., 1986a). Haguenauer-Tsapis and Hinnen (1984) and Schauer et al. (1985) demonstrated that secretory proteins which fail to be processed exhibit dramatically lower rates of transit through the yeast secretory pathway. These results suggested that signal-peptidase mutants would, as a result of the processing dysfunction, exhibit a general delay in protein secretion, rather than a stagespecific secretory block. Interestingly, Bohni et al. (1988) discovered that seclltS mutants exhibited the appropriate behaviour expected for signalpeptidase mutants. Note that secll mutants were the only sec mutants that failed to exaggerate an intracellular organelle when challenged with the restrictive condition (see above). At the non-permissive temperature, pulseradiolabelled polypeptides accumulated as core-glycosylated species that had entered the E R lumen in secll" strains. However, these coreglycosylated species retained their signal peptides. Bohni et d. (1988) recovered SECIl clones, and found that the SECII nucleotide sequence

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predicted an 18.8 kDa polypeptide containing a putative N-terminal signal peptide and one potential asparagine-linked glycosylation site. As a result, the SECl l p exhibited some superficial similarity with the 22-24 kDa henoviduct signal-peptidase glycoprotein subunit and the two canine signalpeptidase glycoprotein subunits of 22 kDa and 23 kDa. These vertebrate glycoprotein subunits uniformly contained a polypeptide backbone of 19 kDa, but there was no significant primary sequence homology detected between the S E C l l p and the canine glycoprotein subunit (Deshaies et al., 1989). It remains to be determined whether or not the yeast signal-peptidase exists in a multisubunit complex.

c. In Vitro SYSTEMS Although “wreck and check” approaches have shown some promise, it is desirable to have functional assays at one’s disposal for a biochemical dissection of protein transport. To this end, powerful in vitro systems for monitoring protein transport into the E R have been developed. The current efforts with these systems follow the classical resolution and reconstitution strategy. The systems for in vitro translocation of yeast secretory proteins into the ER lumen were nearly simultaneously developed by three research groups (Rothblatt and Meyer, 1986a; Hansen et af., 1986; Waters and Blobel, 1986). The basic features of these similar systems involved generation of radiochemically pure precursor substrates by specifically programmed in vitro translation systems, incubation of yeast microsomes with the labelled substrate, and determination of whether translocation into the microsome lumen had occurred. Three operational criteria are generally applied for signifying translocation: (a) core glycosylation of substrate, (b) acquisition by the substrate of resistance to exogenously provided protease, and (c) processing of the substrate by signal peptidase. Under optimal conditions, it was found that some 40% of the total input prepro-a-factor substrate could be translocated into E R microsomes. A truncated form of secretory invertase could also be faithfully translocated into E R microsomes in vitro (Rothblatt and Meyer, 1986a). One of the interesting findings that was forthcoming from characterization of the in vitro system was that, in contrast to mammalian in vitro translocation systems, the prepro-a-factor was capable of being translocated in an entirely post-translational manner in the yeast system, and that the efficiency of the post-translational mode of transport was similar to that of transport measured in the cotranslational reaction (Hansen et al., 1986; Waters and Blobel, 1986). The ability to undergo the post-translational reaction was substrate-specific, however (Rothblatt et al., 1987; Hansen and Walter, 1988). As an illustration of this,

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prepro-carboxypeptidase Y (CPY) and a truncated pre-invertase were efficient post-translational translocation substrates, while full-length preinvertase could be translocated in vitro only in cotranslational reactions (Hansen and Walter, 1988). Regardless, the ability to translocate certain substrates post-translationally effectively uncoupled transport from protein synthesis, and permitted a strict analysis of the energy requirements for transport. It is clear that ATP was required to support transport, perhaps by fuelling an ATP-driven translocase activity (Waters and Blobel, 1986; Hansen et al., 1986; Rothblatt and Meyer, 1986b). Non-hydrolysable analogues of ATP were unable to support transport, while maintenance of a membrane potential was not required for translocation. The process was refractory to challenge by a number of ionophores or other uncouplers (Hansen et al., 1986; Waters and Blobel, 1986). The availability of the in vitro translocation system in Sacch. cerevisiae permitted the pursuit of several different lines of investigation directed at addressing the question of whether the E R transport scheme in yeast operates via the same mechanism as the one described by the mammalian paradigm. One line of investigation involved an attempt to resolve the in vitro system by methods that were successful in resolving key aspects of the mammalian reaction. Another approach has been to characterize the in vitro defects of the appropriate sec mutants in the transport reaction. The first strategy was employed by Rothblatt and Meyer (1986a), who tested whether washing yeast microsomes with solutions containing high concentrations of salt, or proteolytic treatment of such microsomes with elastase, compromised the translocation competence of microsomes. Unfortunately, neither did so. It should be noted that the microsomal salt wash fractionated the mammalian SRP, whereas elastase treatment of microsomal membranes yielded an assay for the mammalian SRP receptor (reviewed in Walter and Lingappa, 1986). However, in contrast to the negative results obtained with these treatments, the experiments of Hansen et al. (1986) demonstrated a requirement for microsomal membrane proteins in the transport reaction. Pretreatment of microsomes with the sulphydryl alkylating agent Nethylmaleimide rendered the microsomes unable to support translocation. The identities of the essential components that are sensitive to the alkylating agent remain to be determined. A combination of the “wreck and check” and in vitro resolution strategies holds considerable potential. It will be of great interest to determine whether depletion of SRP54” from the in vitro reaction has any deleterious consequences for translocation, and whether such consequences are specific for proteins that employ exclusively the cotranslational mode of transport. The in vitro translocation system has also permitted a more complete characterization of mutants that were defective in their ability to translocate

88

A. E. CLEVES AND V. A. BANKAlTlS

proteins from the cytoplasm into the E R lumen. In particular, since the cytosolic components of the reaction are readily separated from the microsomal components of the reaction, it has been possible to assign the relevant translocation defects to one of the two specific components. For instance, the sec62, sec62 and sec63 mutants were defective in ER translocation, while nucleotide sequences of the SEC62 and SEC63 gene products suggested that both of these were integral membrane proteins of the E R (see Section III.B.2). Confirmatory evidence has been obtained from in uitro analysis of the sec62 and sec63 mutants. Rothblatt et al. (1989) demonstrated that, while cytosol fractions prepared from sec63 cells were competent to support translocation of prepro-a-factor into wild-type microsomes, even a short pre-incubation of sec63 microsomes at the nonpermissive temperature markedly decreased the efficiency of prepro-afactor translocation, even with wild-type cytosol. Similar results were reported for sec62 with the added caveat that sec62 microsomes were generally defective in the translocation assay (Deshaies and Schekman, 1989). Chirico et al. (1988) fractionated the cytosolic component of the reaction into two distinct activities, one sensitive and the other resistant to Nethylmaleimide. Both activities were required for efficient post-translational transport of prepro-a-factor into E R microsomes. The N-ethylmaleimideresistant factor consisted of the two constitutively expressed 70 kDa heatshock proteins of yeast, the SSAlp and SSA2p. Deshaies et al. (1988) similarly reproduced in uitro the in uiuo secretory defect associated with SSAp depletion, and demonstrated reconstitution of the translocationsustaining activity of the depleted cytosol by addition of the SSAlp. That the SSA proteins function to maintain the prepro-a-factor translocation substrate in a transport-competent state was suggested by the finding that pretreatment of the substrate with urea increased the translocation rate (Chirico et al., 1988). The cytosolic N-ethylmaleimide-sensitive translocation factor has not yet been suitably resolved for biochemical analysis. IV. Protein Trafficking from the Endoplasmic Reticulum to the Golgi Complex

Once a polypeptide has been translocated into the E R lumen, it has essentially overcome the major topological obstacle. That is, the polypeptide no longer has to traverse a lipid bilayer en route to its ultimate destination. As a result (sorting and retention considerations aside), an analysis of further progression through the secretory pathway becomes a matter of understanding: (a) the biogenesis of transport vesicles on the donor

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89

compartment surface, (b) the targeting of the transport vesicles to the correct acceptor membrane, and (c) fusion of the transport vesicles to the acceptor membrane. Within the broad framework of such a model, one can envisage two general types of components. Firstly, there are those that are commonly employed by various compartments for vesicle metabolism. Secondly, there are those that play compartment-specific roles in vesicular traffic. A . THE MAMMALIAN PARADIGM

A general diagram depicting the prevailing view of steps involved in vesiclemediated intercompartmental protein transport is shown in Fig. 3. The basic form of a single round of vesicle transport was determined by elegant biochemical and morphological studies of protein transport between Golgicomplex compartments in vitro (Orci et af.,1989; Rothman and Orci, 1990). Rothman and his colleagues (for a review, see Rothman and Orci, 1990) proposed that transport vesicles budding from the donor membrane are driven by an ATP-dependent assembly of vesicle-coat protein subunits that ultimately cause pinching off of a coated transport vesicle. Upon docking to the target membrane, the vesicle is uncoated and allowed to enter the fusion pathway. Biochemical studies have revealed that ATP, cytosolic factors, and long-chain fatty-acyl-CoA esters are required for both vesicle budding and for fusion of uncoated vesicles to their target membranes. An N-ethylmaleimide-sensitivefactor (NSF), which is a 76 kDa ATP-binding protein that functions as a tetramer, is required for fusion. The function of this factor requires participation of other protein cofactors, some of which belong to a family of three 36 kDa polypeptides that promote attachment of the factor to membranes known as the soluble NSF attachment proteins (a-, b- and y-SNAPS). Uncoating of transport vesicles occurs at the target membrane and involves GTP-binding proteins, as shown by the finding that a non-hydrolysable analogue of GTP (i.e. GTPyS) inhibited the uncoating reaction. The cytosolic factors identified by resolution of the in vitro transport reaction in the Golgi complex may well represent factors that are commonly employed in budding-fusion reactions. The NSF is already known to be required for fusion of Golgi complex-derived vesicles to Golgicomplex compartments (Malhotra et af., 1988), fusion of ER-derived vesicles to Golgi-complex membranes (Beckers et a f . , 1989) and fusion of endocytic vesicles (Diaz et al., 1989). This will likely prove to be true for the SNAPS as well. That there has been a basic functional conservation in the components that drive intercompartmental protein transport has been demonstrated by Dunphy et af. (1986). Yeast cytosol can substitute for the cytosolic requirement in reactions involving mammalian membranes. As a

90


Acceptor Compartment

I I I

Donor Compartment


91

result, yeast provides a facile system with which to study the intercompartmental protein transport problem. A detailed biochemical dissection of transport from the E R to the Golgi complex in mammalian cells has been carried out by Balch and his colleagues (Beckers et al., 1990). These workers have established that transport can be divided into two stages. The first is considered to involve passage of a secretory protein through an early step that requires ATP, NSF, and GTP-binding proteins. It is thought that this stage describes the budding of transport vesicles from the E R surface, subsequent passage of secretory proteins through a still poorly understood intermediate compartment that is operationally defined as the site at which protein transport is blocked in mammalian cells by challenge with cold temperatures (lS0C), docking of transport vesicles to cis-Golgi-complex membranes, and subsequent uncoating of these docked transport vesicles. The second stage is thought to describe late events associated with fusion of vesicles to the cis-Golgicomplex membranes. Calcium ions were cofactors for this stage. Moreover, a role for GTP-binding proteins in fusion of uncoated vesicles to cis-Golgicomplex membranes was suggested by the sensitivity of this late stage to inhibition by a synthetic peptide homologous to the effector domain of rub proteins, a family of small GTP-binding proteins (Beckers et af., 1990; Pliitner et af., 1990). B . RECONSTITUTION OF THE YEAST PROCESS

In Vitro

I. Characterization of the Assay Reconstitution of transport from the E R to the Golgi complex in vitro was independently developed in the laboratories of Randy Schekman (Baker et af., 1988) and Susan Ferro-Novick (Ruohola et al. , 1988). The development of these in vitro systems rested on two technological advances. First, there FIG. 3 Diagram illustrating a single round of vesicular transport. The paradigm showing how proteins are shuttled from one compartment to another in a transport vesicle-dependent manner has been established by both biochemical and morphological criteria (Rothman and Orci, 1990). The cycle involves an ATP- and longchain acyl-CoA-dependent recruitment of cytosolic transport factors (CF) that catalyse formation and targeting of a transport vesicle. Upon engagement of the transport vesicle with the acceptor membrane, an uncoating reaction occurs in a manner that involves GTP-binding protein function, as shown by the inhibition of this particular step by a non-hydrolysable analogue of GTP (i.e. GTPyS), and vesicle-associated factors are liberated into the cytosol for another round of vesicle transport. Finally, fusion of uncoated vesicles to acceptor membranes requires the function of an N-ethylmaleimide (NEM)-sensitivefusion factor, with ATP and longchain fatty-acyl-CoA acting as cofactors.

92

A . E. CLEVES A N D V. A . BANKAlTlS

was the demonstration that radiochemically pure prepro-a-factor was an efficient post-translational translocation substrate (see above), which yielded an unambiguous method for following transport of labelled substrate from the ER to the Golgi complex. Second, a method for preparing “semi-intact” yeast cells by gentle osmotic lysis of sphaeroplasts was developed. These “semi-intact” cells were essentially intact from a cellular standpoint in that the major organelles remained structurally unperturbed and organized. However, the cytoplasm had been released from the cells through perforations in the plasma membrane. Thus, one could now prepare “semi-intact” yeast cells, add radiolabelled prepro-a-factor as a translocation substrate which had access to the cell interior, and subsequently follow transport of the substrate. In this system, glycosylation was again used as a criterion by which to establish that transport occurred. Core glycosylation of the 19 kDa prepro-a-factor resulted in conversion to a 26 kDa form, and this was evidence for transport into the ER. Conversion of the ER form to a heterogeneous higher molecular-weight form that was recognized by antibodies specifically directed against a-( 1+6)-mannose linkages indicated that transport from the ER to the Golgi complex occurred. Incubation of substrate, washed “semi-intact” cells (i.e. membranes), yeast cytosol, an ATP-regenerating system and GDP-mannose (the sugar donor) resulted in conversion of some 25% of the substrate into an antia-( 1+6)-Man serum-precipitable (i.e. Golgi complex) form. This was evidence for an efficient transport of substrate from the ER to the Golgi complex in the in vitro reaction since only 50% of the input substrate entered the ER and was therefore available for transport from the ER to the Golgi complex (Baker et al., 1988; Ruohola et al., 1988). Further characterization of the in vitro system revealed that low-temperature (10°C) incubation did not significantly affect translocation of substrate into the ER, but did effectively preclude transport from the ER to the Golgi complex (Baker el al., 1988). This was an important finding since it permitted analysis of transport as a two-stage reaction. This involved incorporation of substrate into the ER lumen in a 10°C first-stage reaction, subsequent isolation of the donor compartment in a form that was substantially free from cytosolic factors, followed by a warming of the reaction in the presence of fresh cytosol so that the second-stage ER-to-Golgi complex leg of the reaction could be completed. This effectively uncoupled the translocation reaction from the ER-to-Golgi complex transport reaction, and paved the way for a strict analysis of the requirements for the latter. These can be summarized as follows (Baker et al., 1988). First, ATP was required for transport from the ER to the Golgi complex, as apyrase treatment of the second reaction stage completely inhibited the


