Translocation of Proteins across and Integration of Membrane Proteins into the Rough Endoplasmic Reticulum REID GILMORE AND KENNAN V. KELLARIS Dcpartmcnt of Biochmtiscry and M o h h r B w b ~ Unipersity of Massachusetts Mcdiral School 55 h k c A m u e North Wmster, Massachusetts 01655
One of the major topics addressed in the meeting “Proteases and Protease Inhibitors: Emerging Roles in the Pathogenesis of Alzheimer’s Disease” is the proteolytic processing reactions that lead to the production of the p protein from the amyloid precursor protein (APP). Eukaryotic cells produce numerous proteolytic enzymes that could be potentially responsible for the proteolytic cleavages that liberate the /3 protein from APP. Both the amyloid precursor protein and the proteolytic enzymes are restricted to specific cellular compartments; hence a consideration of the membrane topology and intracellular location of APP may allow investigators to identify proteolytic enzymes that are accessible to the regions of APP that are cleaved during /3 protein formation. The membrane orientation of integral proteins like APP is established during synthesis when the protein is integrated into the rough endoplasmic reticulum (RER). The focus of this paper will be the mechanisms responsible for the asymmetric integration of membrane proteins into the RER. Cotranslational protein modification reactions that occur during glycoprotein biosynthesis will also be described. Newly synthesized polypeptides that are mishandled by the translocation apparatus are probably degraded by proteases that reside within the cytoplasm and the endoplasmic reticulum. Proteins located within the lumen of the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, endosome and lysosome and virtually all secreted proteins are cotranslationally translocated across the rough endoplasmic reticulum (RER). The majority of the integral membrane proteins of the rough and smooth endoplasmic reticulum, Golgi apparatus, endosome, lysosome and plasma membrane are synthesized by the membrane-bound ribosomes of the RER.1,2Integral membrane proteins can be separated into several classes based upon the orientation and number of membrane spanning segment^.^.^ Type I and type I1 membrane proteins both contain a single membrane-spanning segment (FIG.1). When initially integrated into the RER, the amino terminus of type I membrane proteins is located within the membrane lumen, while type I1 integral membrane proteins are integrated in the opposite orientation. Type I membrane proteins can have the bulk of the protein oriented towards either face of the membrane, unlike type I1 proteins, which typically have small Nterminal cytoplasmic domains. Based upon the protein sequence derived from sequencing a cDNA clone, the APP protein appears to be a typical type I membrane protein. Proteins with multiple membrane-spanning segments (type 111) can be oriented with the N-terminus facing either the cytoplasm or the lumen. Proteins anchored to the 27
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Cytoplasm Type II
Type Ill
N
f C
Lumen
FIGURE 1. Topology of integral membrane proteins in the endoplasmic reticulum. The amino (N) terminus of type I integral membrane proteins is located in the lumen, with the carboxyl (C) terminus exposed to the cytoplasm. Type I1 proteins have the opposite orientation. Polytopic or type I11 integral membrane proteins can be oriented so that the N-terminus is located in either the lumen or the cytoplasm. Polytopic integral membrane proteins can have an even or odd number of membrane-spanning segments. N-linked oligosaccharide is designated by the symbol Y, and is only found on lumenally exposed protein domains.
membrane via a phosphatidylinositol glycan anchor are transiently inserted into the membrane as type I membrane proteins. The selective translocation of a protein across, or integration of a protein into, the rough endoplasmic reticulum occurs by a sequence of protein-mediated reactions that can be designated as follows: (i) sorting, (ii) targeting, (iii) insertion, (iv) transport, and (v) nascent chain modification. As described below, researchers have identified topogenic signals within membrane proteins that are recognized by the various components of the protein translocation apparatus. Current models for the function of the protein translocation and integration apparatus provide a molecular explanation for how several of these topogenic signals are decoded.
Signal Sequences and Sorting Reaction Proteins destined for translocation across the RER are selected during synthesis from among all polypeptides that are translated within the cytoplasm by virtue of an RER-specific signal sequence. Secreted proteins and lumenal proteins of both the exocytic and endocytic membrane systems contain a cleavable signal sequence located at the extreme N-terminus of the protein (for a review see A cleavable signal sequence is also located at the N-terminus of type I membrane proteins that have large lumenal domains. In contrast, the first transmembrane spanning segment of type I1 273).
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and type I11 integral membrane proteins functions as a noncleavable signal sequence. Cleavable signal sequences from numerous proteins have been compared to identify sequence motifs of functional s i g n i f i c a n ~ eThese . ~ ~ ~ comparisons did not reveal the presence of a strongly conserved sequence. Instead, the typical 15-25 amino acid residue signal sequence was found to contain the following structural features: (a) a basic amino terminal region, (b) a central core of hydrophobic amino acids and (c) a carboxyl terminal region that specifies cleavage by the enzyme signal ~ e p t i d a s eOf .~~~ these three regions, the central hydrophobic core is the most important determinant as a sorting signal for translocation across the endoplasmic reticulum. The sorting reaction corresponds to binding of the signal recognition particle (SRP) to the signal sequence during translation. The SRP was initially isolated as a peripheral membrane protein complex that was required for the translocation of a secretory polypeptide across mammalian endoplasmic reticulum.6 An SRP-mediated sorting step is the initial event in the translocation of most proteins across the endoplasmic reticulum. Several low molecular weight secretory polypeptides can be translocated across the endoplasmic reticulum by an SRP-independent mechanism.’J Subsequent experiments established that SRP is required for the integration of the three classes of integral membrane proteins depicted in FIGURE 1 .9-11 However, integral membrane proteins with cytoplasmic domains that are anchored to the membrane via a carboxyl terminal tail, such as cytochrome bs, are integrated into the membrane by a pathway that is not dependent upon SRP.’ SRP is a ribonucleoprotein particle consisting of six polypeptide subunits (72, 68, 54, 19, 14 and 9 kDa) and the 7SL RNA.6J2 The SRP binds with high affinity to ribosomes translating a mRNA encoding a protein with a RER-specificsignal sequence. High affinity binding decreases or halts the elongation of the nascent polypeptide. 1 3 ~ 1 4 The functional significance of the altered elongation rate will be discussed in the next section regarding targeting of SRP-ribosome complexes to the RER. SRP particles have been disassembled into protein and RNA components and then reconstituted.I5 The 7SL RNA is essential for the activity of SRP,Is and provides a framework upon which the protein subunits of SRP are assembled into three structurally and functionally distinct domains.16The 9/14 kD heterodimer binds to the Alu-like region of the 7SL RNA to comprise the elongation arrest domain of SRP.’”18 The 54kD subunit of SRP ( i x . SRP54) binds to the signal sequence of a nascent polypeptide shortly after it emerges from the large ribosomal subunit, as shown by photocross-linking SRP54 experirnent~.~ ~ J ~ and SRP19 comprise the signal sequence recognition domain of the SRP particle.16 Homologues of the 7SL RNA and the SRP54 polypeptide have been identified in a variety of eukaryotic organisms including the yeast S~hizosaEchaaromycerp@~-~~ and S. ccrtpljllc.22~24Depletion of the S. cmpiw SRP54 polypeptide results in the cytoplasmic accumulation of precursor forms of polypeptides that are normally se~reted.~‘ This result demonstrates an in viw function for SRP that is entirely consistent with the in vipo requirement for SRP in a protein translocation assay. Aberrant cytoplasmic forms of secretory proteins and integral membrane proteins are not readily detected in mammalian cells. Presumably, recognition of RER signal sequences by SRP is an efficient reaction in vim.
Targeting of SRP-Ribosome Complexes to the Endoplasmiic Reticulum Upon binding ofSRP to the signal sequence, the SRP-ribosomecomplex is targeted to the microsomal membrane25due to the affinity between SRP and a RER membrane protein known as the SRP receptor,26 or docking protein.27 Since SRP receptor is
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localized to the endoplasmic reticulum,28 the SRP ribosome complex is exclusively targeted to the correct intracellular membrane. The SRP receptor is a heterodimer composed of a 68-kDa a subunit (SRa) and a 30-kDa fl subunit (SRp).28SRP receptor reverses the SRP-induced elongation arrest of protein s y n t h e s i by ~ ~displacing ~ ~ ~ ~ SRP from the signal sequence.29However, the targeting reaction does not require ongoing protein synthesis30and can occur after a significant portion of the nascent polypeptide has been s y n t h e ~ i z e d . ~The ’ , ~ ~SRP-induced decrease in the elongation rate of the nascent polypeptide may increase the efficiency of the targeting reaction by preventing the premature folding of protein domains within the cytoplasmic compartment.
