Glycoprotein Synthesis and Secretion: Translation and Targeting 1 , 2 CI. KOCH-BRANDT
The General Problem: Protein Sorting in the Eucaryotic Cell The eucaryotic cell is subdivided into structurally and functionally distinct membranebound compartments that derive their identity from specific sets of proteins and other specialized molecules that they harbor either in the membrane or in the lumen of a given organelle. To understand eucaryotic cell function it is therefore crucial to understand how the compartments are created and maintained. Because almost all protein synthesis is confined to the cytosol, eucaryotic cells must have mechanisms to direct proteins from their site of synthesis to the compartment, where they are supposed to exert their function. Additionally, cells must possess mechanisms that enable them to maintain the identity of organelles, despite the extensive flow of material between them (1). Newly synthesized proteins are sorted in a series of multiple targeting steps. Initially, proteins are segregated into two groups; cytosolic, mitochondrial, nuclear, and peroxisomal proteins are releasedinto the cytosol after their synthesis has been completed. Cytosolic proteins remain here, whereas mitochondrial, nuclear and peroxisomal proteins are translocated from the cytosol directly into their target organelle (figure 1).The specificity of the translocation arises from the interaction of specific structures in the proteins that specify their destination - sorting signals - and translocator proteins in the target organelle membrane, which recognize these signals and mediate the transport. Sorting signals for transport into the nucleus, the mitochondria, and peroxisomes have been identified, and putative nuclear and mitochondrial translocator proteins have been characterized (2, 3). The second group includes proteins of the endoplasmic reticulum (ER), the Golgi complex, the lysosomes, secretory vesicles, and the secretory and plasma membrane proteins. These proteins carry a hydrophobic signal sequence, generally located at their aminoterminal end, which mediates, as soon as it emerges from the ribosome, a translational arrest, the attachment of the ribosome to the ER membrane and the segregation during translation into the ER, either as soluble proteins into the lumen or as transmembrane proteins embedded into the ER membrane. Involved in this complex series of events are the signal recognition particle (SRP), the SRPreceptor, and a variety of other components (4). In the biogenesis of the second group of proteins, the segregation into the ER is only the initial sorting step. Proteins have further options: they may stay in the organelles along the axocytic pathway, they may proceed to the cell surface, or they may be diverted to lyso-
SUMMARY Toestablish and maintain organelle identity, the eucaryotic cell must be able to target newly synthesized proteins to the various cellular compartments. The specificity of this process appears to be generally mediated by the interaction of structural features of the transported proteins (sorting signals) with cellular proteins that bind these structures and mediate the targeted transport (sorting receptors). Although signals involved in the sorting into a variety of intracellular organelles as well as some (putative) receptor proteins have been identified, the sorting signals and receptors involved in the targeted transport of proteins to the cells surface are just begining to evolve. AM REV RESPIR DIS 1991; 144:S29-S32
somes. Irrespective of their final destination, proteins that have been inserted into the ER do not need to cross a lipid bilayer again. Their further transport is mediated by a sequence of specific vesiclebudding and fusion events in which GTP-binding proteins appear to playa key regulatory role (5). Once a protein has been segregated into the ER, its further targeting is dependent on its sorting into the correct vesicle population, which is transported to and fuses with the target membrane (table 1). The Specific Problem: Protein Targeting in the Exocytic Pathway The general pathway of protein secretion outlined above has been clear for many years (6). Although similar in all cells,there may be considerable variability from celltype to cell type. Cells can secrete proteins continuously (constitutive secretion) or discontinuously (regulated secretion) along the entire surface (nonpolarized) or at specific regions of the plasma membrane (polarized) (7). The constitutive pathway is present in all cells (figure 2). The vesicles in this pathway continuously fuse with plasma membrane, releasing the proteins according to their rate of synthesis. In contrast to the continuous release via, the constitutive route, proteins secreted via the regulated exocytic pathway are stored intracellularly in secretory vesicles or granules and are released only when the fusion of the storage vesicleswith the plasma membrane has been triggered by an extracellular stimulus, which alters the intracellular concentration of a second messenger such as calcium. Cells that have evolved a regulated exocytic pathway for the short-term release of high levels of certain proteins also exhibit a constitutive pathway. Consequently, regulated and constitutive secretions may coexist in the same cell (7). There is even evidence that more than one regulated pathway may exist in a single cell (8). The coexistence of constitutive and regulated secretions raises questions about the molecular mechanisms that assemble the vesicles of the regulated and the constitutive pathways, mediate the sorting of specific sets of proteins into either vesi-
cle population, and control the differential fusion of regulated and constitutive secretory vesicles. Examples of regulated transport are the secretion of surfactant protein A by the alveolar type II cells,of mucin by the bronchial mucus cells, and of lysozyme and lactoferrin by the bronchial serous cells (9). Cells cannot only control the time, they can also control the site of exocytosis (7). This is most prominent in polarized epithelial cells (10). These cells are characterized by the differentiation of the plasma membrane into structurally and functionally distinct domains, an apical domain that faces the lumen of a cavity and a basolateral domain that contacts neighboring cells, the basal lamina, and the bloodstream. The two domains, which differ in protein and lipid composition, are separated by circumferential tight junctions. Examples of localized secretion are the release of zymogens at the luminal surface of the pancreatic exocrine cells and of surfactant protein A at the apical surface of the alveolar type II cells.In both celltypes, polarized secretion occurs via the regulated pathway of exocytosis. Cells can, however, also secrete proteins constitutively at a specific cell-surface domain. Examples are the secretion of albumin, transferrin, a2f..l-globulin in the liver parenchymal cells, which occurs at the sinusoidal surface (11), and the polarized secretion of proteins in intestinal and kidney epithelial cells (12, 13). In the latter cells the coexistence of polarized and nonpolarized, respectively, apical and basolateral exocytosis has been demonstrated (13),raising the question of how apical and basolateral transport vesicles are generated, how proteins destined for either cell surface are sorted into these vesicles, and how these vesicles are transported to and fuse at the correct plasma membrane domain. These questions concern the cellular machinery of the generation, transport, and fusion 1 From the Institute fur Biochemie, Universitat Frankfurt, Frankfurt, Germany. 2 Correspondence and requests for reprints should be addressed to Cl. Koch-Brandt, Institut fur Biochemie, J. Gutenberg-Universitat, Becherweg 30, 6500 Mainz, Germany.
