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Ceramide Biosynthesis Is Required for the Formation of the Oligomeric H+-ATPase Pma1p in the Yeast Endoplasmic Reticulum

2002; Elsevier BV; Volume: 277; Issue: 25 Linguagem: Inglês

10.1074/jbc.m200450200

1083-351X

Lee M, Susan Hamamoto, Randy Schekman,

Photosynthetic Processes and Mechanisms

The yeast plasma membrane H+-ATPase Pma1p is one of the most abundant proteins to traverse the secretory pathway. Newly synthesized Pma1p exits the endoplasmic reticulum (ER) via COPII-coated vesicles bound for the Golgi. Unlike most secreted proteins, efficient incorporation of Pma1p into COPII vesicles requires the Sec24p homolog Lst1p, suggesting a unique role for Lst1p in ER export. Vesicles formed with mixed Sec24p-Lst1p coats are larger than those with Sec24p alone. Here, we examined the relationship between Pma1p biosynthesis and the requirement for this novel coat subunit. We show that Pma1p forms a large oligomeric complex of >1 MDa in the ER, which is packaged into COPII vesicles. Furthermore, oligomerization of Pma1p is linked to membrane lipid composition; Pma1p is rendered monomeric in cells depleted of ceramide, suggesting that association with lipid rafts may influence oligomerization. Surprisingly, monomeric Pma1p present in ceramide-deficient membranes can be exported from the ER in COPII vesicles in a reaction that is stimulated by Lst1p. We suggest that Lst1p directly conveys Pma1p into a COPII vesicle and that the larger size of mixed Sec24pLst1p COPII vesicles is not essential to the packaging of large oligomeric complexes. The yeast plasma membrane H+-ATPase Pma1p is one of the most abundant proteins to traverse the secretory pathway. Newly synthesized Pma1p exits the endoplasmic reticulum (ER) via COPII-coated vesicles bound for the Golgi. Unlike most secreted proteins, efficient incorporation of Pma1p into COPII vesicles requires the Sec24p homolog Lst1p, suggesting a unique role for Lst1p in ER export. Vesicles formed with mixed Sec24p-Lst1p coats are larger than those with Sec24p alone. Here, we examined the relationship between Pma1p biosynthesis and the requirement for this novel coat subunit. We show that Pma1p forms a large oligomeric complex of >1 MDa in the ER, which is packaged into COPII vesicles. Furthermore, oligomerization of Pma1p is linked to membrane lipid composition; Pma1p is rendered monomeric in cells depleted of ceramide, suggesting that association with lipid rafts may influence oligomerization. Surprisingly, monomeric Pma1p present in ceramide-deficient membranes can be exported from the ER in COPII vesicles in a reaction that is stimulated by Lst1p. We suggest that Lst1p directly conveys Pma1p into a COPII vesicle and that the larger size of mixed Sec24pLst1p COPII vesicles is not essential to the packaging of large oligomeric complexes. endoplasmic reticulum hemagglutinin blue native 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol detergent-insoluble glycolipid-enriched complex guanyl-5′-yl imidodiphosphate medium speed supernatant phytosphingosine aureobasidin A soluble NSF attachment protein receptor An essential protein of the yeast plasma membrane is the H+-ATPase Pma1p. At steady state, it composes >25% of the total protein at the plasma membrane, where it generates a proton gradient that maintains the intracellular pH and drives the import of nutrients (1Serrano R. Broach J.R. Jones E.W. Pringle J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1991: 523-585Google Scholar). Pma1p spans the lipid bilayer with 10 transmembrane segments and belongs to the family of P2-type ATPases, which includes the Na+,K+-ATPases and Ca2+-ATPases of the mammalian plasma membrane (2Morsomme P. Slayman C.W. Goffeau A. Biochim. Biophys. Acta. 2000; 1469: 133-157Crossref PubMed Scopus (103) Google Scholar,3Ferreira T. Mason A.B. Slayman C.W. J. Biol. Chem. 2001; 276: 29613-29616Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). As with other integral membrane proteins destined for the plasma membrane, Pma1p is translocated into the endoplasmic reticulum (ER)1 and travels through a series of transport vesicles to its final destination (4Holcomb C.L. Hansen W.J. Etcheverry T. Schekman R. J. Cell Biol. 1988; 106: 641-648Crossref PubMed Scopus (47) Google Scholar, 5Chang A. Slayman C.W. J. Cell Biol. 1991; 115: 289-295Crossref PubMed Scopus (151) Google Scholar). En route, Pma1p and the