Membrane Insertion of the Nascent Polypeptide Dissociation of SRP from the signal sequence allows membrane insertion of the nascent polypeptide. Membrane-inserted nascent polypeptides can be extracted from the membrane by water soluble protein denaturants (alkaline pH or 6 M urea), but not by high salt or EDTA solutions, suggesting that nascent polypeptides undergoing transport are in direct contact with integral membrane proteins.30Recent evidence for the transport of proteins through a protein conducting channel has been obtained by Simon and Blobel using electrophysiological techniques. 33 The most direct evidence for contact between nascent polypeptides and specific RER membrane proteins has been provided by cross-linkingof translocation intermediates to specific integral membrane proteins.3c37The latter results will be discussed in detail in a subsequent section of the manuscript. Upon completion ofthe nascent chain insertion reaction, the ribosome is bound to the membrane via the nascent polypeptide, and by attachment to currently unidentified membrane proteins that have been termed “ribosome receptors.” The membrane content of the SRP receptor is approximately 5-fold lower than that of membrane bound ribosomes,z6.28and the SRP receptor lacks afKnity for 80s ribosomes.29Therefore, the SRP receptor does not function as the RER binding site for the ribosome. Nontranslating 80s ribosomes bind to ribosome-stripped microsomal membranes under low ionic strength conditions (k,50 mM KC1),38 in a SRP and SRP receptor independent r e a ~ t i 0 n .Binding j~ of nontranslating 80s ribosomes to membrane vesicles has been interpreted as being indicative of a receptor for ribosomes engaged in translocation. Although ribophorins I and I1 were initially proposed to be the ribosome receptor,40 subsequent research demonstrated that the ribosome-binding activity of rough micro somal membranes was more sensitive to proteolysis than either ribophorin I or II.39 The ribosome binding activity of RER derived vesicles can be solubilized, fractionated, and functionally reconstituted into proteolipo~omes.~’ Although a 180-kD integral several subsequent membrane protein was reported to be the ribosome recept~r,’~ reports have disputed this i d e n t i f i ~ a t i o n . ~ ~ - ~
Asymmetric Integration ofMembrane Proteins into the RER The asymmetric orientation of integral membrane proteins is believed to be established during the nascent chain insertion phase of the translocation reaction. Translocation across the RER membrane is initiated by an amino terminal signal sequence for type I membrane proteins that have large lumenally exposed amino terminal domains. Complete transfer of the protein into the lumen is prevented by a hydrophobic mem brane-spanning segment. For this reason, membrane-spanning segments of type I proteins are referred to as stop-transfer sequences. Membrane integration of APP
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almost certainly occurs by the standard pathway for a type I integral membrane protein. Although stop-transfer sequences are more hydrophobic than signal sequences, a s t o p transfer sequence from a type I integral membrane protein will function as a noncleavable signal sequence when placed at the N-terminus of a protein.45 The hydrophilic sequences that flank the transmembrane span probably play a crucial role in stoptransfer function. Type I1 and type I11 proteins, as well as type I proteins without extensive lumenal domains, lack cleavable signal sequences. The orientation of these integral membrane proteins is determined by the direction of insertion of the hydrophobic segment that is closest to the amino terminus of the protein. Insight into the mechanism of integration of type I1 integral membrane protein was provided by considering the membrane orientation of a cleavablesignal sequence of a secretory protein.2 As originally proposed in the “loop model,”% the signal sequence of a secreted protein appears to be inserted into the RER membrane in a looped configuration with the N-terminus remaining in the cyt~plasm.~’ Cleavage of the signal sequence, which is presumed to occur at the lumenal face of the RER membrane, results in release of the secreted protein into the lumen. Thus, the noncleavable signal sequence of a type I1 membrane protein is inserted in an orientation that is identical to the signal sequence of a secreted protein, thereby anchoring the type I1 protein in the membrane. In contrast, the noncleavable signal-stop transfer sequence of a type I protein that lacks a lumenal domain is inserted in the opposite orientation. A method for predicting the membrane orientation of proteins that lack cleavable signal sequences has been derived by analyzing the distribution of charged amino acids in the sequences that flank the first membrane-spanning s e g ~ n e n t . ~ ~ Tnet h echarge ofthe 15-amino acid residues that flank the first membranespanning segment correlates with the orientation of the protein in the membrane, with the cytoplasmically exposed domain containing the more positive net charge.48
Ribonudeotide-DependentReaction Steps during Translocation The role of ribonucleotides and ribonucleotide hydrolysis in protein transport reactions has been an area of considerable interest in recent years. Ribosome-independent posttranslational translocation requires hydrolysis of ATP,49-51which is at least in part due to the ATP hydrolysis activity of 7 2 - 0 heat shock proteins that act as chaperone^.^*.^^ The ribosome and SRP-dependent translocation of nascent polypeptides requires GTP (or a nonhydrolyzable GTP analogue) in a reaction step that is distinct from elongation of the To date, the only GTP-dependent step in the protein translocation reaction that has been detected is the SRP-receptor-mediated . ~ ~ the displacement of SRP from the signal sequence of the nascent p ~ l y p e p t i d eThus, transition point between the SRP-SRP-receptor-mediatedtargeting reaction and the nascent chain insertion reaction is controlled by a GTP hydrolysis cycle. Surprisingly, the a-subunit of the SRP receptor (SRa),57the 54kDa subunit of SRP ( SRP54)5859and the &subunit of the SRP receptor (SRp)(J. Miller and P. Walter, UCSF; persona) communication) all contain GTP-binding site consensus sequence motifs.60In addition to a GTP binding site, SRP54 contains a C-terminal methioninerich domain ( M - d ~ m a i n that ) ~ ~ is responsible for binding of SRP54 to the 7SL RNA.6’-62Cross-linking experiments show that the M-domain of SRP54 also contains the signal sequence binding site.61 GTP binding proteins cycle between GDP-bound inactive forms and GTP-bound active forms. The hydrolysis cycle of the GTPase (i.e., the GTP binding protein) is controlled by one or more accessory proteins that accelerate either GDP dissociation or hydrolysis of bound GTP (for a review see 63). Regulatory proteins that catalyze
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FIGURE 2. Proposed model for the GTP hydrolysiscycle of the SRP receptor a subunit (SRa). To simplify the diagram, the SRP-ribosome-nascent polypeptide complex is shown as a signal is not shown, as current evidence indicates that SRa is the sequence bound to SRP (a). subunit involved in the SRP-signal sequence displacement reaction. By analogy to other GTP binding proteins, SRa is presumably occupied by GDP before SRP contact (b). Contact between SRP and SRa is proposed to initiate a guanine nucleotide exchange reaction resulting in dissociation of GDP From SRa (c) followed by binding of GTP to SRa (d). The signal sequence then dissociates From SRP54 (e). Hydrolysis of bound GTP by SRa (r) allows dissociation of SRP (9). Possible roles for the GTP hydrolysis cycle of SRP54 are not shown, as they are primarily speculation at this time.
Sw
GDP dissociation are termed guanine nucleotide exchange (release)proteins (GNRPs), while proteins that permit hydrolysis of bound GTP are termed GTPase activating proteins (GAPS).The GTP-bound form of the GTPase typically displays an enhanced affinity for a downstream effector protein that may have GAP activity.H Our current model for the GTP hydrolysis cycle of SRa (FIG. 2) is based upon experimental data and upon analogies to other better characterized GTP hydrolysis cycles.63Binding of the SRP-ribosome-nascentpolypeptide complex to the SRP receptor initiates a guanine nucleotide exchange reaction. Assignment of SRa as the relevant GTP binding site for this hydrolysis cycle is based upon site directed mutagenesis of the GTP binding site in SRa.65Point mutations in SRa that are restricted to the GTP binding site consensus elements either inactivate the SRP receptor, or reduce the affinity of the receptor for GTP, as shown by the reduced translocation activity of microsomal membranes that contain mutant SRa Binding of the triphosphate form of the ribonucleotide results in the formation of a high affinity complex between SRP and SRP receptor that has been analyzed using the nonhydrolyzable analogue Gpp(N H ) P . ~ ~ ,Occupation & of the SRa site by GTP is responsible for initiating the dissociation of the SRP from the signal sequence, perhaps as a direct consequence of the enhanced affinity between the SRP and the SRP receptor. Dissociation of SRP from the signal sequence allows the subsequent insertion ofthe nascent polypeptide into the RER~nembrane.~’ Hydrolysis of SRa-boundGTP decreases the affinity between SRP and SRP receptor to allow
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dissociation of SRP from the membrane for use in subsequent reaction cycles.66 The GTP hydrolysis cycle has also been analyzed using purified preparations of SRP and SRP receptor (Connolly and Gilmore, manuscript in preparation). Under low ionic strength conditions (50 mM KOAc) SRP-SRP receptor complexes hydrolyze GTP. Since these GTP hydrolysis assays contain three protein subunits with GTP binding sites, we cannot be certain which site(s) are active under the conditions used for the hydrolysis assay. We suggest that portions of SRP act both as a guanine nucleotide release protein to initiate GTP binding to SRa, and as a GTPase activating protein (GAP) to control hydrolysis by SRa. Although the function of the GTP binding site in SRP54 is less defined, it appears likely that the GTP hydrolysis cycles ofSRP and SRP receptor have evolved as a mechanism to control the cyclic assembly and disassembly of the components of the translocation apparatus.
