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• W2116122989 abstract "The proton-pumping H+-ATPase, Pma1p, is an abundant and very long lived polytopic protein of the yeast plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as a model to study plasma membrane biogenesis. Pma1p associates with detergent-resistant membrane domains (lipid “rafts”) already in the ER, and a lack of raft association correlates with mistargeting of the protein to the vacuole, where it is degraded. We are analyzing the role of specific lipids in membrane domain formation and have previously shown that surface transport of Pma1p is independent of newly synthesized sterols but that sphingolipids with C26 very long chain fatty acid are crucial for raft association and surface transport of Pma1p (Gaigg, B., Timischl, B., Corbino, L., and Schneiter, R. (2005) J. Biol. Chem. 280, 22515-22522). We now describe a more detailed analysis of the function that sphingolipids play in this process. Using a yeast strain in which the essential function of sphingolipids is substituted by glycerophospholipids containing C26 very long chain fatty acids, we find that sphingolipids per se are dispensable for raft association and surface delivery of Pma1p but that the C26 fatty acid is crucial. We thus conclude that the essential function of sphingolipids for membrane domain formation and stable surface delivery of Pma1p is provided by the C26 fatty acid that forms part of the yeast ceramide. The proton-pumping H+-ATPase, Pma1p, is an abundant and very long lived polytopic protein of the yeast plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as a model to study plasma membrane biogenesis. Pma1p associates with detergent-resistant membrane domains (lipid “rafts”) already in the ER, and a lack of raft association correlates with mistargeting of the protein to the vacuole, where it is degraded. We are analyzing the role of specific lipids in membrane domain formation and have previously shown that surface transport of Pma1p is independent of newly synthesized sterols but that sphingolipids with C26 very long chain fatty acid are crucial for raft association and surface transport of Pma1p (Gaigg, B., Timischl, B., Corbino, L., and Schneiter, R. (2005) J. Biol. Chem. 280, 22515-22522). We now describe a more detailed analysis of the function that sphingolipids play in this process. Using a yeast strain in which the essential function of sphingolipids is substituted by glycerophospholipids containing C26 very long chain fatty acids, we find that sphingolipids per se are dispensable for raft association and surface delivery of Pma1p but that the C26 fatty acid is crucial. We thus conclude that the essential function of sphingolipids for membrane domain formation and stable surface delivery of Pma1p is provided by the C26 fatty acid that forms part of the yeast ceramide. Integral membrane proteins enter the membrane environment in the ER 2The abbreviations used are: ER, endoplasmic reticulum; PHS, phytosphingosine; GFP, green fluorescent protein. and are then transported along a vesicular pathway to their final subcellular destination (1Behnia R. Munro S. Nature. 2005; 438: 597-604Crossref PubMed Scopus (381) Google Scholar). Although much attention has been paid to uncovering the role of protein-encoded signals in determining sorting of the membrane-bound cargo to its final destination, comparatively little is known about how the lipid-protein interface itself affects protein sorting. The integrity of this interface is probably surveilled by components of the protein quality control system to ensure that only functional proteins are delivered to the cell surface when needed. Co-delivery of integral membrane proteins together with their surrounding membrane domain to the cell surface may, in principle, ensure balanced expansion of the plasma membrane itself. The proton-pumping H+-ATPase, Pma1p, is biosynthetically inserted into the ER membrane, where it homo-oligomerizes to form a 1.8-MDa complex that resists detergent extraction (2Lee M.C. Hamamoto S. Schekman R. J. Biol. Chem. 2002; 277: 22395-22401Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). This complex is