93

ability of substrate to acquire precipitability with a-(l+6)-Man antibodies. Second, GDP-mannose stimulated the reaction but was not required to support it. Third, pretreatment of membranes with the non-ionic detergent saponin completely destroyed transport activity, indicating that organelle integrity was necessary for transport. Fourth, transport was stimulated some six-fold by addition of cytosol. This reflected the involvement of cytosolic proteins, as indicated by the finding that treatment of cytosol with N-ethylmaleimide, heat (15 minutes at 95°C) or trypsin destroyed the stimulatory activity. Fifth, transport was greatly inhibited by addition of GTPyS ( K i 5 p ~ to ) the reaction, and this inhibition was overcome by concomitant addition of excess GTP, suggesting the involvement of GTPbinding proteins in the process. Finally, E G T A was found to inhibit a late step in transport from the E R to the Golgi complex, signifying the requirement for calcium ions as a necessary cofactor in the reaction (Baker et a f . , 1990). Thus, transport from the E R to the Golgi complex in Sacch. cerevisiae exhibited similar requirements to those possessed by the mammalian reaction. That the yeast in vitro reaction faithfully reconstituted bonafide transport from the E R to the Golgi complex was indicated by two independent lines of evidence. First, the Golgi-complex form of the substrate was sequestered within a compartment that could only be sedimented at 100,OOOg. In contrast, the core-glycosylated ER form of the substrate was recovered only in membranes that sedimented at much lower gravitational forces. Furthermore, the E R marker, NADPH4ytochrome-c reductase, was almost quantitatively recovered with the rapidly sedimenting membranes (Baker et af., 1988). These data provided a strong indication of intercompartmental transport of the substrate. Second, it was demonstrated that lysates prepared from sec mutants that were conditionally defective for transport from the E R to the Golgi complex in vivo failed to support such transport in vitro (Baker et a f . , 1988; Ruohola et af.,1988). In secl2 and secl8 mutant lysates, transport was defective at all temperatures. In lysates of secl2 mutants, addition of wild-type cytosol failed to have any restorative effect (as might be expected when membranes are defective) whereas, in lysates of secl8 mutants, supplementation with wild-type cytosol exhibited a rather mild stimulatory effect. The behaviour of sec23 mutant lysates in the in vitro reaction was especially noteworthy. The ts defect in transport from the E R to the Golgi complex observed for sec23‘”mutants in vivo was reproduced in vitro (Baker et al., 1988; Ruohola et a f . , 1988). This in vitro defect was complemented by wild-type cytosol (indicating that the SEC23p was a cytosolic factor) and provided a biochemical complementation assay by which the functional SEC23p was partially purified (Hicke and Schekman, 1989). It was found that the SEC23p exibited a monomeric molecular mass

94


of 85 kDa and copurified with a 105 kDa polypeptide in a large complex (approximately 400 kDa) (R. Schekman, personal communication). 2. Isolation of Transitional Vesicles that Execute Transport from the Endoplasmic Reticulum to the Golgi Complex

Biochemical characterization of the transport vesicles that shuttle between the E R and the Golgi complex has been hindered by their elusive nature. Groesch etal. (1990) took advantage of the observation that these transport vesicles were released from “semi-intact” cells under transport-competent conditions, and could be captured prior to their engagement with acceptor Golgi-complex membranes. Operationally, these vesicles were first identified as a population of substrate molecules that had escaped from cell membranes into the supernatant fraction (Ruohola et al., 1988). Subsequent characterization indicated that the released substrate was pelleted upon centrifugation (around 100,000g) and was resistant to digestion by exogenous protease in the absence (but not the presence) of detergent (Groesch et al., 1990). These data indicated sequestration of released substrate in vesicles. That the vesicles were of a homogeneous population was indicated by equilibrium centrifugation experiments that demonstrated recovery of the vesicles in a single, sharp peak corresponding to the 40% ~ ) et al., 1990). The sucrose region of the gradient (1.1764 g ~ m - (Groesch density of the putative transport vesicles was considerably different from that of the heavier E R membranes, thereby distinguishing the two compartments. Moreover, the yeast E R marker enzyme hydroxymethylglutarylCoA reductase (isozyme 1) was not enriched in the vesicle fraction. This finding eliminated the possibility that such vesicles arose by adventitious fragmentation of the ER. Finally, these vesicles behaved as true transport intermediates by yet another criterion. They were competent to undergo fusion with acceptor Golgi-complex membranes in an ATP- and cytosoldependent manner. Detailed characterization of these transport vesicles now awaits their purification in sufficient quantities so that the protein components that define the vesicle surface can be identified and their precise functions determined. C. MOLECULAR ANALYSIS OF GENES WHOSE PRODUCTS STIMULATE TRANSPORT FROM THE ENDOPLASMIC RETICULUM TO T H E GOLGl COMPLEX

As indicated above, transport from the E R to the Golgi complex can be categorized into three steps: (a) budding of transport vesicles from the cell surface, (b) targeting of the vesicles to the Golgi complex, and (c) fusion of the transport vesicles to the acceptor membrane. A complementary


95

approach to an in vitro resolution of the various factors that govern such events has been the detailed analysis of sec mutants that are defective in the transport process in vivo. Nine sec mutants (secl2,secl3, secl6, secl7, secl8, sec20, sec21, sec22 and sec23) and two bet mutants exhibit such a defect. A genetic analysis of these mutants has yielded key insights into which of the three events associated with intercompartmental protein transport is likely to be defective. 1. Early SEC Gene Products Define Functions Involved in Vesicle Budding

and Fusion

Kaiser and Schekman (1990) undertook a careful morphological analysis of intracellular membrane structure in wild-type Sacch. cerevisiae and ERblockedsectsmutants of this yeast. The aim was to look for subtle differences in the terminal phenotypes of these mutants to assess the nature of their block in transport from the E R to the Golgi complex. It was found that these nine sec mutants could be categorized into two general categories on the basis of the terminal phenotype. The class-I mutants (secl2,secl3, secl6 and sec23) exhibited an exaggeration of the E R , but did not exhibit any significant proliferation of small vesicles. Class-I1 mutants (secl7, secl8, sec20, sec21 and sec22), in addition to the exaggerated E R phenotype, also exhibited an approximately five-fold increase in the average number of cytoplasmic vesicles with respect to the average number of such vesicles measured in wild-type and class-I mutant strains. The mean diameter of these vesicles was approximately 50 nm, in contrast to the dimensions of Golgi complex-derived secretory vesicles that measured some 80 nm in diameter. The terminal phenotypes were not allele-specific for the gene in question, suggesting that the 50 nm-vesicle proliferation was a gene-specific property. Moreover, class-I phenotypes were epistatic to class-I1 (i.e. vesicle-proliferating) terminal phenotypes. These epistatic relationships suggested that the class-I sec gene products acted at an execution point that preceded the execution point of the class-I1 gene products. On the basis of these data, Kaiser and Schekman (1990) suggested that the class-I sec blocks were exerted at the level of vesicle formation on the E R surface, whereas the class-I1 sec blocks were exerted at the level of transport-vesicle consumption. It should be noted, however, that vesicle proliferation was not as exaggerated for the sec20 and sec21 mutants compared with that observed for the other class-I1 mutants. The distinction between class-I and class-I1 ER-blocked sec mutants on the basis of morphological criteria was reflective of a fundamental difference between the execution points of the corresponding gene products. Construction of a number of haploid sec" double mutants indicated that combinations

96

A. E. CLEVES A N D V . A . B A N K A l T l S

of class-I sec" and class-I1 sectsmutations in haploids did not compromise cell viability and did not cause any exaggerated ts phenotypes. As a general rule, however, haploid cells were unable to tolerate double-mutant combinations restricted to class-I or class-I1 sectsalleles (Kaiser and Schekman, 1990). This intolerance was usually manifested by non-viability but, in a few instances, caused a significant depression in restrictive temperature and general sickliness of the double mutants. In the latter cases, the general morbidity of the double mutants was directly correlated to dramatic decreases in the efficiency of protein transport from the E R to the Golgi complex in vivo. Thus, these synthetic lethalities revealed a set of genetic interactions that redefined precisely the same two sect.* mutant classes that were established on morphological grounds. These findings suggested the occurrence of intraclass functional interactions between members of the class-I gene products and between members of the class-I1 gene products. An extreme extension of this reasoning leads to the suggestion that the intraclass interactions are of a direct nature and reflect the formation of a transport vesicle-budding and fusion machinery, respectively. The two bet mutants (betl and bet2) were isolated via a ['Hlmannose suicide selection and found to exhibit ts defects in protein transport in vivo from the E R to the Golgi complex, and to exaggerate the E R when challenged with restrictive conditions (Newman and Ferro-Novick, 1987). Subsequent characterization of the relationship between BETI and early SEC gene function revealed a pattern of genetic interactions that suggested a direct role for the B E T l p in transport from the E R to the Golgi complex. The relevant findings are summarized as follows (Newman et al., 1990). Genetic crosses revealed that betl sec22 double-mutant haploids were nonviable. That this synthetic lethality was reflective of some real functional relationship between these genes was further supported by the observation that overproduction of the BETI product suppressed the sec22'" defect. Overproduction of the B E T l p was also noted to have a weak suppressive effect on sec2ItS. Another novel gene ( B O S I ) was identified by virtue of its high dosage-mediated suppression of betl'", but not lethal betl null, mutations. Furthermore, BOSlp overproduction also suppressed se~22'~ (Newman et al., 1990). Deciding whether or not BET1 and SEC22 gene products interact directly awaits co-immunoprecipitation experiments. Regardless, the observed genetic interactions of BETl with a class-I1 SEC gene implies a class-I1 designation for BETl as well.

2. Characterization of Early SEC Gene Products A detailed molecular analysis has been carried out for three of the nine SEC genes whose products stimulate transport from the E R to the Golgi


97

complex; these are SEC12, SEC23 and SEC18. In at least one case, such an analysis has provided a penetrating insight into the role of the SEC gene product in the transport process. Yeast genomic SECI2 clones were obtained by complementation of the sec12-4'' mutation, and verified by integrative genetic mapping strategies (Nakano et af., 1988). Nucleotide-sequence analysis demonstrated that SECZ2 contained an open reading frame with the potential to encode a 471residue hydrophilic protein, and that the SEC12p exhibited one potential membrane-spanning domain located towards the C-terminus of the protein. Unfortunately, protein similarity searches have not provided any meaningful homologies that might provide some insight into the function of the SEC12p. Nonetheless, the SEC12p plays an essential role in yeast vegetative growth as shown by the lethality associated with secl2 null mutations (Nakano et af., 1988). Immunodetection of the SEC12p has revealed some interesting features concerning biogenesis of this protein (Nakano et af.,1988). First, the protein could only be detected in yeast cells that were overproducing it. These data indicated that the SEC12p was not an abundant protein, and this conclusion was consistent with the estimate that there were only two to four SEC12 mRNA transcripts in each haploid yeast cell. Second, the observed SEC12p molecular mass (70 kDa) was considerably larger than the one predicted from the SEC12 nucleotide sequence (52 kDa). This discrepancy was due to glycosyl modification of the gene product at one or two N-linked sites, and additional glycosylations of presumably the O-linked variety. Interestingly, even though the SEC12p exhibited both types of glycosyl modification at short times after synthesis (as a 65 kDa form), the protein experienced a slow but progressive glycosylation with time until it finally achieved its mature apparent molecular mass of 70 kDa some three hours after synthesis. This slow maturation did not involve acquisition of N-linked oligosaccharide chains, was blocked by cycloheximide treatment (thereby revealing a role for ongoing protein synthesis) and was also blocked by the secZ8'' defect, suggesting participation of the Golgi complex in the slow terminal maturation process. A role for the Golgi complex in SEC12p biogenesis was further suggested by immunoelectron-microscopy experiments that demonstrated a specific labelling of exaggerated sec7 Golgi-complex stacks (see below) with gold-labelled SEC12p antibodies. The significance of this finding was somewhat diluted, however, by the inability to visualize the gene product in wild-type cells. Therefore it remains unclear as to what fraction of the total cellular SEC12p is localized in the Golgi complex at any given time. Finally, subcellular fractionation experiments indicated an integral membrane location for the SEC12p, and that the product was predominantly localized in a rapidly sedimenting membrane fraction that contained the bulk of the

98


cellular E R (Nakano et al., 1988). The finding that the SEC12p is an integral membrane protein was consistent with the in vitro data which indicated that secZ2 membranes were defective in transport from the E R to the Golgi complex (Section 1V.B). However, the class I designation for secZ2 mutants is more readily incorporated into a scenario where the SEC12p is found in ER rather than exclusively Golgi-complex membranes. It should be noted that the SEC12p could be functionally localized in both Golgi-complex and ER membranes, a situation that might be indicative either of a recycling of the gene product between the E R and Golgi-complex membranes or the existance of multiple execution points with only the first one being phenotypically apparent. This last point is treated in more detail later in this review. Genomic clones of SEC23 have also been obtained and characterized (Hicke and Schekman, 1989). Sequence analysis demonstrated that SEC23 had the potential to encode a hydrophilic protein of 768 amino-acid residues (M,85,400) that did not exhibit any obvious candidate signal peptides or membrane-spanning regions. While protein similarity searches failed to reveal any informative homologies, it was noted that the SEC23 sequence was identical with that of NUCZ, a yeast gene identified by mutants defective in nuclear transport. While the complete ramifications of this identity remain unclear, some general remarks to this effect were made for the analogous SEC63-NPLZ identity (see Section III.B.2). In any event, the SEC23p was essential for yeast vegetative growth, as shown by the lethality associated with sec23 null mutations (Hicke and Schekman, 1989). During the course of these gene-disruption experiments it was noted that the heterozygous sec23'"l~ec23~ condition caused death of diploid cells, even though the mutant protein exhibited a wild-type abundance. On the basis of this intriguing observation, Hicke and Schekman (1989) suggested that the SEC23p was a limiting factor for cell growth. Immunodetection of the SEC23p confirmed the basic inferences that were forthcoming from the SEC23 nucleotide-sequence data. The protein was found to be unglycosylated with a monomeric molecular mass of some 84 kDa. Although subcellular fractionation experiments indicated that the SEC23p from both wild-type and mutant strains sedimented with membranes at 100,000g in p H 6.5 buffer, subsequent analyses demonstrated that the association of the SEC23p with intracellular membranes was of a peripheral nature (Hicke and Schekman, 1989). First, the SEC23p was completely digested by protease in cell-free lysates where the integrity of intracellular organelles was maintained. These data indicated a cytoplasmic location for the SEC23p. Second, the SEC23p was rendered at least partially soluble by treatment of the lysate with 2.5 M urea, 0.5 M potassium acetate, 25 mM EDTA or buffer (pH 7.5). Release of the SEC23p under these


99

conditions was also consistent with the behaviour of a peripheral, rather than integral, membrane protein. While the precise biochemical function of the SEC23p is presently unresolved, on the basis of purification it seems likely that it functions in a multicomponent complex (Section IV.B.1), perhaps at the level of stimulation of transport-vesicle formation (i.e. SEC23 is a class-I gene: see Section IV.C.1). While molecular analysis of the SEC12 and SEC23 genes has not yet yielded remarkable insights into SEC12p and SEC23p function, molecular analysis of SECZ8 has converged with in vitro studies of intercompartmental protein transport in mammalian systems to provide several key findings relating to the enzymology of transport-vesicle metabolism (Rothman and Orci, 1990). The SECI8gene was characterized and its product identified by Eakle et af. (1988). This gene was found to encode potentially for an 84 kDa polypeptide that was essential for cell growth, as shown by the haploid-lethal nature of secZ8 null mutations. Immunoprecipitation experiments utilizing SEC18p-specific antisera revealed two cross-reacting species in yeast lysates. One exhibited a molecular mass of 84 kDa whereas the other was an 82 kDa polypeptide. These SEC18p species appeared to arise from differential translation-initiation events rather than heterogeneity in transcription-start sites or some other sort of processing events. Both SEC18p species fractionated identically in subcellular fractionation experiments; these forms exhibited essentially equal distributions between the 100,OOOg pellet and supernatant fractions. Thus, the SEC18p also behaved as a peripheral membrane protein. Although initial homology searches failed to provide any insight into SEC18p function (Eakle et af.,1988), such an end was realized by Rothman and his colleagues who undertook a molecular characterization of the structural gene for the mammalian NSF (Wilson et al., 1989). The nucleotide sequence data predicted a hydrophilic polypeptide of some 83 kDa, which was consistent with the monomeric molecular mass of the NSF as determined by SDS-PAGE (76 kDa). The striking finding was that the NSF and SEC18p shared a 48% identity and 63% similarity (allowing for conservative substitutions) over their entire primary sequence. This homology suggested that the NSF and SEC18p exhibited similar biochemical activities and, on the basis of several criteria, this was conclusively established by Wilson et af. (1989). First, both the NSF and SEC18p were shown to be ATP-binding proteins, and it was determined that both proteins were stabilized by ATP. Second, overproduction of the SEC18p in yeast cells resulted in a concomitant elevation of NSF activity in yeast lysates. Finally, secl8‘” mutants failed to produce any NSF activity that could be assayed in vitro. Taken together, these data indicated that the SEC18p was the yeast NSF,