glycosylphosphatidylinositol-anchored protein Gas1p become associated with lipid rafts (6Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (503) Google Scholar, 7Bagnat M. Chang A. Simons K. Mol. Biol. Cell. 2001; 12: 4129-4138Crossref PubMed Scopus (178) Google Scholar). Interestingly, raft association of Gas1p initiates in the ER (6Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (503) Google Scholar), unlike in mammalian cells, where glycosylphosphatidylinositol-anchored proteins enter rafts in post-ER compartments (8Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar, 9Muniz M. Riezman H. EMBO J. 2000; 19: 10-15Crossref PubMed Scopus (139) Google Scholar). Exit out of the ER is mediated by COPII (coat protein complex II) vesicles, a universal mechanism in eukaryotes employing a set of cytoplasmic coat proteins (10Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (819) Google Scholar). Budding is regulated by the small G-protein Sar1p, which recruits two complexes, Sec23p-Sec24p and Sec13p-Sec31p (10Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (819) Google Scholar, 11Matsuoka K. Orci L. Amherdt M. Bednarek S.Y. Hamamoto S. Schekman R. Yeung T. Cell. 1998; 93: 263-275Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 12Antonny B. Madden D. Hamamoto S. Orci L. Schekman R. Nat. Cell Biol. 2001; 3: 531-537Crossref PubMed Scopus (232) Google Scholar). Assembly of these three components on the surface of liposomes is sufficient to deform the lipid bilayer to generate small coated vesicles (11Matsuoka K. Orci L. Amherdt M. Bednarek S.Y. Hamamoto S. Schekman R. Yeung T. Cell. 1998; 93: 263-275Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). On the ER membrane, Sar1p and Sec23p-Sec24p promote the capture of a number of cargo proteins, suggesting that Sec23p-Sec24p may function to selectively engage cargo proteins during coat assembly (13Kuehn M.J. Herrmann J.M. Schekman R. Nature. 1998; 391: 187-190Crossref PubMed Scopus (328) Google Scholar, 14Aridor M. Weissman J. Bannykh S. Nuoffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (247) Google Scholar). Homologs of Sec24p have been identified in yeast and mammals (15Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (131) Google Scholar, 16Pagano A. Letourneur F. Garcia-Estefania D. Carpentier J.L. Orci L. Paccaud J.P. J. Biol. Chem. 1999; 274: 7833-7840Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and may act to diversify the range of cargo proteins recruited into a nascent vesicle. In yeast, the Sec24p homolog Lst1p was discovered in a screen for synthetic interactions with the sec13-1 allele (lethal with s ec-thirteen) (15Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (131) Google Scholar). A null mutant of lst1 is viable, although sensitive to low pH resulting from a reduced flux of Pma1p out of the ER (15Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (131) Google Scholar). Efficient incorporation of Pma1p into COPII vesicles requires a combination of both Sec24p and Lst1p complexes, unlike other cargo proteins studied to date (16Pagano A. Letourneur F. Garcia-Estefania D. Carpentier J.L. Orci L. Paccaud J.P. J. Biol. Chem. 1999; 274: 7833-7840Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In addition to the enhancement of Pma1p packaging, Lst1p-positive vesicles are ∼15% larger than vesicles generated with standard COPII subunits (17Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (117) Google Scholar). In this study, we investigated the role of Pma1p oligomerization in Lst1p-dependent Pma1p transport. Both oligomeric and monomeric Pma1p are packaged into COPII vesicles, and both forms of Pma1p require Lst1p for efficient transport from the ER. Reagents were purchased from Sigma unless otherwise noted. Aureobasidin A was purchased from Takara Bio Inc. (Shiga, Japan). Rich medium (yeast extract/peptone/dextrose) and minimal medium containing either 2% glucose (synthetic dextrose medium) or 2% raffinose were prepared as described (17Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (117) Google Scholar). The yeast strains used in this study were as follows: YPH499 (MAT a ade2-101oc his3-Δ200 leu2-Δ1 lys2-801am trp1-63 ura3-52), RSY1578 (YPH499pma1::HA-PMA1::LEU2), MLY1 (MATα lcb1-100 leu2 ura3 his4 pma1::HA-PMA1::LEU2) (HR2607, H. Riezman, University of Basel, Basel, Switzerland), RSY372 (MATα ura3-52 leu2-3,112 sec18-1), MLY18 (RSY372pma1::HA-PMA1::LEU2), MLY4 (MATα ade2-1 his3-11,15 leu2-3,112 trp1-Δ1 ura3-1 pep4::TRP1 pma1::HA-PMA1::LEU2), and RSY1801 (RSY1578 lst1::HIS3). Galactose induction of hemagglutinin (HA)-tagged Pma1p carried out with pFP302 (18Portillo F. FEBS Lett. 1997; 402: 136-140Crossref PubMed Scopus (28) Google Scholar) in either YPH499 or RSY372. Microsomal membranes were prepared as described (19Wuestehube L.J. Schekman R.W. Methods Enzymol. 