Protein Sorting in the Late Golgi of Saccharomyces cerevisiae Does Not Require Mannosylated Sphingolipids
2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês
10.1074/jbc.m306119200
ISSN1083-351X
AutoresQuirine Lisman, Thomas Günther Pomorski, Chantal Vogelzangs, Dorothy Urli-Stam, William de Cocq van Delwijnen, Joost C. M. Holthuis,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoGlycosphingolipids are widely viewed as integral components of the Golgi-based machinery by which membrane proteins are targeted to compartments of the endosomal/lysosomal system and to the surface domains of polarized cells. The yeast Saccharomyces cerevisiae creates glycosphingolipids by transferring mannose to the head group of inositol phosphorylceramide (IPC), yielding mannosyl-IPC (MIPC). Addition of an extra phosphoinositol group onto MIPC generates mannosyldi-IPC (M(IP)2C), the final and most abundant sphingolipid in yeast. Mannosylation of IPC is partially dependent on CSG1, a gene encoding a putative sphingolipidmannosyltransferase. Here we show that open reading frame YBR161w, renamed CSH1, is functionally homologous to CSG1 and that deletion of both genes abolishes MIPC and M(IP)2C synthesis without affecting protein mannosylation. Csg1p and Csh1p are closely related polytopic membrane proteins that co-localize with IPC synthase in the medial-Golgi. Loss of Csg1p and Csh1p has no effect on clathrin- or AP-3 adaptor-mediated protein transport from the Golgi to the vacuole. Moreover, segregation of the periplasmic enzyme invertase, the plasma membrane ATPase Pma1p and the glycosylphosphatidylinositol-anchored protein Gas1p into distinct classes of secretory vesicles occurs independently of Csg1p and Csh1p. Our results indicate that protein sorting in the late Golgi of yeast does not require production of mannosylated sphingolipids. Glycosphingolipids are widely viewed as integral components of the Golgi-based machinery by which membrane proteins are targeted to compartments of the endosomal/lysosomal system and to the surface domains of polarized cells. The yeast Saccharomyces cerevisiae creates glycosphingolipids by transferring mannose to the head group of inositol phosphorylceramide (IPC), yielding mannosyl-IPC (MIPC). Addition of an extra phosphoinositol group onto MIPC generates mannosyldi-IPC (M(IP)2C), the final and most abundant sphingolipid in yeast. Mannosylation of IPC is partially dependent on CSG1, a gene encoding a putative sphingolipidmannosyltransferase. Here we show that open reading frame YBR161w, renamed CSH1, is functionally homologous to CSG1 and that deletion of both genes abolishes MIPC and M(IP)2C synthesis without affecting protein mannosylation. Csg1p and Csh1p are closely related polytopic membrane proteins that co-localize with IPC synthase in the medial-Golgi. Loss of Csg1p and Csh1p has no effect on clathrin- or AP-3 adaptor-mediated protein transport from the Golgi to the vacuole. Moreover, segregation of the periplasmic enzyme invertase, the plasma membrane ATPase Pma1p and the glycosylphosphatidylinositol-anchored protein Gas1p into distinct classes of secretory vesicles occurs independently of Csg1p and Csh1p. Our results indicate that protein sorting in the late Golgi of yeast does not require production of mannosylated sphingolipids. Correct sorting of membrane proteins and lipids is essential for establishing and maintaining the identity and function of the different cellular organelles. Although much progress has been made in uncovering the transport machinery for delivering endosomal/lysosomal proteins (1.Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1331) Google Scholar, 2.Burd C.G. Babst M. Emr S.D. Semin. Cell Dev. Biol. 1998; 9: 527-533Crossref PubMed Scopus (42) Google Scholar), the mechanisms for cargo sorting to the cell surface are still poorly defined. Exocytic cargo can reach the cell surface by multiple pathways in most, if not all eukaryotic cells (3.Keller P. Simons K. J. Cell Sci. 1997; 110: 3001-3009Crossref PubMed Google Scholar). For example, the polarized organization of epithelial cells relies on the sorting of both proteins and lipids into distinct classes of Golgi-derived vesicles that are targeted to the apical or basolateral surface (4.Mostov K.E. Verges M. Altschuler Y. Curr. Opin. Cell Biol. 