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and these data demonstrated that the SEC18p was involved in fusion of ER-derived transport vesicles to the acceptor Golgi complex. It should be noted that this conclusion was entirely consistent with the general interpretation of class-I1 sec mutants, namely that those mutants were defective in consumption of ER-derived transport vesicles (Kaiser and Schekman, 1990). The discovery that the SEC18p is the yeast NSF has yielded significant insight into the biochemical function of at least one other SECgene product. As already noted (Section IV.A), NSF function required participation of other protein cofactors, most notably the three SNAPS (a, P and y) which promoted association of the NSF with Golgi-complex membranes. Clary et a f .(1990) found that yeast cytosol elaborated SNAP activity and, moreover, that yeast SNAP was responsive to the SEC18p but not to the mammalian NSF. A functional in vitro assay for yeast SNAP activity was devised and cytosol preparations from wild-type secl6", secl 7 ts ,sec2@, sec21ts,sec22" and sec23" strains were screened. The sec17" cytosol was uniformly defective in in vitro SNAP activity at all temperatures, and could be complemented by wild-type cytosol in a SEC18p-independent fashion. Moreover, purified bovine a-SNAP corrected the transport defect associated with secl7" cytosol. It was noteworthy that inclusion of purified P-SNAP or y-SNAP failed to complement the secl7" defect in the transport reaction. Although it has not yet been proven that SECZ7is the structural gene for the yeast a-SNAP, it is tempting to believe that this will prove to be correct. The biochemical data are in striking agreement with the genetic arguments for a functional interaction of the SEC18p with the SEC17p, and with the class-I1 morphological categorization of these two activities as being required for consumption of transport vesicles at the acceptor membrane (Section 1V.C.l). Thus, the present data argue strongly for an adapter role for the SEC17p in delivery of the SEC18p to Golgi-complex membranes, perhaps as a crucial event in the building of a transport-vesicle "fusion machine". The finding that the SEC18p is the yeast NSF, and that the SEC17p probably represents the yeast analogue of a-SNAP, is indicative of one final point. This is that many (and perhaps all) of the SECgene products assigned to the ER-to-Golgi complex stage of transport participate at many steps along the secretory pathway and therefore represent general factors involved in transport-vesicle metabolism. Assignment of these gene products to an early stage of the secretory pathway was based upon an analysis that could only reveal the first execution point (Novick e t a f . ,1980). In this regard, we note that the SEC23p and the SEC18p are also required for fluid-phase endocytosis in yeast (Riezman, 1985).

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3. GTP-Binding Proteins

To date, three GTP-binding proteins have been implicated in transport from the ER to the Golgi complex in Sacch. cerevisiae; these are the SA R l , Y P T l and ARF gene products. The data suggest that at least this stage of the secretory pathway requires the action of multiple GTP-binding proteins. The SARI gene was initially obtained in attempts to clone the SEC12 gene, where it was recognized as a multicopy suppressor of secl2‘” (Nakano etal., 1988). Overproduction of the S A R l p was not capable of suppressing the lethality of secl2 null mutations, indicating that increased S A R l p function diminished, but did not bypass, the need for SEC12p function (Nakano and Muramatsu, 1989). Interestingly, as little as a presumed twofold increase in SARl gene dosage was sufficient for efficient suppression of secl2”. Nucleotide-sequence analysis suggested that the SARlp was a polypeptide of 190 residues that exhibited significant homology to the ras family of proteins, while gene-disruption experiments demonstrated that SARl was essential for cell growth and viability. To test whether the function of S A R l p was relevant to secretory pathway function (as implied by the genetic interaction with secl2‘”), Nakano and Muramatsu (1989) engineered SARI expression that was under G A L promoter control, and investigated the consequences of depletion of the S A R l p in cells. These workers found that S A R l p deficiency was specifically manifested by a secretory defect. In particular, core-glycosylated forms of the secretory prepro-a-factor and vacuolar proCPY were found to accumulate intracellularly. Genetic interactions of SARl with SEC12 implicate a class-I (i.e. transport-vesicle formation) execution point for the SARlp. A more precise understanding of the function of the S A R l p awaits localization of the protein to some clearly definable membrane structure. The gene Y P T l was originally identified by Gallwitz et al. (1983) as an open reading frame situated between the actin and j3-tubulin structural genes. Interest in Y P T l was stimulated by virtue of the finding that the inferred 207-residue YPTlp exhibited significant sequence identity with the human ras proto-oncogene products. Subsequent gene-disruption experiments revealed Y P T l to be essential for yeast-cell viability (Schmitt et al., 1986; Segev and Botstein, 1987), and the study of temperatureconditional mutants (both ts and cs) indicated that loss of YPTlp function had profound effects on secretory pathway function, calcium-ion metabolism, microtubule organization, and nuclear metabolism (Schmitt et a l . , 1988; Segev et al., 1988). Schmitt et al. (1988) proposed that the primary defect in yptl‘” strains was related to impaired homeostasis of calcium ions, in part because the yptl‘” growth phenotype was calcium-ion remedial and uptake of these ions was greatly increased in these mutants. Segev et al. (1988),

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however, argued for a primary role for the YPTlp in stimulating secretory pathway function. This hypothesis was supported by their findings that: (a) the YPTlp was located in the yeast Golgi complex in indirect immunofluorescence experiments utilizing wild-type cells, (b) a mammalian YPTlplike protein was also associated with the Golgi complex in mouse L-cells, (c) loss of YPTlp function resulted in a proliferation of yeast Golgi-complex membranes reminiscent of that observed for Golgi complex-blocked sec7’” mutants, and (d) abnormally high intracellular pools of secretory invertase were accumulated under restrictive conditions. Curiously, the secretory defect was only partial and it was noted that the secreted invertase was remarkably undermodified, regardless of whether restrictive or permissive conditions were imposed. T o resolve some of the major questions regarding YPTlp function, and the role of calcium-ion metabolism, Bacon et al. (1989) and Baker et al. (1990) analysed the dependence of the in vitro ER-to-Golgi complex transport reaction (Section 1V.B) on the function of the YPTlp. It was found that the system was dependent on this function, as shown by lack of transport detected in cell lysates prepared from yptl mutants and the inhibitory effect of addition of anti-YPTlp Fab fragments to the reaction mixture. More detailed fractionations revealed that it was the acceptor Golgi-complex membranes that were defective for transport in the yptl mutants. Titration of the free calcium ions present in the in vitro reactions, over a wide range of concentrations, failed to remedy yptl defects, and consumption of the calcium ion-requiring intermediate was not inhibited by anti-YPTlp Fab fragments. These data indicated that YPTlp and calcium ions acted at functionally independent execution points (Baker et al., 1990). Thus, it seems unlikely that YPTlp functions primarily to control intracellular concentrations of calcium ions. Rather, the YPTlp functions essentially to stimulate protein transport through the secretory pathway. At present, the YPTlp is considered to be involved in protein transport through the Golgi complex, largely on the inference that the undermodified invertase elaborated by yptl mutants possessed early Golgi-complex carbohydrate modifications (for a discussion, see Bacon et al., 1989). It remains less clear whether the YPTlp is also involved in fusion of ERderived vesicles to the Golgi complex. This problem will be resolved once the nature of the secretory block associated with depletion of the functional YPTlp in vivo is determined. The yeast ARFl gene product was recognized as a homologue to the mammalian ADP-ribosylation factor (ARF), a ubiquitous and highly conserved 21 kDa GTP-binding protein that is related to both ras and GTPbinding protein a-subunit families (Sewell and Kahn, 1988). The gene ARFl was shown not to be essential for vegetative growth because of a redundant

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function that was expressed at low levels from the ARF2 gene, which encodes a polypeptide that exhibits 96% primary sequence identity with the ARFlp (Stearns et al., 1990). Whereas arfl" arf2' double mutants were inviable, arfl" mutants exhibited a number of phenotypes, including cold sensitivity for growth. Analysis of the secretory competence of arfl" yeast strains revealed that they were partially impaired in secretion ability at both permissive and restrictive temperatures, and that the accumulated and secreted invertase populations were uniformly undermodified. These invertase-undermodificationand partial secretory block phenotypes mimicked the defects seen in yptl strains. Stearns et al. (1990) also analysed the consequences of depleting cells of all A R F activity. The data showed an ERto-Golgi complex secretory block in those cells. Finally, immunocytochemistry demonstrated a Golgi-complex localization of the mammalian ARF in NIH3T3 cells, and it appeared that there may have been some enrichment of A R F on the cytoplasmic face of cis-Golgi-complex membranes. Bourne (1988) proposed a mechanism for the involvement of GTPbinding proteins in regulation of vesicular transport. The basic tenets of this model are discussed in Section V1.A. D. THE RETENTION PROBLEM

The E R provides the proteins and phospholipids that sustain bulk secretory flow through the secretory pathway. As a result, the E R experiences a massive efflux of proteins and phospholipids of such magnitude that Wieland et al. (1987) estimated that the lumenal E R volume is turned over within approximately 10 minutes in mammalian cells. That the integrity of the ER is maintained under these conditions is testimony to the remarkably efficient mechanisms for retention of resident E R proteins in the face of such massive protein efflux from the reticulum, and retrieval of phospholipids back to the ER. The latter point is discussed in the following section of this review (Section V.C). Finally, the E R also plays a quality-control function in the sense that it is within this compartment that unfolded or malfolded polypeptides are withheld until the appropriate conformations are achieved and further transit through the secretory pathway is permitted.

I . Retention of Incompletely Assembled Polypeptides

How does the E R monitor the assembly state of polypeptides within its

lumen? Although polypeptide properties that determine their ability to be assembled remain mysterious, studies on mammalian cells have identified a resident E R protein termed BiP (binding protein) that is intimately involved in recognition of the state of polypeptide assembly, or perhaps even catalysis

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of the assembly process itself (Munro and Pelham, 1986; Gething et al., 1986; Bole et al., 1986). As such, BiP fulfills some general criteria associated with a sorting function. That is, it sorts polypeptides not yet ready to leave the ER from those that have assembled and are therefore competent to enter the default pathway for exit (Pfeffer and Rothman, 1987; Rothman, 1989). Saccharomyces cerevisiae has a BiP homologue and it is encoded by the KAR2 gene (Rose et al., 1989). Originally, KAR2 was identified in a search for mutants that failed to undergo karyogamy (i.e. nuclear fusion) during the mating process (Polaina and Conde, 1982). The recognition that KAR2 encodes yeast BiP came from a molecular analysis of KAR2 and studies of its transcriptional regulation. Authentic KAR2 clones were recovered by Rose et al. (1989) and the nucleotide sequence was determined. The KAR2 gene was inferred to encode a 682-residue protein that exhibited a 67% identity with mammalian BiP, and similar homologies to members of the yeast, amphibian, insect and bacterial HSP70 family. Several other structural features were noted that were suggestive of equivalence of BiP and the KAR2p. These included the findings that: (a) the KAR2p (like BiP) contained a functional signal peptide that directed its entry into the secretory pathway in a SEC6lp-dependent fashion, a feature not exhibited by any of the other HSP70 cognates, and (b) the KAR2p contained a Cterminal H D E L sequence that functions in retention by the E R in yeast (Section IV.D.2), implying a lumenal E R location for this gene product (as with BiP in mammalian cells). This latter point was confirmed by immunofluorescence experiments, coupled with protease protection experiments that independently assigned a lumenal location for the protein. Finally, the demonstration that KAR2 is an essential gene (Rose et al., 1989; Normington et al., 1989), coupled with the genetic demonstration that KAR2 was not allelic to SSCZ (the sole member of the eight previously identified yeast HSP70 cognate-gene family that is essential for growth; Craig et al., 1987), identified KAR2 as a ninth member of the yeast HSP70 family. The inability of the other HSP70 cognates to supply redundant functions is probably related to the unique compartmentalization of the KAR2p within the E R lumen. Mammalian BiP and the KAR2p were also found to exhibit several common features with respect to their transcriptional regulation (Rose et al., 1989; Normington et al., 1989). Challenging of yeast cells with either 2deoxyglucose or tunicamycin, a drug that inhibits addition of N-linked glycosyl chains to secretory proteins, resulted in a five- to 10-fold elevation in the level of KAR2 mRNA. Treatment with the calcium ionophore A23187 resulted in a slight elevation. These same conditions induce expression of the mammalian BiP gene, and are specific for KAR2 as these fail to induce expression of other HSP70 cognates. However, heat shock also induced

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KAR2 expression. This induction occurred rapidly, as a measurable increase in the levels of the KAR2 message was apparent after some five minutes of heat shock, and maximal induction (about seven-fold over basal level) was reached some 30 minutes after imposition of heat shock. Interestingly, this induced level of expression was not sustained. It decayed rapidly after achievement of peak induction, and basal levels of KAR2 expression were re-established after one hour of heat shock. It is similarly noteworthy that BiP synthesis is not heat-shock inducible. To investigate further the role of the KAR2p in vivo, Vogel et al. (1990) generated a number of kar2" alleles and characterized the tightest of these, kar2-159, in detail. Imposition of non-permissive conditions onto kar2-159 cells led to a rapid and irreversible loss of viability. The particular defect associated with the kar2-159 lesion involved defects in bud emergence and growth, suggestive of defects in the secretory pathway. Analysis of the biogenesis of two secretory proteins (invertase and prepro-a-factor), and a vacuolar proteinase (carboxypeptidase Y), yielded a surprising result. This was that these polypeptides failed to be translocated into the E R lumen under conditions of the kar2-159 block. This was surprising because the kar2-159 defect was observed for events involving the cytosolic face of the ER, whereas the KAR2p is a lumenal E R component. This translocation defect was reproduced when functional KAR2p was depleted in yeast, confirming that this defect was the result of loss of KAR2p function rather than some sort of disruptive action of the defective kar2-159 gene product. It was also noteworthy that nuclear fusion could be uncoupled from translocation using these mutants. Thus, the karyogamy defect associated with kar2159 occurred under conditions that were entirely permissive for E R translocation. The kar2-1 lesion also lacked secretory effect, even though nuclear fusion was unconditionally defective in these strains. The unanticipated E R translocation defect in kar2-159 strains is not completely understood. Vogel et al. (1990) proposed two general models for KAR2p function that seek to account for this unexpectedly early execution point. First, the KAR2p may be required to maintain polypeptides in an unfolded state during the translocation process. In strains unable to synthesize the KAR2p, an unscheduled and premature folding of polypeptides in transit might somehow inactivate the secretion machinery, resulting in an ER translocation defect. A broadly similar function has been alluded to with the SEC63 gene product (Section III.B.2). The second model suggests that the process of protein translocation through the E R consumes some component of the secretion machinery, perhaps by a stable association of such a component with the secretory polypeptide during transit. In this scenario, the KAR2p would function in a dissociation event that permits recycling of the secretion-machinery component. Further

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experimentation is required to determine which, if either, of these possibilities is correct. 2. Retention of Resident Endoplasmic-Reticulum Proteins