then packaged into a larger subclass of COPII transport vesicles that contain Lst1p in addition to Sec24p (3Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (130) Google Scholar) and is transported to the cell surface by a branch of the secretory pathway that does not intersect with endosomes (4Harsay E. Schekman R. J. Cell Biol. 2002; 156: 271-285Crossref PubMed Scopus (120) Google Scholar, 5Gurunathan S. David D. Gerst J.E. EMBO J. 2002; 21: 602-614Crossref PubMed Scopus (87) Google Scholar). Once at surface, Pma1p becomes stabilized and occupies domains that are distinct from those occupied by the arginine/H+ symporter Can1p (6Malinska K. Malinsky J. Opekarova M. Tanner W. Mol. Biol. Cell. 2003; 14: 4427-4436Crossref PubMed Scopus (224) Google Scholar). A relationship between Pma1p biogenesis and lipid synthesis is indicated by the observations that long-chain base or ceramide synthesis is required for oligomerization and raft association of Pma1p in the ER (2Lee M.C. Hamamoto S. Schekman R. J. Biol. Chem. 2002; 277: 22395-22401Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 7Wang Q. Chang A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12853-12858Crossref PubMed Scopus (65) Google Scholar). Oligomerization of Pma1p, however, is not required for ER export or surface delivery but might be important for stabilization of the protein at the cell surface (2Lee M.C. Hamamoto S. Schekman R. J. Biol. Chem. 2002; 277: 22395-22401Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 7Wang Q. Chang A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12853-12858Crossref PubMed Scopus (65) Google Scholar). Raft association of Pma1p, on the other hand, is required for surface delivery and subsequent stabilization of the protein (7Wang Q. Chang A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12853-12858Crossref PubMed Scopus (65) Google Scholar, 8Bagnat M. Chang A. Simons K. Mol. Biol. Cell. 2001; 12: 4129-4138Crossref PubMed Scopus (178) Google Scholar, 9Gong X. Chang A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9104-9109Crossref PubMed Scopus (61) Google Scholar). We previously observed that newly synthesized Pma1p is mistargeted to the vacuole in an elo3Δ mutant that affects acyl chain elongation and hence the synthesis of the ceramide-bound C26 very long-chain fatty acid (10Eisenkolb M. Zenzmaier C. Leitner E. Schneiter R. Mol. Biol. Cell. 2002; 13: 4414-4428Crossref PubMed Scopus (97) Google Scholar). Further characterization of the role of lipids in Pma1p biogenesis revealed that neither sterols nor the head group modifications on the sphingolipids are important for raft association and surface transport of Pma1p (11Gaigg B. Timischl B. Corbino L. Schneiter R. J. Biol. Chem. 2005; 280: 22515-22522Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Instead, the results suggested that ceramide levels and/or their substitution with saturated very long-chain fatty acids are crucial for the biogenesis of Pma1p. These results also indicated that the lipid requirement of Pma1p to form membrane microdomains is distinct from the classical sterol and sphingolipid-rich domains that are normally referred to as detergent-resistant membranes/lipid rafts (12Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2551) Google Scholar, 13Munro S. Cell. 2003; 115: 377-388Abstract Full Text Full Text PDF PubMed Scopus (1329) Google Scholar, 14Simons K. Vaz W.L. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 269-295Crossref PubMed Scopus (1361) Google Scholar). The aim of this study is to discriminate between the role of ceramide and that of the very long-chain fatty acid in Pma1p sorting. Such discrimination is made possible by the use of a mutant that bypasses the essential requirement for sphingolipids by producing unusual inositol-containing glycerophospholipids carrying a C26 fatty acid in the sn-2 position (15Lester R.L. Wells G.B. Oxford G. Dickson R.C. J. Biol. Chem. 1993; 268: 845-856Abstract Full Text PDF PubMed Google Scholar). Surprisingly, Pma1p is raft-associated and stably transported to the cell surface if sphingolipids are replaced by C26-containing glycerophospholipids. Reduction of the length of the