1992; 219: 124-136Crossref PubMed Scopus (80) Google Scholar) unless otherwise noted. Both35S-labeled and unlabeled cells were converted to semi-intact spheroplasts as described (17Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (117) Google Scholar). Purification of Sec23p-Lst1p (17Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (117) Google Scholar) and Sar1p, Sec23p-Sec24p, and Sec13p-Sec31p (20Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1058) Google Scholar) is described elsewhere. Membranes were solubilized with detergents at 4 °C (except SDS at room temperature) for 30 min in 50 mm Tris-HCl, 150 mm NaCl, and 1 mmEDTA, pH 7.5, and then centrifuged for 5 min at 15,000 ×g. BN-PAGE sample buffer (0.1 volume of 5% Coomassie Brilliant Blue G-250, 50 mm BisTris, 750 mm 6-aminocaproic acid, and 50% glycerol, pH 7.0) was added to the supernatant, which was separated on 4–10% polyacrylamide gradient gels as described (21Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1918) Google Scholar) with high molecular mass markers fromAmersham Biosciences. Cells grown overnight at 24 °C in yeast extract/peptone/dextrose medium were harvested, washed twice, and resuspended in synthetic dextrose medium at ∼1 A 600/ml. After 15 min at 30 °C, cells were pulsed with 60 μCi of35S-Promix/A 600 (AmershamBiosciences) for 3 min. Chase was initiated by addition of 50 mm methionine, 10 mm cysteine, 4% yeast extract, and 2% dextrose. At each time point, cells (1A 600 unit) were removed to ice and arrested with 20 mm NaN3/KF. Cells converted to spheroplasts were lysed with 1% SDS at 55 °C for 5 min, and lysates were diluted with 1 ml of immunoprecipitation buffer (15 mmTris-HCl, 1% Triton X-100, 150 mm NaCl, and 0.05% SDS, pH 7.5) and centrifuged for 5 min at 15,000 × g, and the supernatant was incubated with anti-HA antibodies (HA.11, Berkeley Antibody Co., Berkeley, CA) and protein G-Sepharose (AmershamBiosciences). Immunoprecipitates were analyzed by SDS-PAGE and with a PhosphorImager using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). Microsomes and spheroplasts were centrifuged at 15,000 × g for 5 min, and vesicles were centrifuged at 100,000 × g for 20 min and resuspended in buffer A (1% Triton X-100, 50 mm Tris-HCl, 150 mm NaCl, 5 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride, pH 7.4) at 4 °C for 30 min. Extracts were centrifuged at 1000 × g for 5 min, and the supernatant (330 μl) was adjusted to 35% Optiprep (Nycomed, Oslo, Norway) with 770 μl of 50% Optiprep in buffer A, overlaid with 1.4 ml of 30% Optiprep in buffer A and 0.5 ml of buffer A, and centrifuged at 4 °C for 16 h at 40,000 rpm in a Beckman SW 55 rotor. Fractions (200 μl) were analyzed by either immunoblotting or immunoprecipitation. The indicated fractions (400 μl) were immunoprecipitated by addition of SDS to 1% at 55 °C for 5 min and dilution with 4 ml of immunoprecipitation buffer; immunoprecipitations were performed sequentially using anti-HA, anti-Gas1p, and anti-Sec22p antisera. Vesicle budding from microsomal membranes and semi-intact cells was performed as described (17Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (117) Google Scholar, 20Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1058) Google Scholar). Membranes were first washed with buffer B (20 mm Hepes, 250 mm sorbitol, 150 mm KOAc, and 5 mmMg(OAc)2, pH 6.8); and unless otherwise noted, budding reactions contained 20 μg/ml Sar1p and Sec13p-Sec31p and either 20 μg/ml Sec23p-Sec24p or 10 μg/ml each Sec23p-Sec24p and Sec23p-Lst1p as well as the non-hydrolyzable GTP analog GMP-PMP (Roche Molecular Biochemicals) and an ATP regeneration system. Budding reactions for BN-PAGE used 100 μg of microsomes (125-μl reaction), and those for Fig. 3 B used 400 μg of microsomes (500-μl reaction). Budding from labeled semi-intact cells used cells atA 600 = 1.25 (125-μl reaction). For electron microscopy, vesicles were generated from 1.5 mg of microsomes washed to remove endogenous COPII proteins (2× buffer B + 2.5 murea, 2× buffer B + 1 mm GTP, and 3× buffer