Identification of Integral Membrane Proteins Involved in Nascent Polypeptide Transport Several different experimental strategies have been used to identify integral membrane proteins that act during the insertion and transport phases of the translocation reaction. Membrane-inserted nascent polypeptides containing photoactivatable amino acid analogues can be cross-linked to a 35-kD integral membrane glycoprotein that has been named the signal sequence receptor ( SSR).34Other cross-linking experiments demonstrated that contact between the nascent polypeptide and an integral membrane glycoprotein of approximately 39 kD (mp39) is maintained at early and late stages of transport.35Based upon this finding, Krieg etal.j5suggest that mp39 does not discriminate between signal sequences and other portions of the nascent polypeptide. Although mp39 and SSR may be the same polypeptide, this has yet to be established. In Piho synthesized integral membrane proteins can be cross-linked to SSR, mp3936p67and to a novel 37-kD protein.36 Purification of SSR in a native form revealed a second glycoprotein subunit of 21 kD.6s Both the 35-kD (SSRaw) and 21-kD (SSRp‘I) subunits have been cloned and sequenced. Sequence analysis of SSR suggests that both subunits contain a single membrane-spanning segment. The location of the lumenally exposed asn-X-ser/thr sites for N-linked glycosylation in the protein sequence relative to the predicted membrane-spanning segment indicates that the majority of both SSRa and SSRp are located within the RER lumen.6s-69The chemical cross-linker disuccinimidyl suberate has been used to identify integral membrane proteins adjacent to partially translocated nascent polypeptide^.^^ A major cross-linked product of 45 kD was obtained that is comprised of the 11-kD nascent polypeptide linked to an integral membrane protein of approximately 34 kD (designated imp-34). As imp-34 lacks N-linked carbohydrate, it is not identical to SSRa or 1np39,~’but may be identical to the 37-kD protein identified by High et al. Could integral membrane proteins other than SSR, mp39 and imp-34 be required for the nascent polypeptide insertion and transport phases of the translocation reaction? Mutations have been isolated in three different yeast genes (Sec61, Sec62, and Sec63 (a.k.a. ptl-1)) that impart a translocation defective phenotype at nonpermissive t e m p e r a t ~ r e s . ~Protein ~ - ~ * sequence analysis indicates that all three of these gene products are membrane proteins, based upon the presence of potential membrane-spanning segment^.^^-'^ Sequence comparisons did not reveal any relationship between Sec61, Sec62, Sec63 and any mammalian translocation protein sequenced thus far, including SSRa and SSRp. Recent experiments show that Sec61, Sec62, Sec63 plus two additional polypeptides form a complex in the yeast endoplasmic reticulum.75
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Biochemical evidence for an additional unidentified membrane-bound translocation component has been provided by N-ethylmaleimide alkylation of canine microsomal membrane vesicle^.^^.^^ Although one target of NEM is the SRP receptor a a second currently unidentified NEM-sensitive protein functions after membrane insertion of the signal sequence.76 Neither subunit of SSR contains a cysteine residue; consequently, SSR does not correspond to this second NEM-sensitive NEM-alkylation of this unidentified protein blocks formation of the crosslink between imp-34 and the nascent p o l y p e ~ t i d eThe . ~ ~ latter observation indicates that the NEM-sensitiveprotein is required for assembly of the translocation intermediate in which imp-34 is adjacent to the nascent polypeptide.
Cotranslational Protein Modification Reaction in the Lumen of the Endoplasmk Reticulum Newly integrated proteins undergo protein modification reactions that include signal peptide removal, disulfide bond formation and N-linked glycosylation. The signal peptidase complex has been isolated from canine pancreas microsomes and consists of five polypeptide subunits (12, 18, 21, 22/23 and 25 kDa).78 Different extents of mannose trimming of the N-linked carbohydrate presumably account for the heterogeneous migration of the glycosylated (22/23) kDa subunit. Although signal peptidase preparations isolated from hen oviduct contain fewer subunits,r) the 18 and 23 kDa hen oviduct subunits are probably homologous to the 18 and 22/23 kD canine subunits. The signal peptidase complex is an abundant component of the RER that has been estimated to be present in roughly equal stoichiometry to membrane bound ribosomes.78The amino acid sequences of the 18, 21 and 22/23 kD subunits have been deduced by sequencing cDNA clones.80-82Two of the protein subunits (18 and 21 kD)are homologous to the yeast Secl 1 protein.s0.81The Secl 1 protein was a presumed component of the yeast signal peptidase, as yeast with temperature-sensitive defects in the Secl 1 gene are defective in signal sequence cleavage.83 Partially purified preparations of yeast signal peptidase contain the 18-kD Secll protein along with polypeptides of 13, 20 and 25 kD.84None of the signal peptidase subunits sequenced to date appears to be related to the E. cofi leader peptidase, or to other proteases.8' Consequently, the enzymatically active subunit(s) of eukaryotic signal peptidase remains unidentified. Conceivably, the enzymatic reaction mechanism of signal peptidase may be distinct from other classes of proteases. Signal peptide removal appears to be an efficient reaction, as translocated precursors do not accumulate in tiw. However, mutations near the signal sequence cleavage site can interfere with signal peptide cleavage, leading to the production of nonfunctional proteins.85 N-linked glycoprotein biosynthesis is initiated in the RER by the transfer of a high mannose (Glc3MwGlcNAc2)oligosaccharide onto an asparagine residue in the consensus sequence asn-X-ser/thr where X can be any amino acid other than proline.= The enzyme that catalyzes transfer of high mannose oligosaccharide to asn-X-sedthr sites is the oligosaccharyltransferase. N-linked glycosylation is a cotranslational reaction, with transfer occurring immediately after transport of the nascent polypeptide into the RER lumen.87The oligosaccharide donor for the transferase reaction is the When o l . ~ the ~ initial reaction dolichol-linkedcompound G l ~ ~ M ~ G l c N A c , - P P - d o l i c h in lipid-linked oligosaccharide assembly is blocked by treatment of cells with tunicarnycin, malfolded nonglycosylated proteins accumulate in the lumen of the endoplasrnic reticulum.w Recently, the oligosaccharyltransferasewas purified from canine pancreas microsomal membranes as a protein complex composed of 66-,63- and 48-kD subunits.w Protein immunoblot analysis revealed that the 66-and 63-kD subunits of
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the protein complex correspond to ribophorin I and ribophorin 11, respectively. Antibodies that recognize the carboxyl-terminal cytoplasmic domain of ribophorin I could be used to immunodeplete the oligosaccharyltransferase activity from detergent extracts and to immunopurify the heteroligomeric complex. The ribophorins are relatively abundant integral membrane glycoproteins that are restricted to the rough endoplasmic r e t i ~ u l u r n .The ~ ~ ~protein ' sequences of ribophorins I and I1 have been determined from both rat and human cDNA clone^.^^-^ A consensus sequence motif for a dolichol binding site was recently proposed based upon a comparison of several glyco~yltransferases.~~ A search for this sequence in ribophorins I and I1 revealed a related protein sequence in ribophorin I, so we suggest that this subunit of the oligosaccharyltransferasecontains the binding site for the lipid-linked oligosaccharide substrate.
CONCLUSIONS Considerable information is now available concerning the reaction steps that occur during transport of proteins across and integration of proteins into the endoplasmic reticulum. The majority of proteins transported across the endoplasmic reticulum are delivered to the membrane through the combined action of SRP and the SRP receptor. We are now beginning to understand how the interaction between SRP,SRP receptor and signal sequences is controlled by a complex GTP hydrolysis cycle. It is now clear that the transport phase of the translocation reaction is mediated by a protein complex that may include SSR,mp39, imp-34 and perhaps others that have yet to be identified. Whether these polypeptides are assembled into a pore-like transport apparatus remains to be established. The protein modification enzymes signal peptidase and oligosaccharyltransferase have been isolated, and correspond to abundant RER protein complexes. Together all of these proteins comprise a membrane bound translocation apparatus that has been termed the translocon.% REFERENCES 1 . BLOBEL,G. 1980. Proc. Natl. Acad. Sci. USA 77: 1496-1500. 2. SABATINI,D. D., G. KREIBICH, T. MORIMOTO & M. ADESNICK. 1982. J. Cell Biol. 92: 1-22. 3. WICKNER,W. T. & H. F. LODISH.1985. Science 230: 400-407. 4. VON HEIJNE, G . 1983. Eur. J. Biochem. 133: 17-21. G . 1986. Nucieic Acids Res. 14: 4683-4690. 5. VON HEIJNE, 6 . WALTER,P. & G. BLOBEL.1980. Proc. Natl. Acad. Sci. USA 77: 7112-7116. R. & C. MOLIAY.1986. J. Biol. Chem. 261: 12889-12895. 7. ZIMMERMANN, 8. SCHLENSTEDT, G. & R. ZIMMERMANN.1987. EMBO J. 6: 699-703. 9. ANDERSON,D. J., K. E. MOSTOV& G. BLOBEL.1983. Proc. Natl. Acad. Sci. USA 80: 7249- 7253. E. C. & K. DRICKAMER. 1985. J. Biol. Chem. 261: 1286-1292. 10. HOLLAND, 1 1 . HULL,J. D., R. GILMORE & R. A. LAMB. 1988. J. Cell Biol. 106: 1489-1498. P. & G. BLOBEL.1982. Nature 299: 691-698. 12. WALTER, 13. WALTER, P. & G. BLOBEL.1981. J. Cell Biol. 91: 577-561. 1988. EMBO J. 7: 3559-3569. 14. WOLIN,S. L. & P. WALTER. P. & G. BLOBEL.1983. Cell 3 4 525-533. 15. WALTER, V. & P. WALTER. 1988. Cell 52: 39-49. 16. SIEGEL, 17. SIEGEL,V. & P. WALTER. 1985. J. Cell Bioi. 100: 1913-1921. 1986. Nature 320: 81-84. 18. SIEGEL,V. & P. WALTER.