Two basic mechanisms have been considered for retention of resident ER proteins, namely a receptor-mediated mechanism and a mechanism that involves a structural exclusion of resident proteins from packaging into transport vesicles. In the former mechanism, it is considered that resident ER proteins are sorted from the secretory pathway by means of a retention signal that is recognized by a specific receptor. In the mammalian system, Pelham and others have shown that a C-terminal peptide sequence (KDEL) is necessary and sufficient for retention in the E R (Munro and Pelham, 1987; Pelham, 1988; Ceriotti and Colman, 1988). Interestingly, the KDELdependent retention system appears to involve an efficient retrieval from a compartment of the secretory pathway located after the E R (Pelham, 1988; Ceriotti and Colman, 1988). Pelham et al. (1988) have shown that yeast also exhibits an analogous sorting system. The KAR2p has a C-terminal HDEL sequence that closely resembles the mammalian KDEL retention signal, although these signals are not interchangeable. Attachment of the FEHDEL sequence directly to the C-terminus of secretory invertase failed to cause retention of the tagged secretory protein within the cell. However, when an appreciable linker (55 residues) was engineered onto the C-terminus of invertase, followed by FEHDEL, efficient retention was observed (Pelham et al., 1988). The results with prepro-a-factor were more convincing. Engineering of an 11residue linker, followed by the FEHDEL sequence, to the C-terminus of prepro-a-factor resulted in efficient retention of the secretory protein within the cell (Dean and Pelham, 1990). Efficient retention required that the HDEL sequence be at the extreme C-terminus of the protein. An FEHDELS sequence was a poor retention signal, albeit a signal nonetheless (Pelham et al., 1988). Furthermore, efficient retention required a low rate of tagged secretory protein synthesis, suggesting some saturability in the retention system. This saturability was confirmed in experiments in which it was demonstrated that high-level synthesis of HDEL-tagged prepro-afactor not only resulted in its own secretion, but also secretion of an endogenous HDEL-containing protein (i.e. the KAR2p) (Dean and Pelham, 1990). The saturable nature of the retention system suggested the existence of an HDEL receptor, the identity of which has probably been determined (see below). Does HDEL-dependent retention of proteins involve a true retention mechanism or does it reflect an efficient retrieval system? Current data


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strongly suggest the latter. Subcellular fractionation experiments have indicated that the retained HDEL-tagged secretory protein was localized in the yeast E R on the basis of its sedimentability with membranes at 10,000g and cosedimentation in sucrose gradients with core-glycosylated proCPY (used as a marker for E R lumen; Dean and Pelham, 1990). This was a pleasing result as it was consistent with retention of the tagged secretory protein in the appropriate intracellular compartment. However, analysis of the glycosyl modifications of the tagged secretory protein revealed that the glycosyl chains were not of the core variety but, rather, had been more extensively modified. Specifically, the HDEL-tagged secretory proteins had acquired at least some mannose residues in a-(1+6) linkage, an early Golgicomplex modification, in a SEC18p-dependent manner. However, the HDEL-containing reporter protein did not acquire late Golgi-complex glycosyl modifications (i.e. mannose residues in a-(1-+3) linkage). A discussion of N-glycosylation and its compartmentalization in yeast is presented in Section V.A.2. Taken together, these data were incorporated into a model in which H D E L is recognized by a specific receptor that functions in retrieval of resident E R proteins from an early Golgi-complex compartment (Dean and Pelham, 1990). The ability of H D E L to confer E R retention on secretory invertase provided a basis for direct selection for mutants that fail to retain tagged invertase but, rather, secrete it. This selection was based on a general scheme that was first employed to isolate mutants that were defective in targeting of proteins to the yeast vacuole (Bankaitis et af.,1986b). A total of 50 such erd (ER retention defective) mutants were isolated, and two well-defined complementation groups (i.e. E R D l and ERD2) identified (Hardwick et af., 1990). Cells lacking either functional E R D l p or ERD2p secreted significant amounts of HDEL-tagged invertase and native HDELcontaining polypeptides (i.e. the KAR2p) (Hardwick et al., 1990; Semenza et af., 1990). However, the rate of KAR2p secretion was very slow in erdl strains (the half-life was seven hours as opposed to less than three minutes for normal secretory proteins), and these strains exhibited normal intracellular levels of the KAR2p as well. This homeostasis was possibly the result of elevated KAR2p synthesis in erdl cells. A further, an puzzling, curiosity associated with erdl mutants was that their erd phenotypes were nutritionally remedial, in that growth in medium containing low concentations of sulphate suppressed the KAR2p secretion phenotype (Hardwick et af., 1990). Clones of E R D l were recovered by Hardwick and his coworkers (1990) and studied in some detail. The nucleotide sequence revealed a single long open reading frame that had the potential to encode a 362-residue polypeptide whose primary sequence was not homologous with that of any

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other known protein. Furthermore, the ERDlp was inferred to be an integral membrane protein since it exhibited several candidate transmembrane domains. Some indirect support for this conclusion was forthcoming from gene-fusion experiments demonstrating that the first 101 residues of the ERDlp were sufficient to permit transfer of invertase into the ER lumen. Unfortunately it was not determined whether or not the ERDlpinvertase fusion polypeptide was an integral membrane protein. Gene-disruption experiments revealed ERDl to be a non-essential gene, and all erdl phenotypes were reproduced in the null mutant. Interestingly, erdl null mutants exhibited severe defects in Golgi complex-dependent modifications of N-linked oligosaccharide chains for at least three glycoproteins, namely CPY, invertase and a polypeptide that exhibited immunological cross-reactivity to invertase. Taken together, the data suggested a Golgi-complex defect in erdl mutants. However, neither sorting of CPY to the vacuole (a late Golgi-complex event) nor protein transport from the Golgi complex to the cell surface appeared to be affected. Furthermore, loss of ERDlp function did not result in morphologically aberrant Golgi complexes. At present, it seems most likely that erdl defects result in a general perturbation of Golgi-complex function. Although the collective data imply an indirect effect of the E R D l p on the HDEL-dependent ER retention system, they are nevertheless strongly supportive of the idea that the HDEL system involves an efficient retrieval of protein from the Golgi complex back to the ER. Given such a scenario, it seems probable that the ERDlp will be found to be in the Golgi complex. Current evidence strongly suggests that the ERD2p is the HDEL receptor. Other than the initial observation that erd2 mutants fail to retain HDEL-containing polypeptides, there are two additional lines of evidence that identify the ERD2p as the HDEL receptor. First, levels of ERDZ expression are directly proportional to the capacity of the HDEL-retention system. Overproduction of the ERD2p suppressed secretion of HDELtagged prepro-a-factor or the endogenous KAR2p, caused by high levels of synthesis of the tagged species (Semenza et al., 1990). Second, the specificity of the retention system was determined by the ERD2p. The budding yeast Kluyveromyces lactis apparently exhibits two ER retention signals, namely HDEL and DDEL, and Sacch. cerevisiae is incapable of recognizing DDEL. Exchange of ERDZ from K . lactis to Sacch. cerevisiae for the native ERD2 conferred upon the recipient cells the ability to recognize both HDEL and DDEL (Lewis et al., 1990). Cloning of ERDZ revealed an open reading frame of 219 residues that could encode a hydrophobic 26 kDa polypeptide, an inference that was confirmed by identification of the ERD2p as a 26 kDa integral membrane protein. Immunofluorescence studies indicated that the ERD2p was not in the ER but rather in some other cytoplasmic organelle


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that may represent the Golgi complex. In any event, disruption of ERD2 proved to be a lethal event, although depletion experiments indicated that arrested cells remained viable for extended periods of time. The primary defect associated with depletion of the ERD2p appeared to be at the level of Golgi-complex function, as with dysfunction of the E R D l p . This erd2 Golgi-complex dysfunction was manifested by likely defects in transport of vacuolar CPY from the late Golgi complex, and by abnormal elaboration of intracellular membranes, although it remains unclear as to whether these proliferating membranes were Golgi-complex derived (Lewis et at., 1990). Thus, as anticipated, the H D E L receptor appears to act at the level of the Golgi complex, most likely by a retrieval mechanism. It remains unclear, however, as to why erd2 defects affect late events in the Golgi complex (i.e. transport of CPY) when previous data suggested that retrieval occurred from an early Golgi-complex compartment (see above). Perhaps the ERD2p has multiple functions, only one of which is involved in HDELdependent retrieval of polypeptides from the Golgi complex back to the E R (Lewis et at., 1990).

3. Speculations on the Role of Calcium Ions in Endoplasmic-Reticulum Retention

A second independent mechanism for retaining resident proteins in the ER could involve their exclusion from those areas of the reticulum that participate in formation of transport vesicles. Sambrook (1990) suggested a model for how calcium-ion sequestration within the E R lumen could participate in such an exclusion mechanism. This model was based on two lines of evidence. The first of these was the observation that treatment of mammalian cells with the calcium ionophore A23187 resulted in a transient secretion of resident E R proteins (i.e. BiP, protein disulphide isomerase and CRP55) and a striking dispersion of the reticulum itself (Booth and Koch, 1989). Since the E R lumen serves as the major intracellular reservoir for calcium ions, these data suggested a role for elevated lumenal calcium-ion concentrations in retention of resident E R proteins. The second line of evidence was obtained from a study of yeast mutants defective in a Ca2+-ATPase pump, the product of the PMRI gene, which probably functions in mobilization of calcium ions into the ER lumen (Rudolph et al., 1989). Interestingly, this gene was originally identified in a search for mutants that exhibited more efficient secretion of heterologous proteins engineered for expression in yeast, and was termed SSCl (Smith et al., 1985). This gene is not to be confused with the structural gene for the mitochondria1 HSP70 (Section 1V.D). Detailed analyses of wild-type and pmrl strains revealed

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that native secretory proteins, although efficiently secreted from pmrl strains, were remarkably underglycosylated in a manner similar to that observed foryptl mutants (Section IV.C.3). Moreover, P M R l was found to be a non-essential gene while p m r l null mutants grew poorly under conditions of calcium-ion stress (Rudolph et al., 1989). That loss of Ca2+-ATPase function could alleviate secretory difficulties experienced by heterologous proteins engineered for secretion by yeast implied an unscheduled export of protein from the E R , a point that was consistent with the underglycosylation of secretory proteins in p m r l mutants. However, it remains to be determined whether p m r l mutants exhibit true erd phenotypes. Nevertheless, the yeast prnrl data were at least broadly consistent with the conclusions concerning calcium ions and E R retention in mammalian cells. How then might high levels of calcium ions in the E R lumen contribute to retention of resident ERproteins? Sambrook (1990) proposed that the ERis loaded with proteins (such as BiP) having reasonable calcium ion-binding capacities. These could form an extensive calcium-co-ordinated matrix involving interactions with each other and with the appropriate anionic phospholipid head groups in the inner leaflet of the E R membrane. Transport-vesicle formation at the E R surface might involve fluxes of calcium ions across the E R membrane, and resident E R proteins would largely be excluded from being packaged into such transport vesicles because of their being immobilized in the lumenal calcium-ion-protein matrix. Any residents escaping such an immobilization would be retrieved from the Golgi complex via the receptor-mediated H D E L system, while wholesale depletion of calcium ions from the E R lumen, induced by either the presence of A23187 in mammalian cells or absence of a functional Ca2+-ATPase in yeast, would lead to disintegration of the calcium-ion-protein matrix and subsequent disorganization and vesicularization of the E R . It is this disruption of the calcium-ion-protein matrix that is thought to lead to unscheduled secretion of resident E R proteins (Sambrook, 1990). A final point regarding the effect of p m r l null mutations on yeast secretory function deserves comment. Rudolph et al. (1989) observed that pmrl null mutations suppressed conditional lethal yptl defects. These workers then speculated that loss of Ca2+-ATPasefunction may bypass the YPTlp requirement. This was an interesting suggestion since the YPTlp had, at one point, been considered to play a role in calcium-ion homeostasis in yeast (Schmitt etal., 1988). Given our present understanding, however, it seems unlikely that bypass of the YPTlp is a consequence of defects in P M R l . Rather, p m r l null mutations more likely cause yeast to fail to sequester calcium ions in the E R lumen. This may lead to an elevated


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cytosolic calcium-ion concentration that exerts a remedial effect on a labile Yptlcs activity (see Section IV.C.3). V. The Golgi Complex as a Secretory Organelle It has recently become appreciated that the Golgi complex occupies a unique position in the cellular scheme for protein-traffic control. This organelle plays a fundamentally important role in regulating several aspects of protein and membrane movement throughout the cell. For example, the Golgi complex is the compartment for sorting of proteins from the constitutive secretory pathway to the lysosomal and regulated secretory pathways. Furthermore, the Golgi complex is involved in regulation of membrane recycling from the cell surface and in communication with aspects of the endocytic pathway (Griffith and Simons, 1986). Finally, it must be stressed that the Golgi complex represents a segmentally structured and inherently compartmentalized organelle across which secretory protein traffic must flow. In order to understand fully how the Golgi complex functions, two basic informational requirements must be met. First, one must appreciate the architecture of the organelle from the perspective of knowing the identity and function of the polypeptides that are indigenous to the complex. Second, one must know the identity and function of polypeptides that drive, or otherwise regulate, Golgi-complex secretory functions. Although significant progress has been made on both counts, our understanding of each remains limited. Electron microscopy has indicated a conservation of Golgi-complex morphology across the eukaryotic kingdom. This organelle consists of a set of some five o r more cisternae that exhibit flattened centres and dilated rims (Farquhar, 1978; Tartakoff, 1980). The aspect of the Golgi complex that lies in apposition to the transitional ER has been designated as the cis-face while the aspect oriented towards the plasma membrane has been designated as the trans-face (Ehrenreich et al., 1973). The intermediate cisternae lying between cis- and trans-Golgi-complex aspects define the medial aspect of the Golgi-complex. The observations of Bergmann and Singer (1983) and Saraste and Hedman (1983), which indicated a vectorial transport of glycoproteins across the Golgi complex in the cis- to trans-direction, suggested that the complex was not composed simply of repeating units of compositionally identical cisternae but, rather, was organized in a functionally asymmetric fashion. Some of the key advances in Golgi-complex research in recent years have confirmed demonstrations of the compartmental organization of the Golgi complex. More specifically, four lines of research have converged to a

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point where it is now clear that the three regions of the complex are biochemically distinct, and that operational criteria can be applied to which cisternae constitute cis-, medial or trans-aspects. These lines of investigation included: (a) classical histochemical studies, (b) analysis of oligosaccharideproduct distribution within the Golgi complex, (c) subfractionation of biochemically distinct Golgi-complex membranes, and (d) in situ localization of defined marker enzymes (or undefined polypeptide antigens) to distinct regions of the complex (reviewed in Dunphy and Rothman, 1985). As already discussed (Section IV.A), Rothman and his colleagues devised a cell-free system which reconstituted intercisternal protein transport through the Golgi complex with the aim of identifying compartment-specific transport components by the classical strategy of resolution and reconstitution. While the biochemical approach has yielded rich dividends in identifying the basic concepts involved in transport-vesicle biogenesis and consumption, the transport components resolved to date (i.e. the NSF and the SNAPS) probably represent general fusion components involved in a number of stages on the secretory pathway (see Section IV.C.2). With the isolation and characterization of sec mutants, an independent means for recognizing compartment-specific functions has become available. These mutants permitted identification of two genes, SEC7 and SECII, whose products are essential for secretory function by the Golgi complex. Analysis of the roles of these two gene products in the function of the Golgi complex has yielded substantial insights into its organization and secretory function. In particular, characterization of the SEC14p has provided new perspectives on several long-standing issues in cell biology. A . FUNCTIONAL COMPARTMENTALIZATION OF THE YEAST GOLGI COMPLEX

I . Identification of the Organelle Saccharomyces cerevisiae appears to exhibit only a constitutive secretory pathway that transports proteins from the E R to the cell surface extremely rapidly. This characteristic, coupled with the high density of cytoplasmic ribosomes in this yeast, has obscured all but the largest organelles (i.e. nucleus, vacuoles and mitochondria) from a consistent view by electron microscopy. As a result, cells of this yeast do not display the morphologically correct Golgi complex typical of most other eukaryotic cells. Thus, identification of yeast Golgi complexes by cytochemistry remains somewhat problematic. The first evidence for Golgi complex-like structures in yeast was obtained from thin-section electron-microscopic analysis of the terminal phenotypes exhibited by sec7’’ and s e c l B strains. These mutants revealed a proliferation