B). All reactions were performed for 30 min at 25 °C. The vesicle-enriched medium speed supernatant (MSS) was centrifuged at 100,000 × g for 20 min. The vesicle pellet was processed as described (12Antonny B. Madden D. Hamamoto S. Orci L. Schekman R. Nat. Cell Biol. 2001; 3: 531-537Crossref PubMed Scopus (232) Google Scholar). Vesicle diameter was measured from scanned negatives (×30,000) using the Photoshop measuring tool. A sample of 500 vesicles was measured for each condition. We used BN-PAGE to examine the oligomeric state of Pma1p in microsomal membranes. Membranes generated from a strain expressing a chromosomal HA-tagged form of Pma1p were solubilized with different detergents, and the proteins were separated by BN-PAGE (Fig.1 A). Pma1p migrated as a distinct high molecular mass complex of ∼1.8 MDa in extracts solubilized with Triton X-100, digitonin, or n-dodecyl maltoside. Pma1p solubilized with SDS migrated with an apparent molecular mass of 160 kDa, most likely corresponding to monomeric protein, which has a predicted molecular mass of ∼100 kDa. Interestingly, two bands of intermediate size were observed inn-dodecyl maltoside extracts; the apparent sizes of these intermediates corresponded closely to those of predicted trimeric and hexameric complexes of Pma1p (Fig. 1 B) and may represent partial dissociation of a more abundant dodecameric species. Although the size of the 1.8-MDa species is suggestive of a homo-oligomeric complex of 12 Pma1p molecules (possibly arranged as four trimers), we cannot yet rule out the presence of additional proteins in the complex. To determine whether the Pma1p multimer originates in the ER, we examined newly synthesized Pma1p in the sec18-1 strain, which is rapidly blocked in ER-to-Golgi transport at 37 °C. Cells were shifted to 37 °C for 5 min, and expression of epitope-tagged Pma1p was induced with galactose for 30 min. Oligomerization of the induced Pma1p was observed in both wild-type and sec18-1extracts (Fig. 1 C), whereas no protein was detected in the absence of galactose (data not shown). Although Pma1p in wild-type cells was found predominantly as the oligomeric species, imposition of a sec18 block resulted in approximately equal amounts of oligomeric and monomeric Pma1p. Thus, Pma1p can oligomerize in the ER, but oligomerization is somewhat impeded by a block in ER-to-Golgi transport. The observation that Pma1p associates with DIGs or lipid rafts (6Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (503) Google Scholar) prompted us to investigate whether oligomerization is correlated with sphingolipid biosynthesis. To deplete cell membranes of sphingolipids, we used the lcb1-100 strain, which has a temperature-sensitive mutation in a subunit of serine palmitoyltransferase and is unable to synthesize sphingoid base, ceramide, and sphingolipids (22Buede R. Rinker-Schaffer C. Pinto W.J. Lester R.L. Dickson R.C. J. Bacteriol. 1991; 173: 4325-4332Crossref PubMed Scopus (157) Google Scholar). Bagnat et al. (6Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (503) Google Scholar) found that Pma1p isolated from lcb1-100 cells grown at restrictive temperature is no longer raft-associated. We examined the oligomeric state of Pma1p in wild-type and lcb1-100 cells grown either at 24 °C or after a 2-h shift to 37 °C. Unlike wild-type membranes, Pma1p isolated from lcb1-100 cells migrated almost exclusively as the monomeric form, regardless of the temperature at which the cells were grown (Fig.2 A). This is consistent with the observation that sphingolipid levels in lcb1-100 cells are considerably lower than those in wild-type cells even at 24 °C (23Zanolari B. Friant S. Funato K. Sutterlin C. Stevenson B.J. Riezman H. EMBO J. 2000; 19: 2824-2833Crossref PubMed Scopus (210) Google Scholar). We investigated whether addition of exogenous sphingoid base in the form of phytosphingosine (PHS) could restore the levels of sphingolipid sufficiently to allow oligomerization of Pma1p. Both wild-type and lcb1-100 cells grown at 24 °C were supplemented with various levels of PHS for 2 h and analyzed by BN-PAGE (Fig. 2 B). Addition of 1–10 μm PHS tolcb1-100 cells promoted increased oligomerization of Pma1p. Interestingly, the proportion of oligomeric Pma1p in wild-type cells was also increased by addition of low levels of PHS, suggesting that oligomerization is intimately linked with sphingolipid levels in the cell. Depletion of sphingolipid levels