ANNALS NEW YORK ACADEMY OF SCIENCES
36
U. C., P.WALTER& A. E. JOHNSON. 1986. Proc.Natl. Acad. Sci. USA 83: 860419. KIUEG, 8608. 20. KURZCHALIA,T. V., M. WIEDMANN, A. S. GIRSHOVICH, E. S. BOCHKAREVA, H. BIELKA & T. A. RAPOPORT. 1986. Nature 320: 634-636. 21. BRENNWALD,P., X. LIAO,K. HOLM,G. PORTER& J. A. WISE.1988. Mol. Cell. Biol. 8: 1580-1590. 22. HA", B. C., M. A. PORITZ& P. WALTER. 1989. J. Cell Biol. 1 0 9 3223-3230. 23. PORITZ,M. A., V. SIEGEL, W. HANSEN & P.WALTER. 1988. Proc.Natl. Acad. Sci. USA 85: 4315-4319. 1991. Cell 67: 131-144. 24. HA", B. C. & P. WALTER. 25. WALTER, P.& G. BLOBEL. 1981. J. Cell Biol. 91: 551-556. & G. BLOBEL.1982. J. Cell Biol. 95: 470-477. 26. GILMORE, R., P. WALTER 27. MEYER,D. I., E. KRAUSE& B. DOBBERSTEIN. 1982. Nature 297: 647-650. 28. TAJIMA, S.,L. LUFFER, V. L. RATH& P.WALTER. 1986. J. Cell Biol. 103: 1167-1178. 29. GILMORE,R. & G. BLOBEL.1983. Cell 35: 677-685. 30. GILMORE,R. & G. BLOBEL.1985. Cell 42: 497-505. 1986. EMBO J. 5: 951-955. 31. AINGER,K. J. & D. I. MEYER. 32. SIEGEL,V. & P. WALTER. 1988. EMBO J. 7: 1769-1775. S. M. & G. BLOBEL.1991. Cell 65: 371-380. 33. SIMON, 34. WIEDMA",M., T. V. KURZCHALIA,E. HARTMANN & T. A. RAPOPORT. 1987. Nature 328: 830-833. 1989. J. Cell Biol. 109 2033-2043. 35. KIUEG,U. C., A. E. JOHNSON & P. WALTER. 36. HIGHS., D. GORLICH, M. WIEDMAN, T. A. WOPORT & B. DOBBERSTEIN. 1991. J. Cell Biol. 113: 35-44. 1991. J. Cell Biol. 114: 21-33. 37. KELLARIS, K. V., S. BOWEN & R. GILMORE. 1974. J. Mol. Biol. 88: 55938. BORGESE, N., W. MOK,G. KREIBICH & D. D. SABATINI. 580. 1986. J. Cell Biol. 103: 241-253. 39. HORTSCH, M., D. AVOSSA& D. I. MEYER. 40. KREIBICH,G., B. L. ULRICH & D. D. SABATINI. 1978. J. Cell Biol. 77: 464-487. Y. ASANO,K. MIZUSAWA, R. YAMAGISHI, T. HORIGOME 41. YOSHIDA, H., N. TONDOKORO, & H. SUGANO.1987. Biochern. J. 245: 811-819. 1990. Nature 346: 540-544. 42. SAVITZA. J. & D. I. MEYER. 43. NUNNARI, J. M., D. L. ZIMMERMAN, S. C. OGG& P.WALTER. 1991. Nature 352: 638640.
44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.
P. & R. GILMORE.1991. J. Cell Biol. 114: 639-649. COLLINS, 1986. Cell 47: 711-719. MIZE,N. K., D. W. ANDREW& V. R. LINGAPPA. INOUYE, M. & S. HALEGOUA. 1980. CRC Crit. Rev. Biochern. 7: 339-371. A. S., P. J. M. ROTITER & J. K. ROSE.1988. Proc.Natl. Acad. Sci. USA 85: 7592SHAW, 7596. HARTMANN, E., T. A. RAPOPORT& H. F. LODISH.1989. Proc. Natl. Acad. Sci. USA 86: 5786- 5790. ROTHBLATT,J. A. & D. I. MEYER. 1986. EMBO J. 5: 1031-1036. HANSEN, W., P. D. GARCIA & P. WALTER. 1986. Cell 45: 397-406. M. G. & G. BLOBEL.1986. J. Cell Biol. 102: 1543-1550. WATERS, DESHAIES, R J., B. D. KOCH, M. WERNER-WASHBURNE, E. A. CRAIG & R. SCHEKMAN. 1988. Nature 332: 800-805. & G. BLOBEL.1988. Nature 322: 805-810. CHIRICO, W. J., M. G. WATERS 1986. J. Cell Biol. 103: 2253-2261. CONNOLLY, T. & R. GILMORE. HOFFMAN, K. & R. GILMORE. 1988. J. Biol. Chern. 263: 4381-4385. WILSON,C., T. CONNOLLY, T. MORRISON& R. GILMORE. 1988. J. Cell Biol. 107: 6977. 1989. Cell 57: 599-610. CONNOLLY, T. & R. GILMORE. BERNSTEIN,H. D., M. A. PORITZ,K. STRUB, P. J. HOBEN,S. BRENNER& P.WALTER. 1989. Nature 340: 482-486. ROMISH, K., J. WEBB,J. HERZ,S. PREHN,R. FRANK, M. VINGRON& B. DOBBERSTEIN. 1989. Nature 340: 478-482.
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& W. C. MERRICK.1987. Proc. Natl. Acad. Sci. USA 84: 60. DEVER.T. E.. M. J. GLYNIAS 1814-1818: A. E. JOHNSON & P.WALTER.1990. EMBO J. 9 451161. ZOPF, D., H. D. BERNSTEIN, 4517. H. GAUSEPOHL & B. DOBBERSTEIN. 1990. J. 62. ROMISCH,K., J. WEBB,K. LINGELBACH, Cell Biol. 111: 1793-1802. & F. MCCORMICK. 1990. Nature 348: 125-132. 63. BOURNE,H. R, D. A. SANDERS M. & F. MCCORMICK.1987. Science 238: 542-545. 64. TRAHEY, P.& R GILMORE.1992. J. Cell Biol. In press. 65. RAPIEJKO, T., P. J. RAPIEJKO & R GILMORE.1991. Science 252: 1171-1173. 66. CONNOLLY, & A. E. JOHNSON. 1991. J. Cell Biol. 112: 67. THRIIT,R N., D. W. ANDmws, P.WALTER 809-821. 68. GORLICH,D., S. PREHN,E. HARTMA”,J. HERZ, A. Ono, R KRAIT, M. WIEDMAN, S. KNEPSEL, B. DOBBERSTEIN & T. A. RAPOPORT. 1990. J. Cell Biol. 111:2283-2294. R FRANK,K. ROMISCH,B. 69. PREHN,S., J. HERZ, E. HARTMA”,T. V. KURZCHALIA, & T. A. RAPOPORT.1990. Eur. J. Biochem. 188: 439-445. DOBBERSTEIN 1987. J. Cell. Biol. 105: 633-645. 70. DESHAIES,R J. & R SCHEKMAN. G. DAUM& R SCHEKMAN. 1989. J. 71. ROTHBLAIT,J.A., R J. DESHAIES,S. L. SANDERS, Cell Biol. 109: 2641-2652. J., A. R HIBBS,P. SANZ,J. CROWE& D. I. MEYER.1988. EMBO J. 7: 434772. TOYN, 4353. J. ROTHBLAIT,J. WAY& P.SILVER.1989. J. Cell 73. SADLER,I., A. CHIANG,T. KURIHARA, Biol. 109: 2665-2675. 1989. J. Cell Biol. 109 2653-2664. 74. DESHAIES,R J. & R SCHEKMAN. D. A. FELDHEIM & R SCHEKMAN.1991. Nature (Lon75. DESHAIES,R J., S. L. SANDERS,
don) 349: 806-808. 76. NICCHITTA, C. V. & G. BLOBEL.1989. J. Cell Biol. 108: 789-795. & P. WALTER.1982. J. Cell Biol. 95: 463-469. 77. GILMORE,R.,G. BLOBEL & G. BLOBEL.1986. Proc. Natl. Acad. Sci. USA 83: 58178. EVANS,E. A,, R GILMORE 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91.
585. BAKER,R. K. & M. 0. LIVELY. 1987. Biochem. 26: 8561-8567. GREENBERG, G.,G. S.SHELNESS&G. BLOBEL.1989. J. Biol. Chem. 264:15762-15765. SHELNESS, G. S.& G. BLOBEL.1990. J. Biol. Chem. 265: 9512-9519. SHELNESS, G. S., Y. S. KANWm & G. BLOBEL.1988. J. Biol. Chem. 263: 17063-17070. BOHNI,P. C., R J. DESHAIES& R SCHEKMAN. 1988. J. Cell Biol. 106: 1035-1042. YADEAU,J. T., C. KLEIN& G. BLOBEL.1991. Proc.Natl. Acad. Sci. USA 88: 517-521. ARNOLD,A., S. A. HORST,T. J. GARDELLA,H. BABA,M. A. LJ”E& H. M. K m NENBERG. 1990. J. Clin. Invest. 86 1084-1087. KORNFELD, R & S. KORNFELD. 1985. Ann. Rev. Biochem. 5 4 631-664. ROTHMAN, J. E.& H. F. LODISH.1977. Nature 269 775-780. Lw, T., B. STETSON,S. J. TVRCO,S. C. HUBBARD& P. W. ROBBINS.1979. J. Biol. Chem. 254: 4554-4559. ELBEIN,A. D. 1987. Annu. Rev. Biochem. 56: 497-534. KELLEHER,D. J., G. KREIBICH & R GILMORE.1992. Cell. In press. MARCANTONIO, E.E., A. A~URCOSTESEC & G. KREIBICH. 1984. J. Cell. Biol. 99:2254-
2259. 92. HARNIK-ORT,V., K. WH, E. ~ C A N T O N I O ,D. R C o w , M. G. ROSENFELD, & G. KREIBICH. 1987. J. Cell Biol. 104: 855-863. M. ADESNIK,D. D. SABATINI 93. CRIMAUDO, C., M. HORTSCH,H. GAUSEPOHL & D. I. MEYER.1987. EMBO J. 6: 7582. & G. KREIBICH.1991. Bio94. PIROZZI,G., Z. ZHOU, P. D’EUSTACHIO,D. D. SABATINI chem. Biophys. Res. Commun. 176: 1482-1486. & P. W. ROBBINS. 1989. Proc. Natl. Acad. Sci. USA 86: 95. ALBRIGHT,C. F., P. ORLEAN 7366-7369. 96. WALTER,P. & V. R LINGAPPA.1986. Ann. Rev. Cell Biol. 2: 499-516.
