The Yeast Pma1 Proton Pump: a Model for Understanding the Biogenesis of Plasma Membrane Proteins

Abstract

endoplasmic reticulum The Pma1 H+-ATPase ofSaccharomyces cerevisiae, which functions physiologically to pump protons out of the cell, is one of the most abundant proteins in the yeast plasma membrane (1Morsomme P. Slayman C.W. Goffeau A. Mutagenic study of the structure, function, and biogenesis of the yeast plasma membrane H+-ATPase..Biochim. Biophys. Acta. 2000; 1469 (review): 133-157Crossref PubMed Scopus (103) Google Scholar). It is a 100-kDa polypeptide, anchored in the membrane by 10 hydrophobic α-helices (Fig.1) (2Auer M. Scarborough G.A. Kuhlbrandt W. Three-dimensional map of the plasma membrane H+-ATPase in the open conformation..Nature. 1998; 392: 840-843Crossref PubMed Scopus (185) Google Scholar) and belonging to a widespread family of cation transporters known as the P2-type ATPases (6Lutsenko S. Kaplan J.H. Organization of P-type ATPases: significance of structural diversity..Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (418) Google Scholar). Members of the P2 family in animal cells include the plasma membrane Na+,K+- and Ca2+-ATPases, gastric mucosal H+, K+-ATPase, and sarcoplasmic reticulum Ca2+-ATPase. In recent years, the yeast H+-ATPase has emerged as a valuable prototype for studies of plasma membrane biogenesis. Several complementary approaches have been taken, all drawing on the power of yeast genetics. (i) Strains with temperature-sensitive blocks at successive steps in the secretory pathway have made it possible to map the route by which the H+-ATPase travels to the plasma membrane (7Brada D. Schekman R. Coincident localization of secretory and plasma membrane proteins in organelles of the yeast secretory pathway..J. Bacteriol. 1988; 1780: 2775-2783Crossref Google Scholar, 8Holcomb C.L. Hansen W.J. Etcheverry T. Schekman R. Secretory vesicles externalize the major plasma membrane ATPase in yeast..J. Cell Biol. 1988; 106: 641-648Crossref PubMed Scopus (47) Google Scholar, 9Chang A. Slayman C.W. Maturation of the yeast plasma membrane [H+]ATPase involves phosphorylation during intracellular transport..J. Cell Biol. 1991; 115: 289-295Crossref PubMed Scopus (151) Google Scholar). (ii) Point mutations in the PMA1 gene have given insights into the structural requirements for proper folding and trafficking of the H+-ATPase (10Harris S.L. Na S. Zhu X. Seto-Young D. Perlin D.S. Teem J.H. Haber J.E. Dominant lethal mutations in the plasma membrane H+-ATPase gene of Saccharomyces cerevisiae..Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10531-10535Crossref PubMed Scopus (62) Google Scholar, 11Portillo F. Characterization of dominant lethal mutations in the yeast plasma membrane H+-ATPase gene..FEBS Lett. 1997; 402: 136-140Crossref PubMed Scopus (28) Google Scholar, 12Nakamoto R.K. Verjovski-Almeida S. Allen K.E. Ambesi A. Rao R. Slayman C.W. Substitutions of aspartate 378 in the phosphorylation domain of the yeast PMA1 H+-ATPase disrupt protein folding and biogenesis..J. Biol. Chem. 1998; 273: 7338-7344Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 13DeWitt N.D. Tourinho dos Santos C.F. Allen K.E. Slayman C.W. Phosphorylation region of the yeast plasma membrane H+-ATPase. Role in protein folding and biogenesis..J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). (iii) Suppressors and enhancers of biogenesis-defective pma1 mutants have revealed new components of the secretory process (14Chang A. Fink G.R. Targeting of the yeast plasma membrane [H+]ATPase: a novel gene AST1 prevents mislocalization of the ATPase to the vacuole..J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar, 15Na S. Hincapie M. McCusker J.H. Haber J.E. MOP2 (SLA2) affects the abundance of the plasma membrane H+-ATPase of Saccharomyces cerevisiae..J. Biol. Chem. 1995; 270: 6815-6823Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 16Luo W. Chang A. Novel genes involved in endosomal traffic in yeast revealed by suppression of a targeting-defective plasma membrane ATPase mutant..J. Cell Biol. 1997; 138: 731-746Crossref PubMed Scopus (91) Google Scholar, 17Wang Q. Chang A. Eps1, a novel PDI-related protein involved in ER quality control in yeast..EMBO J. 1999; 18: 5972-5982Crossref PubMed Scopus (88) Google Scholar, 18Luo W. Chang A. An endosome-to-plasma membrane pathway involved in trafficking of a mutant plasma membrane ATPase in yeast..Mol. Biol. Cell. 2000; 11: 579-592Crossref PubMed Scopus (64) Google