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of toroid membaneous structures or, under appropriate nutritional conditions (low concentrations of glucose), stack-like structures reminiscent of mammalian Golgi-complex membranes (Novick et al., 1980). That such membranes represented defective forms of a functional Golgi complex was indicated by the structure of the N-linked oligosaccharides of the secretory glycoproteins trapped within them (see below). A second cytochemical method for identification of yeast Golgi complexes has recently been developed; this is indirect immunofluorescence microscopy using the KEX2p as a Golgi-complex marker. The KEX2p is an integral membrane endoprotease involved in proteolytic maturation of the yeast a-factor mating pheromone. Based on the a-factor precursor species that accumulated within sects mutants at 37”C, Julius et al. (1984) assigned the KEX2p to the yeast Golgi complex, and it now appears that the KEX2p functions in a late Golgi-complex compartment (Franzusoff and Schekman, 1989). Indirect immunofluorescence experiments employing affinity-purified anti-KEX2p primary sera revealed a punctate cytoplasmic staining of some four to six bodies in each focal plane of a cell (Franzusoff etal., 1991). This staining was quite similar to that observed for the YPTlp (Section IV.C.3). At present, yeast Golgi complexes can be most readily detected in wildtype cells by immunofluorescence microscopy, a low-resolution method. Visualization of yeast Golgi complexes by high-resolution electronmicroscopy methods unfortunately requires imposition of secretory blocks that may well distort Golgi-complex structure to an extent such that structural inferences gleaned from such exaggerated organelles may be inaccurate. Thus, cytochemical methods have failed to provide any solid evidence for a segmental and functionally compartmentalized organization for the yeast Golgi complex. Clearly, methods for morphological detection of Golgi-complex bodies in wild-type strains of yeast need to be vastly improved before the full complement of direct evidence to this effect can be obtained. 2. Compartmental Organization of the Yeast Golgi Complex Presently, the notion that the yeast Golgi complex posseses a functional compartmentalization akin to that in mammalian Golgi complexes has been based on evidence that the oligosaccharide modifications experienced by yeast glycoproteins in transit through the Golgi complex are executed in a sequential and biochemically separable manner. This evidence is somewhat analogous to localization of defined carbohydrate-assembly reactions to distinct regions of the mammalian Golgi complex (reviewed by Dunphy and Rothman, 1985). To extend analyses of carbohydrate assembly to the yeast

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Golgi complex, it was necessary to know the precise structure of the N-linked oligosaccharides on yeast glycoproteins. The core oligosaccharides of Sacch. cerevisiae were identical in composition with those of mammalian cells (Li et al., 1978; Lehle, 1980). However, in contrast to the complex carbohydrate of mammalian glycoproteins, it was demonstrated that the yeast outer-chain carbohydrate consisted entirely of a-D-mannose residues in a-(1+6), a-(1+2) and a-(1+3) linkages together with a limited number of mannobiosylphosphate groups in diester linkage (Ballou, 1976). Furthermore, the order of events that occur in conversion of the core oligosaccharide to fully matured N-linked glycans has been partially deduced (Flores-Carreon, 1990). Raschke et al. (1973) isolated mutants (termed mnn) that exhibited alterations in the oligosaccharide-residue composition of the yeast cell-wall mannoprotein. Further analyses of these mnn mutants revealed that specific linkages of mannose residues were absent from the outer chain and some cell-free extracts derived from mnn strains were deficient in particular mannosyltransferase activities. For example, mnnl cells did not possess terminal a-(1+3)-mannose residues in their cell-wall glycoproteins. This defect correlated directly with the absence of a-(1+3)-mannosyltransferase activity from mnnl cell extracts (Nakajima and Ballou, 1975). It was subsequently established that the mnnl locus encoded an a-(1-3)mannosyltransferase (Ballou, 1982). However, this enzyme has not yet been localized to a specific subcellular compartment. Although these studies provided a detailed structural analysis of the yeast outer-chain mannoprotein, no evidence for the compartmentalized assembly of oligosaccharides was obtained. The first evidence for the compartmentalization of mannosyltransferase activities in yeast was provided by Esmon et al. (1981), who examined the oligosaccharide modifications of secretory invertase that had accumulated in the appropriate intracellular compartments in particular sec mutants. A comparison of the electrophoretic mobilities of the invertase retained in the ER-blocked, Golgi complex-blocked and late secretory vesicle-blocked mutants showed that modification of N-linked oligosaccharides occurred in at least two distinct compartments. A t 37"C, the ER-blocked mutants contained intracellular invertase with immature glycosyl chains, while the Golgi complex-blocked and vesicle-blocked mutants accumulated invertase species that migrated on non-denaturing gels with a profile similar to the invertase obtained from wild-type strains. To verify that the differences in the electrophoretic mobilities of invertase from the sec mutants were due solely to differences in carbohydrate content, it was shown that endoglycosidase H treatment converted the various forms of invertase into a single 61 kDa polypeptide species.


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Some additional evidence for functional compartmentation of the yeast Golgi complex was obtained by Franzusoff and Schekman (1989), who characterized the glycosylation states of the invertase, CPY and a-factor precursors accumulated in secF strains at 37°C. These studies employed antisera raised against mannose residues of specific linkages and which could be used, in conjunction with electrophoretic mobilities, to characterize more precisely the outer-chain composition of yeast glycoproteins. The intracellular invertase obtained from secF mutants represented three populations that differed only in the number and linkage type of mannose residues added to the core oligosaccharide. One species of the accumulated invertase migrated with the same mobility on SDS gels as the coreglycosylated form and was not precipitable with anti-a-(1-+3)-Man or antia-(1+6)-Man antisera. A second population displayed a migration that was intermediate between the core-glycosylated protein and invertase from an mnn9 mutant. The mnn9 strain failed to mature completely the outer chain of glycoproteins, resulting in N-linked carbohydrates with only four or five mannose residues added to each core oligosaccharide. The third invertase population migrated similarly to fully matured invertase. However, these species lacked the terminal a-(1+3)-mannose residues. The p l (i.e. core-glycosylated) form of proCPY contains four Winked core oligosaccharide units, and its conversion to the p2 form is dependent upon addition of outer-chain and terminal a-(1+3)-mannose residues. It was previously reported that, after one hour at 37”C, 88% of the CPY in sec7‘ strains was in the p l form (Stevens et al., 1982). Following further analyses of the proCPY accumulated in sec7” mutants, it was shown that, in addition to p l CPY, a new species with a slightly lower electrophoretic mobility than p l CPY was also present. The thought was that this novel intermediate had experienced addition of a few outer-chain a-(1-6)mannose residues to the core oligosaccharide chains on the basis of the following observations. Both p2 CPY and mCPY from wild-type yeast were precipitable with anti+( 1-6)-Man and anti-a-( 1+3)-Man sera, while p l CPY was recognized by neither of these antisera. The new proCPY species found in the secF strain at 37°C was precipitated by anti-a-(1+6)-Man sera but not anti-a-(1+3)-Man sera. In addition to invertase and CPY, immature forms of the yeast matingpheromone a-factor accumulated in the sec7’ strains (Julius et al., 1984). Normal maturation of a-factor involves specific glycosylation and proteolytic processing events. As with invertase, a-factor was accumulated in various populations of precursor forms, one of which was heterogeneously glycosylated. The relevant aspect was that these species were precipitable with anti-a-( 1+6)-Man sera but not with anti-a-(1+3)-Man sera. Taking into account the variety of incompletely glycosylated precursors

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I

CELL SURFACE

I

SECRETORY VESICLES

Asn-Core t 6 M C [ 6 M C 6 M C 6 M + 6 M ] n + 6 M

Asn - Core 1‘M

(

ER Core Glycosylation

FIG. 4. Diagram illustrating compartmental organization of the yeast Golgi complex. Franzusoff and Schekman (1989) proposed this general model for biochemical compartmentation of the yeast Golgi complex-associated mannosyltransferase activities. Although this model enjoys considerable experimental support (see text), Franzusoff and Schekman (1989) point out that the depiction of one activity in each cisterna is rather arbitrary. The illustration of the yeast Golgi complex as an organelle that physically resembles a mammalian Golgi complex is merely convenient, and has not been unambiguously established by any direct means. Asn indicates the asparagine residue which is linked to the glycosyl chains. Mannose residues (M) in the outer glycosyl chain are indicated. Numbers indicate the linkage through the a-1 carbon atom to the preceding mannose residue in the outer-chain carbohydrate.

that accumulated in secF strains at 37”C, Franzusoff and Schekman (1989) proposed the model shown in Fig. 4 for compartmentation of the yeast Golgi complex. This model suggests a sequential, compartment-specific assembly


117

of N-linked oligosaccharides based on the previously determined structure of the yeast outer chain. Also, assignment of multiple execution points for the SEC7p accounted for the pleiotropic defects observed in s e c P mutants at 37°C. The suggestion of a-( 1+6)-mannose-residue addition occurring prior to a-( 1+3)-mannose-residue addition was further supported by in vitro ER+Golgi-complex assays that monitored glycosylation of the prepro-a-factor substrate (Section 1V.B.1). Transported prepro-a-factor acquired a-(l-+6)-mannoseresidues but not a-( 1+3)-mannose residues. These data suggested that this system reconstituted transport to the early Golgi-complex compartments but not subsequent early Golgi-complex+late Golgi-complex transport events. The SEC7 gene has been characterized at the nucleotide level and shown to be essential for yeast vegetative growth (Achstetter et af., 1988). The SEC7 gene was found potentially to encode a 2008 amino acid-residue hydrophilic protein predicted to be of some 230 kDa. In accordance with the nucleotide-sequence data, anti-SEC7p serum was used to probe extracts from radiolabelled yeast and precipitated a polypeptide that migrated on SDS gels with an apparent molecular mass of 227 kDa (Franzusoff et af., 1991). Fractionation experiments demonstrated that 60% of the total SEC7p was soluble while the remaining 40% was associated with pellet fractions. Sedimentable SEC7p was solubilized from pellet fractions by treatment with urea or alkaline sodium carbonate, but not by treatment with non-ionic detergents. Thus, the SEC7p behaved as a peripheral membrane protein. Indirect immunofluorescence microscopy revealed a punctate staining pattern of four to six particles in each cell and focal plane for the SEC7p. That at least some of these structures represented authentic yeast Golgi complexes was supported by two lines of evidence. First, sec24" cells, known to accumulate Golgi complex-like cisternae at 37"C, displayed a temperature-dependent exaggeration of the punctate structures that were seen in cells grown at the permissive temperature. Second, in doublelabelling experiments, some 58% of the structures that stained with antiSEC7p serum also stained with anti-KEX2p serum. Conversely, 81% of the KEX2p-positive structures were also SEC7p-positive. Taken together, the data suggested that the SEC7p was a peripheral membrane protein of the yeast Golgi complex that exhibited multiple execution points in intercisternal Golgi-complex transport. As yet, there are no obvious clues as to the biochemical function of the SEC7p although, interestingly, it has been found to be a phosphoprotein (Franzusoff et af., 1991). B . INVOLVEMENT OF A PHOSPHOLIPID-TRANSFER PROTEIN

Whereas the SEC7p is considered to be involved in a number of execution points in protein transport through the Golgi complex, current data suggest

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that the SEC14p is specifically involved in stimulating protein transport from a late Golgi-complex compartment. This inference is based on two lines of evidence. First, it has been observed that the secretory invertase accumulated in sec14-ltsstrains was quantitatively immunoprecipitated with anti-a(1+3)-Man serum, indicating that the intracellular invertase had experienced a complete maturation of its N-linked glycosyl chains (Franzusoff and Schekman, 1989). Moreover, the kinetics of a-( 1+3)-mannose acquisition by invertase in ~ e c l 4 - lstrains '~ under non-permissive conditions appeared to exhibit wild-type rates (A. E. Cleves and V. A. Bankaitis, unpublished data). Second, analysis of biogenesis of CPY in se~14-1~'mutants also indicated that the secl4 block was exerted at the step at which the ultimate proCPY precursor form (a late Golgi-complex species) was processed to the mature vacuolar form (Stevens et al., 1982). Thus, the SEC14p appears to represent a truly compartment-specific transport factor. A study of the SEC14p has revealed a precise biochemical activity for this polypeptide, and some penetrating insights into the role of the SEC14p in stimulation of yeast Golgi-complex secretory function have been obtained. In particular, these studies have indicated an active role for phospholipids in the secretory process, a concept that is generally ignored in current models of intercompartmental protein transport.

I . The SEC14p is the Yeast PhosphatidylinositoNPhosphatidylcholineTransfer Protein The yeast SECl4 gene and its gene product have been extensively characterized by Bankaitis and his coworkers (Bankaitis et al., 1989, 1990; Salama et al., 1990; Cleves et al., 1991). Nucleotide-sequence analysis of the SECl4 revealed a 304-codon open reading frame that was interrupted between the third and fourth codons by a 156-nucleotide intron. The primary sequence of the inferred SEC14p indicated a hydrophilic 35 kDa polypeptide that exhibited no remarkable hydrophobic character, no canonical sites for addition of N-linked oligosaccharide chains, and a general acidic character ( p l 5.3) (Bankaitis et al., 1989). These basic conclusions were confirmed by immunoprecipitation of the SEC14p from cell-free yeast lysates. The protein was found to be a non-glycosylated polypeptide with an apparent molecular mass of some 37 kDa. Subcellular fractionation experiments demonstrated that, as expected, the SEC14p was predominantly a cytosolic species. Some 60% of the total SEC14p was recovered from the 100,OOOg supernatant fraction. However, significant fractions of the total SEC14p were also found to be associated with membranes that pelleted at 12,000gand lOO,OOOg(Bankaitisetal.,1989). Although it wasnot initially clear that these membrane associations were of any physiological

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relevance, subsequent experiments demonstrated that such was indeed so (Cleves et al., 1991). Treatment of permeabilized yeast cells with fluorescein-labelled SEC14p antibodies revealed a staining of four to eight ovoid cytoplasmic bodies in each cell that were superimposed upon a diffuse cytoplasmic staining. That the SEC14p-positive structures represented yeast Golgi complexes was confirmed by double-label immunofluorescence experiments that showed colocalization of the punctate SEC14p with the yeast Golgi-complex marker KEX2p. Independent corroboration of the cytochemical data was obtained from quantitative subcellular fractionation experiments. It was demonstrated that sedimentable SEC14p codistributed with the KEX2p throughout a rigorous fractionation regimen. The collective data indicated a remarkable enrichment of sedimentable SEC14p to yeast Golgi-complex membranes, a minor class of intracellular membrane, thereby establishing a direct link between the SEC14p and the organelle that is dysfunctional in secZ4'" mutants. Taken together, the available evidence suggested that the SEC14p was a cytosolic factor directly required for yeast Golgi-complex secretory function. The essential nature of the SEC14p was revealed by gene-disruption experiments that demonstrated a recessive lethality of secl4 disruption mutations (Bankaitis et al., 1989). Although initial protein similarity searches failed to provide any meaningful homologies between the SEC14p and other known protein sequences (Bankaitis et al., 1989), subsequent searches provided the first clues as to the biochemical nature of SEC14p function. Salama et al. (1990) discovered a significant homology between the SEC14p and the human retinaldehydebinding protein (HRBP) at the primary sequence level. The SEC14p exhibited a 25% identity with the HRBP over an uninterrupted stretch of 219 amino-acid residues, which was statistically significant. On the basis of this homology, Salama et al. (1990) proposed that the SEC14p served as a carrier that delivered a hydrophobic ligand to yeast Golgi-complex membranes. This hypothesis was confirmed by the finding that the SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein (Bankaitis et al., 1990). This discovery represented a convergence of work by Bankaitis and his colleagues on SEC14p function and the independent analysis of phospholipid-transfer protein function by Dowhan and his coworkers (Aitken et al., 1990). It was noted that the N-terminal30 residues of the purified yeast PI/PC-transfer protein corresponded exactly to the SEC14p N-terminus (inferred from the nucleotide sequence) when the initiator methionine residue was excluded, and that the DNA sequence of the 5' end of the transfer-protein structural gene (PZTZ) was also identical with that of SECZ4 (intron inclusive). The ultimate proof of identity of SECZ4 and PIT1 involved completion of the PITI nucleotide