One of the major topics addressed in the meeting “Proteases and Protease Inhibitors: Emerging Roles in the Pathogenesis of Alzheimer’s Disease” is the proteolytic processing reactions that lead to the production of the p protein from the amyloid precursor protein (APP). Eukaryotic cells produce numerous proteolytic enzymes that could be potentially responsible for the proteolytic cleavages that liberate the /3 protein from APP. Both the amyloid precursor protein and the proteolytic enzymes are restricted to specific cellular compartments; hence a consideration of the membrane topology and intracellular location of APP may allow investigators to identify proteolytic enzymes that are accessible to the regions of APP that are cleaved during /3 protein formation. The membrane orientation of integral proteins like APP is established during synthesis when the protein is integrated into the rough endoplasmic reticulum (RER). The focus of this paper will be the mechanisms responsible for the asymmetric integration of membrane proteins into the RER. Cotranslational protein modification reactions that occur during glycoprotein biosynthesis will also be described. Newly synthesized polypeptides that are mishandled by the translocation apparatus are probably degraded by proteases that reside within the cytoplasm and the endoplasmic reticulum. Proteins located within the lumen of the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, endosome and lysosome and virtually all secreted proteins are cotranslationally translocated across the rough endoplasmic reticulum (RER). The majority of the integral membrane proteins of the rough and smooth endoplasmic reticulum, Golgi apparatus, endosome, lysosome and plasma membrane are synthesized by the membrane-bound ribosomes of the RER.1,2Integral membrane proteins can be separated into several classes based upon the orientation and number of membrane spanning segment^.^.^ Type I and type I1 membrane proteins both contain a single membrane-spanning segment (FIG.1). When initially integrated into the RER, the amino terminus of type I membrane proteins is located within the membrane lumen, while type I1 integral membrane proteins are integrated in the opposite orientation. Type I membrane proteins can have the bulk of the protein oriented towards either face of the membrane, unlike type I1 proteins, which typically have small Nterminal cytoplasmic domains. Based upon the protein sequence derived from sequencing a cDNA clone, the APP protein appears to be a typical type I membrane protein. Proteins with multiple membrane-spanning segments (type 111) can be oriented with the N-terminus facing either the cytoplasm or the lumen. Proteins anchored to the 27
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Cytoplasm Type II
Type Ill
N
f C
Lumen
FIGURE 1. Topology of integral membrane proteins in the endoplasmic reticulum. The amino (N) terminus of type I integral membrane proteins is located in the lumen, with the carboxyl (C) terminus exposed to the cytoplasm. Type I1 proteins have the opposite orientation. Polytopic or type I11 integral membrane proteins can be oriented so that the N-terminus is located in either the lumen or the cytoplasm. Polytopic integral membrane proteins can have an even or odd number of membrane-spanning segments. N-linked oligosaccharide is designated by the symbol Y, and is only found on lumenally exposed protein domains.
membrane via a phosphatidylinositol glycan anchor are transiently inserted into the membrane as type I membrane proteins. The selective translocation of a protein across, or integration of a protein into, the rough endoplasmic reticulum occurs by a sequence of protein-mediated reactions that can be designated as follows: (i) sorting, (ii) targeting, (iii) insertion, (iv) transport, and (v) nascent chain modification. As described below, researchers have identified topogenic signals within membrane proteins that are recognized by the various components of the protein translocation apparatus. Current models for the function of the protein translocation and integration apparatus provide a molecular explanation for how several of these topogenic signals are decoded.
Signal Sequences and Sorting Reaction Proteins destined for translocation across the RER are selected during synthesis from among all polypeptides that are translated within the cytoplasm by virtue of an RER-specific signal sequence. Secreted proteins and lumenal proteins of both the exocytic and endocytic membrane systems contain a cleavable signal sequence located at the extreme N-terminus of the protein (for a review see A cleavable signal sequence is also located at the N-terminus of type I membrane proteins that have large lumenal domains. In contrast, the first transmembrane spanning segment of type I1 273).
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and type I11 integral membrane proteins functions as a noncleavable signal sequence. Cleavable signal sequences from numerous proteins have been compared to identify sequence motifs of functional s i g n i f i c a n ~ eThese . ~ ~ ~ comparisons did not reveal the presence of a strongly conserved sequence. Instead, the typical 15-25 amino acid residue signal sequence was found to contain the following structural features: (a) a basic amino terminal region, (b) a central core of hydrophobic amino acids and (c) a carboxyl terminal region that specifies cleavage by the enzyme signal ~ e p t i d a s eOf .~~~ these three regions, the central hydrophobic core is the most important determinant as a sorting signal for translocation across the endoplasmic reticulum. The sorting reaction corresponds to binding of the signal recognition particle (SRP) to the signal sequence during translation. The SRP was initially isolated as a peripheral membrane protein complex that was required for the translocation of a secretory polypeptide across mammalian endoplasmic reticulum.6 An SRP-mediated sorting step is the initial event in the translocation of most proteins across the endoplasmic reticulum. Several low molecular weight secretory polypeptides can be translocated across the endoplasmic reticulum by an SRP-independent mechanism.’J Subsequent experiments established that SRP is required for the integration of the three classes of integral membrane proteins depicted in FIGURE 1 .9-11 However, integral membrane proteins with cytoplasmic domains that are anchored to the membrane via a carboxyl terminal tail, such as cytochrome bs, are integrated into the membrane by a pathway that is not dependent upon SRP.’ SRP is a ribonucleoprotein particle consisting of six polypeptide subunits (72, 68, 54, 19, 14 and 9 kDa) and the 7SL RNA.6J2 The SRP binds with high affinity to ribosomes translating a mRNA encoding a protein with a RER-specificsignal sequence. High affinity binding decreases or halts the elongation of the nascent polypeptide. 1 3 ~ 1 4 The functional significance of the altered elongation rate will be discussed in the next section regarding targeting of SRP-ribosome complexes to the RER. SRP particles have been disassembled into protein and RNA components and then reconstituted.I5 The 7SL RNA is essential for the activity of SRP,Is and provides a framework upon which the protein subunits of SRP are assembled into three structurally and functionally distinct domains.16The 9/14 kD heterodimer binds to the Alu-like region of the 7SL RNA to comprise the elongation arrest domain of SRP.’”18 The 54kD subunit of SRP ( i x . SRP54) binds to the signal sequence of a nascent polypeptide shortly after it emerges from the large ribosomal subunit, as shown by photocross-linking SRP54 experirnent~.~ ~ J ~ and SRP19 comprise the signal sequence recognition domain of the SRP particle.16 Homologues of the 7SL RNA and the SRP54 polypeptide have been identified in a variety of eukaryotic organisms including the yeast S~hizosaEchaaromycerp@~-~~ and S. ccrtpljllc.22~24Depletion of the S. cmpiw SRP54 polypeptide results in the cytoplasmic accumulation of precursor forms of polypeptides that are normally se~reted.~‘ This result demonstrates an in viw function for SRP that is entirely consistent with the in vipo requirement for SRP in a protein translocation assay. Aberrant cytoplasmic forms of secretory proteins and integral membrane proteins are not readily detected in mammalian cells. Presumably, recognition of RER signal sequences by SRP is an efficient reaction in vim.
Targeting of SRP-Ribosome Complexes to the Endoplasmiic Reticulum Upon binding ofSRP to the signal sequence, the SRP-ribosomecomplex is targeted to the microsomal membrane25due to the affinity between SRP and a RER membrane protein known as the SRP receptor,26 or docking protein.27 Since SRP receptor is
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localized to the endoplasmic reticulum,28 the SRP ribosome complex is exclusively targeted to the correct intracellular membrane. The SRP receptor is a heterodimer composed of a 68-kDa a subunit (SRa) and a 30-kDa fl subunit (SRp).28SRP receptor reverses the SRP-induced elongation arrest of protein s y n t h e s i by ~ ~displacing ~ ~ ~ ~ SRP from the signal sequence.29However, the targeting reaction does not require ongoing protein synthesis30and can occur after a significant portion of the nascent polypeptide has been s y n t h e ~ i z e d . ~The ’ , ~ ~SRP-induced decrease in the elongation rate of the nascent polypeptide may increase the efficiency of the targeting reaction by preventing the premature folding of protein domains within the cytoplasmic compartment.