acyl chain on the suppressor lipids, however, impairs the essential function of these lipids and results in conditional degradation of Pma1p, strongly indicating that lipids with a C26 fatty acid either bound to a glycerophospholipid or to ceramide are crucial for the vital function of sphingolipids and for surface delivery and stability of Pma1p. These observations are discussed in the context of a possible hydrophobic match between the length of a protein transmembrane domain and the thickness of its surrounding lipid membrane as a parameter that may affect Pma1p sorting to or retention at the plasma membrane of yeast. Yeast Strains and Growth Conditions—Yeast strains used in this study are listed in Table 1. Strains bearing single deletions of nonessential genes were obtained from EUROSCARF (available on the World Wide Web at www.rz.uni-frankfurt.de/FB/fb16/mikro/euroscarf/index.html) (16Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Liebundguth N. Lockhart D.J. Lucau-Danila A. Lussier M. M'Rabet N. Menard P. Mittmann M. Pai C. Rebischung C. Revuelta J.L. Riles L. Roberts C.J. Ross-MacDonald P. Scherens B. Snyder M. Sookhai-Mahadeo S. Storms R.K. Veronneau S. Voet M. Volckaert G. Ward T.R. Wysocki R. Yen G.S. Yu K. Zimmermann K. Philippsen P. Johnston M. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3191) Google Scholar). Strains were cultivated at 24, 30, or 37 °C in YPD-rich medium (1% Bacto yeast extract, 2% Bacto peptone (USBiological, Swampscott, MA), 2% glucose) or in minimal medium. Cells lacking sphingolipids were cultivated in complete synthetic medium or complete complex medium as described (17Pinto W.J. Srinivasan B. Shepherd S. Schmidt A. Dickson R.C. Lester R.L. J. Bacteriol. 1992; 174: 2565-2574Crossref PubMed Google Scholar). Phytosphingosine (PHS) was supplemented in 0.5% Tergitol at a concentration of 25 μm. The presence of the kanMX marker was selected for by growing the cells on medium containing 200 μg/ml G418 (Invitrogen). Chemicals were purchased from Sigma, unless otherwise noted. Aureobasidin A was obtained from Takara Bio Inc. (Shiga, Japan); myriocin and fumonisin B1 were from Alexis Corp. (Lausen, Switzerland).TABLE 1S. cerevisiae strains used in this studyStrainRelevant genotypeSource or referenceYRS1878MATa ura3-52 leu2-3, 112 ade122YRS1877MATa ura3-52 leu2-3, 112 ade1 lcb1::URA3 SLC1-122YRS1029MATa ura3 leu2 his4 bar1 end8-149YRS1118MATα his3Δ200 leu2Δ1 ura3-52 elo3::his510YRS2915MATa ura3-52 leu2-3, 112 ade1 pep4::LEU2This workYRS2138MATa ura3-52 leu2-3, 112 ade1 lcb1::URA3 SLC1-1 pep4::LEU2This workYRS2569MATa ura3-52 leu2-3, 112 ade1 p[PMA-GFP-URA3]This workYRS2942MATa ura3-52 leu2-3, 112 ade1 lcb1::URA3 SLC1-1p[PMA1-GFP-LEU2]This workYRS2914MATa ura3-52 leu2-3, 112 ade1 lcb1::URA3 SLC1-1 elo3::kanMX4This workYRS2943MATa ura3-52 leu2-3, 112 ade1 lcb1::URA3 SLC1-1 elo3::kanMX4 p[PMA1-GFP-LEU2]This work Open table in a new tab For DNA cloning and propagation of plasmids, Escherichia coli strain XL1-blue (Stratagene, La Jolla, CA) was used. To generate double mutants with pep4Δ, a pep4::LEU2 disruption cassette (pTS17; Tom Stevens, University of Oregon, Eugene, OR) was cut with BamHI and used for transformation. PEP4 disruption was confirmed by PCR and a plate assay for carboxypeptidase Y activity. The plasmid containing a GFP-tagged version of Pma1p was kindly provided by A. Breton (Institut de Biochimie et Genetique Cellulaires, Bordeaux, France) (18Balguerie A. Bagnat M. Bonneu M. Aigle M. Breton A.M. Eukaryot. Cell. 2002; 1: 1021-1031Crossref PubMed Scopus (42) Google Scholar). The URA3 marker on this plasmid was switched to LEU2 using the marker swap plasmid pUL9 (19Cross F.R. Yeast. 