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plasma membrane ~+-~__~~ secretory vesicle ___--endosome ~~-*----lysosome
mitochondrion
----~-
~----~---Golgi
peroxisome
apparatus
•
je ~-----H----
endoplasmatic reticulum
nucleus cytosol
Fig. 1. Schematic drawing of an animal cell emphasizing the intracellular compartments and transport routes of newly synthesized proteins
of specific sets of exocytic vesicles, the dependence on cytoskeletal elements, and on low molecular weight modulators such as Ca 2 +, ATP, OTP and their respective binding proteins as well as the structures in the proteins that specify their final destination and transport route and the receptor and effector proteins that decipher this information and mediate the transport. The Experimental Approaches Most of the experiments designed to characterize cellular components of the transport machinery exploit the simplicity of model systems. These may be established cell lines (10), semipermeabilized cells (14), distinct subcellular fractions (15), or reconstituted vesicles (16). The use of lower eucaryotic organisms with a well-characterized genetic background such as yeast has greatly advanced our knowledge about proteins involved in the exocytic pathway (17). Recombinant DNA techniques and gene transfection experiments that have proved to be very successful in the identifica-tion of sorting signals for transport into intracellular organelles such as the nucleus, mitochondria, peroxisomes; and the- ER are widely used in an effort to characterize sort-
ing signals effective in the exocytic pathway (18). Chemical (photo-crosslinking [19, 20], affinity chromatography [21]) and immunologic (antiidiotype antibodies [3, 22]) approaches are exploited to identify the cellular receptors that recognizethe sorting signals. Data and Hypotheses
The Site of Sorting of Secretory and Plasma Membrane Proteins Although it is clear that the sorting of secretory proteins has to occur intracellularly, plasma membrane proteins may be sorted intracellularly on the exocytic route as well as after arrival at the cell surface on the endocytic route (figure 3). Recent evidence suggests that sorting on the exocytic route takes place upon exit of the proteins from tile trans-Golgi network (TON) (23). This has most clearly been established for cells with a regulated pathway of secretion (24, 25). By analogy, it has been proposed that sorting into the apical and basolateral constitutive exocytic pathways in polarized epithelial cells might also occur in this compartment (26). This, however, still needs to be shown. Sorting on the endocytic route occurs at the plasma membrane. Theoretically, one can
envisage that plasma membrane proteins are initially inserted either randomly along the entire cell surface or into a single domain (e.g., the basolateral). Plasma membrane proteins destined for the opposite domain would subsequently be internalized and routed by transcytosis to the correct domain. It has been shown that exocytic and endocytic sorting may be operative in the same cell and that various epithelial cell types differ in the extent to which they use either route for sorting (26). The Madin-Darby canine kidney (MOCK) cells efficiently sort proteins on the exocytic route. Different sets of secretory proteins are vectorially released at either cell surface (27, 28), and plasma membrane proteins are sorted intracellularly into the apical or the basolateral pathway and are directly delivered at the correct plasma membrane domain (10,26). That the cells can also sort plasma membrane proteins via the endocytic route has been shown by the studies of Casanova and coworkers (29) on the transport of the polymeric Ig receptor expressed from recombinant DNA in these cells. In the intestinal cell line CaCo-2, some resident apical membrane proteins such as sucrase-isomaltase are sorted in the TON and routed directly to the apical surface, whereas others such as aminopeptidase and dipeptidylpeptidase IV use both the direct route and the transcytotic pathway via the basolateral membrane (30). Evidently, these cellsuse both pathways, the extent being dependent on the individual protein. This is supported by results on protein secretion in these cells. Exogenous secretory proteins expressed in CaCo-2 cells are released predominantly into the basolateral medium, including the immunoglobulin K light chain and growth hormone. Both proteins are secreted in equal amounts at both surfaces when expressed in MOCK cells (12,31). In contrast to MOCK and CaCo-2 cells, hepatocytes rely exclusivelyon endocytic sorting to target membrane proteins to the apical surface domain. In these cells apical and basolateral plasma membrane proteins are initially inserted into the basolateral membrane from where the apical proteins are segregated and transported via the transcytotic route to the apical domain (11). Secretory proteins are released from these cells exclusively at the basolateral surface (11).
TABLE 1 SORTING SIGNALS IN THE EXOCYTIC
Examples of Structural Signals Directing (+) a Protein to or Retarding (-) in an Organelle
Location of the Signal
Reference No.
KDEL (in soluble proteins) Sequences in the cytoplasmic domain of the E19 glycoprotein of adenovirus 1. Transmembrane domain of the E1 glycoprotein of coronavirus Sequences in the stem region of galactosylsialytransferase Propeptides of some secretory proteins, not of others ( +) 17 amino acids in the cytoplasmic domain of the polyimmunoglobulin receptor Tyrosine residues in the cytoplasmic domain of two viral membrane proteins (+) Glycophospholipid-anchor
Luminal Cytoplasmic Transmembrane Luminal Luminal Cytoplasmic Cytoplasmic Membrane anchor
43 44 45 46 34-37
Organelles Endoplasmic reticulum cis-Golgi trans-Golgi Regulated secretory pathway Basolateral plasma-domain membrane in MOCK cells Apical plasma-membrane domain in some epithelial cells Definition of abbreviations: KOEL
=
PATHW~Y
( -) (- ) ( -) (- ) (+)
lysine-aspartic acid-glutamic acid-leucine; MOCK
=
Madin-Oarby canine kidney.
38 41
39
GLYCOPROTEIN SYNTHESIS AND SECRETION: TRANSLATION AND TARGETING
Fig. 2. Schematic drawing of a fibroblast cell with a single constitutive exocytic pathway (a), a secretory cell with a constitutive and a regulated exocytic pathway (b), and an epithelial cell with direct exocytic routes to the apical and the basolateral plasma membrane domain (c).
Fig. 3. Established sorting pathways in epithelial cells. Proteins are sorted intracellularly and delivered directly at the correct plasma membrane domain (left). Alternatively or additionally, proteins are initially delivered at the basolateral cell surface domain, and the apical proteins are routed by transcytosis to their target membrane (right).