Membrane Insertion of the Nascent Polypeptide Dissociation of SRP from the signal sequence allows membrane insertion of the nascent polypeptide. Membrane-inserted nascent polypeptides can be extracted from the membrane by water soluble protein denaturants (alkaline pH or 6 M urea), but not by high salt or EDTA solutions, suggesting that nascent polypeptides undergoing transport are in direct contact with integral membrane proteins.30Recent evidence for the transport of proteins through a protein conducting channel has been obtained by Simon and Blobel using electrophysiological techniques. 33 The most direct evidence for contact between nascent polypeptides and specific RER membrane proteins has been provided by cross-linkingof translocation intermediates to specific integral membrane proteins.3c37The latter results will be discussed in detail in a subsequent section of the manuscript. Upon completion ofthe nascent chain insertion reaction, the ribosome is bound to the membrane via the nascent polypeptide, and by attachment to currently unidentified membrane proteins that have been termed “ribosome receptors.” The membrane content of the SRP receptor is approximately 5-fold lower than that of membrane bound ribosomes,z6.28and the SRP receptor lacks afKnity for 80s ribosomes.29Therefore, the SRP receptor does not function as the RER binding site for the ribosome. Nontranslating 80s ribosomes bind to ribosome-stripped microsomal membranes under low ionic strength conditions (k,50 mM KC1),38 in a SRP and SRP receptor independent r e a ~ t i 0 n .Binding j~ of nontranslating 80s ribosomes to membrane vesicles has been interpreted as being indicative of a receptor for ribosomes engaged in translocation. Although ribophorins I and I1 were initially proposed to be the ribosome receptor,40 subsequent research demonstrated that the ribosome-binding activity of rough micro somal membranes was more sensitive to proteolysis than either ribophorin I or II.39 The ribosome binding activity of RER derived vesicles can be solubilized, fractionated, and functionally reconstituted into proteolipo~omes.~’ Although a 180-kD integral several subsequent membrane protein was reported to be the ribosome recept~r,’~ reports have disputed this i d e n t i f i ~ a t i o n . ~ ~ - ~
Asymmetric Integration ofMembrane Proteins into the RER The asymmetric orientation of integral membrane proteins is believed to be established during the nascent chain insertion phase of the translocation reaction. Translocation across the RER membrane is initiated by an amino terminal signal sequence for type I membrane proteins that have large lumenally exposed amino terminal domains. Complete transfer of the protein into the lumen is prevented by a hydrophobic mem brane-spanning segment. For this reason, membrane-spanning segments of type I proteins are referred to as stop-transfer sequences. Membrane integration of APP
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almost certainly occurs by the standard pathway for a type I integral membrane protein. Although stop-transfer sequences are more hydrophobic than signal sequences, a s t o p transfer sequence from a type I integral membrane protein will function as a noncleavable signal sequence when placed at the N-terminus of a protein.45 The hydrophilic sequences that flank the transmembrane span probably play a crucial role in stoptransfer function. Type I1 and type I11 proteins, as well as type I proteins without extensive lumenal domains, lack cleavable signal sequences. The orientation of these integral membrane proteins is determined by the direction of insertion of the hydrophobic segment that is closest to the amino terminus of the protein. Insight into the mechanism of integration of type I1 integral membrane protein was provided by considering the membrane orientation of a cleavablesignal sequence of a secretory protein.2 As originally proposed in the “loop model,”% the signal sequence of a secreted protein appears to be inserted into the RER membrane in a looped configuration with the N-terminus remaining in the cyt~plasm.~’ Cleavage of the signal sequence, which is presumed to occur at the lumenal face of the RER membrane, results in release of the secreted protein into the lumen. Thus, the noncleavable signal sequence of a type I1 membrane protein is inserted in an orientation that is identical to the signal sequence of a secreted protein, thereby anchoring the type I1 protein in the membrane. In contrast, the noncleavable signal-stop transfer sequence of a type I protein that lacks a lumenal domain is inserted in the opposite orientation. A method for predicting the membrane orientation of proteins that lack cleavable signal sequences has been derived by analyzing the distribution of charged amino acids in the sequences that flank the first membrane-spanning s e g ~ n e n t . ~ ~ Tnet h echarge ofthe 15-amino acid residues that flank the first membranespanning segment correlates with the orientation of the protein in the membrane, with the cytoplasmically exposed domain containing the more positive net charge.48
Ribonudeotide-DependentReaction Steps during Translocation The role of ribonucleotides and ribonucleotide hydrolysis in protein transport reactions has been an area of considerable interest in recent years. Ribosome-independent posttranslational translocation requires hydrolysis of ATP,49-51which is at least in part due to the ATP hydrolysis activity of 7 2 - 0 heat shock proteins that act as chaperone^.^*.^^ The ribosome and SRP-dependent translocation of nascent polypeptides requires GTP (or a nonhydrolyzable GTP analogue) in a reaction step that is distinct from elongation of the To date, the only GTP-dependent step in the protein translocation reaction that has been detected is the SRP-receptor-mediated . ~ ~ the displacement of SRP from the signal sequence of the nascent p ~ l y p e p t i d eThus, transition point between the SRP-SRP-receptor-mediatedtargeting reaction and the nascent chain insertion reaction is controlled by a GTP hydrolysis cycle. Surprisingly, the a-subunit of the SRP receptor (SRa),57the 54kDa subunit of SRP ( SRP54)5859and the &subunit of the SRP receptor (SRp)(J. Miller and P. Walter, UCSF; persona) communication) all contain GTP-binding site consensus sequence motifs.60In addition to a GTP binding site, SRP54 contains a C-terminal methioninerich domain ( M - d ~ m a i n that ) ~ ~ is responsible for binding of SRP54 to the 7SL RNA.6’-62Cross-linking experiments show that the M-domain of SRP54 also contains the signal sequence binding site.61 GTP binding proteins cycle between GDP-bound inactive forms and GTP-bound active forms. The hydrolysis cycle of the GTPase (i.e., the GTP binding protein) is controlled by one or more accessory proteins that accelerate either GDP dissociation or hydrolysis of bound GTP (for a review see 63). Regulatory proteins that catalyze
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FIGURE 2. Proposed model for the GTP hydrolysiscycle of the SRP receptor a subunit (SRa). To simplify the diagram, the SRP-ribosome-nascent polypeptide complex is shown as a signal is not shown, as current evidence indicates that SRa is the sequence bound to SRP (a). subunit involved in the SRP-signal sequence displacement reaction. By analogy to other GTP binding proteins, SRa is presumably occupied by GDP before SRP contact (b). Contact between SRP and SRa is proposed to initiate a guanine nucleotide exchange reaction resulting in dissociation of GDP From SRa (c) followed by binding of GTP to SRa (d). The signal sequence then dissociates From SRP54 (e). Hydrolysis of bound GTP by SRa (r) allows dissociation of SRP (9). Possible roles for the GTP hydrolysis cycle of SRP54 are not shown, as they are primarily speculation at this time.
Sw
GDP dissociation are termed guanine nucleotide exchange (release)proteins (GNRPs), while proteins that permit hydrolysis of bound GTP are termed GTPase activating proteins (GAPS).The GTP-bound form of the GTPase typically displays an enhanced affinity for a downstream effector protein that may have GAP activity.H Our current model for the GTP hydrolysis cycle of SRa (FIG. 2) is based upon experimental data and upon analogies to other better characterized GTP hydrolysis cycles.63Binding of the SRP-ribosome-nascentpolypeptide complex to the SRP receptor initiates a guanine nucleotide exchange reaction. Assignment of SRa as the relevant GTP binding site for this hydrolysis cycle is based upon site directed mutagenesis of the GTP binding site in SRa.65Point mutations in SRa that are restricted to the GTP binding site consensus elements either inactivate the SRP receptor, or reduce the affinity of the receptor for GTP, as shown by the reduced translocation activity of microsomal membranes that contain mutant SRa Binding of the triphosphate form of the ribonucleotide results in the formation of a high affinity complex between SRP and SRP receptor that has been analyzed using the nonhydrolyzable analogue Gpp(N H ) P . ~ ~ ,Occupation & of the SRa site by GTP is responsible for initiating the dissociation of the SRP from the signal sequence, perhaps as a direct consequence of the enhanced affinity between the SRP and the SRP receptor. Dissociation of SRP from the signal sequence allows the subsequent insertion ofthe nascent polypeptide into the RER~nembrane.~’ Hydrolysis of SRa-boundGTP decreases the affinity between SRP and SRP receptor to allow
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dissociation of SRP from the membrane for use in subsequent reaction cycles.66 The GTP hydrolysis cycle has also been analyzed using purified preparations of SRP and SRP receptor (Connolly and Gilmore, manuscript in preparation). Under low ionic strength conditions (50 mM KOAc) SRP-SRP receptor complexes hydrolyze GTP. Since these GTP hydrolysis assays contain three protein subunits with GTP binding sites, we cannot be certain which site(s) are active under the conditions used for the hydrolysis assay. We suggest that portions of SRP act both as a guanine nucleotide release protein to initiate GTP binding to SRa, and as a GTPase activating protein (GAP) to control hydrolysis by SRa. Although the function of the GTP binding site in SRP54 is less defined, it appears likely that the GTP hydrolysis cycles ofSRP and SRP receptor have evolved as a mechanism to control the cyclic assembly and disassembly of the components of the translocation apparatus.