can also be achieved by addition of drugs such as myriocin, which inhibits serine palmitoyltransferase activity (24Horvath A. Sutterlin C. Manning-Krieg U. Movva N.R. Riezman H. EMBO J. 1994; 13: 3687-3695Crossref PubMed Scopus (184) Google Scholar), and aureobasidin A (AbA), which blocks synthesis of the sphingolipid inositol phosphorylceramide from ceramide (25Nagiec M.M. Nagiec E.E. Baltisberger J.A. Wells G.B. Lester R.L. Dickson R.C. J. Biol. Chem. 1997; 272: 9809-9817Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). To determine whether myriocin influences Pma1p oligomerization, we treated cells with up to 40 μg/ml myriocin for 2 h. Under these conditions, we were unable to observe a significant shift of the steady-state levels of oligomeric Pma1p to monomer (data not shown), suggesting either that depletion of sphingolipid was not sufficient during the 2-h treatment or that, once formed, Pma1p oligomers are resistant to a decrease in sphingolipid levels. To analyze the effects of myriocin and AbA on newly synthesized Pma1p, we used the galactose-inducible HA-tagged Pma1p plasmid described above. Wild-type cells expressing endogenous untagged Pma1p were grown in non-inducing medium at 24 °C and treated with myriocin or AbA for 15 min before induction of HA-tagged Pma1p. Unlike control cells, myriocin-treated cells showed a defect in the ability of newly synthesized Pma1p to oligomerize (Fig. 2 C). However, oligomerization was not prevented in cells treated with AbA (Fig. 2 C), suggesting that either ceramide or sphingoid base (but not sphingolipids per se) is required for Pma1p oligomerization. Under these conditions, AbA prevented the incorporation of 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))ceramide into inositol phosphorylceramide as determined by TLC (26Levine T.P. Wiggins C.A. Munro S. Mol. Biol. Cell. 2000; 11: 2267-2281Crossref PubMed Scopus (134) Google Scholar). 2P. Malkus and R. Schekman, personal communication. We assessed the effect of ceramide depletion on the stability of Pma1p by a 35S pulse-chase analysis. In wild-type cells, Pma1p remained stable throughout the chase period (Fig. 2 D), consistent with its long half-life (5Chang A. Slayman C.W. J. Cell Biol. 1991; 115: 289-295Crossref PubMed Scopus (151) Google Scholar). In both lcb1-100cells and myriocin-treated wild-type cells, however, Pma1p was markedly destabilized. Myriocin failed to induce degradation of Pma1p inpep4Δ cells, suggesting that monomeric Pma1p is permitted to exit the ER, but is subsequently rerouted to the vacuole. A block in ceramide synthesis by myriocin treatment or by growth oflcb1-100 cells at nonpermissive temperature prevents the ER-to-Golgi transport of the glycosylphosphatidylinositol-anchored protein Gas1p (24Horvath A. Sutterlin C. Manning-Krieg U. Movva N.R. Riezman H. EMBO J. 1994; 13: 3687-3695Crossref PubMed Scopus (184) Google Scholar, 27Sutterlin C. Doering T.L. Schimmoller F. Schroder S. Riezman H. J. Cell Sci. 1997; 110: 2703-2714Crossref PubMed Google Scholar), which associates with DIGs before leaving the ER (6Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (503) Google Scholar). To determine whether oligomerization of Pma1p in the ER coincides with its entry into Triton X-100-insoluble DIGs, we isolated DIGs from pulse-labeled sec18-1 cells. At permissive temperature, the proportion of Pma1p associated with DIGs increased markedly after prolonged chase (Fig.3 A). Gas1p displayed a similar pattern, with a proportion of both the ER form (2 min) and the Golgi-modified form (15 min) in the detergent-insoluble fraction (Fig.3 A). At 37 °C, the pool of DIG-associated Gas1p increased after extended chase, whereas Pma1p was no longer observed in DIGs (Fig. 3 A). This suggests that, unlike Gas1p, Pma1p does not associate with lipid rafts in the ER or, alternatively, that a block in ER export diminishes the stability of the association of Pma1p with lipid rafts. To distinguish between these possibilities, we analyzed the detergent solubility of Pma1p in ER-derived COPII vesicles. Microsomal membranes were used in a vesicle budding reaction using purified COPII proteins (Sar1p, Sec23p-Sec24p, and Sec13p-Sec31p) together with the Sec24p homolog Lst1p. The vesicle-enriched MSS was subjected to centrifugation at 100,000 × g to pellet vesicles, from which DIGs were isolated by flotation as described above. Vesicles generated in the presence of nucleotide showed