1997; 13: 647-653Crossref PubMed Scopus (140) Google Scholar). Isolation of Detergent-insoluble Membrane Domains—Detergent-insoluble membrane domains were isolated after flotation on Optiprep gradients (Axis-Shield, Huntingdon, UK) as previously described (10Eisenkolb M. Zenzmaier C. Leitner E. Schneiter R. Mol. Biol. Cell. 2002; 13: 4414-4428Crossref PubMed Scopus (97) Google Scholar, 20Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (502) Google Scholar). Proteins were precipitated with trichloroacetic acid (10%), dissolved in sample buffer, and subjected to SDS-PAGE and Western blot analyses using rabbit antibodies against Pma1p (1:10,000), Gas1p (1:2000; a kind gift from A. Conzelmann, University Fribourg, Switzerland), and Wbp1p (1:1000; a kind gift from C. Jakob and M. Aebi, ETH Zurich, Switzerland). Pulse-Chase Analysis—For pulse-chase analysis, cells were grown to A600 nm ∼1 in minimal medium lacking cysteine and methionine, unless otherwise noted, and the culture was then split and preincubated at either 24 or 37 °C for 15 min. Cells were pulsed with 100 μCi/ml EXPRE35S35S protein labeling mix (∼1175 Ci/mmol; PerkinElmer Life Sciences) for 5 min. Chase was initiated by the addition of chase solution (100×; 0.3% cysteine, 0.3% methionine, 300 mm ammonium sulfate). At each time point, cells were removed (1 A600 nm unit), placed on ice, and arrested with 20 mm NaN3/NaF. Cells were centrifuged; resuspended in 50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 10 μg/ml leupeptin A, 10 μg/ml pepstatin; and disrupted by vortexing with glass beads. SDS was added to 1%, and the lysate was incubated at 45 °C for 10 min. The lysate was diluted by the addition of 800 μl of TNET (30 mm Tris-HCl, pH 7.5, 120 mm NaCl, 5 mm EDTA, 1% Triton X-100) and centrifuged at 15,000 × g for 10 min. The supernatant was incubated with anti-Pma1p antibody and protein A-Sepharose. Immunoprecipitates were analyzed by SDS-PAGE, visualized with a PhosphorImager, and quantified using Quantify One software (Bio-Rad). Detergent resistance of newly synthesized Pma1p was examined as previously described (9Gong X. Chang A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9104-9109Crossref PubMed Scopus (61) Google Scholar, 10Eisenkolb M. Zenzmaier C. Leitner E. Schneiter R. Mol. Biol. Cell. 2002; 13: 4414-4428Crossref PubMed Scopus (97) Google Scholar, 11Gaigg B. Timischl B. Corbino L. Schneiter R. J. Biol. Chem. 2005; 280: 22515-22522Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Lysates of labeled cells (3-4 A600 nm equivalents) were extracted with 1% Triton X-100 for 30 min at 4 °C. Samples were centrifuged at 100,000 × g for 1 h. Pellets were resuspended in 1% SDS. Detergent concentrations in aliquots of total, supernatant, and pellet samples were adjusted for immunoprecipitation. Fluorescence Microscopy—in vivo localization of green fluorescent protein (GFP)-tagged Pma1p was performed by fluorescence microscopy using a Zeiss Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with an AxioCam CCD camera and AxioVision 3.1 software. Lucifer yellow uptake was examined as described (21Dulic V. Egerton M. Elguindi I. Raths S. Singer B. Riezman H. Methods Enzymol. 1991; 194: 697-710Crossref PubMed Scopus (162) Google Scholar). Lipid Analysis—For sphingolipid analysis, 10 A600 nm units of cells (1 × 108 cells) grown in SC-inositol were harvested, resuspended in 1 ml of fresh SC-inositol, and labeled by the addition of 40 μCi of [3H]inositol (1 mCi/ml; American Radiolabeled Chemicals Inc., St. Louis, MO). Cells were incubated for 40 min at 30 °C, diluted with prewarmed fresh medium, and incubated for another 80 min. Labeling was terminated by adding NaF and NaN3 to a final concentration of 20 mm. Cells were harvested and broken with glass beads, and lipids were extracted with chloroform/methanol (1:1; v/v). Phospholipids were deacylated by treatment with mild base, and sphingolipids were analyzed by thin layer chromatography on silica gel 60 plates (Merck) using chloroform, methanol, 0.25% KCl (55:45:5; v/v/v) as the solvent system. Radioactivity was detected and quantified by two-dimensional radioscanning on a Berthold Tracemaster 40, and plates were visualized using a PhosphorImager (Bio-Rad). For lipid analysis by mass spectrometry, 40,000 × g microsomal membranes were extracted three times with ethanol/water/diethylether/pyridine/NH4OH (15:15:5:1:0.018; v/v/v/v/v) at 60 °C, for 15 min each. Lipids were resuspended in chloroform/methanol (1:1; v/v) and analyzed in the negative ion mode on a Bruker Esquire HCT ion trap mass spectrometer (ESI) with a flow rate of 120 μl/h and a capillary tension of -250 V. Ion fragmentation was induced with argon as