Identification of Integral Membrane Proteins Involved in Nascent Polypeptide Transport Several different experimental strategies have been used to identify integral membrane proteins that act during the insertion and transport phases of the translocation reaction. Membrane-inserted nascent polypeptides containing photoactivatable amino acid analogues can be cross-linked to a 35-kD integral membrane glycoprotein that has been named the signal sequence receptor ( SSR).34Other cross-linking experiments demonstrated that contact between the nascent polypeptide and an integral membrane glycoprotein of approximately 39 kD (mp39) is maintained at early and late stages of transport.35Based upon this finding, Krieg etal.j5suggest that mp39 does not discriminate between signal sequences and other portions of the nascent polypeptide. Although mp39 and SSR may be the same polypeptide, this has yet to be established. In Piho synthesized integral membrane proteins can be cross-linked to SSR, mp3936p67and to a novel 37-kD protein.36 Purification of SSR in a native form revealed a second glycoprotein subunit of 21 kD.6s Both the 35-kD (SSRaw) and 21-kD (SSRp‘I) subunits have been cloned and sequenced. Sequence analysis of SSR suggests that both subunits contain a single membrane-spanning segment. The location of the lumenally exposed asn-X-ser/thr sites for N-linked glycosylation in the protein sequence relative to the predicted membrane-spanning segment indicates that the majority of both SSRa and SSRp are located within the RER lumen.6s-69The chemical cross-linker disuccinimidyl suberate has been used to identify integral membrane proteins adjacent to partially translocated nascent polypeptide^.^^ A major cross-linked product of 45 kD was obtained that is comprised of the 11-kD nascent polypeptide linked to an integral membrane protein of approximately 34 kD (designated imp-34). As imp-34 lacks N-linked carbohydrate, it is not identical to SSRa or 1np39,~’but may be identical to the 37-kD protein identified by High et al. Could integral membrane proteins other than SSR, mp39 and imp-34 be required for the nascent polypeptide insertion and transport phases of the translocation reaction? Mutations have been isolated in three different yeast genes (Sec61, Sec62, and Sec63 (a.k.a. ptl-1)) that impart a translocation defective phenotype at nonpermissive t e m p e r a t ~ r e s . ~Protein ~ - ~ * sequence analysis indicates that all three of these gene products are membrane proteins, based upon the presence of potential membrane-spanning segment^.^^-'^ Sequence comparisons did not reveal any relationship between Sec61, Sec62, Sec63 and any mammalian translocation protein sequenced thus far, including SSRa and SSRp. Recent experiments show that Sec61, Sec62, Sec63 plus two additional polypeptides form a complex in the yeast endoplasmic reticulum.75
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Biochemical evidence for an additional unidentified membrane-bound translocation component has been provided by N-ethylmaleimide alkylation of canine microsomal membrane vesicle^.^^.^^ Although one target of NEM is the SRP receptor a a second currently unidentified NEM-sensitive protein functions after membrane insertion of the signal sequence.76 Neither subunit of SSR contains a cysteine residue; consequently, SSR does not correspond to this second NEM-sensitive NEM-alkylation of this unidentified protein blocks formation of the crosslink between imp-34 and the nascent p o l y p e ~ t i d eThe . ~ ~ latter observation indicates that the NEM-sensitiveprotein is required for assembly of the translocation intermediate in which imp-34 is adjacent to the nascent polypeptide.
Cotranslational Protein Modification Reaction in the Lumen of the Endoplasmk Reticulum Newly integrated proteins undergo protein modification reactions that include signal peptide removal, disulfide bond formation and N-linked glycosylation. The signal peptidase complex has been isolated from canine pancreas microsomes and consists of five polypeptide subunits (12, 18, 21, 22/23 and 25 kDa).78 Different extents of mannose trimming of the N-linked carbohydrate presumably account for the heterogeneous migration of the glycosylated (22/23) kDa subunit. Although signal peptidase preparations isolated from hen oviduct contain fewer subunits,r) the 18 and 23 kDa hen oviduct subunits are probably homologous to the 18 and 22/23 kD canine subunits. The signal peptidase complex is an abundant component of the RER that has been estimated to be present in roughly equal stoichiometry to membrane bound ribosomes.78The amino acid sequences of the 18, 21 and 22/23 kD subunits have been deduced by sequencing cDNA clones.80-82Two of the protein subunits (18 and 21 kD)are homologous to the yeast Secl 1 protein.s0.81The Secl 1 protein was a presumed component of the yeast signal peptidase, as yeast with temperature-sensitive defects in the Secl 1 gene are defective in signal sequence cleavage.83 Partially purified preparations of yeast signal peptidase contain the 18-kD Secll protein along with polypeptides of 13, 20 and 25 kD.84None of the signal peptidase subunits sequenced to date appears to be related to the E. cofi leader peptidase, or to other proteases.8' Consequently, the enzymatically active subunit(s) of eukaryotic signal peptidase remains unidentified. Conceivably, the enzymatic reaction mechanism of signal peptidase may be distinct from other classes of proteases. Signal peptide removal appears to be an efficient reaction, as translocated precursors do not accumulate in tiw. However, mutations near the signal sequence cleavage site can interfere with signal peptide cleavage, leading to the production of nonfunctional proteins.85 N-linked glycoprotein biosynthesis is initiated in the RER by the transfer of a high mannose (Glc3MwGlcNAc2)oligosaccharide onto an asparagine residue in the consensus sequence asn-X-ser/thr where X can be any amino acid other than proline.= The enzyme that catalyzes transfer of high mannose oligosaccharide to asn-X-sedthr sites is the oligosaccharyltransferase. N-linked glycosylation is a cotranslational reaction, with transfer occurring immediately after transport of the nascent polypeptide into the RER lumen.87The oligosaccharide donor for the transferase reaction is the When o l . ~ the ~ initial reaction dolichol-linkedcompound G l ~ ~ M ~ G l c N A c , - P P - d o l i c h in lipid-linked oligosaccharide assembly is blocked by treatment of cells with tunicarnycin, malfolded nonglycosylated proteins accumulate in the lumen of the endoplasrnic reticulum.w Recently, the oligosaccharyltransferasewas purified from canine pancreas microsomal membranes as a protein complex composed of 66-,63- and 48-kD subunits.w Protein immunoblot analysis revealed that the 66-and 63-kD subunits of
GILMORE 8c KELLARIS: TRANSLOCATION OF PROTEINS
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the protein complex correspond to ribophorin I and ribophorin 11, respectively. Antibodies that recognize the carboxyl-terminal cytoplasmic domain of ribophorin I could be used to immunodeplete the oligosaccharyltransferase activity from detergent extracts and to immunopurify the heteroligomeric complex. The ribophorins are relatively abundant integral membrane glycoproteins that are restricted to the rough endoplasmic r e t i ~ u l u r n .The ~ ~ ~protein ' sequences of ribophorins I and I1 have been determined from both rat and human cDNA clone^.^^-^ A consensus sequence motif for a dolichol binding site was recently proposed based upon a comparison of several glyco~yltransferases.~~ A search for this sequence in ribophorins I and I1 revealed a related protein sequence in ribophorin I, so we suggest that this subunit of the oligosaccharyltransferasecontains the binding site for the lipid-linked oligosaccharide substrate.
CONCLUSIONS Considerable information is now available concerning the reaction steps that occur during transport of proteins across and integration of proteins into the endoplasmic reticulum. The majority of proteins transported across the endoplasmic reticulum are delivered to the membrane through the combined action of SRP and the SRP receptor. We are now beginning to understand how the interaction between SRP,SRP receptor and signal sequences is controlled by a complex GTP hydrolysis cycle. It is now clear that the transport phase of the translocation reaction is mediated by a protein complex that may include SSR,mp39, imp-34 and perhaps others that have yet to be identified. Whether these polypeptides are assembled into a pore-like transport apparatus remains to be established. The protein modification enzymes signal peptidase and oligosaccharyltransferase have been isolated, and correspond to abundant RER protein complexes. Together all of these proteins comprise a membrane bound translocation apparatus that has been termed the translocon.% REFERENCES 1 . BLOBEL,G. 1980. Proc. Natl. Acad. Sci. USA 77: 1496-1500. 2. SABATINI,D. D., G. KREIBICH, T. MORIMOTO & M. ADESNICK. 1982. J. Cell Biol. 92: 1-22. 3. WICKNER,W. T. & H. F. LODISH.1985. Science 230: 400-407. 4. VON HEIJNE, G . 1983. Eur. J. Biochem. 133: 17-21. G . 1986. Nucieic Acids Res. 14: 4683-4690. 5. VON HEIJNE, 6 . WALTER,P. & G. BLOBEL.1980. Proc. Natl. Acad. Sci. USA 77: 7112-7116. R. & C. MOLIAY.1986. J. Biol. Chem. 261: 12889-12895. 7. ZIMMERMANN, 8. SCHLENSTEDT, G. & R. ZIMMERMANN.1987. EMBO J. 6: 699-703. 9. ANDERSON,D. J., K. E. MOSTOV& G. BLOBEL.1983. Proc. Natl. Acad. Sci. USA 80: 7249- 7253. E. C. & K. DRICKAMER. 1985. J. Biol. Chem. 261: 1286-1292. 10. HOLLAND, 1 1 . HULL,J. D., R. GILMORE & R. A. LAMB. 1988. J. Cell Biol. 106: 1489-1498. P. & G. BLOBEL.1982. Nature 299: 691-698. 12. WALTER, 13. WALTER, P. & G. BLOBEL.1981. J. Cell Biol. 91: 577-561. 1988. EMBO J. 7: 3559-3569. 14. WOLIN,S. L. & P. WALTER. P. & G. BLOBEL.1983. Cell 3 4 525-533. 15. WALTER, V. & P. WALTER. 1988. Cell 52: 39-49. 16. SIEGEL, 17. SIEGEL,V. & P. WALTER. 1985. J. Cell Bioi. 100: 1913-1921. 1986. Nature 320: 81-84. 18. SIEGEL,V. & P. WALTER.