Sorting Signals and Receptors In analogy to the sorting into other cellular compartments, the sorting of secretory and plasma membrane proteins has been proposed to rely on the interaction of specific structures in the transported proteins (targeting addresses, sorting signals) with cellular receptors that recognize these structures and initiate the segregation (1). Specific aggregation and condensation of proteins has been suggested to be involved in the sorting at least into the regulated pathway of exocytosis (25). When the problem of sorting signals and receptors is considered, three general considerations have to be taken into account. (1) Sorting signals may either act positively and direct a protein to a compartment such as the mannose-6-phosphate signal, which diverts lysosomal enzymes from the exocytic pathway into the lysosomes, or act negatively and retain it in an organelle along the exocytic pathway such as the KDEL sequence (HDEL in yeast) at the carboxyterminus of soluble ER proteins (32). (2) Sorting signals may be short contiguous sequences of amino acids such as the signals for transport into the ER, nucleus, mitochondria, and peroxisomes. They may, however, also be composed of discontinuous sequences that are juxtaposed only by the proper folding of the protein. Such signals might exert no obvious sequence homology since it would be a feature of the overall stucture rather than a specific stretch of amino acids that mediates the binding to a receptor. As a consequence, these signals might be difficult to identify (18). (3) Not all exocytic traffic needs to be signal mediated. One can easily envisage that in a fibroblast cell with only a single pathway to the cell surface, proteins segregated into the ER are delivered as bulk flow material at the cell surface unless they are specifically retained or diverted from the exocytic route. Experimental evidence for this model exists (33). In cells with alternative cell surface pathways, constitutive and regulated, polarized
and nonpolarized, one has to propose that at least one pathway needs to be signaldependent. In these cells it has to be defined which pathway is signal mediated and which is the default pathway (7).
Protein Sorting in Regulated Secretory Cells Because all cells possess a constitutive exocytic pathway and only cells specialized for rapid, high-level secretion of proteins have evolved a regulated pathway, it has been suggested that constitutive secretion may be the normal pathway involving passive, bulk-flow mechanisms, whereas the regulated pathway would be the specialized, signal-dependent route (7). In some proteins it may be the propeptide sequences that are involved in the targeting into the regulated pathway (34, 35), whereas in others they are not (36, 37). Using mature prolactin, insulin, and growth hormone rather than the propeptide forms as affinity ligands, Chung and coworkers (21)identified a set of 25-kD proteins that might be candidates for receptors that segregate proteins into the regulated pathway. The sorting machinery seems to be conserved among endocrine and exocrine cells of diverse tissue and species origins as shown by DNA transfection experiments (7). Protein Sorting in Polarized Epithelial Cells In polarized epithelial cells the plasma membrane is differentiated into distinct domains. The basolateral domain resembles in its protein and lipid composition the plasma membrane of nonpolarized cells. The apical membranes on the other hand have a unique protein composition that is specific to the cell type and function (26). The organization of the two plasma membrane domains might suggest that protein transport to the apical cell surface is a specialized function of the celland might require specificsignals, whereas
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traffic to the basolateral domain is the normal pathway present in all cells and might be signal independent (26). Although there is general agreement that transport to the specialized apical cell surface is signal mediated' it is not yet clear whether nonsorted protein traffic is generally directed exclusivelyto the basolateral surface or whether there might be cell-type-specific differences. In hepatocytes, which indiscriminately deliver all proteins at the basolateral domain and target apical proteins by transcytosis to their final destination, basolateral delivery may need no signal. In MDCK cells, however, exogenous secretory proteins expressed from cDNA are released in similar amounts at both cell surfaces (12, 13). These include the liver form of a2J..l-globulin, which is secreted basolaterally in hepatocytes. This result suggests that nonsorted proteins randomly enter both basolateral and apical transport vesicles in MDCK cells. The first approaches to characterize sorting signals concentrated on the question: Which domain of membrane spanning proteins harbors the sorting signal? The typical experiment would involvedomain deletion or domain swapping between marker proteins of the apical and the basolateral plasma membrane domain. Although these studies did not offer an unambiguous answer, they strongly suggested that it is the luminal domain that determines the final destination (32). Recent evidence suggests that in addition to targeting addresses located in the luminal domain, sorting signals also may reside in the transmembrane and/or cytoplasmic domain (29, 38). Because the signals located in the different protein domains may be contradictory, as is the case in the polyimmunoglobulin receptor (29, 38), a hierarchy of multiple sorting information has been proposed. Because no conserved sequences can be found in the primary structure of either apical or basolateral proteins, it has been suggested that it may be three-dimensional motifs formed by noncontiguous protein sequences that act as sorting signals. Lisanti and Rodriguez-Boulan (39)werethe first to point out that the presence of a glycosyl-phosphatidyl-inositol (GPI) membrane anchor is exclusively found in apical proteins and to propose that this structure might act as an apical targeting signal. When this hypothesis was tested by the construction and expression of hybrid proteins in the kidney epithelial cell line MDCK, it was shown that the GPI-anchor indeed directs proteins to the apical cell surface (39). In this study, however,it became evident that the glycolipid is not the only structure conveying apical targeting information since a protein deleted in this anchor was still transported in a polarized fashion to the apical cell surface. Interestingly, in a thyroid epithelial cell line, the polarity of GPI-anchored proteins is reversed; they are found predominantly in the basolateral plasma membrane domain (40). A structure essential for basolateral targeting in MDCK cells has been shown to reside