collision gas at a pressure of 8 millibars. Characterization of the SLC1-1 Suppressor Strain—Sphingolipids or intermediates of the sphingolipid biosynthetic pathway are essential lipids in yeast, as indicated by the fact that long-chain base synthesis mediated by serine palmitoyltransferase (LCB1,2) is vital (22Dickson R.C. Wells G.B. Schmidt A. Lester R.L. Mol. Cell. Biol. 1990; 10: 2176-2181Crossref PubMed Scopus (61) Google Scholar, 23Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 2002; 1583: 13-25Crossref PubMed Scopus (198) Google Scholar). However, the essential requirement for long-chain base synthesis is bypassed in a suppressor strain that produces novel C26 fatty acid-containing inositol glycerophospholipids that structurally mimic sphingolipids (Fig. 1) (15Lester R.L. Wells G.B. Oxford G. Dickson R.C. J. Biol. Chem. 1993; 268: 845-856Abstract Full Text PDF PubMed Google Scholar). The semidominant SLC1-1 mutation results in a Q44L substitution in Slc1p, an 1-acyl-sn-glycerol-3-phosphate acyltransferase (24Nagiec M.M. Wells G.B. Lester R.L. Dickson R.C. J. Biol. Chem. 1993; 268: 22156-22163Abstract Full Text PDF PubMed Google Scholar). The semidominant mutation is thus thought to confer altered substrate specificity to Slc1-1p, enabling the transfer of C26 to the sn-2 position of glycerophospholipids (24Nagiec M.M. Wells G.B. Lester R.L. Dickson R.C. J. Biol. Chem. 1993; 268: 22156-22163Abstract Full Text PDF PubMed Google Scholar). To determine the relative fitness of the SLC1-1 suppressor strain, we first analyzed its growth and viability in the presence or absence of exogenous phytosphingosine (PHS) as a longchain base that restores sphingolipid synthesis in this strain (Fig. 2, A and B). This analysis revealed that the SLC1-1 strain grew more slowly than a corresponding wild-type strain but that the strain remained fully viable even in the absence of PHS. PHS itself did not appear to significantly improve growth of the suppressor strain but inhibited growth of both SLC1-1 and wild-type cells. Labeling of cells with [3H]inositol confirmed the absence of sphingolipids, since there were no detectable mild base-resistant lipids present in SLC1-1 when grown without PHS. The addition of PHS, however, restored sphingolipid synthesis in the suppressor strain (Fig. 2C). The presence of C26-containing inositol gylcerophospholipids in SLC1-1 was furthermore confirmed by mass spectrometry, which revealed two major C26-containing inositol glycerophospholipid species in SLC1-1 that were not detectable in wild-type cells (Fig. 2D). Presence of the C26 fatty acid in these two lipid species was confirmed by collision induced fragmentation, which yielded an ion of m/z 395, characteristic for C26 (Fig. 2E). Taken together, these results indicate that the SLC1-1 strain is viable in the absence of sphingolipids and that the strain synthesizes C26-containing inositol glycerophospholipids, consistent with the original characterization of the lipids made by the suppressor strain (15Lester R.L. Wells G.B. Oxford G. Dickson R.C. J. Biol. Chem. 1993; 268: 845-856Abstract Full Text PDF PubMed Google Scholar). Association of Pma1p and Gas1p with Detergent-resistant Membranes in the Absence of Sphingolipids—We next examined whether the association of plasma membrane proteins with detergent-resistant membranes was affected by the absence of sphingolipids in SLC1-1. The glycosylphosphatidylinositol-anchored Gas1p and the polytopic proton-pumping ATPase Pma1p are two yeast plasma membrane proteins that associate with detergent-resistant membranes (20Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (502) Google Scholar). Steadystate association of these two proteins with detergent-resistant membranes depends on sphingolipids and sterols (20Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (502) Google Scholar). To examine raft association of Gas1p and Pma1p in cells lacking sphingolipids, membranes were prepared from wild-type and SLC1-1 cells that were grown in the presence or absence of PHS. These membranes were then extracted with detergent and floated in an Optiprep density gradient to separate detergent-resistant membranes in the top