ANNALS NEW YORK ACADEMY OF SCIENCES
36
U. C., P.WALTER& A. E. JOHNSON. 1986. Proc.Natl. Acad. Sci. USA 83: 860419. KIUEG, 8608. 20. KURZCHALIA,T. V., M. WIEDMANN, A. S. GIRSHOVICH, E. S. BOCHKAREVA, H. BIELKA & T. A. RAPOPORT. 1986. Nature 320: 634-636. 21. BRENNWALD,P., X. LIAO,K. HOLM,G. PORTER& J. A. WISE.1988. Mol. Cell. Biol. 8: 1580-1590. 22. HA", B. C., M. A. PORITZ& P. WALTER. 1989. J. Cell Biol. 1 0 9 3223-3230. 23. PORITZ,M. A., V. SIEGEL, W. HANSEN & P.WALTER. 1988. Proc.Natl. Acad. Sci. USA 85: 4315-4319. 1991. Cell 67: 131-144. 24. HA", B. C. & P. WALTER. 25. WALTER, P.& G. BLOBEL. 1981. J. Cell Biol. 91: 551-556. & G. BLOBEL.1982. J. Cell Biol. 95: 470-477. 26. GILMORE, R., P. WALTER 27. MEYER,D. I., E. KRAUSE& B. DOBBERSTEIN. 1982. Nature 297: 647-650. 28. TAJIMA, S.,L. LUFFER, V. L. RATH& P.WALTER. 1986. J. Cell Biol. 103: 1167-1178. 29. GILMORE,R. & G. BLOBEL.1983. Cell 35: 677-685. 30. GILMORE,R. & G. BLOBEL.1985. Cell 42: 497-505. 1986. EMBO J. 5: 951-955. 31. AINGER,K. J. & D. I. MEYER. 32. SIEGEL,V. & P. WALTER. 1988. EMBO J. 7: 1769-1775. S. M. & G. BLOBEL.1991. Cell 65: 371-380. 33. SIMON, 34. WIEDMA",M., T. V. KURZCHALIA,E. HARTMANN & T. A. RAPOPORT. 1987. Nature 328: 830-833. 1989. J. Cell Biol. 109 2033-2043. 35. KIUEG,U. C., A. E. JOHNSON & P. WALTER. 36. HIGHS., D. GORLICH, M. WIEDMAN, T. A. WOPORT & B. DOBBERSTEIN. 1991. J. Cell Biol. 113: 35-44. 1991. J. Cell Biol. 114: 21-33. 37. KELLARIS, K. V., S. BOWEN & R. GILMORE. 1974. J. Mol. Biol. 88: 55938. BORGESE, N., W. MOK,G. KREIBICH & D. D. SABATINI. 580. 1986. J. Cell Biol. 103: 241-253. 39. HORTSCH, M., D. AVOSSA& D. I. MEYER. 40. KREIBICH,G., B. L. ULRICH & D. D. SABATINI. 1978. J. Cell Biol. 77: 464-487. Y. ASANO,K. MIZUSAWA, R. YAMAGISHI, T. HORIGOME 41. YOSHIDA, H., N. TONDOKORO, & H. SUGANO.1987. Biochern. J. 245: 811-819. 1990. Nature 346: 540-544. 42. SAVITZA. J. & D. I. MEYER. 43. NUNNARI, J. M., D. L. ZIMMERMAN, S. C. OGG& P.WALTER. 1991. Nature 352: 638640.
44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.
P. & R. GILMORE.1991. J. Cell Biol. 114: 639-649. COLLINS, 1986. Cell 47: 711-719. MIZE,N. K., D. W. ANDREW& V. R. LINGAPPA. INOUYE, M. & S. HALEGOUA. 1980. CRC Crit. Rev. Biochern. 7: 339-371. A. S., P. J. M. ROTITER & J. K. ROSE.1988. Proc.Natl. Acad. Sci. USA 85: 7592SHAW, 7596. HARTMANN, E., T. A. RAPOPORT& H. F. LODISH.1989. Proc. Natl. Acad. Sci. USA 86: 5786- 5790. ROTHBLATT,J. A. & D. I. MEYER. 1986. EMBO J. 5: 1031-1036. HANSEN, W., P. D. GARCIA & P. WALTER. 1986. Cell 45: 397-406. M. G. & G. BLOBEL.1986. J. Cell Biol. 102: 1543-1550. WATERS, DESHAIES, R J., B. D. KOCH, M. WERNER-WASHBURNE, E. A. CRAIG & R. SCHEKMAN. 1988. Nature 332: 800-805. & G. BLOBEL.1988. Nature 322: 805-810. CHIRICO, W. J., M. G. WATERS 1986. J. Cell Biol. 103: 2253-2261. CONNOLLY, T. & R. GILMORE. HOFFMAN, K. & R. GILMORE. 1988. J. Biol. Chern. 263: 4381-4385. WILSON,C., T. CONNOLLY, T. MORRISON& R. GILMORE. 1988. J. Cell Biol. 107: 6977. 1989. Cell 57: 599-610. CONNOLLY, T. & R. GILMORE. BERNSTEIN,H. D., M. A. PORITZ,K. STRUB, P. J. HOBEN,S. BRENNER& P.WALTER. 1989. Nature 340: 482-486. ROMISH, K., J. WEBB,J. HERZ,S. PREHN,R. FRANK, M. VINGRON& B. DOBBERSTEIN. 1989. Nature 340: 478-482.
GILMORE & KEUARIS TRANSLOCATION OF PROTEINS
37
& W. C. MERRICK.1987. Proc. Natl. Acad. Sci. USA 84: 60. DEVER.T. E.. M. J. GLYNIAS 1814-1818: A. E. JOHNSON & P.WALTER.1990. EMBO J. 9 451161. ZOPF, D., H. D. BERNSTEIN, 4517. H. GAUSEPOHL & B. DOBBERSTEIN. 1990. J. 62. ROMISCH,K., J. WEBB,K. LINGELBACH, Cell Biol. 111: 1793-1802. & F. MCCORMICK. 1990. Nature 348: 125-132. 63. BOURNE,H. R, D. A. SANDERS M. & F. MCCORMICK.1987. Science 238: 542-545. 64. TRAHEY, P.& R GILMORE.1992. J. Cell Biol. In press. 65. RAPIEJKO, T., P. J. RAPIEJKO & R GILMORE.1991. Science 252: 1171-1173. 66. CONNOLLY, & A. E. JOHNSON. 1991. J. Cell Biol. 112: 67. THRIIT,R N., D. W. ANDmws, P.WALTER 809-821. 68. GORLICH,D., S. PREHN,E. HARTMA”,J. HERZ, A. Ono, R KRAIT, M. WIEDMAN, S. KNEPSEL, B. DOBBERSTEIN & T. A. RAPOPORT. 1990. J. Cell Biol. 111:2283-2294. R FRANK,K. ROMISCH,B. 69. PREHN,S., J. HERZ, E. HARTMA”,T. V. KURZCHALIA, & T. A. RAPOPORT.1990. Eur. J. Biochem. 188: 439-445. DOBBERSTEIN 1987. J. Cell. Biol. 105: 633-645. 70. DESHAIES,R J. & R SCHEKMAN. G. DAUM& R SCHEKMAN. 1989. J. 71. ROTHBLAIT,J.A., R J. DESHAIES,S. L. SANDERS, Cell Biol. 109: 2641-2652. J., A. R HIBBS,P. SANZ,J. CROWE& D. I. MEYER.1988. EMBO J. 7: 434772. TOYN, 4353. J. ROTHBLAIT,J. WAY& P.SILVER.1989. J. Cell 73. SADLER,I., A. CHIANG,T. KURIHARA, Biol. 109: 2665-2675. 1989. J. Cell Biol. 109 2653-2664. 74. DESHAIES,R J. & R SCHEKMAN. D. A. FELDHEIM & R SCHEKMAN.1991. Nature (Lon75. DESHAIES,R J., S. L. SANDERS,
don) 349: 806-808. 76. NICCHITTA, C. V. & G. BLOBEL.1989. J. Cell Biol. 108: 789-795. & P. WALTER.1982. J. Cell Biol. 95: 463-469. 77. GILMORE,R.,G. BLOBEL & G. BLOBEL.1986. Proc. Natl. Acad. Sci. USA 83: 58178. EVANS,E. A,, R GILMORE 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91.
585. BAKER,R. K. & M. 0. LIVELY. 1987. Biochem. 26: 8561-8567. GREENBERG, G.,G. S.SHELNESS&G. BLOBEL.1989. J. Biol. Chem. 264:15762-15765. SHELNESS, G. S.& G. BLOBEL.1990. J. Biol. Chem. 265: 9512-9519. SHELNESS, G. S., Y. S. KANWm & G. BLOBEL.1988. J. Biol. Chem. 263: 17063-17070. BOHNI,P. C., R J. DESHAIES& R SCHEKMAN. 1988. J. Cell Biol. 106: 1035-1042. YADEAU,J. T., C. KLEIN& G. BLOBEL.1991. Proc.Natl. Acad. Sci. USA 88: 517-521. ARNOLD,A., S. A. HORST,T. J. GARDELLA,H. BABA,M. A. LJ”E& H. M. K m NENBERG. 1990. J. Clin. Invest. 86 1084-1087. KORNFELD, R & S. KORNFELD. 1985. Ann. Rev. Biochem. 5 4 631-664. ROTHMAN, J. E.& H. F. LODISH.1977. Nature 269 775-780. Lw, T., B. STETSON,S. J. TVRCO,S. C. HUBBARD& P. W. ROBBINS.1979. J. Biol. Chem. 254: 4554-4559. ELBEIN,A. D. 1987. Annu. Rev. Biochem. 56: 497-534. KELLEHER,D. J., G. KREIBICH & R GILMORE.1992. Cell. In press. MARCANTONIO, E.E., A. A~URCOSTESEC & G. KREIBICH. 1984. J. Cell. Biol. 99:2254-
2259. 92. HARNIK-ORT,V., K. WH, E. ~ C A N T O N I O ,D. R C o w , M. G. ROSENFELD, & G. KREIBICH. 1987. J. Cell Biol. 104: 855-863. M. ADESNIK,D. D. SABATINI 93. CRIMAUDO, C., M. HORTSCH,H. GAUSEPOHL & D. I. MEYER.1987. EMBO J. 6: 7582. & G. KREIBICH.1991. Bio94. PIROZZI,G., Z. ZHOU, P. D’EUSTACHIO,D. D. SABATINI chem. Biophys. Res. Commun. 176: 1482-1486. & P. W. ROBBINS. 1989. Proc. Natl. Acad. Sci. USA 86: 95. ALBRIGHT,C. F., P. ORLEAN 7366-7369. 96. WALTER,P. & V. R LINGAPPA.1986. Ann. Rev. Cell Biol. 2: 499-516.