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sequence and the finding that it was identical with the published SEC14 sequence. Phospholipid-transfer proteins (PL-TPs) are cytosolic factors that catalyse exchange of phospholipids between natural or synthetic membranes in vitro (reviewed by Helmkamp, 1986). These proteins exhibit a range of substrate specificities. Some are quite substrate non-specific whereas others are specific for a very few, or even a single, phospholipid species. The SEC14p catalyses transfer to both PI and P C in vitro, but exhibits a marked preference for PI (Daum and Paltauf, 1984). Although PL-TPs have been detected in the cytoplasm of every eukaryotic cell type tested, and have been the subject of intense biochemical and biophysical characterization over the past 25 years, the relevance of their in vitro activities with respect to in vivo function has remained unclear. This has not only been due to the lack of an in vivo experimental system, but also due to the peculiar properties of the in vitro transfer activities that these proteins catalyse. For instance, they generally catalyse an in vitro exchange of phospholipids, rather than a net vectorial transfer of phospholipid. Second, they have the curious ability to utilize essentially any natural or synthetic membrane as a phospholipid donor or acceptor in vitro. It has been difficult to propose any useful model for in vivo function for such seemingly indiscriminate activities. Yet, the data of Cleves et al. (1991), which demonstrated a clear enrichment of the SEC14p in yeast Golgi-complex membranes, indicates an in vivo specificity for membrane targeting that is not apparent in the in vitro system. The clear implication from this work is that the current notions of PL-TP function are fundamentally incomplete. As a result, the discovery that the SEC14p is the yeast PI-PC-transfer protein had a dual significance. First, it assigned a biochemical activity to a compartment-specific transport factor. Second, it provided the first experimental system with which the function of a PL-TP could be studied in vivo. 2. SEC14p Controls the Phosphatidylinositol: Phosphatidylcholine Ratio of Yeast Golgi-Complex Membranes The identification of S E C l 4 as the structural gene for the yeast PI-PCtransfer protein raised the possibility that the primary biochemical defect in secl4-1‘”strains might correlate with a phospholipid transfer defect in v i m . Bankaitis et al. (1990) tested this hypothesis by assaying the PI-PC-transfer activities in cytosol prepared from secZ4-Z‘” strains. Cytosols prepared from either SECZ4 or ~ecl4-1‘~’ strains displayed comparable PI-transfer activities when assayed at 25°C. Pre-incubation of wild-type cytosol at 3 T C , followed by assay at 37”C, had no effect on PI-transfer activity. However, the secZ4-Z‘”cytosol exhibited no measurable PI-transfer activity under the same


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conditions. Thus, the PI-transfer activity of secZ4-Z'" strains was thermolabile in vitro. This thermolability was completely relieved by incorporation of SECZl on a single-copy centromere plasmid into secZ4-Z'" strains, while the PI-transfer activity of secZ4-1'" strains was partially thermoreversible in vitro. It was noted that the biochemical properties of the PI-transfer activity displayed bysecl4-Z'" strains correlated quite well with the in vivo behaviour of secZ4-1'" mutants. Interestingly, PC-transfer activity was absent from the cytosol of secZ4-1'" mutants, regardless of assay temperature, even though this activity was readily detected in wild-type cytosol preparations (Bankaitis et al., 1990). Taken together, these data provided a direct correlation between the in vivo ~ e c l 4 - 1 'defect ~ and the in vitro defect in phospholipid transfer. Moreover, the data indicated that the SEC14p represented by far the major, if not the only, PI-PC-transfer activity in the yeast cell, Bankaitis et al. (1990) proposed two distinct models for SEC14p function in vivo. First, they considered the possibility that the in vitro PI-PC-transfer activity of the SEC14p might accurately reflect SEC14p function in vivo. Within the framework of this model, the SEC14p would transfer PI and/or PC among intracellular membranes. The second model considered a scenario in which the in vitro PI-PC-transfer activity of the SEC14p might be an artifact of PI or PC binding by the SEC14p as a means for allosterically regulating some unrecognized SEC14p biochemical activity (much like guanine nucleotides regulate the effector functions of GTP-binding proteins, for example). To distinguish between these two models, Cleves et al. (1991) undertook a detailed analysis of SEC14p function in vivo and concluded that the SEC14p does indeed catalyse PI and/or PC transfer in vivo. Current evidence suggests that the in vivo function of the SEC14p is to generate and maintain an appropriate P1:PC ratio in late Golgi-complex membranes. The salient evidence to this effect is now summarized. Key insights into the role that the SEC14p plays in stimulation of yeast Golgi-complex function came from an analysis of mutations that suppressed the secZ4-Z'" defect (Cleves et al., 1991). These suppressor mutations were unlinked to the secZ4-Z'" locus and led to the indentification of six genes. Two of these genes, BSDl and BSD2, were defined by dominant mutations whereas the other four (SACZ, BSR2, BSR3 and BSR4) were defined by recessive mutations. Mutations in all of these genes were capable of efficiently suppressing normally lethal secl4 null mutations without activation of some cryptic PI-PC-transfer activity. Furthermore, secretory pathway function was fully restored in these suppressor mutants, and secretion involved all of the appropriate compartments, as shown by: (a) the proper glycosyl modifications experienced by secretory glycoproteins in these strains, and (b) a requirement for the 10 late-acting SECgene products

122


for secretion to occur. The cumulative data indicated that these suppressor mutations rendered yeast cells independent of the requirement for an essential PL-TP, and did so without elaboration of some other PTPCtransfer activity. Moreover, suppression was essentially limited to secZ4 defects (Cleves et al., 1991). The sacl mutations were exceptional in this regard as they also suppressed certain actin defects and several other lateactingsecdefects (Clevesetal., 1989). TheSACZ gene, its product, andsacl suppressor properties are discussed in detail later in this review (Section V.D). To date, the most penetrating insights into SEC14p function in vivo have been derived from analysis of the recessive suppressors, in particular BSR gene products. Molecular analysis of BSR4 revealed its identity to a previously recognized gene, CKI, the structural gene for yeast choline kinase (Cleves et al., 1991). This is the enzyme that catalyses the first step in synthesis of PC from free choline via the CDP-choline pathway (Carman and Henry, 1989). The identity of BSR4 with CKI was an intriguing result that linked biosynthesis of a SEC14p ligand to SEC14p function in vivo. Moreover, cki null mutations were found to suppress efficiently normally lethal secZ4 null alleles, a result that was in complete agreement with the recessive nature of the original bsr4 alleles. That suppression was a general consequence of dysfunction of the CDP-choline pathway was indicated by: (a) the observation that disruptions of CPTZ, the structural gene for the enzyme that catalyses the ultimate step in biosynthesis of PC via the CDP-choline pathway (i.e. choline phosphotransferase), exhibited bsr phenotypes (Cleves et al., 1991), and (b) the finding that bsr2 and bsr3 mutants failed to incorporate in vivo choline into PC, thereby indicating that these mutants were also defective in functioning of the CDP-choline pathway. Current data suggest that BSR2 and CPTZ are allelic (M. K. Fung and V. A. Bankaitis, unpublished observation). Thus, the absence of PC biosynthesis by the CDP-choline pathway bypassed the normally essential SEC14p requirement. In marked contrast, lack of PC synthesis by the only other alternative mechanism available to yeast, the methylation pathway (Carman and Henry, 1989), in no way relieved the cellular requirement for SEC14p function (Cleves et al., 1991). The demonstration that the SEC14p requirement could be specifically and efficiently bypassed by PC biosynthetic defects argued strongly for a phospholipid equilibration function for the SEC14p. Moreover, the work of Cleves et al. (1991) provided the first link between biosynthetic protein transport, intracellular phospholipid transport and phospholipid biosynthesis. Cleves et al. (1991) proposed two general models to reconcile the suppression data, in particular the striking feature that inactivation of the CDP-choline pathway for PC synthesis resulted in a bypass of the SEC14p

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whereas inactivation of the methylation pathway did not; these are illustrated in Fig. 5. Both of these models assumed that the SEC14p catalyses mobilization of phospholipids in vivo and, in doing so, maintains an appropriate P1:PC ratio in Golgi-complex membranes that is a critical feature of the secretory competence of these membranes (Fig. 5(a)). The compartmentalized PC-synthesis model suggests that PC synthesis by the CDP-choline pathway occurs in the Golgi complex whereas PC synthesis by the methylation pathway does not. The resulting net PC synthesis in the Golgi complex would lower the P1:PC ratio of Golgi-complex membranes below some critical level if it were not countered by removal of PC from the complex. In this model, the SEC14p is proposed to function in such a removal of PC (Fig. 5(b)). Bypass of the SEC14p would then be the expected result of inactivation of the CDP-choline pathway. Clearly, localization of enzymes of the CDP-choline pathway in specific membranes will provide a crucial test of this hypothesis. The second model, the regulation model, suggests that inactivation of the CDP-choline pathway results in an altered balance of P1:PC in bulk membranes, in particular the E R membranes where PC synthesis by the methylation pathway would be expected to occur. Thus, an appropriate P1:PC ratio would be imposed upon the Golgi complex by bulk-membrane flow from the E R (Fig. 5(c)). In this scenario, the SEC14p would raise the P1:PC ratio of Golgi-complex membranes above that of bulk membranes, and the achievement of an appropriate P1:PC ratio in the E R would obviate the need for SEC14p function. The key prediction of the regulation model is that bsr mutants will have elevated P1:PC ratios in bulk-membrane fractions relative to the P1:PC ratio of bulk membranes from wild-type cells. The various predictions of these two models are testable, and provide a precise experimental framework for a further investigation of SEC14p function in vivo. Outstanding questions still remain and must be addressed. These include: (a) is there a SEC14p receptor in Golgi-complex membranes that imposes a specificity of membrane interaction, (b) does the SEC14p catalyse phospholipid exchange or net phospholipid transfer in vivo and, if net transfer, which phospholipid is the ligand, and (c) which membranes serve as partners in the transfer reaction in vivo? Finally, in vivo analyses of SEC14p function were informative from the perspective of the models for SEC14p function that were rejected by the data. One such example has already been discussed. Another important model rejected by the data dealt with the role of PL-TPs in recycling of bulk intracellular membrane. Wieland et al. (1987) had argued that the rate of phospholipid removal from the E R by bulk-membrane flow was far greater than the rate at which E R phospholipid could possibly be regenerated by synthesis. A convincing argument was made for phospholipid retrieval back

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(a) Golgi complex (inactive 1

Golgi complex (active)


125

to the E R as a necessary mechanism to compensate for this imbalance, and preserve the integrity of the E R . Wieland et a f .(1987) proposed that PL-TPs were the determining participants in such a retrieval, and Rothman (1990) offered a highly speculative model for the manner in which the SEC14p might be involved in such a bulk phospholipid retrieval in yeast. Current data do not support this hypothesis. Salama et a f .(1990) demonstrated that, in wild-type cells, the SEC14p was present at levels some 10-fold in excess of those required for efficient Golgi-complex secretory function. This was not immediately consistent with a bulk phospholipid movement function for the SEC14p. Moreover, the findings of Cleves et al. (1991) that indicated a bypass of SEC14p function could occur without activation of some cryptic PIPC transfer activity, coupled with the fact that the SEC14p ligands (PI and PC) represent the two most abundant phospholipids in yeast, rendered such a bulk phospholipid mobilization model for the SEC14p entirely untenable. Other mechanisms for phospholipid retrieval must operate in vivo.

3. Conservation of SEC14p Structure and Function The ubiquity of PI/PC transfer-protein activities in eukaryotic cells appears to be accompanied by a conservation of transfer-protein structure across wide evolutionary distances. Cytosolic fractions prepared from various mammalian, insect, amphibian, reptilian and avian cells all harbour polypeptides of some 35 kDa that exhibit an immunological cross-reactivity with the bovine PI/PC-transfer protein ( M , 36 kDa) (Wirtz, 1982). FIG. 5. Diagram illustrating a phospholipid mobilization model for the SEC14p. (a) Cleves et al. (1991) proposed that the SEC14p elevates the phosphatidylinositol: phosphatidylcholine(P1:PC) ratio in yeast Golgi-complexmembranes above that of bulk membranes, a function that is critical for maintaining the secretory competence of Golgi-complex membranes. Two models were offered in an attempt to reconcile the observation that mutations in the CDP-choline pathway, but not the methylation pathway, for PC synthesis bypass the SEC14p requirement: (b) PC synthesis by the CDP-cholinepathway is localized in the Golgi complex whereas PC synthesis via the methylation pathway is not. As the SEC14p functions to remove PC from the Golgi complex, a block in PC synthesis by the CDP-choline pathway results in bypass of SEC14p function. (c) Neither PC biosynthetic pathway operates in the Golgi complex, but inactivation of the CDP-choline pathway results in an altered balance of PI:PC in the endoplasmic reticulum (ER) (the most likely biosynthetic compartment for PI and PC). This favourable PI:PC ratio is then imposed upon the Golgi complex by bulk membrane flow in a SEC14p-independent fashion. CDP-DG, CDPdiacylglycerol; PDME, phosphatidyldimethylethanolamine; PE, phosphatidylethanolamine; PMME, phosphatidylmonomethylethanolamine.

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Moreover, Helmkamp and his colleagues reported hybridization of rat PI/PC transfer-protein cDNA to genomic sequences of a number of higher eukaryotic organisms including Drosophila melanogaster (Dickeson et al., 1989). Whether these structural homologues exhibit a functional relatedness in vivo is still an open question. Recent studies in the yeast system have firmly established that the SEC14p is structurally and functionally conserved in lower eukaryotes that are separated by vast evolutionary distances. Bankaitis et al. (1989) identified SEC14p-immunoreactive polypeptides in cell-free lysates prepared from two widely divergent yeasts, namely Kluyveromyces lactis and Schiz. pombe. These SEC14p homologues had apparent molecular weights (as judged by SDS-PAGE) that were similar to, but not identical with, that of the SEC14p in Sacch. cerevisiae. The functional identity of these SEC14p species was unambiguously established by recovery of genomic clones from K. lactis and cDNA clones from Schiz. pombe that complemented secI4 defects in Sacch. cerevisiue, and the demonstration that these clones encoded the corresponding SEC14p structural homologues. The SECI4 gene (SEC14KL)in K . lactis and its product were characterized by Salama et al. (1990). It was shown that the SEC14pKL could functionally substitute for the SEC14p (SEC14pSC) in Sacch. cerevisiae, as shown by the patently wild-type kinetics for protein secretion and vacuolar protein biogenesis exhibited by strains that carried a normally lethal secl4 null mutation and SEC14KL in an ectopic single-copy configuration. Moreover, the level of the SEC14pKL in such strains was very much decreased relative to normal intracellular levels of the SEC14pSC, thereby arguing for a functional identity for these SEC14p species. Nucleotide sequence analysis of SEC14KLrevealed a single, uninterrupted 301-codon open reading frame that encoded a polypeptide with an inferred molecular weight of 34,165. The SEC14pKLexhibited a 77% identity at the primarysequence level with the SEC14pSC,a result that was immediately consistent with the structural and functional homology between these two polypeptides. Clones of cDNA from the SECl4 gene from Schiz. pombe (SEC14SP) were recovered by H . B. Skinner and V. A . Bankaitis (unpublished observation). The SEC14pSP exhibited a functional identity with the SEC14pSCon the basis of the ability of the former to complement fully the growth and secretory defects associated with normally lethal secl4 null mutations. The SEC14SP nucleotide sequence has been determined and inferred to encode a 284-residue polypeptide ( M , 32,788) that displayed almost a 60% identity with the SEC14pSC at the primary sequence level. This striking structural and functional relatedness to the SEC14pSCwas even the more remarkable when one considers that Sacch. cerevisiae and Schiz. pombe exhibit as wide a phylogenetic divergence from each other as each of