efficient budding of Pma1p, whereas the ER resident Sec61p was not packaged into vesicles (Fig.3 B). A significant proportion of Pma1p in COPII vesicles was in a detergent-insoluble pool (fractions 5 and 6) (Fig. 3 B). In contrast, the SNARE proteins Sec22p, Bet1p, and Bos1p were efficiently incorporated into COPII vesicles, but were not DIG-associated (Fig. 3 B and data not shown). To confirm that the DIG-associated Pma1p observed in the in vitro budding reaction corresponded to newly synthesized protein from the ER, we performed a budding reaction using semi-intact cells generated after a brief period of radiolabeling. Vesicle DIGs were isolated by flotation, and Pma1p was immunoprecipitated from fractions representative of detergent-insoluble and detergent-soluble material. A significant fraction (11%) of Pma1p in COPII vesicles was DIG-associated (Fig. 3 C). Thus, Pma1p association with DIGs either is stabilized by entry into COPII vesicles or occurs initially in the ER, but is disrupted by the imposition of a secretion block. The dependence of Pma1p oligomerization on ceramide biosynthesis, together with the observation that Pma1p is associated with DIGs in COPII vesicles, prompted us to investigate the effect of ceramide depletion on the oligomeric state of Pma1p packaged into vesicles. COPII vesicles were generated from wild-type microsomes or microsomes derived fromlcb1-100 cells and analyzed by BN-PAGE. In vesicles generated from wild-type microsomes, Pma1p was packaged as the large oligomeric species (Fig. 4 A). Interestingly, COPII vesicles generated from lcb1-100microsomes packaged Pma1p, but the protein remained in the monomeric form (Fig. 4 A). We investigated whether oligomerization might enhance the uptake of Pma1p into vesicles, possibly by allowing more efficient recruitment of vesicle coat components to the ER membrane. We performed budding reactions with 35S-labeled cells grown in the presence of myriocin for 2 h, a treatment that renders Pma1p monomeric (see Fig. 2 C). lst1-null cells were used to allow an accurate titration of Pma1p budding by addition of increasing amounts of purified Lst1p and Sec24p complexes in the presence of constant levels of Sar1p and Sec13p-Sec31p. In both myriocin-treated and control membranes, Pma1p budding was stimulated by increasing concentrations of Sec24p and Lst1p complexes (Fig. 4 B). No significant differences in budding efficiency were observed between wild-type and myriocin-treated membranes, regardless of the concentration of COPII components provided. We next investigated more directly the relationship between the oligomeric state of Pma1p and the requirement for Lst1p in ER export. Shimoni et al. (17Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (117) Google Scholar) proposed two possible mechanisms for the Lst1p-mediated stimulation of Pma1p budding. Lst1p may act as a "cargo-adapter," binding directly to Pma1p to efficiently recruit it into a nascent vesicle. Alternatively, Lst1p may form larger vesicles with a lower membrane curvature and greater surface area, thus indirectly facilitating the packaging of oligomeric Pma1p. If so, the ability of high levels of Sec24p to overcome the requirement for Lst1p in Pma1p packaging (17Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (117) Google Scholar) may reflect an increase in the incorporation of monomeric Pma1p into nascent vesicles. To address this issue, we compared the oligomeric state of Pma1p in vesicles that had been generated with either a combination of Sec24p and Lst1p complexes or with high levels of the Sec24p complex alone (Fig. 4 C). Analysis of vesicles by BN-PAGE revealed that the presence of Lst1p was not essential for packaging oligomeric Pma1p, which could be accommodated in Lst1p-negative vesicles given a sufficient quantity of coat proteins (Fig. 4 C). We next considered whether Pma1p rendered monomeric by ceramide depletion may bypass the requirement for Lst1p and be incorporated into the smaller vesicles generated with the standard COPII proteins. Membranes were isolated from lst1Δ cells treated with myriocin and incubated with low-to-intermediate concentrations of Sec24p and Lst1p complexes. Under these conditions, Pma1p was not significantly packaged when only the Sec24p complex was added (Fig.4 D); however, addition of equivalent amounts of both Sec24p and Lst1p complexes stimulated budding ∼3-fold in both myriocin-treated and control