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in a minimal sequence of 17 amino acids in the cytoplasmic domain of the poly-IgAreceptor (38).Tyrosine-phosphorylation in the cytoplasmic domain has been suggested to be involved in the basolateral targeting of two viral membrane proteins in the same cells(41). Despite the fact that sorting signals for vectorial exocytosis are not yet clearly defined, one can analyze how general or specific to a particular epithelial cell they might be. Studies of the transport of amino peptidase N, which reaches the apical surface in CaCo-2 cells both by the direct and the transcytotic route, and when expressed in MOCK cells is sorted directly to the apical surface, argue that sorting signals might be general for all epithelial cells. Furthermore, the demonstration that viral marker proteins for the apical and the basolateral plasma membrane domain in MOCK cells are transported to the axonal, respectively, to the dendrite membrane in neurons suggests that common mechanisms of plasma membrane protein segregation might exist in epithelial and neuronal cells (42). The results we have so far on protein sorting in the exocytic pathway are pieces in a mosaic (table 1). They demonstrate both a high degree of conservation as well as flexibility in the ways cells target proteins to the cell surface. As more and more data become available, they will hopefully add up to reveal a complete and maybe unifying picture of protein sorting in the exocytic pathway. References 1. Caplan M, Matlin KS. Sorting of membrane and secretory proteins in polarized epithelial cells. New York: Alan R. Liss, 1989; 71-127. 2. von Heinje G. Protein targeting signals. Curr Opin Cell BioI 1990; 2:604-8. 3. Pain D, Murakami H, Blobel G. Identification of a receptor for protein import into mitochondria. Nature 1990; 347:444-9. 4. Rapoport TA. Protein transport across the ER membane. Trends Biochem Sci 1990; 15:355-8. 5. Bourne HR. Do GTPases direct membrane traffic in secretion? Cell 1988; 53:669-71. 6. Palade G. Intracellular aspects of the process of protein sorting. Science 1975; 189:347-57. 7. Kelly RB. Pathways of protein secretion in eukaryotes. Science 1985; 230:25-32. 8. Miller SG, Moore HP. Regulated secretion. Curr Opin Cell BioI 1990; 2:642-7. 9. Basbaum CB, Carlson D, Davidson E, Verdugo P, Gail DB. Cellular mechanisms of airwaysecretion. Am Rev Respir Dis 1988; 137:479-85. 10. Simons K, Fuller SD. Cell surface polarity in epithelia. Annu Rev Cell BioI 1985; 1:243-88. 11. Bartles JR, Hubbard AL. Plasma membrane protein sorting in epithelial cells: Do secretory pathways hold the key? Trends Biochem Sci 1988;
13:181-4. 12. Rindler MJ, Traber MG. A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells. J Cell BioI 1988; 107:471-9. 13. Kondor-Koch C, Bravo R, Fuller SD, Cutler D, Garoff H. Exocytotic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell MDCK. Cell 1985; 43:297-306. 14. Featherstone C. Perforated cell systemsto study membrane transport. Trends Biochem Sci 1988; 13:284-6. 15. Balch WE. Biochemistry of interorganelle transport. J BioI Chern 1989; 264:16965-8. 16. Hurtley SM. Protein translocation into reconstituted vesicles.TrendsBiochem Sci 1990;15:211-2. 17. Balch WE. Molecular dissection of early stages of the eukaryotic secretory pathway. Curr Opin Cell BioI 1990; 2:634-41. 18. Garoff H. Using recombinant DNA techniques to study protein targeting in the eukaryotic cell. Annu Rev Cell BioI 1985; 1:403-45. 19. Wiedmann M, Kurzchalia TV, Hartmann E, Rapoport TA. A signal sequence receptor in the endoplasmic reticulum membrane. Nature 1987; 328:830-3. 20. Krieg UC, Johnson AE, Walter P. Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as a part of a putative translocation tunnel. J BioI Chern 1989; 109:2033-43. 21. Chung KN, Walter P, Aponte GW, Moore HPH. Molecular sorting in the secretory pathway. Science 1989; 243:192-7. 22. Vaux D, Tooze J, Fuller S. Identification by antitype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal. Nature 1990; 345: 495-502. 23. Griffiths G, Simmons K. The trans-Golgi network: sorting at the exit site of the Golgi complex. Science 1986; 234:438-43. 24. Tooze J, Tooze S, Fuller SD. Sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-Golgi network of AtT-20 cells. J Cell BioI 1987; 101:949-64. 25. Orci L, Ravazzola M, Amherdt M, et ale The trans-most cisternae of the Golgi complex: a compartment for sorting of secretory and plasma membrane proteins. Cell 1987; 511:1039-51. 26. Simons K, Wandinger-Ness A. Polarized sorting in epithelia. Cell 1990; 62:207-10. 27. Urban J, Parczyk K, Leutz A, Kayne M, Kondor-Koch C. Constitutive apical secretion of an 80-kD sulfated glycoproteincomplexin the polarized epithelial Madin Darby canine kidney cellline. J Cell BioI 1987; 105:2735-43. 28. Caplan MJ, Stow JL, Newman AP, et ale Dependence on pH of polarized sorting of secreted proteins. Nature 1987; 329:632-5. 29. Casanova JE, Breitfeld PP, Mostov KE. Phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis. Science 1990; 248:742-5. 30. Matter K, Brauchbar M, Bucher K, Hauri HP.
Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2). Cell 1990; 60:429-37. 31. Hughson EJ, Cutler DF, Hopkins CR. Basolateral secretion of kappa light chain in the polarised epithelial cell line Caco-2. J Cell Sci 1989; 94:327-32. 32. Breitfeld PP, Casanova JE, Simister NE, Ross SA, McKinnon WC, Mostov KE. Sorting signals. Curr Opin Cell BioI 1989; 1:617-23. 33. Wieland FT, Gleason ML, Serafini TA, Rothman JR. The rate of bulkflow from the endoplasmic reticulum to the cell surface. Cell 1987; 50:289-300. 34. Sevarino KA, Stork P, Ventimiglia R, Mandel G, Goodman RH. Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity. Cell 1989; 57:11-9. 35. Stoller TJ, Shields D. The propeptide of preposomatostatin mediates intracellular transport and secretion of a-globin from mammalian cells. J Cell BioI 1989; 108:1647-55. 36. BurgessTL, Craik CS, Matsuuchi L, KellyRB. In vitromutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules. J Cell BioI 1987; 105:659-68. 37. Powell SK, Orci L, Craik CS, Moore HPH. Efficient targeting to storage granules of human proinsulins with altered propeptide domain. J Cell BioI 1988; 106:1843-51. 38. Apodaca G, Casanova JE, Mosto KE. Identification of a signal in the cytoplasmic tail of the poly-immunoglobin receptor that targets the normally apical protein placental alkaline phosphatase to the basolateral cell surface of MDCK cells. J Cell BioI 1990; 111:138a. 39. Lisanti MP, Rodriguez-Boulan E. Glycophospholipid membrane anchoring provides clues to the mechanism of protein sorting in polarized epithelial cells. Trends Biochem Sci 1990; 15:113-8. 40. Zurzolo C, Nitsch L, Rodriguez-Boulan E, Lisanti M. Reversed polarity of GPI -anchored proteins in a polarized thyroid epithelial cell line (abstract). J Cell BioI 1990; 111:327a. 41. Brewer CB, Thomas D, Roth MG. A signal of basolateral sorting of proteins in MDCK cells (abstract). J Cell BioI 1990; 111:327a. 42. Dotti CO, Simons K. Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons. Cell 1990; 62:63-72. 43. Pelham HRB. The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci 1990; 15:483-6. 44. Nilsson T, Jackson M, Peterson PA. Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. Cell 1989; 58:707-18. 45. Machamer CE, Mentone SA, Rose JK, Farquhar MO. The E1 glycoprotein of an avian coronaviruses targeted to the cisgolgi complex. Proc Natl Acad Sci USA 1990; 87:6944-8. 46. Colley KJ, Paulson JC. A Golgi apparatus localization/retention signal is located in the stem region of the ~-galactosidase 2,6-sialyhransferase (abstract). J Cell BioI 1990; 111:5a.