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these yeasts exhibits from mammals (Russell and Nurse, 1986). Taken together, the data strongly suggest that the corresponding in vivo roles for the SEC14pKLand SEC14pSPwill be very similar to that of the SEC14pSC, namely a role in Golgi-complex secretory function. It will be of singular interest to determine the subcellular localization of the SEC14pSPin Schiz. pombe and in the heterologous Sacch. cerevisiae. Finally, it is of some interest to note that these three yeast SEC14p species exhibit a significant relatedness to the human retinaldehyde-binding protein (HRBP), a carrier for the hydrophobic ligands 11-cis-retinol and ll-cisretinaldehyde (Salama et al., 1990). Thus, it appears likely that the molecular architecture of these carrier proteins may share some common ancestry, even though the SEC14p and HRBP ligands bear little similarity. It is interesting to note that the SEC14p shows no significant homology to its mammalian biochemical counterpart, the rat PI/PC-transfer protein. This lack of relatedness is especially intriguing in light of the possible conservation of P I P C transfer-protein structure in higher eukaryotes (see above). The very real possibility that the higher eukaryotic PI/PC-transfer proteins serve fundamentally different in vivo functions than do yeast SEC14ps must still be considered seriously in the absence of any direct evidence to the contrary. C. THE RETENTION PROBLEM: THE ROLE OF CLATHRIN

Like the E R , the Golgi complex also contains unique resident proteins that must be retained in face of the massive bulk flow of biosynthetic secretory traffic. The KEX2p is an example of such a Golgi-complex resident in yeast, while Fuller et al. (1989) have shown that the C-terminal cytosolic tail of the KEX2p is required for its retention in the Golgi complex. Although the signals for retention remain undefined, it appears that yeast clathrin plays an entirely unanticipated role as a critical component of a yeast Golgi-complex retention machinery. Clathrin coats are polyhedral lattices that are thought to function in protein sorting, the clustering of receptors and formation of vesicles from the plasma membrane and trans-Golgi-complex network in mammalian cells (Goldstein etal., 1985; Griffiths and Simons, 1986). In yeast and mammalian cells, the major components of clathrin cages are units of three cytosolic clathrin heavy-chain ( M , about 180 kDa) and three clathrin light-chain ( M , about 30-40 kDa) polypeptides (Pearse, 1976; Kirchhausen and Harrison, 1981; Mueller and Branton, 1984; Lemmon et al., 1988) which can selfassemble into basket-like structures in vitro (Keen et al. , 1979; Woodward and Roth, 1979; Lemmon et al., 1988). In order to determine more precisely the role of clathrin in intracellular

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A. E. CLEVES AND V . A . BANKAITIS

protein traffic in vivo, studies of clathrin function were initiated in Sacch. cerevisiae. Using antibodies raised against yeast-clathrin heavy chain, clones of the gene encoding this polypeptide, CHCl , were recovered (Payne and Schekman, 1985; Lemmon and Jones, 1987). Haploid cells bearing a deletion of the CHCl gene were sickly, but nonetheless viable, and exhibited essentially wild-type rates of secretion. Furthermore, no clathrin heavy-chain homologues were identified in strains with a deletion in chcl, designated Achcl , by low-stringency DNA hybridization or antibody crossreactivity (Payne and Schekman, 1985). These data suggested that yeast clathrin plays no role in protein transport to the cell surface. Also, because protein secretion in yeast was virtually unaffected in the absence of the clathrin heavy chain, and yeast showed no obvious substitute for the CHClp, models that invoked an essential role for clathrin in protein transport were rejected. It should be pointed out, however, that the nonessential nature of chcl null mutations remains controversial. Lemmon and Jones (1987) found such lesions to be lethal in other yeast genetic backgrounds. Nevertheless, the ability to generate Achcl mutants, in at least some genetic backgrounds, has yielded startling clues as to the role of clathrin in yeast. Further insights into the role of clathrin in yeast were obtained by Payne and Schekman (1989) who observed that Achcl cells secreted a precursor form of a-factor that had not been proteolytically processed. This precursor comigrated on SDS gels with the unprocessed form of a-factor secreted by cells that lacked the KEX2p endoprotease that is required for a-factor maturation. It was subsequently demonstrated that secretion of a-factor precursor in Achcl strains was due to a failure of such strains to retain the KEX2p in the Golgi complex, the normal location of this protease. It was found that the KEX2p could be specifically radio-iodinated in Achcl but not CHCl cells under conditions of exclusive surface labelling. Moreover, the KEX2p enzymic activity could be measured at the cell surface of Achcl but not CHCl cells. About 75% of the total KEX2p was estimated to be mislocalized to the cell surface of Achcl cells at the steady-state level (Payne and Schekman, 1989). Based on these data, it was proposed that clathrin serves to: (a) retain certain proteins within the Golgi complex, or (b) retrieve escaped resident proteins from the plasma membrane to the complex. Localization of clathrin to a specific subcellular compartment should discriminate between these two general possibilities. In either case, clathrin is a critical component of the mechanism by which organelle identity is maintained in yeast. This retention model for clathrin function will be further supported if other resident Golgi-complex proteins are found to be mislocalized in clathrindeficient yeast.


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D. COUPLING OF GOLGI-COMPLEX AND ACTIN-CYTOSKELETON FUNCTIONS

Cells of Sacch. cerevisiae show a polarized mode of secretion that is an essential feature of cell growth and division in this organism. Golgi complexderived secretory vesicles are targeted almost exclusively to the growing bud. As a result, the bud grows whereas the mother cell largely does not. Based on the structural changes exhibited by actin during the yeast cell cycle and the phenotypes displayed by actin-deficient mutants, it was proposed that the polarized mode of cell growth is mediated by the actin cytoskeleton (Kilmartin and Adams, 1984; Adams and Pringle, 1984; Novick and Botstein, 1985). Fluorescence-microscopy analyses revealed a yeast filamentous actin cytoskeleton comprised of two basic components: (a) actin cables oriented along the mother-bud axis, and (b) membrane-associated cortical patches preferentially distributed to the bud in regions associated with cell-surface growth (Kilmartin and Adams, 1984; Adams and Pringle, 1984). Thus, the orientation of the actin cytoskeleton correlated with the polarity of cell growth. Analysis of the terminal phenotypes of mutants bearing conditionally lethal mutations in ACT1 (the essential, single-copy structural gene for yeast actin) provided confirmatory evidence for involvement of actin in organization of the late stages of the yeast secretory pathway. Under non-permissive conditions, the actlts mutants contained increased intracellular pools of fully glycosylated secretory invertase, a proliferation of intracellular membranes that appeared to represent Golgi complex-derived secretory vesicles and, in the case of actl-lfS,Golgicomplex bodies, and a wholesale disorganization of cell-surface growth. This last characteristic was shown by the arrest of growth at the nonpermissive temperature of actl" mutants as swollen unbudded cells and their aberrant deposition of cell-wall chitin (Novick and Botstein, 1985). Taken together, these data argued for an actin involvement in the late stages of the secretory pathway and in the establishment and maintenance of correct secretory polarity. Recent work has indicated that the yeast cell co-ordinates Golgi-complex secretory and actin cytoskeleton function, and some insights have been obtained with regard to the identity of the cellular machinery by which such a coupling might be achieved. These findings represented the convergence of two independent lines of inquiry, one involving a study of mutations that suppress actin defects and the other a study of mutations that bypass the normally essential SEC14p requirement for Golgi-complex function (Section V.B). The SAC1 gene product is an excellent candidate for a component of a cellular machinery that couples the activities of the late stages of the secretory pathway and those of the actin cytoskeleton. This gene was independently identified in suppressor analyses of the nctl-1" and secl4-1'" defects, respectively. Novick et al. (1989) attempted to identify gene

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A. E. CLEVES AND V. A. BANKAITIS

products that interact with yeast actin in vivo by isolating recessive mutations that suppressed the actl-l'" defect and, additionally, exhibited a new cs phenotype. The phenotypic properties of sacZCSmutants fulfilled several genetic criteria that might be expected of a mutant that is defective in an actin-binding protein. First, sacZcssuppressed actl'" defects and did so in an allele-specific manner. That is, while sacZCSsuppressed actl-Z'", the combination of sadCSand actl-2'" was a haploid-lethal event. Allele-specific interactions of this sort are often used as genetic diagnoses for interactions between the corresponding gene products (Botstein and Maurer, 1982). Second, the terminal phenotypes of sacZCS mutants at the restrictive temperature of 14°C redisplayed some of the actZ-ItSterminal phenotypes. At 14"C, sacZCSmutants failed to exhibit visible actin cables and showed a randomization of actin cortical patches between mother and bud. An aberrant pattern of chitin deposition was also strikingly apparent. Novick et al. (1989) recovered SACZ clones and demonstrated that the s a d null phenotype was cold-sensitive for growth. As discussed in Section V.C.3, extragenic suppressors of secZ4-Z'" were isolated and characterized. The rsdZ complementation group was recognized amongst these suppressors as a class of recessive mutations that uniformly exhibited an unselected cs phenotype (Cleves et al., 1989, 1991). The terminal rsdl phenotypes were characterized at 14°Cand were found to coincide exactly with those observed for sucZcsmutants. Interestingly (even though rsdl" suppressed sec14-Zts),rsdl'" mutants failed to exhibit any significant secretory defects or any significant exaggeration of intracellular membranes. Because the actin cytoskeleton was thought to be involved in the later stages of the secretory pathway, and the ~ecZ4-Z'~ block was most likely exerted at the level of a late Golgi-complex compartment, Cleves et al. (1989) considered the possibility that RSDZ and SACZ were allelic. From several lines of genetic evidence this allelism was proven and the sad nomenclature was adopted. Strikingly, sacZCSalleles, generated as suppressors of actl-l'", were also shown to suppress secZ4-Z'". In fact, sacZCSalleles bypassed the SEC14p requirement altogether as sacla suppressed normally lethal secl4 null mutations (Cleves et al., 1989). To determine if the sacZCSmutants exhibited genetic interactions with any of the other sects mutations, the appropriate double mutants were constructed and analysed for growth at a variety of temperatures. The s a ~ Z - 6 ~ ~ allele was chosen because it had been shown to suppress actZ-Z'" and se~Z4-Z~". Interestingly, s a ~ Z - 6exhibited ~~ a partial suppression of sec6" and sec9" as well. Since sec6'" and sec9" represented defects at the level of Golgi complex-derived secretory vesicle function, and actin had been implicated in the later stages of the secretory pathway, it was suggested that the suppression data provided additional evidence for a late execution point


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for the SEC14p, perhaps at the level of secretory protein exit from the Golgi complex (Cleves et al., 1989). The SAC1 gene and its gene product have been characterized (Cleves et al., 1989; A . E. Cleves and V. A . Bankaitis, unpublished observation). Sequence analysis of the DNA revealed a 623-codon open reading frame that potentially encoded a polypeptide with a molecular mass of 71,132. Inspection of the SAClp primary sequence revealed several features. First, a run of 23 uncharged amino-acid residues, which constituted a good candidate for a membrane-spanning region, divided the SAClp into a large 521-residue N-terminal domain and a small 79 residue C-terminal domain. Second, there were no obvious signal-peptide structures that might direct the SAClp into the secretory pathway. Finally, the C-terminal 95-residue region of the SAClp identified a very basic domain (PI 9.8). Immunoprecipitation experiments using anti-SAClp monoclonal antibodies showed the SAClp to be an unglycosylated polypeptide with an apparent molecular mass of some 65 kDa. The inconsistency between the predicted molecular mass and the value estimated from the mobility on SDS-PAGE gels is considered to be most likely due to the basic character of the SAClp. In subcellular fractionation experiments, the SAClp was quantitatively recovered in pellet fractions and was not extracted from membranes by treatment with 0.1 M sodium carbonate (pH 11.5), indicating the SAClp to be an integral membrane protein. Protease protection experiments employing lysates from gently lysed sphaeroplasts, in which the integrity of small organelles was maintained, suggested that the SAClp was oriented such that the large N-terminal domain was lumenally disposed while the small C-terminal domain was exposed to the cytosol. Preliminary subcellular fractionation and immunofluorescence microscopy data suggest a localization of the SAClp to the Golgi complex and the ER. In summary, the discovery that mutations in a single gene, SACZ, could suppress secretory and cytoskeletal defects provided the first genetic link between actin-cytoskeleton assembly and secretory pathway activities, in particular late Golgi-complex function. How might the SAClp co-ordinate such seemingly disparate activities as Golgi-complex and yeast actin function? We can now offer a speculative model. It has recently been demonstrated that the actin-binding proteins profilin and gelsolin also bind the phospholipid phosphatidylinositol4,5-bisphosphate(PIP2) with a higher affinity than that which these proteins exhibit for actin, and these proteins have been proposed to cluster PIP2 on the cytoplasmic leaflet of membranes (Yin et al., 1988; Hartwig et al., 1989; Goldschmidt-Clermont et al., 1990). Since the C-terminal tail of the SAClp, which appears to be disposed to the cytosol , exhibits some primary-sequence homology to several known actin-binding proteins, the question of whether the SAClp binds

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phospholipids becomes relevant. The fact that: (i) the SEC14p is the PI-transfer protein (Bankaitis et a f . , 1990), (ii) the SEC14p appears to function in modulating the P1:PC ratio of yeast Golgi-complex membranes (Cleves et a f . , 1991), and (iii) PI is the precursor to PIP2, provides an intriguing possibility as to how the SAClp might influence both SEC14p and actin function. Perhaps the SAClp exerts these comodulatory effects by a phospholipid (PI, PIP or PIP2)-sequestration mechanism. Clearly, in vitro phospholipid-binding analyses should provide some further insight. In any event, we note that the idea of some sort of coupling of actin and Golgicomplex function is an attractive one. If actin cables do indeed direct secretory vesicles to the bud, it seems that regulating aspects of actin assembly in the immediate vicinity of the late Golgi-complex membranes, from which such transport vesicles will be derived, is a reasonable strategy. VI. Fusion of Golgi Complex-Derived Vesicles with the Plasma Membrane

Ten genes whose products participate in targeting and/or fusion of Golgi complex-derived secretory vesicles with the plasma membrane have been identified in yeast (Novick et al., 1980). Mutations in SECZ, SEC2, SEC3, SEC4, SECS, SEC6, SEC8, SEC9, SECIO and SECIS result in the accumulation of these vesicles. Molecular and genetic analyses of these lateacting SEC gene products have categorized these genes into two classes, namely those that exhibit genetic interactions with SEC4 and those that do not. Little is known about the latter class of gene products and these will not be discussed in this review. A study of the yeast SEC4p, a ras-like GTPbinding protein, has yielded valuable insights into how vesicular transport is regulated. In fact, the work of Novick and his colleagues represented the first direct demonstration of GTP-binding protein involvement in protein transport, and has spawned a great deal of current effort, in numerous laboratories, directed at analysing how such proteins regulate secretory processes. As the subject of GTP-binding protein function has been exhaustively reviewed elsewhere (Gilman, 1987; Bourne, 1988; Hall, 1990), we will limit our treatment of this subject to those topics that relate directly to secretion in yeast. A. INVOLVEMENT OF A GTP-BINDING PROTEIN

The initial demonstration that a small GTP-binding protein plays a role in secretory pathway function was provided by Novick and his colleagues who extensively characterized the SEC4 gene product (Salminen and Novick, 1987; Goud et af., 1988; Walworth et af., 1989; Kabcenell et a f . , 1990).