membranes. We conclude that the oligomeric state of Pma1p does not enhance budding and that both the oligomeric complex and the monomeric protein require Lst1p for efficient incorporation into COPII vesicles. The requirement for Lst1p to package both oligomeric and monomeric Pma1p suggests a function as a cargo-specific adapter, rather than an indirect role in accommodating large complexes. We considered the possibility that the larger size of COPII vesicles produced with the Lst1p complex was influenced by the incorporation of large DIG-associated Pma1p oligomers. Pma1p is a particularly abundant protein; thus, it is conceivable that its presence alone in Lst1p-positive vesicles may influence vesicle size. Large-scale budding reactions were performed using either wild-type or lcb1-100 microsomes prepared from cells grown at 24 °C and shifted to 37 °C for 1 h. Under these conditions, Pma1p appeared monomeric in lcb1-100 membranes (see Fig. 2 A). Microsomal membranes were washed to remove endogenous COPII components and then supplied with Sar1p, Sec13p-Sec31p, and either the Sec24p complex alone or a mixture of both Sec24p and Lst1p complexes, in addition to the non-hydrolyzable GTP analog GMP-PMP to preserve vesicle coats. The high speed vesicle pellet was analyzed by electron microscopy. Vesicles with a discernible coat were generated under all conditions, and no obvious differences in coat morphology were detected (data not shown). Lst1p-positive vesicles produced from wild-type andlcb1-100 membranes were of comparable size and were ∼10–15% larger in diameter than Lst1p-negative vesicles (TableI). Thus, incorporation of large Pma1p complexes into Lst1p-positive COPII vesicles does not explicitly determine the size of the vesicle produced.Table ILst1p generates larger COPII vesicles independently of Pma1p oligomerizationMembranes−Lst1p+Lst1p1-an = 500;p < 0.001. nm nmWild-type75 ± 1384 ± 14 lcb1–10077 ± 1485 ± 161-a n = 500;p < 0.001. Open table in a new tab Pma1p is a particularly abundant secretory protein that uniquely requires Lst1p for packaging into COPII vesicles for ER export. We have investigated the nature of this requirement with respect to the quaternary structure of Pma1p. We found that, upon BN-PAGE, Pma1p migrated as an oligomeric complex. Our estimate that this complex represents a homododecamer of Pma1p is consistent with observations in related yeast suggesting that H+-ATPases form higher order structures. Formation of a multimer of 8–10 monomers was reported for the Schizosaccharomyces pombe H+-ATPase (28Dufour J.P. Goffeau A. Eur. J. Biochem. 1980; 105: 145-154Crossref PubMed Scopus (58) Google Scholar), whereas the Neurospora crassa H+-ATPase was observed as a hexamer upon gradient centrifugation and crystallization (29Chadwick C.C. Goormaghtigh E. Scarborough G.A. Arch. Biochem. Biophys. 1987; 252: 348-356Crossref PubMed Scopus (48) Google Scholar, 30Auer M. Scarborough G.A. Kuhlbrandt W. Nature. 1998; 392: 840-843Crossref PubMed Scopus (185) Google Scholar). Oligomerization does not appear to be essential for activity, however, as monomeric protein isolated from N. crassa was reconstituted into proteoliposomes as fully active enzyme (31Goormaghtigh E. Chadwick C. Scarborough G.A. J. Biol. Chem. 1986; 261: 7466-7471Abstract Full Text PDF PubMed Google Scholar). Recently, Bagnat et al. (7Bagnat M. Chang A. Simons K. Mol. Biol. Cell. 2001; 12: 4129-4138Crossref PubMed Scopus (178) Google Scholar) reported that a fraction ofSaccharomyces cerevisiae Pma1p behaves as an oligomer of ∼400 kDa on SDS/Triton X-100 velocity gradients. In contrast, we observed by BN-PAGE that the majority of Pma1p migrated as an ∼1.8-MDa complex under a variety of detergent conditions. However, a small proportion of Pma1p migrated at ∼490 kDa when membranes were solubilized with SDS (Fig. 1 A), which may correspond to the complex observed by Bagnat et al. (7Bagnat M. Chang A. Simons K. Mol. Biol. Cell. 2001; 12: 4129-4138Crossref PubMed Scopus (178) Google Scholar). Interestingly, overexpression of a peripheral membrane protein (Ast1p) was found to substantially increase the proportion of the ∼400-kDa Pma1p complex, in addition to conferring increased raft association on a mutant Pma1p protein (7Bagnat M. Chang A. Simons K. Mol. Biol. Cell. 2001; 12: 4129-4138Crossref PubMed Scopus (178) Google Scholar). Upon BN-PAGE, Ast1p did not co-migrate with the 1.8-MDa Pma1p complex and thus does not