The General Problem: Protein Sorting in the Eucaryotic Cell The eucaryotic cell is subdivided into structurally and functionally distinct membranebound compartments that derive their identity from specific sets of proteins and other specialized molecules that they harbor either in the membrane or in the lumen of a given organelle. To understand eucaryotic cell function it is therefore crucial to understand how the compartments are created and maintained. Because almost all protein synthesis is confined to the cytosol, eucaryotic cells must have mechanisms to direct proteins from their site of synthesis to the compartment, where they are supposed to exert their function. Additionally, cells must possess mechanisms that enable them to maintain the identity of organelles, despite the extensive flow of material between them (1). Newly synthesized proteins are sorted in a series of multiple targeting steps. Initially, proteins are segregated into two groups; cytosolic, mitochondrial, nuclear, and peroxisomal proteins are releasedinto the cytosol after their synthesis has been completed. Cytosolic proteins remain here, whereas mitochondrial, nuclear and peroxisomal proteins are translocated from the cytosol directly into their target organelle (figure 1).The specificity of the translocation arises from the interaction of specific structures in the proteins that specify their destination - sorting signals - and translocator proteins in the target organelle membrane, which recognize these signals and mediate the transport. Sorting signals for transport into the nucleus, the mitochondria, and peroxisomes have been identified, and putative nuclear and mitochondrial translocator proteins have been characterized (2, 3). The second group includes proteins of the endoplasmic reticulum (ER), the Golgi complex, the lysosomes, secretory vesicles, and the secretory and plasma membrane proteins. These proteins carry a hydrophobic signal sequence, generally located at their aminoterminal end, which mediates, as soon as it emerges from the ribosome, a translational arrest, the attachment of the ribosome to the ER membrane and the segregation during translation into the ER, either as soluble proteins into the lumen or as transmembrane proteins embedded into the ER membrane. Involved in this complex series of events are the signal recognition particle (SRP), the SRPreceptor, and a variety of other components (4). In the biogenesis of the second group of proteins, the segregation into the ER is only the initial sorting step. Proteins have further options: they may stay in the organelles along the axocytic pathway, they may proceed to the cell surface, or they may be diverted to lyso-
SUMMARY Toestablish and maintain organelle identity, the eucaryotic cell must be able to target newly synthesized proteins to the various cellular compartments. The specificity of this process appears to be generally mediated by the interaction of structural features of the transported proteins (sorting signals) with cellular proteins that bind these structures and mediate the targeted transport (sorting receptors). Although signals involved in the sorting into a variety of intracellular organelles as well as some (putative) receptor proteins have been identified, the sorting signals and receptors involved in the targeted transport of proteins to the cells surface are just begining to evolve. AM REV RESPIR DIS 1991; 144:S29-S32
somes. Irrespective of their final destination, proteins that have been inserted into the ER do not need to cross a lipid bilayer again. Their further transport is mediated by a sequence of specific vesiclebudding and fusion events in which GTP-binding proteins appear to playa key regulatory role (5). Once a protein has been segregated into the ER, its further targeting is dependent on its sorting into the correct vesicle population, which is transported to and fuses with the target membrane (table 1). The Specific Problem: Protein Targeting in the Exocytic Pathway The general pathway of protein secretion outlined above has been clear for many years (6). Although similar in all cells,there may be considerable variability from celltype to cell type. Cells can secrete proteins continuously (constitutive secretion) or discontinuously (regulated secretion) along the entire surface (nonpolarized) or at specific regions of the plasma membrane (polarized) (7). The constitutive pathway is present in all cells (figure 2). The vesicles in this pathway continuously fuse with plasma membrane, releasing the proteins according to their rate of synthesis. In contrast to the continuous release via, the constitutive route, proteins secreted via the regulated exocytic pathway are stored intracellularly in secretory vesicles or granules and are released only when the fusion of the storage vesicleswith the plasma membrane has been triggered by an extracellular stimulus, which alters the intracellular concentration of a second messenger such as calcium. Cells that have evolved a regulated exocytic pathway for the short-term release of high levels of certain proteins also exhibit a constitutive pathway. Consequently, regulated and constitutive secretions may coexist in the same cell (7). There is even evidence that more than one regulated pathway may exist in a single cell (8). The coexistence of constitutive and regulated secretions raises questions about the molecular mechanisms that assemble the vesicles of the regulated and the constitutive pathways, mediate the sorting of specific sets of proteins into either vesi-
cle population, and control the differential fusion of regulated and constitutive secretory vesicles. Examples of regulated transport are the secretion of surfactant protein A by the alveolar type II cells,of mucin by the bronchial mucus cells, and of lysozyme and lactoferrin by the bronchial serous cells (9). Cells cannot only control the time, they can also control the site of exocytosis (7). This is most prominent in polarized epithelial cells (10). These cells are characterized by the differentiation of the plasma membrane into structurally and functionally distinct domains, an apical domain that faces the lumen of a cavity and a basolateral domain that contacts neighboring cells, the basal lamina, and the bloodstream. The two domains, which differ in protein and lipid composition, are separated by circumferential tight junctions. Examples of localized secretion are the release of zymogens at the luminal surface of the pancreatic exocrine cells and of surfactant protein A at the apical surface of the alveolar type II cells.In both celltypes, polarized secretion occurs via the regulated pathway of exocytosis. Cells can, however, also secrete proteins constitutively at a specific cell-surface domain. Examples are the secretion of albumin, transferrin, a2f..l-globulin in the liver parenchymal cells, which occurs at the sinusoidal surface (11), and the polarized secretion of proteins in intestinal and kidney epithelial cells (12, 13). In the latter cells the coexistence of polarized and nonpolarized, respectively, apical and basolateral exocytosis has been demonstrated (13),raising the question of how apical and basolateral transport vesicles are generated, how proteins destined for either cell surface are sorted into these vesicles, and how these vesicles are transported to and fuse at the correct plasma membrane domain. These questions concern the cellular machinery of the generation, transport, and fusion 1 From the Institute fur Biochemie, Universitat Frankfurt, Frankfurt, Germany. 2 Correspondence and requests for reprints should be addressed to Cl. Koch-Brandt, Institut fur Biochemie, J. Gutenberg-Universitat, Becherweg 30, 6500 Mainz, Germany.