Scholar). (iv) Finally, by screening for mutations that exacerbate a temperature-sensitive defect in one of the standard COPII coat subunits, a specialized coat protein has been identified that helps to mediate the exit of newly synthesized H+-ATPase from the ER1 (19Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum..J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (130) Google Scholar, 20Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae..J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (116) Google Scholar). In the following sections, recent results from all four approaches are woven together into a stepwise description of H+-ATPase biogenesis. A comprehensive review of earlier work can be found in a chapter by de Kerchove d'Exaerde et al. (21Kerchove d'Exaerde A. Supply P. Goffeau A. Subcellular traffic of the plasma membrane H+-ATPase in Saccharomyces cerevisiae..Yeast. 1996; 12: 907-916Crossref PubMed Scopus (30) Google Scholar). As expected, Pma1 H+-ATPase is synthesized and integrated into the membrane in the rough endoplasmic reticulum. Pulse-chase experiments suggest that it achieves a fully folded structure very rapidly, because it can be protected against trypsinolysis by physiological concentrations of ligands even at the earliest time points (22Chang A. Rose M.D. Slayman C.W. Folding and intracellular transport of the yeast plasma membrane H+-ATPase: effects of mutations in KAR2SEC65..Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5808-5812Crossref PubMed Scopus (13) Google Scholar). The ATPase then travels to the cell surface via the secretory pathway (Fig. 2), as shown by the fact that its biogenesis can be blocked by temperature-sensitive mutations in genes governing successive steps of the pathway: SEC18 (ER to Golgi), SEC7 (Golgi to secretory vesicles), and SEC6 (secretory vesicles to plasma membrane) (8Holcomb C.L. Hansen W.J. Etcheverry T. Schekman R. Secretory vesicles externalize the major plasma membrane ATPase in yeast..J. Cell Biol. 1988; 106: 641-648Crossref PubMed Scopus (47) Google Scholar, 9Chang A. Slayman C.W. Maturation of the yeast plasma membrane [H+]ATPase involves phosphorylation during intracellular transport..J. Cell Biol. 1991; 115: 289-295Crossref PubMed Scopus (151) Google Scholar). Interestingly, the 100-kDa H+-ATPase undergoes post-translational phosphorylation on multiple Ser and Thr residues during its transit from the ER to the cell surface (9Chang A. Slayman C.W. Maturation of the yeast plasma membrane [H+]ATPase involves phosphorylation during intracellular transport..J. Cell Biol. 1991; 115: 289-295Crossref PubMed Scopus (151) Google Scholar). The functional reason for these stepwise phosphorylations is unknown, although there is good evidence that the last one, occurring at or near the plasma membrane, plays a role in the activation of the ATPase by glucose (9Chang A. Slayman C.W. Maturation of the yeast plasma membrane [H+]ATPase involves phosphorylation during intracellular transport..J. Cell Biol. 1991; 115: 289-295Crossref PubMed Scopus (151) Google Scholar). The oligomeric state of the mature H+-ATPase is not yet certain. Monomers of the closely related Neurospora crassaenzyme are fully active after reconstitution into proteoliposomes (23Scarborough G.A. Addison R. On the subunit composition of the Neurospora plasma membrane H+-ATPase..J. Biol. Chem. 1984; 259: 9109-9114Abstract Full Text PDF PubMed Google Scholar), but radiation inactivation experiments give a target size of 230 kDa, consistent with a functional dimer (24Bowman B.J. Berenski C.J. Jung C.Y. Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation..J. Biol. Chem. 1985; 260: 8726-8730Abstract Full Text PDF PubMed Google Scholar). Hexameric complexes are recovered on glycerol or sucrose gradients after detergent solubilization (25Chadwick C.C. Goormaghtigh E. Scarborough G.A. A hexameric form of the Neurospora crassa plasma membrane H+-ATPase..Arch. Biochem. Biophys. 1987; 252: 348-356Crossref PubMed Scopus (48) Google Scholar) and have made it possible to produce two-dimensional crystals for structural studies (2Auer M. Scarborough G.A. Kuhlbrandt W. Three-dimensional map of the plasma membrane H+-ATPase in the open conformation..Nature. 