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Nucleotide sequence analysis of SEC4 revealed a 215-codon open reading frame that had the potential to encode a hydrophilic polypeptide with an apparent molecular mass of 23.5 kDa (Salminen and Novick, 1987). Inspection of the inferred SEC4p primary sequence was immediately informative since the SEC4p primary sequence displayed a 32% and a 48% homology with the human H-ras and yeast YPTZ gene products, respectively. Moreover, sec4 disruption alleles proved to represent haploid-lethal events. Thus, there is no redundancy in SEC4p function in yeast. This is in contrast to yeast RAS function, which is genetically redundant (Kataoka et al., 1984). Also, in spite of the extensive homology between the SEC4p and the YPTlp, these polypeptides exhibited no detectable functional overlap (Salminen and Novick, 1987). That the SEC4p is indeed a GTP-binding protein was established by Goud et a f . (1988) who employed a ligandblotting assay to demonstrate that, although the SEC4p bound GTP in this assay, the sec4-8" gene product did not. However, as pointed out by these workers, these data did not unambiguously indicate that the ~ec4-8~' mutation directly affects GTP binding by the SEC4p. The demonstration that GTP binding was required for SEC4p function was provided by Walworth et al. (1989), who created mutant Sec4p derivatives that failed to bind GTP and showed that such mutant proteins were non-functional in vivo. Using the combined techiques of differential centrifugation and densitygradient fractionation, Goud et al. (1988) determined the subcellular localization of the SEC4p in both wild-type and secretory vesicleaccumulating yeast strains. In wild-type cells, the SEC4p was located predominantly in a 10,OOOg pellet fraction, while a smaller amount was recovered from a 100,OOOgfraction, and some 10% of the total SEC4p was found in the cytosolic fraction. In marked contrast, after imposition of the secretory-vesicle block, the majority of the SEC4p recovered from a sec6" yeast strain was recovered from the 100,000gpellet fraction. Only some 10% of the SEC4p sedimented at 10,OOOg under these conditions. Subsequent sucrose density-gradient fractionations demonstrated that the SEC4p recovered from the 10,OOOg pellets in both wild-type and sec6'' lysates cofractionated with a plasma-membrane marker. The SEC4p recovered from the 100,000g pellet in the sec6" lysate cofractionated with secretory invertase, a lumenal secretory vesicle marker under those conditions. It is presumed that the minor SEC4p fraction found in the 100,OOOg pellet from wild-type cells also represented secretory vesicle-associated material. Unfortunately, there do not as yet exist any secretory vesicle markers in wild-type cells that can be used to establish this point unambiguously. Nevertheless, Goud et a f . (1988) further demonstrated that the membranebound SEC4p was exposed to the yeast cytosol, as judged by its sensitivity to

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exogenous protease challenge of intact secretory vesicles, and that the SEC4p could be chased from the plasma membrane to secretory vesicles upon imposition of the sec6" block. These data indicated a redistribution of the SEC4p and raised the possibility that the SEC4p recycled from the plasma membrane to secretory vesicles, perhaps via a soluble intermediate. The SEC4p primary sequence indicated a hydrophilic protein that was devoid of significant hydrophobic character (Salminen and Novick, 1987). Yet, the kinetics of the association of the SEC4p with membranes in vivo were extremely rapid, exhibiting a half-life of less than one minute (Goud et al., 1988). To investigate further the nature of the association of the SEC4p with membranes, Goud et al. (1988) attempted to extract the SEC4p from membranes by a variety of treatments. The results clearly showed that the SEC4p behaved as an integral membrane protein. It was not liberated by treatment with an alkaline sodium carbonate solution and it partitioned into the detergent phase following solubilization in an aqueous solution of Triton X-114. Overproduction of the SEC4p specifically increased the soluble pool of this protein, however, and this soluble pool partitioned into the aqueous phase following Triton X-114 solubilization. Thus, it seems that the SEC4p post-translationally acquires a hydrophobic modification that promotes membrane attachment, much like the farnesyl and palmityl modifications that are experienced by ras and other GTP-binding proteins, and that SEC4p attachment to membranes is specific and saturable. Inspection of the SEC4p primary sequence reveals that, as with the YPTlp, the polypeptide terminates with two cysteine residues (Salminen and Novick, 1987). This sequence resembles the canonical CAAX box that is characteristic of the C-termini of GTP-binding proteins. Molenaar et al. (1988) showed that these terminal cysteine residues were required for YPTlp function. To determine if the same was true for the SEC4p, Walworth et al. (1989) constructed a SEC4 allele from which the terminal cysteine residues were deleted (sec4-CCA).Gene-replacement experiments showed that sec4-CCA did not encode a functional protein as the deletion failed to complement the sec4-8" growth defect and represented a recessivelethal mutation. Subcellular fractionation experiments indicated that the Sec4p-CCA was a stable protein whose GTP-binding properties remained intact, but which was located exclusively in the cytosol. These data demonstrated that the C-terminus of the SEC4p was involved in determining the membrane-binding capacity of the gene product, and that membrane binding was required for SEC4p function. A direct demonstration of a posttranslational modification of SEC4p is, however, not yet available. Based on the collective data, Walworth et al. (1989) and Bourne (1988) argued that SEC4p function can be viewed as being analogous to that of the translation elongation factor EF-Tu in E. coli (Kaziro, 1978). An adaptation


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Nucleotide

FIG. 6. Diagram showing a Cycling model for function of the SEC4p. Soluble SEC4p exchanges GDP for GTP and binds to a newly formed secretory vesicle, thereby rendering the labelled vesicle competent for interaction with a plasmamembrane effector protein. Engagement of the vesicle with the effector triggers the fusion event. Guanine nucleotide hydrolysis by the SEC4p results in disengagement of the SEC4p.GDP from the effector so that both components can be re-utilized for another round of secretory vesicle targeting and fusion. From Walworth etaf.(1989).

of this model is shown in Fig. 6. In this scheme, G D P is exchanged for GTP by soluble SEC4p. This SEC4p-activation step may require an ancillary guanine-nucleotide exchange activity. The SEC4p.GTP then engages a newly formed secretory vesicle, and renders the “labelled” vesicle competent for docking with the plasma membrane. Such a docking event may require participation of an effector protein that recognizes the SEC4p. Following the subsequent fusion event, release of the SEC4p is coupled to a conformational change induced by GTP hydrolysis. The released

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SEC4peGDP is then available for participation in another round of secretory vesicle docking and fusion. This model provides a satisfactory explanation for the need for the SEC4p in secretory vesicle consumption. It also makes a powerful prediction, namely that an irreversibly activated SEC4p would serve as a potent inhibitor of the vesicle-fusion process by virtue of its progressive and irreversible occupation of the effector. Important support for this model, in particular from the standpoint of the last prediction, is forthcoming from a study of sec4-Ife133. This allele was designed to mimic an oncogenic H-ras mutation that resulted in constitutive activation of RAS function in the absence of any GTP binding (Walworth et a f ., 1989; Walter et af.,1986). Expression of sec4-ZfeZ33 resulted in a specific and dose-dependent block at the level of secretory vesicle fusion to the plasma membrane in vivo. At low levels, the S e ~ 4 p " " caused ' ~ ~ a dominant ts phenotype that correlated with a late-acting vesicle block in secretion. At high levels, the S e ~ 4 p " " caused ' ~ ~ a dominant-lethal secretory block. These secretory blocks were uniformly characterized by accumulation of secretory vesicles within the yeast cell (Walworth et a f . , 1989). Deletion of the terminal cysteine residues of the S e ~ 4 p " " 'completely ~~ suppressed its dominant-negative effects on secretion without destabilizing the protein. Thus, one can incorporate the S e ~ 4 p ' " ' ~ data ~ directly into the model of Walworth et a f . (1989) (Fig. 6). The S e ~ 4 p " most ~ ' ~ ~likely represents a protein that is permanently locked into the active state, and is thereby liberated from the guanine-nucleotide switch that normally regulates SEC4p function. In this scenario, the dominant-negative S e ~ 4 p " " 'catalyses ~~ the first round of secretory vesicle fusion, but fails to disengage from the effector once fusion has occurred. The net consequence of this behaviour would be a dose-dependent and irreversible occupation of the effector, thereby precluding participation of the effector in more than a single round of S e ~ 4 p " ~ ' ~ ~ - c a t a l yvesicle s e d fusion. We note that the inhibitory role of GTPdS in a number of intercompartmental transport systems in v i m is also very easily incorporated into such a scenario. Evidence obtained from mammalian systems indicates that small GTPbinding proteins similar to the SEC4p are likely to be involved in regulating vesicular traffic at several stages of interorganelle transport. Chavrier et af. (1990) identified a number of novel GTP-binding proteins (termed rab proteins) that appear to be involved in endocytic as well as exocytic transport (Chavrier et a f . , 1990). At this point, it seems likely that the current model for SEC4p function can be extended to the function of other small GTP-binding proteins in secretion, and serve as a paradigm for describing one level of regulation of vesicular traffic in the eukaryotic secretory pathway.


B. OTHER GENE PRODUCTS THAT POTENTIATE

137

GTP-BINDING PROTEIN FUNCTION

The SEC4p has been purified and its biochemical properties that relate to nucleotide binding and GTP hydrolysis examined (Kabcenell et al., 1990). This polypeptide was found to have high affinities for GTP and GDP, but release of GTP from the SEC4p (0.0012 min-') was exceedingly slow relative to that of GDP (0.21 min-'). Given these kinetics, an unregulated SEC4p would be expected to exist predominantly in a GTP-bound state, a condition that would not be favourable for sustained secretory vesicle fusion to the plasma membrane. Thus, auxiliary factors must participate in regulation of nucleotide binding, exchange and hydrolysis by the SEC4p. Moreover, there remains the question of the identity of the effector through which the SEC4p operates. Elucidation of the identities of these auxiliary factorsand of the effector will be the next step in attaining a more complete understanding of the SEC4p cycle. The remaining nine late-acting S E C gene products are good candidates for factors that either modulate or respond to SEC4 function in vivo. This view was strongly supported by the observation that SEC4 exhibited striking genetic interactions with several of the other late-acting S E C genes (Salminen and Novick, 1987). Duplication of SEC4 elicited a clear suppression of the seclSS,sec2" and sec8" defects, a weaker suppression of seclts,sec5" and secl@' defects, and no detectable suppression of the ~ e c 3 ' ~ , sec6" and secP defects. With respect to this suppression, it is of interest to note that SEC4 clones were originally recovered in attempts to recover SECl5 clones (Salminen and Novick, 1987). These suppression data suggested a functional relationship between the SEC4p and the late gene products whose defects were dosage compensated by the SEC4p. Further support for a functional interaction between the SEC4p and a subset of the late SEC gene products was obtained by an independent line of evidence. The sec4-8" mutation exhibited a synthetic lethality when combined with either of the sec2", sec3", secF, secSts,s e c l p and ~ e c l.5mutations. '~ Thus, SEClS, SEC2 and SEC8 exhibited consistent genetic interactions with SEC4 whereas the significance of the SEC4 interactions with S E C l , SEC3, SECS and SEClO are less clear. There are, at present, no genetic grounds for considering SEC6 or SEC9 gene function to have any particular relationship to SEC4 function. As a result of the particularly strong genetic interactions between SEC4, SEC2 and SECl.5, the latter two genes have been a primary focus of study. The SECZS gene has been characterized at the molecular level and shown to be an essential gene that could potentially encode a hydrophilic polypeptide of 911 amino-acid residues (Salminen and Novick, 1989). The SEC15p has been identified and shown to exhibit an apparent molecular

138


mass of 116 kDa that was consistent with the predicted molecular mass of 105 kDa. Subcellular fractionation experiments revealed that, in spite of its hydrophilic character, the SEC15p behaved as a peripheral membrane protein which could be extracted from membranes with salt or urea or at increased pH values. Although the SEC15p was not detected in wild-type cells by indirect immunofluorescence without resorting to SEC15p overproduction (Salminen and Novick, 1989), subsequent fractionation experiments indicated that some 23% of the total gene product was associated with the plasma membane while the remainder fractionated as a high molecularmass soluble species (1000-2000 kDa) (Bowser and Novick, 1990). Further insight into SEC15p function was obtained by analysing yeast cells that overproduced the SEC15p (Salminen and Novick, 1989). Increased amounts of the SEC15p interfered with secretory vesicle fusion with the plasma membrane. Indeed, secretory vesicles aggregated into a striking patch within the growing bud. That this patching was a manifestation of some physiologically relevant aspect of SEC15p function was indicated by the findings that: (a) overproduction of the Sec15pt”failed to induce vesiclepatch formation, (b) overproduction of the SEC15p resulted in a coincident patching of the SEClSp, as judged by indirect immunofluorescence, and (c) SEC15p patch formation was dependent on the SEC4p and SEC2p. On the basis of these data, Salminen and Novick (1989) suggested that one interpretation of the data was that the SEC15p may facilitate docking of SEC4p-labelled secretory vesicles to the plasma membrane. Patching would simply reflect incorporation of the SEClSp onto vesicles, thereby resulting in a target-membrane identity crisis which causes inappropriate vesiclevesicle docking reactions (i.e. patching). The data also suggest that the SEC2p and SEC4p act upstream of the SEC15p execution point in the secretory vesicle fusion pathway, although the order of action of the SEC2p and SEC4p is not revealed by these data. At present, the collective SEC15p data suggest an involvement for this protein in effector function. It will be of great interest to determine whether the SEC4p exhibits a direct physical interaction with the SEC15p in vivo. Clones of the SEC2 gene have also been recovered and characterized (Nair et al., 1990). The SEC2p was inferred to be a hydrophilic polypeptide of 759 amino-acid residues ( M , 84 kDa), which gene-disruption experiments have indicated to be essential for yeast viability. Homology searches have revealed that the N-terminal one-third of the SEC2p exhibited a 25% identity with the rod region of the myosin heavy chain, and similar homologies to other cytoskeletal proteins. Nair et al. (1990) suggested that these homologies reflect the presence of an a-helical, coiled-coil domain (a general feature of the rod region of the myosin heavy chains) in the SEC2p, rather than some cytoskeletal association of the SEC2p. This suggestion is

SECKETOKY PATHWAY FlJNLTION IN SACCHAROMYCES CEREVISIAE

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supported by subcellular fractionation experiments that indicated a predominantly cytosolic localization of the SEC2p in a high molecular-mass complex (500-700 kDa). A striking confirmation for the coiled-coil domain of the SEC2p playing a crucial role in the SEC2p function was forthcoming from analysis of sec2" mutations. Two such alleles were determined to be nonsense mutations, specifically opal mutations. These data suggested that the C-terminus of the SEC2p was at least partially dispensible, and this idea was further supported by gene-disruption experiments. Truncation of the last 251 SEC2p residues, a removal of one-third of the primary sequence, did not detectably affect SEC2p function. Truncation of the last 368 residues rendered SEC2p function thermosensitive. Deletion of the N-terminus of the SEC2p was a haploid-lethal event (Nair et al., 1990). Clearly, the means now exist for a detailed study of how the function of the SEC15p and SEC2p, in concert with the SEC4p, regulate secretory vesicle fusion to the plasma membrane. It remains less clear, at this time, what roles the other late-acting SEC gene products play at this step. Continued molecular analysis of these genes should eventually permit an incorporation of those corresponding gene products into a model that fully describes secretory vesicle targeting to, and fusion with, the plasma membrane. VII. Summary A genetic analysis of secretory pathway function in yeast was initiated some 12 years ago in the laboratory of Randy Schekman. These mutants held great promise in terms of providing an experimental system with which molecular participants of secretory pathway function could be investigated. This early promise has not failed. For the last five years, analysis of yeast secretory pathway function has been at the cutting edge of our understanding of the mechanisms by which proteins travel between intracellular compartments. In some cases, Sacch. cerevisiae has provided a valuable in vivo corroboration of the concepts derived from biochemical studies of mammalian intercompartmental protein transport in vitro. In other cases, studies conducted in the yeast system have defined previously unanticipated involvements for known catalytic activities in the secretory process. It is clear that yeast will continue to play a major role in setting the pace of research directed towards a detailed molecular understanding of protein secretion. Since it is now apparent that the basic strategies that underlie secretory pathway function have been conserved among eukaryotes, further exploitation of the powerful and complementary yeast and mammalian experimental systems guarantees that the next decade will see even greater progress towards

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our understanding of protein secretion in eukaryotic cells than did the first. VIII. Acknowledgements

We wish to thank all of our colleagues for their cooperation in providing us with results in advance of publication. The expert assistance of Sandy Henson in the preparation of this manuscript is also greatly appreciated. Work in the authors’ laboratory was supported by grants from the National Science Foundation (DCB-9003750) and the American Heart Association Illinois Affiliate (880752).

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