fractions of the gradient from the solubilized material. This analysis revealed that Pma1p and Gas1p were both enriched in the detergent-resistant membrane fraction even in cells lacking sphingolipids but making suppressor lipids (Fig. 3A). The ER membrane protein and component of the oligosaccharyltransferase complex, Wbp1p, on the other hand, was solubilized upon detergent treatment and stayed in the bottom fractions of the gradient. Depletion of sphingolipids by using a temperature-sensitive mutant in long-chain base synthesis, lcb1-100 (end8-1), impaired the association of Pma1p and Gas1p with detergent-resistant membranes at the nonpermissive temperature, as has been observed before (20Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (502) Google Scholar, 25Sutterlin C. Horvath A. Gerold P. Schwarz R.T. Wang Y. Dreyfuss M. Riezman H. EMBO J. 1997; 16: 6374-6383Crossref PubMed Scopus (87) Google Scholar) (Fig. 3B). These results thus indicate that the function of sphingolipids in the formation of detergent-resistant membrane domains at the plasma membrane can be substituted by C26-containing inositol gylcerophospholipids made in the SLC1-1 suppressor strain. This substitution, however, appears to be slightly compromised, as indicated by the fact that the flotation behavior of both Pma1p and Gas1p when isolated from the SLC1-1 suppressor strain differs slightly from that of wild-type cells. When isolated from the suppressor strain, Pma1p and Gas1p displayed a trailing population of protein that did not exclusively fractionate in the top fraction, as was the case in wild type. Similarly, the addition of PHS to wild-type cells induced the appearance of a population of Gas1p that was detergent-soluble. PHS addition has previously been shown to induce ubiquitin-dependent internalization and down-regulation of the uracil permease Fur4p (26Chung N. Jenkins G. Hannun Y.A. Heitman J. Obeid L.M. J. Biol. Chem. 2000; 275: 17229-17232Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Based on this observation, it is conceivable that PHS also induces down-regulation of Gas1p and that this down-regulation is accompanied by the exclusion of Gas1p from detergentresistant membranes. Suppressor Lipids Functionally Substitute Sphingolipids in Surface Transport of Newly Synthesized Pma1p—Sphingolipid synthesis is required for the association of newly synthesized Pma1p with detergent-resistant membranes already in the ER and for proper routing of Pma1p to the cell surface (2Lee M.C. Hamamoto S. Schekman R. J. Biol. Chem. 2002; 277: 22395-22401Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 7Wang Q. Chang A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12853-12858Crossref PubMed Scopus (65) Google Scholar, 8Bagnat M. Chang A. Simons K. Mol. Biol. Cell. 2001; 12: 4129-4138Crossref PubMed Scopus (178) Google Scholar). In the absence of sphingolipids, newly synthesized Pma1p is rapidly degraded in the vacuole. Given that C26-containing suppressor lipids can substitute for sphingolipids in raft association of steady-state levels of Pma1p at the plasma membrane, we examined whether these lipids also protected newly synthesized Pma1p from missorting to the vacuole. Therefore, the stability of newly synthesized Pma1p in the SLC1-1 suppressor strain grown in the presence or absence of PHS was examined by pulse-chase analysis and immunoprecipitation. These experiments revealed that newly synthesized Pma1p was stable in the SLC1-1 suppressor strain independent of whether suppressor lipids or sphingolipids were synthesized (Fig. 4A). This result would thus indicate that suppressor lipids functionally substitute for sphingolipids also in surface transport of newly synthesized Pma1p. Moreover, the suppressor lipids appeared to provide full functionality as indicated by the fact that Pma1p was stable even when synthesized at 37 °C. The stability of Pma1p at 37 °C allows for discrimination between fully functional sphingolipids with a mature C26 fatty acid and those that are conditionally impaired in function due to the presence of a C22 fatty acid, as is the case in acyl chain elongase mutant cells, elo3Δ (10Eisenkolb M. Zenzmaier C. Leitner