1998; 392: 840-843Crossref PubMed Scopus (185) Google Scholar), but there is no clear evidence that such complexes exist in vivo. Even though the ER is not as clearly differentiated in yeast as in many mammalian cells, immunofluorescence and immunoelectron microscopy have revealed two morphologically distinct parts: (i) prominent perinuclear elements, continuous with the outer nuclear membrane, and (ii) peripheral tubules, extending outward through the cytoplasm and concentrated in the region immediately beneath the plasma membrane (26Preuss D. Mulholland J. Kaiser C.A. Orlean P. Albright C. Rose M.D. Robbins P.W. Botstein D. Structure of the yeast endoplasmic reticulum: localization of ER proteins using immunofluorescence and immunoelectron microscopy..Yeast. 1991; 7: 891-911Crossref PubMed Scopus (147) Google Scholar). Both are studded with ribosomes and can be labeled by antibodies against ER markers, and it seems likely that they form a single interconnected network (27Klumperman J. Transport between ER and Golgi..Curr. Opin. Cell Biol. 2000; 12: 445-449Crossref PubMed Scopus (110) Google Scholar). If so, one would like to learn where specific plasma membrane proteins are synthesized within the network and where they are packaged into vesicles for shipment to the Golgi. It would also be useful to know where and how such proteins are screened for proper folding before being allowed to leave the ER. Partial answers to both questions have come from the use of Pma1 H+-ATPase as a model plasma membrane protein. Of nearly 300 site-directed mutations that have been introduced throughout the ATPase, amino acid substitutions at 45 positions have led to defects in biogenesis (Fig. 1) (reviewed in Ref. 1Morsomme P. Slayman C.W. Goffeau A. Mutagenic study of the structure, function, and biogenesis of the yeast plasma membrane H+-ATPase..Biochim. Biophys. Acta. 2000; 1469 (review): 133-157Crossref PubMed Scopus (103) Google Scholar). Most (and perhaps all) of these polypeptides are poorly folded, as evidenced by their abnormal sensitivity to trypsin (12Nakamoto R.K. Verjovski-Almeida S. Allen K.E. Ambesi A. Rao R. Slayman C.W. Substitutions of aspartate 378 in the phosphorylation domain of the yeast PMA1 H+-ATPase disrupt protein folding and biogenesis..J. Biol. Chem. 1998; 273: 7338-7344Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 13DeWitt N.D. Tourinho dos Santos C.F. Allen K.E. Slayman C.W. Phosphorylation region of the yeast plasma membrane H+-ATPase. Role in protein folding and biogenesis..J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Confocal and immunoelectron microscopy have shown that such mutations trigger a dramatic proliferation of ER-derived membranes, in which misfolded H+-ATPase accumulates along with standard ER markers such as Kar2p (10Harris S.L. Na S. Zhu X. Seto-Young D. Perlin D.S. Teem J.H. Haber J.E. Dominant lethal mutations in the plasma membrane H+-ATPase gene of Saccharomyces cerevisiae..Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10531-10535Crossref PubMed Scopus (62) Google Scholar, 11Portillo F. Characterization of dominant lethal mutations in the yeast plasma membrane H+-ATPase gene..FEBS Lett. 1997; 402: 136-140Crossref PubMed Scopus (28) Google Scholar, 13DeWitt N.D. Tourinho dos Santos C.F. Allen K.E. Slayman C.W. Phosphorylation region of the yeast plasma membrane H+-ATPase. Role in protein folding and biogenesis..J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar,21Kerchove d'Exaerde A. Supply P. Goffeau A. Subcellular traffic of the plasma membrane H+-ATPase in Saccharomyces cerevisiae..Yeast. 1996; 12: 907-916Crossref PubMed Scopus (30) Google Scholar). When wild-type H+-ATPase is co-expressed with a mutant of this type, it becomes arrested in the same membranes and growth stops; thus, the mutation acts genetically in a dominant lethal fashion (10Harris S.L. Na S. Zhu X. Seto-Young D. Perlin D.S. Teem J.H. Haber J.E. Dominant lethal mutations in the plasma membrane H+-ATPase gene of Saccharomyces cerevisiae..Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10531-10535Crossref PubMed Scopus (62) Google Scholar, 11Portillo F. Characterization of dominant lethal mutations in the yeast plasma membrane H+-ATPase gene..FEBS Lett. 1997; 402: 136-140Crossref PubMed Scopus (28) Google Scholar, 12Nakamoto R.K. Verjovski-Almeida S. Allen K.E. Ambesi A. Rao R. Slayman C.W. Substitutions of aspartate 378 in the phosphorylation domain of the yeast PMA1 H+-ATPase disrupt protein folding and biogenesis..J. Biol. Chem. 1998; 273: 7338-7344Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 13DeWitt N.D. Tourinho dos Santos C.F. Allen K.E. Slayman C.W. Phosphorylation region of the yeast plasma membrane H+-ATPase. Role in protein folding