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plasma membrane ~+-~__~~ secretory vesicle ___--endosome ~~-*----lysosome
mitochondrion
----~-
~----~---Golgi
peroxisome
apparatus
•
je ~-----H----
endoplasmatic reticulum
nucleus cytosol
Fig. 1. Schematic drawing of an animal cell emphasizing the intracellular compartments and transport routes of newly synthesized proteins
of specific sets of exocytic vesicles, the dependence on cytoskeletal elements, and on low molecular weight modulators such as Ca 2 +, ATP, OTP and their respective binding proteins as well as the structures in the proteins that specify their final destination and transport route and the receptor and effector proteins that decipher this information and mediate the transport. The Experimental Approaches Most of the experiments designed to characterize cellular components of the transport machinery exploit the simplicity of model systems. These may be established cell lines (10), semipermeabilized cells (14), distinct subcellular fractions (15), or reconstituted vesicles (16). The use of lower eucaryotic organisms with a well-characterized genetic background such as yeast has greatly advanced our knowledge about proteins involved in the exocytic pathway (17). Recombinant DNA techniques and gene transfection experiments that have proved to be very successful in the identifica-tion of sorting signals for transport into intracellular organelles such as the nucleus, mitochondria, peroxisomes; and the- ER are widely used in an effort to characterize sort-
ing signals effective in the exocytic pathway (18). Chemical (photo-crosslinking [19, 20], affinity chromatography [21]) and immunologic (antiidiotype antibodies [3, 22]) approaches are exploited to identify the cellular receptors that recognizethe sorting signals. Data and Hypotheses
The Site of Sorting of Secretory and Plasma Membrane Proteins Although it is clear that the sorting of secretory proteins has to occur intracellularly, plasma membrane proteins may be sorted intracellularly on the exocytic route as well as after arrival at the cell surface on the endocytic route (figure 3). Recent evidence suggests that sorting on the exocytic route takes place upon exit of the proteins from tile trans-Golgi network (TON) (23). This has most clearly been established for cells with a regulated pathway of secretion (24, 25). By analogy, it has been proposed that sorting into the apical and basolateral constitutive exocytic pathways in polarized epithelial cells might also occur in this compartment (26). This, however, still needs to be shown. Sorting on the endocytic route occurs at the plasma membrane. Theoretically, one can
envisage that plasma membrane proteins are initially inserted either randomly along the entire cell surface or into a single domain (e.g., the basolateral). Plasma membrane proteins destined for the opposite domain would subsequently be internalized and routed by transcytosis to the correct domain. It has been shown that exocytic and endocytic sorting may be operative in the same cell and that various epithelial cell types differ in the extent to which they use either route for sorting (26). The Madin-Darby canine kidney (MOCK) cells efficiently sort proteins on the exocytic route. Different sets of secretory proteins are vectorially released at either cell surface (27, 28), and plasma membrane proteins are sorted intracellularly into the apical or the basolateral pathway and are directly delivered at the correct plasma membrane domain (10,26). That the cells can also sort plasma membrane proteins via the endocytic route has been shown by the studies of Casanova and coworkers (29) on the transport of the polymeric Ig receptor expressed from recombinant DNA in these cells. In the intestinal cell line CaCo-2, some resident apical membrane proteins such as sucrase-isomaltase are sorted in the TON and routed directly to the apical surface, whereas others such as aminopeptidase and dipeptidylpeptidase IV use both the direct route and the transcytotic pathway via the basolateral membrane (30). Evidently, these cellsuse both pathways, the extent being dependent on the individual protein. This is supported by results on protein secretion in these cells. Exogenous secretory proteins expressed in CaCo-2 cells are released predominantly into the basolateral medium, including the immunoglobulin K light chain and growth hormone. Both proteins are secreted in equal amounts at both surfaces when expressed in MOCK cells (12,31). In contrast to MOCK and CaCo-2 cells, hepatocytes rely exclusivelyon endocytic sorting to target membrane proteins to the apical surface domain. In these cells apical and basolateral plasma membrane proteins are initially inserted into the basolateral membrane from where the apical proteins are segregated and transported via the transcytotic route to the apical domain (11). Secretory proteins are released from these cells exclusively at the basolateral surface (11).
TABLE 1 SORTING SIGNALS IN THE EXOCYTIC
Examples of Structural Signals Directing (+) a Protein to or Retarding (-) in an Organelle
Location of the Signal
Reference No.