contribute to the increased size of the Pma1p oligomer we observed (data not shown). The association of Pma1p with lipid rafts and with Ast1p was proposed by Bagnat et al. (7Bagnat M. Chang A. Simons K. Mol. Biol. Cell. 2001; 12: 4129-4138Crossref PubMed Scopus (178) Google Scholar) to occur in the Golgi; however, our observation that raft-associated Pma1p oligomers can be isolated from COPII vesicles suggests that entry into rafts and oligomerization begin before arrival at the Golgi. The formation of an oligomeric Pma1p complex in the ER is consistent with the observation that expression of a number of dominant-negative mutants of Pma1p results in the retention of both the mutant and wild-type proteins in the ER (18Portillo F. FEBS Lett. 1997; 402: 136-140Crossref PubMed Scopus (28) Google Scholar, 32Harris S.L., Na, S. Zhu X. Seto-Young D. Perlin D.S. Teem J.H. Haber J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10531-10535Crossref PubMed Scopus (62) Google Scholar). Formation of mixed oligomers between the normal and mutant proteins could lead to the recognition of the entire complex as misfolded and subject to ER quality control. This model is supported by our observations that assembly of the multimeric Pma1p complex is dependent on the level of ceramide biosynthesis and that a block in this pathway relieves the dominant lethal phenotype of a Pma1p mutant. 3Q. Wang and A. Chang, personal communication. The association of Pma1p with DIGs suggests one possible mechanism for oligomerization whereby newly synthesized Pma1p preferentially partitions into lipid microdomains, creating a local concentration of protein sufficient to drive oligomerization. A decrease in the flux of ceramide through the ER may result in the fragmentation of these microdomains such that oligomerization is prevented. Alternatively, oligomerization may proceed concurrently but independently of the formation of nascent lipid rafts, requiring only a product of the ceramide biosynthesis pathway. We are currently examining the oligomeric state of Pma1p in membranes depleted of ergosterol. In contrast with the transport of Gas1p, which fails to reach the Golgi if ceramide biosynthesis is interrupted (24Horvath A. Sutterlin C. Manning-Krieg U. Movva N.R. Riezman H. EMBO J. 1994; 13: 3687-3695Crossref PubMed Scopus (184) Google Scholar, 27Sutterlin C. Doering T.L. Schimmoller F. Schroder S. Riezman H. J. Cell Sci. 1997; 110: 2703-2714Crossref PubMed Google Scholar), ER export of Pma1p is not dependent on the presence of ceramide or on its oligomeric state. However, the stability of Pma1p is dramatically altered by a disruption in the ceramide biosynthesis pathway, resulting in a rerouting to the vacuole for degradation. A consequence of the formation of large oligomeric cargo such as Pma1p may be the requirement for a more spacious transport vehicle (33Antonny B. Schekman R. Curr. Opin. Cell Biol. 2001; 13: 438-443Crossref PubMed Scopus (162) Google Scholar). The observation that oligomeric Pma1p is packaged into COPII vesicles has allowed us to address the unique requirement for Lst1p in this process. Our finding that efficient export of Pma1p requires Lst1p regardless of cargo size suggests, however, that Lst1p functions to actively promote Pma1p packaging, possibly by direct recruitment of Pma1p during polymerization of the nascent vesicle coat. Once in COPII vesicles, Pma1p can be co-immunoprecipitated with both the Lst1p and Sec24p complexes (17Shimoni Y. Kurihara T. Ravazzola M. Amherdt M. Orci L. Schekman R. J. Cell Biol. 2000; 151: 973-984Crossref PubMed Scopus (117) Google Scholar); however, to confirm a direct interaction, it will be necessary to reconstitute these associations in purified proteoliposomes. Such a system may help to explain the contribution of unique coat components such as Lst1p to vesicle formation. Our observation that Lst1p generates larger vesicles from membranes depleted of DIGs and oligomeric Pma1p and potentially other sphingolipid-dependent protein complexes suggests that the size of COPII vesicles is not driven by the nature of their cargo, but by the intrinsic properties of their coats. We thank members of the Schekman laboratory, in particular E. Miller and F. Portillo, for generous help. Special thanks to C. Chan and R. Lesch for preparation of reagents. We are grateful to A. Chang and P. Malkus for communicating unpublished results and H. Riezman for providing yeast strains.