and biogenesis..J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Recent experiments have suggested that the exact point of arrest in the ER may vary with the severity of the pma1 mutation. Least seriously affected among those studied to date is G381A, only three residues downstream from the critical Asp that is phosphorylated by ATP during the catalytic cycle. The G381A polypeptide displays an intermediate folding defect and is transiently arrested in the ER; it then escapes via the secretory vesicles to the plasma membrane, from which it is recycled to the vacuole for degradation (13DeWitt N.D. Tourinho dos Santos C.F. Allen K.E. Slayman C.W. Phosphorylation region of the yeast plasma membrane H+-ATPase. Role in protein folding and biogenesis..J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Confocal microscopy has shown that the temporary arrest of G381A occurs in punctate ER-derived bodies, located predominantly near the periphery of the cell. 2T. Ferreira, A. B. Mason, and C. W. Slayman, manuscript in preparation. Upon closer examination by electron microscopy, the punctate bodies look markedly similar to the well known vesicular-tubular compartment of mammalian cells (28Balch W.E. McCaffery J.M. Plutner H. Farquhar M.G. Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum..Cell. 1994; 76: 841-852Abstract Full Text PDF PubMed Scopus (333) Google Scholar) and may therefore correspond to specific exit sites from the ER. Consistent with the intermediate behavior of G381A in biogenesis experiments, it is not a fully dominant mutation but instead allows slow growth of cells co-expressing wild-type H+-ATPase (13DeWitt N.D. Tourinho dos Santos C.F. Allen K.E. Slayman C.W. Phosphorylation region of the yeast plasma membrane H+-ATPase. Role in protein folding and biogenesis..J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). D378N, in which the catalytically important Asp residue has been replaced by Asn, exhibits a more severe biogenetic defect, leading to a dominant lethal phenotype. As judged by its extreme sensitivity to trypsin, this ATPase is very poorly folded and becomes arrested in invasive “sausage-like” structures, which are derived from the ER (10Harris S.L. Na S. Zhu X. Seto-Young D. Perlin D.S. Teem J.H. Haber J.E. Dominant lethal mutations in the plasma membrane H+-ATPase gene of Saccharomyces cerevisiae..Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10531-10535Crossref PubMed Scopus (62) Google Scholar, 13DeWitt N.D. Tourinho dos Santos C.F. Allen K.E. Slayman C.W. Phosphorylation region of the yeast plasma membrane H+-ATPase. Role in protein folding and biogenesis..J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and may represent proliferation of the vesicular-tubular elements. Unlike G381A, D378N fails to escape to the Golgi and is translocated back into the cytoplasm for degradation by the proteasome (17Wang Q. Chang A. Eps1, a novel PDI-related protein involved in ER quality control in yeast..EMBO J. 1999; 18: 5972-5982Crossref PubMed Scopus (88) Google Scholar). Insight into the nature of the ER quality control process has come from screening an insertional genomic library for suppressors of the dominant lethal behavior of D378N (17Wang Q. Chang A. Eps1, a novel PDI-related protein involved in ER quality control in yeast..EMBO J. 1999; 18: 5972-5982Crossref PubMed Scopus (88) Google Scholar). This approach has yielded a gene called EPS1 (ER-retainedp ma1 suppressing) that, when disrupted, prevents degradation of the D378N polypeptide and allows it to reach the cell surface. The product of the EPS1 gene belongs to the protein disulfide isomerase family and may act as a membrane-bound chaperone. Its specificity for misfolded H+-ATPase is not fully understood, but deletingEPS1 has little or no effect on the biogenesis of the wild-type H+-ATPase or on the retention of other proteins that normally reside in the ER. Like secreted proteins, proteins destined for the plasma membrane are carried from the ER to the Golgi by COPII vesicles, whose coats contain heterodimeric protein complexes known as Sec23/24p and Sec13/31p (reviewed in Ref. 29Barlowe C. COPII and selective export from the endoplasmic reticulum..Biochim. Biophys. Acta. 1998; 1404 (review): 67-76Crossref PubMed Scopus (102) Google Scholar). Recent work has turned up another coat component (Lst1p) that helps to package Pma1 ATPase into COPII vesicles (19Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum..J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (130) Google