KDEL (in soluble proteins) Sequences in the cytoplasmic domain of the E19 glycoprotein of adenovirus 1. Transmembrane domain of the E1 glycoprotein of coronavirus Sequences in the stem region of galactosylsialytransferase Propeptides of some secretory proteins, not of others ( +) 17 amino acids in the cytoplasmic domain of the polyimmunoglobulin receptor Tyrosine residues in the cytoplasmic domain of two viral membrane proteins (+) Glycophospholipid-anchor
Luminal Cytoplasmic Transmembrane Luminal Luminal Cytoplasmic Cytoplasmic Membrane anchor
43 44 45 46 34-37
Organelles Endoplasmic reticulum cis-Golgi trans-Golgi Regulated secretory pathway Basolateral plasma-domain membrane in MOCK cells Apical plasma-membrane domain in some epithelial cells Definition of abbreviations: KOEL
=
PATHW~Y
( -) (- ) ( -) (- ) (+)
lysine-aspartic acid-glutamic acid-leucine; MOCK
=
Madin-Oarby canine kidney.
38 41
39
GLYCOPROTEIN SYNTHESIS AND SECRETION: TRANSLATION AND TARGETING
Fig. 2. Schematic drawing of a fibroblast cell with a single constitutive exocytic pathway (a), a secretory cell with a constitutive and a regulated exocytic pathway (b), and an epithelial cell with direct exocytic routes to the apical and the basolateral plasma membrane domain (c).
Fig. 3. Established sorting pathways in epithelial cells. Proteins are sorted intracellularly and delivered directly at the correct plasma membrane domain (left). Alternatively or additionally, proteins are initially delivered at the basolateral cell surface domain, and the apical proteins are routed by transcytosis to their target membrane (right).
Sorting Signals and Receptors In analogy to the sorting into other cellular compartments, the sorting of secretory and plasma membrane proteins has been proposed to rely on the interaction of specific structures in the transported proteins (targeting addresses, sorting signals) with cellular receptors that recognize these structures and initiate the segregation (1). Specific aggregation and condensation of proteins has been suggested to be involved in the sorting at least into the regulated pathway of exocytosis (25). When the problem of sorting signals and receptors is considered, three general considerations have to be taken into account. (1) Sorting signals may either act positively and direct a protein to a compartment such as the mannose-6-phosphate signal, which diverts lysosomal enzymes from the exocytic pathway into the lysosomes, or act negatively and retain it in an organelle along the exocytic pathway such as the KDEL sequence (HDEL in yeast) at the carboxyterminus of soluble ER proteins (32). (2) Sorting signals may be short contiguous sequences of amino acids such as the signals for transport into the ER, nucleus, mitochondria, and peroxisomes. They may, however, also be composed of discontinuous sequences that are juxtaposed only by the proper folding of the protein. Such signals might exert no obvious sequence homology since it would be a feature of the overall stucture rather than a specific stretch of amino acids that mediates the binding to a receptor. As a consequence, these signals might be difficult to identify (18). (3) Not all exocytic traffic needs to be signal mediated. One can easily envisage that in a fibroblast cell with only a single pathway to the cell surface, proteins segregated into the ER are delivered as bulk flow material at the cell surface unless they are specifically retained or diverted from the exocytic route. Experimental evidence for this model exists (33). In cells with alternative cell surface pathways, constitutive and regulated, polarized
and nonpolarized, one has to propose that at least one pathway needs to be signaldependent. In these cells it has to be defined which pathway is signal mediated and which is the default pathway (7).
Protein Sorting in Regulated Secretory Cells Because all cells possess a constitutive exocytic pathway and only cells specialized for rapid, high-level secretion of proteins have evolved a regulated pathway, it has been suggested that constitutive secretion may be the normal pathway involving passive, bulk-flow mechanisms, whereas the regulated pathway would be the specialized, signal-dependent route (7). In some proteins it may be the propeptide sequences that are involved in the targeting into the regulated pathway (34, 35), whereas in others they are not (36, 37). Using mature prolactin, insulin, and growth hormone rather than the propeptide forms as affinity ligands, Chung and coworkers (21)identified a set of 25-kD proteins that might be candidates for receptors that segregate proteins into the regulated pathway. The sorting machinery seems to be conserved among endocrine and exocrine cells of diverse tissue and species origins as shown by DNA transfection experiments (7). Protein Sorting in Polarized Epithelial Cells In polarized epithelial cells the plasma membrane is differentiated into distinct domains. The basolateral domain resembles in its protein and lipid composition the plasma membrane of nonpolarized cells. The apical membranes on the other hand have a unique protein composition that is specific to the cell type and function (26). The organization of the two plasma membrane domains might suggest that protein transport to the apical cell surface is a specialized function of the celland might require specificsignals, whereas
531
traffic to the basolateral domain is the normal pathway present in all cells and might be signal independent (26). Although there is general agreement that transport to the specialized apical cell surface is signal mediated' it is not yet clear whether nonsorted protein traffic is generally directed exclusivelyto the basolateral surface or whether there might be cell-type-specific differences. In hepatocytes, which indiscriminately deliver all proteins at the basolateral domain and target apical proteins by transcytosis to their final destination, basolateral delivery may need no signal. In MDCK cells, however, exogenous secretory proteins expressed from cDNA are released in similar amounts at both cell surfaces (12, 13). These include the liver form of a2J..l-globulin, which is secreted basolaterally in hepatocytes. This result suggests that nonsorted proteins randomly enter both basolateral and apical transport vesicles in MDCK cells. The first approaches to characterize sorting signals concentrated on the question: Which domain of membrane spanning proteins harbors the sorting signal? The typical experiment would involvedomain deletion or domain swapping between marker proteins of the apical and the basolateral plasma membrane domain. Although these studies did not offer an unambiguous answer, they strongly suggested that it is the luminal domain that determines the final destination (32). Recent evidence suggests that in