E. Schneiter R. Mol. Biol. Cell. 2002; 13: 4414-4428Crossref PubMed Scopus (97) Google Scholar, 11Gaigg B. Timischl B. Corbino L. Schneiter R. J. Biol. Chem. 2005; 280: 22515-22522Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 27Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Given that suppressor lipids can substitute for sphingolipids in surface transport of Pma1p, one would expect that Pma1p biogenesis in the suppressor strain becomes resistant against degradation induced by drugs that inhibit sphingolipid biosynthesis. To test this prediction, we compared the stability of newly synthesized Pma1p in the SLC1-1 strain with that of wild-type cells upon treatment with three different inhibitors of the sphingolipid biosynthetic pathway. Myriocin (20 μg/ml) was used to block long-chain base synthesis by serine palmitoyltransferase, fumonisin B1 (72 μg/ml) to block ceramide synthase, and aureobasidin A (2 μg/ml) to inhibit the conversion of ceramide to inositolphosphorylceramide (28Horvath A. Sütterlin C. Manning-Krieg U. Movva N.R. Riezman H. EMBO J. 1994; 13: 3687-3695Crossref PubMed Scopus (184) Google Scholar, 29Merrill Jr., A.H. van Echten G. Wang E. Sandhoff K. J. Biol. Chem. 1993; 268: 27299-27306Abstract Full Text PDF PubMed Google Scholar, 30Nagiec 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 (325) Google Scholar). This analysis revealed that Pma1p was rapidly turned over in drug-treated wild-type cells but that Pma1p was stable in the SLC1-1 suppressor strain (Fig. 4B). This observation is thus consistent with the notion that the C26-containing suppressor lipids functionally substitute for sphingolipids in surface transport of newly synthesized Pma1p. In addition, these data indicate that surface transport of Pma1p is one of the essential pathways that is blocked by these three inhibitors of the sphingolipid pathway in wild-type cells. To confirm that Pma1p reaches the cell surface in the absence of sphingolipids rather than stably accumulating within an intracellular compartment, we examined the steadystate distribution of a GFP-tagged version of Pma1p by fluorescent microscopy. In cells lacking sphingolipids, Pma1p-GFP displayed a characteristic ringlike staining at the cell periphery, consistent with surface transport of Pma1p in the absence of sphingolipids. No fluorescence was observed to accumulate in the vacuole, even in SLC1-1 cells that were shifted to 37 °C, indicating that Pma1p is stable at the cell surface in the absence of sphingolipids (Fig. 5). Drug-induced inhibition of sphingolipid synthesis resulted in vacuolar localization of Pma1p-GFP in wild-type cells but not in the SLC1-1 suppressor strain (Fig. 5). Taken together, these results indicate that the C26-containing suppressor lipids that are synthesized in the semidominant 1-acyl-sn-glycerol-3-phosphate acyltransferase mutant SCL1-1 strain fully substitute for sphingolipids in surface transport and stabilization of Pma1p. Long-chain Base Synthesis Is Dispensable for Endocytosis in the Suppressor Strain—Long-chain base synthesis is required for the internalization step of endocytosis and for the organization of the actin cytoskeleton (31Zanolari B. Friant S. Funato K. Sutterlin C. Stevenson B.J. Riezman H. EMBO J. 2000; 19: 2824-2833Crossref PubMed Scopus (210) Google Scholar). We previously showed that the accelerated turnover of Pma1p in an elo3Δ mutant at 37 °C can be rescued by preventing endocytosis, indicating that turnover of Pma1p in elo3Δ occurs after the protein has reached the cell surface (10Eisenkolb M. Zenzmaier C. Leitner E. Schneiter R. Mol. Biol. Cell. 2002; 13: 4414-4428Crossref PubMed Scopus (97) Google Scholar). To examine whether the apparent stability of newly synthesized Pma1p in the suppressor strain was due to a block in endocytosis in this strain, we examined whether the suppr" @default.
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• W2116122989 title "Very Long-chain Fatty Acid-containing Lipids rather than Sphingolipids per se Are Required for Raft Association and Stable Surface Transport of Newly Synthesized Plasma Membrane ATPase in Yeast" @default.
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