Scholar) (Fig. 2). As suggested by its name (lethal withsec thirteen), the LST1 gene was found in a screen for mutations that block growth when combined with a mutation in one of the known coat protein genes, SEC13. Deletions of LST1 were also lethal in combination with mutations in SEC23, SEC24, or SEC31(19Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum..J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (130) Google Scholar). This kind of genetic behavior, in which a pair of mutations leads to a more severe defect than either one alone, is known as “synthetic lethality” and can frequently be traced to a physical interaction between the corresponding proteins. Indeed, Lst1p shares 23% sequence identity with Sec24p (19Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum..J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (130) Google Scholar), and it has recently been purified from yeast lysates as a Sec23/Lst1p heterodimer (20Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae..J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (116) Google Scholar). At a functional level, there is good evidence that Sec23/Lst1p mediates but is not absolutely required for the recruitment of Pma1 ATPase into COPII vesicles. Thus, in an lst1 deletion mutant, transport of the ATPase out of the ER is inhibited but not completely blocked (19Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum..J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (130) Google Scholar). Furthermore, although Sec23/Lst1p and Sec23/24p can work together to package the ATPase in vitro, Lst1p can be eliminated as long as a 10-fold excess of Sec24p is present (20Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae..J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (116) Google Scholar). Based on these and other results, Shimoni et al. (20Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae..J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (116) Google Scholar) have suggested that Pma1 ATPase may contain a cytoplasmically exposed sorting signal that interacts weakly with Sec24p and more strongly with Lst1p and serves to direct newly synthesized ATPase into ER-derived vesicles. An alternative model will be discussed at the end of the next section. Lipid rafts, consisting of tightly packed sphingolipids and cholesterol, were first observed in mammalian cells, where they form in the Golgi and are believed to play a role in membrane trafficking and cell signaling (reviewed in Ref. 30Brown D.A. London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts..J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar). Recently, Bagnat et al. (31Bagnat M. Keranen S. Shevchenko A. Shevchenko A. Simons K. Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast..Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (502) Google Scholar) have isolated lipid rafts from yeast cells by flotation as detergent-insoluble glycolipid-enriched complexes. The rafts, which appear as early as the ER in yeast, resemble the plasma membrane in having a high content of sphingolipids, ergosterol, and saturated phospholipids; and mass spectrometry has shown them to contain Pma1 ATPase and at least one other protein (Gas1p, a glycophospholipid-anchored protein of unknown function) bound for the cell surface. Conspicuously absent are ER-resident proteins and proteins destined for the sphingolipid- and ergosterol-poor vacuolar membrane. Thus, it seems reasonable to think of lipid rafts as the point at which proteins accumulate for delivery to the plasma membrane (31Bagnat M. Keranen S. Shevchenko A. Shevchenko A. Simons K. Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast..Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (502) Google Scholar). Because Pma1 ATPase and other plasma membrane proteins possess relatively long hydrophobic transmembrane segments (32Munro S. An investigation of the role of transmembrane domains in Golgi protein retention..EMBO J. 1995; 14: 4695-4704Crossref PubMed Scopus (343) Google Scholar, 33Rayner J.C. Pelham H.R. Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast..EMBO J. 1997; 16: 1832-1841Crossref PubMed Scopus (151) Google Scholar), they may sort spontaneously into thick bilayers of the kind found in lipid rafts (34Bretscher M.S. Munro S. Cholesterol and the Golgi apparatus..Science. 