addition to targeting addresses located in the luminal domain, sorting signals also may reside in the transmembrane and/or cytoplasmic domain (29, 38). Because the signals located in the different protein domains may be contradictory, as is the case in the polyimmunoglobulin receptor (29, 38), a hierarchy of multiple sorting information has been proposed. Because no conserved sequences can be found in the primary structure of either apical or basolateral proteins, it has been suggested that it may be three-dimensional motifs formed by noncontiguous protein sequences that act as sorting signals. Lisanti and Rodriguez-Boulan (39)werethe first to point out that the presence of a glycosyl-phosphatidyl-inositol (GPI) membrane anchor is exclusively found in apical proteins and to propose that this structure might act as an apical targeting signal. When this hypothesis was tested by the construction and expression of hybrid proteins in the kidney epithelial cell line MDCK, it was shown that the GPI-anchor indeed directs proteins to the apical cell surface (39). In this study, however,it became evident that the glycolipid is not the only structure conveying apical targeting information since a protein deleted in this anchor was still transported in a polarized fashion to the apical cell surface. Interestingly, in a thyroid epithelial cell line, the polarity of GPI-anchored proteins is reversed; they are found predominantly in the basolateral plasma membrane domain (40). A structure essential for basolateral targeting in MDCK cells has been shown to reside
832
CI. KOCH-BRANDT
in a minimal sequence of 17 amino acids in the cytoplasmic domain of the poly-IgAreceptor (38).Tyrosine-phosphorylation in the cytoplasmic domain has been suggested to be involved in the basolateral targeting of two viral membrane proteins in the same cells(41). Despite the fact that sorting signals for vectorial exocytosis are not yet clearly defined, one can analyze how general or specific to a particular epithelial cell they might be. Studies of the transport of amino peptidase N, which reaches the apical surface in CaCo-2 cells both by the direct and the transcytotic route, and when expressed in MOCK cells is sorted directly to the apical surface, argue that sorting signals might be general for all epithelial cells. Furthermore, the demonstration that viral marker proteins for the apical and the basolateral plasma membrane domain in MOCK cells are transported to the axonal, respectively, to the dendrite membrane in neurons suggests that common mechanisms of plasma membrane protein segregation might exist in epithelial and neuronal cells (42). The results we have so far on protein sorting in the exocytic pathway are pieces in a mosaic (table 1). They demonstrate both a high degree of conservation as well as flexibility in the ways cells target proteins to the cell surface. As more and more data become available, they will hopefully add up to reveal a complete and maybe unifying picture of protein sorting in the exocytic pathway. References 1. Caplan M, Matlin KS. Sorting of membrane and secretory proteins in polarized epithelial cells. New York: Alan R. Liss, 1989; 71-127. 2. von Heinje G. Protein targeting signals. Curr Opin Cell BioI 1990; 2:604-8. 3. Pain D, Murakami H, Blobel G. Identification of a receptor for protein import into mitochondria. Nature 1990; 347:444-9. 4. Rapoport TA. Protein transport across the ER membane. Trends Biochem Sci 1990; 15:355-8. 5. Bourne HR. Do GTPases direct membrane traffic in secretion? Cell 1988; 53:669-71. 6. Palade G. Intracellular aspects of the process of protein sorting. Science 1975; 189:347-57. 7. Kelly RB. Pathways of protein secretion in eukaryotes. Science 1985; 230:25-32. 8. Miller SG, Moore HP. Regulated secretion. Curr Opin Cell BioI 1990; 2:642-7. 9. Basbaum CB, Carlson D, Davidson E, Verdugo P, Gail DB. Cellular mechanisms of airwaysecretion. Am Rev Respir Dis 1988; 137:479-85. 10. Simons K, Fuller SD. Cell surface polarity in epithelia. Annu Rev Cell BioI 1985; 1:243-88. 11. Bartles JR, Hubbard AL. Plasma membrane protein sorting in epithelial cells: Do secretory pathways hold the key? Trends Biochem Sci 1988;
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Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2). Cell 1990; 60:429-37. 31. Hughson EJ, Cutler DF, Hopkins CR. Basolateral secretion of kappa light chain in the polarised epithelial cell line Caco-2. J Cell Sci 1989; 94:327-32. 32. Breitfeld PP, Casanova JE, Simister NE, Ross SA, McKinnon WC, Mostov KE. Sorting signals. Curr Opin Cell BioI 1989; 1:617-23. 33. Wieland FT, Gleason ML, Serafini TA, Rothman JR. The rate of bulkflow from the endoplasmic reticulum to the cell surface. Cell 1987; 50:289-300. 34. Sevarino KA, Stork P, Ventimiglia R, Mandel G, Goodman RH. Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity. Cell 1989; 57:11-9. 35. Stoller TJ, Shields D. The propeptide of preposomatostatin mediates intracellular transport and secretion of a-globin from mammalian cells. J Cell BioI 1989; 108:1647-55. 36. BurgessTL, Craik CS, Matsuuchi L, KellyRB. In vitromutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules. J Cell BioI 1987; 105:659-68. 37. Powell SK, Orci L, Craik CS, Moore HPH. Efficient targeting to storage granules of human proinsulins with altered propeptide domain. J Cell BioI 1988; 106:1843-51. 38. Apodaca G, Casanova JE, Mosto KE. Identification of a signal in the cytoplasmic tail of the poly-immunoglobin receptor that targets the normally apical protein placental alkaline phosphatase to the basolateral cell surface of MDCK cells. J Cell BioI 1990; 111:138a. 39. Lisanti MP, Rodriguez-Boulan E. Glycophospholipid membrane anchoring provides clues to the mechanism of protein sorting in polarized epithelial cells. Trends Biochem Sci 1990; 15:113-8. 40. Zurzolo C, Nitsch L, Rodriguez-Boulan E, Lisanti M. Reversed polarity of GPI -anchored proteins in a polarized thyroid epithelial cell line (abstract). J Cell BioI 1990; 111:327a. 41. Brewer CB, Thomas D, Roth MG. A signal of basolateral sorting of proteins in MDCK cells (abstract). J Cell BioI 1990; 111:327a. 42. Dotti CO, Simons K. Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons. Cell 1990; 62:63-72. 43. Pelham HRB. The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci 1990; 15:483-6. 44. Nilsson T, Jackson M, Peterson PA. Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. Cell 1989; 58:707-18. 45. Machamer CE, Mentone SA, Rose JK, Farquhar MO. The E1 glycoprotein of an avian coronaviruses targeted to the cisgolgi complex. Proc Natl Acad Sci USA 1990; 87:6944-8. 46. Colley KJ, Paulson JC. A Golgi apparatus localization/retention signal is located in the stem region of the ~-galactosidase 2,6-sialyhransferase (abstract). J Cell BioI 1990; 111:5a.