1993; 261: 1280-1281Crossref PubMed Scopus (735) Google Scholar). According to this model, there would be no need for a cytoplasmically exposed sorting signal on the ATPase itself; rather, the Sec23/Lst1p heterodimer would somehow tailor the budding vesicle to accommodate the bulky lipid raft (20Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae..J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (116) Google Scholar). Recent studies by Chang and co-workers have pointed to a Golgi-based quality control process, which recognizes abnormal H+-ATPases that have avoided proteasomal degradation and delivers them to the vacuole for proteolysis. One such mutant,pma1–7, carries two amino acid substitutions (P434A in the central cytoplasmic loop and G789S at the extracytoplasmic end of transmembrane segment 8) and displays a temperature-sensitive defect in H+-ATPase biogenesis (14Chang A. Fink G.R. Targeting of the yeast plasma membrane [H+]ATPase: a novel gene AST1 prevents mislocalization of the ATPase to the vacuole..J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar). At 25 °C, Pma1–7p is able to reach the plasma membrane and support growth, whereas at 37 °C, it is degraded rapidly in the vacuole. Two different approaches have been used to isolate suppressors ofpma1–7 with the aim of uncovering novel components of the vacuolar degradation pathway. In the first, screening with a high-copy genomic library yielded a pair of related genes (AST1 andAST2, for ATPase stabilizing) whose products, when overexpressed, cause Pma1–7p to bypass the degradation pathway and travel to the plasma membrane (14Chang A. Fink G.R. Targeting of the yeast plasma membrane [H+]ATPase: a novel gene AST1 prevents mislocalization of the ATPase to the vacuole..J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar). Further examination showed Ast1p to be a peripheral membrane protein, which co-fractionates with detergent-insoluble material that may correspond to the recently identified lipid rafts (see above). In a subsequent study, screening with an insertional library yielded 16 different SOP (suppressors ofp ma1–7) genes, which, when disrupted, could re-route Pma1–7p to the plasma membrane (16Luo W. Chang A. Novel genes involved in endosomal traffic in yeast revealed by suppression of a targeting-defective plasma membrane ATPase mutant..J. Cell Biol. 1997; 138: 731-746Crossref PubMed Scopus (91) Google Scholar). Eight of theSOP suppressors match known VPS(vacuolar protein sorting) genes that control the biogenesis of newly synthesized vacuolar proteins; others are not absolutely required for vacuolar biogenesis but still have noticeable effects on Golgi-to-endosome or endosome-to-vacuole protein trafficking (16Luo W. Chang A. Novel genes involved in endosomal traffic in yeast revealed by suppression of a targeting-defective plasma membrane ATPase mutant..J. Cell Biol. 1997; 138: 731-746Crossref PubMed Scopus (91) Google Scholar, 18Luo W. Chang A. An endosome-to-plasma membrane pathway involved in trafficking of a mutant plasma membrane ATPase in yeast..Mol. Biol. Cell. 2000; 11: 579-592Crossref PubMed Scopus (64) Google Scholar). Based on these results, which point to a central and complex role of the endosomal system in dictating the fate of the mutant Pma1–7 H+-ATPase, the authors have proposed the existence of an alternative endosome-to-surface pathway (16Luo W. Chang A. Novel genes involved in endosomal traffic in yeast revealed by suppression of a targeting-defective plasma membrane ATPase mutant..J. Cell Biol. 1997; 138: 731-746Crossref PubMed Scopus (91) Google Scholar, 18Luo W. Chang A. An endosome-to-plasma membrane pathway involved in trafficking of a mutant plasma membrane ATPase in yeast..Mol. Biol. Cell. 2000; 11: 579-592Crossref PubMed Scopus (64) Google Scholar) (Fig. 2). Like most other cell surface or secreted proteins, wild-type H+-ATPase travels from the Golgi to the cell surface via secretory vesicles, which bud from the Golgi to fuse with the plasma membrane. In fact, yeast contains at least two subpopulations of secretory vesicles, similar in size (100 nm) but separable by equilibrium isodensity sedimentation (35Harsay E. Bretscher A. Parallel secretory pathways to the cell surface in yeast..J. Cell Biol. 1995; 131: 297-310Crossref PubMed Scopus (209) Google Scholar). The ATPase is found in the major vesicle population, together with the cell wall form of endoglucanase (Bgl2p); the minor vesicle population contains periplasmic enzymes such as invertase and acid phosphatase, as well as secreted exoglucanase (Exg1p) (35Harsay E. Bretscher A. Parallel secretory pathways to the cell surface in yeast..J. Cell Biol. 1995; 131: 297-310Crossref PubMed Scopus (209) Google Scholar). As described above, there is no clear evidence that the H+-ATPase is catalytically active in the ER, although it can be protected from trypsinolysis there by ligands such as MgADP and orthovanadate. In the secretory vesicles, however, the ATPase is clearly able to hydrolyze ATP and pump protons at rates comparable with those seen in the plasma membrane; this property allows isolated secretory vesicles to be used as a convenient expression system for site-directed pma1 mutants (36Nakamoto R.K. Rao R. Slayman C.W. Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles: a new strategy for directed mutagenesis..J. Biol. Chem. 1991; 266: 7940-7949Abstract Full Text PDF PubMed Google Scholar). Wild-type Pma1 H+-ATPase turns over with a half-life of 11 h, making it one of the most stable constituents of the yeast plasma membrane (37Benito B. Moreno E. Laguna R. Half-life of the plasma membrane ATPase and its activating system in resting yeast cells..Biochim. Biophys. Acta. 1991; 1063: 265-268Crossref PubMed Scopus (63) Google Scholar). By contrast, the G381A mutant polypeptide has a half-life of only 1 h, leaving the plasma membrane by way of the endocytic pathway to undergo degradation in the vacuole.2 This finding suggests the existence of a third quality control mechanism at the yeast cell surface. The precise mechanism for recognizing misfolded H+-ATPase is unknown, but a protein known variously as End4p/Sla2p/Mop2p has been shown to be required for efficient endocytosis of G381A.2 End4p/Sla2p/Mop2p is a component of the actin cytoskeleton and may help in the formation of endocytic vesicles by stimulating actin depolymerization at the site of vesicle budding (Fig. 2) (reviewed in Ref. 38Wendland B. Emr S.D. Riezman H. Protein traffic in the yeast endocytic and vacuolar sorting pathways..Curr. Opin. Cell Biol. 1998; 10: 513-522Crossref PubMed Scopus (150) Google Scholar). Interestingly, mutations in theEND4/SLA2/MOP2gene have been selected by their ability to lower the abundance of wild-type ATPase at the cell surface (15Na S. Hincapie M. McCusker J.H. Haber J.E. MOP2 (SLA2) affects the abundance of the plasma membrane H+-ATPase of Saccharomyces cerevisiae..J. Biol. Chem. 1995; 270: 6815-6823Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). There is considerable evidence that ubiquitination plays an essential role in the endocytosis of short-lived plasma membrane proteins (reviewed in Refs. 39Hicke L. Ubiquitin-dependent internalization and down-regulation of plasma membrane proteins..FASEB J. 1997; 11: 1215-1226Crossref PubMed Scopus (229) Google Scholar and 40Hicke L. Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels..Trends Cell Biol. 1999; 9: 107-112Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar) but whether it is similarly involved in the endocytosis of mutant or wild-type H+-ATPase remains to be established. Taken together, the research described above has begun to define the path by which a highly abundant plasma membrane protein, the yeast H+-ATPase, travels from the ER to the cell surface. Further work should clarify the relationship between the sorting of newly synthesized ATPase into lipid rafts and the packaging of the ATPase into the appropriate subset of COPII vesicles. The location of these events within the ER will also be of interest, as well as the functional role of the exaggerated vesicular-tubular elements that form in pma1 mutants such as G381A. In parallel, further research is needed to understand the significance of the stepwise phosphorylation events that accompany movement of Pma1 ATPase along the secretory pathway. Finally, Pma1p can serve as a valuable model for understanding quality control during biogenesis because abnormal forms are recognized and removed at three successive points along the pathway. These and other aspects of ATPase biogenesis promise to be active subjects for study in the years to come. For further information on members of the P-type ATPase family, the reader is directed to the accompanying review of Na+,K+- and H+,K+-ATPases by Dunbar and Caplan (41Dunbar L.A. Caplan M.J. Ion pumps in polarized cells: sorting and regulation of the Na+,K+- and H+,K+-ATPases..J. Biol. Chem. 2001; 276: 29617-29620Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). We thank Dr. Michael Caplan for critical reading of this manuscript.