2000; 12: 483-490Crossref PubMed Scopus (331) Google Scholar). Apical and basolateral proteins expressed in fibroblasts are also sorted into different vesicles (5.Yoshimori T. Keller P. Roth M.G. Simons K. J. Cell Biol. 1996; 133: 247-256Crossref PubMed Scopus (201) Google Scholar), and it appears that the Golgi-based sorting machinery for apical and basolateral cargo operates both in polarized and non-polarized cell types (6.Musch A. Xu H. Shields D. Rodriguez-Boulan E. J. Cell Biol. 1996; 133: 543-558Crossref PubMed Scopus (147) Google Scholar). Characterization of secretory vesicles that accumulate in late (post-Golgi-blocked) secretory yeast mutants has identified two vesicle populations with different densities and unique cargo proteins (7.Harsay E. Bretscher A. J. Cell Biol. 1995; 131: 297-310Crossref PubMed Scopus (206) Google Scholar, 8.David D. Sundarababu S. Gerst J.E. J. Cell Biol. 1998; 143: 1167-1182Crossref PubMed Scopus (114) Google Scholar, 9.Harsay E. Schekman R. J. Cell Biol. 2002; 156: 271-285Crossref PubMed Scopus (120) Google Scholar). Hence, transport of exocytic cargo by independent routes seems a conserved feature of eukaryotic cells.There are numerous indications that lipid microheterogeneity plays a role in cargo sorting along the secretory pathway. Importantly, sphingolipids and in particular glycosphingolipids have the propensity to segregate from glycerolipids and to cluster with sterols into lateral microdomains with physicochemical properties distinct from those of the bulk membrane (10.Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2043) Google Scholar). Glycosphingolipid/sterol-rich microdomains were first conceived in polarized MDCK cells as Golgi-based sorting platforms for apically directed proteins and lipids (11.van Meer G. Stelzer E.H.K. Wijnaendts-van-Resandt R.W. Simons K. J. Cell Biol. 1987; 105: 1623-1635Crossref PubMed Scopus (302) Google Scholar, 12.Simons K. van Meer G. Biochem. 1988; 27: 6197-6202Crossref PubMed Scopus (1079) Google Scholar). In support of this model, inhibition of sphingolipid synthesis with fumonisin B randomizes the cell surface distribution of apical GPI 1The abbreviations used are: GPI, glycosylphosphatidylinositol; IPC, inositol phosphorylceramide; MIPC, mannosyl-IPC; HA, hemagglutinin; ORF, open reading frame; GFP, green fluorescent protein; ER, endoplasmic reticulum; CPY, carboxypeptidase Y; ALP, alkaline phosphatase. 1The abbreviations used are: GPI, glycosylphosphatidylinositol; IPC, inositol phosphorylceramide; MIPC, mannosyl-IPC; HA, hemagglutinin; ORF, open reading frame; GFP, green fluorescent protein; ER, endoplasmic reticulum; CPY, carboxypeptidase Y; ALP, alkaline phosphatase.-anchored proteins in MDCK cells (13.Mays R.W. Siemers K.A. Fritz B.A. Lowe A.W. van Meer G. Nelson W.J. J. Cell Biol. 1995; 130: 1105-1115Crossref PubMed Scopus (192) Google Scholar). A similar glycosphingolipid-based sorting mechanism is held responsible for axonal delivery of GPI-anchored proteins in neurons (14.Ledesma M.D. Simons K. Dotti C.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3966-3971Crossref PubMed Scopus (199) Google Scholar), regulated apical secretion of zymogens from pancreatic acinar cells (15.Schmidt K. Schrader M. Kern H.F. Kleene R. J. Biol. Chem. 2001; 276: 14315-14323Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), apical trafficking of thyroglobulin in thyrocytes (16.Martin-Belmonte F. Alonso M.A. Zhang X. Arvan P. J. Biol. Chem. 2000; 275: 41074-41081Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), and cell surface delivery of plasma membrane ATPase, Pma1p, and diverse GPI-anchored proteins in yeast (17.Skrzypek M. Lester R.L. Dickson R.C. J. Bacteriol. 1997; 179: 1513-1520Crossref PubMed Google Scholar, 18.Bagnat M. Keranen S. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (501) Google Scholar, 19.Bagnat M. Chang A. Simons K. Mol. Biol. Cell. 2001; 12: 4129-4138Crossref PubMed Scopus (178) Google Scholar). Glycosphingolipids are also required for transport of melanosomal proteins from the Golgi to melanososomes in melanoma cells (20.Sprong H. Degroote S. Claessens T. van Drunen J. Oorschot V. Westerink B.H. Hirabayashi Y. Klumperman J. van der Sluijs P. van Meer G. J. Cell Biol. 2001; 155: 369-380Crossref PubMed Scopus (99) Google Scholar), but the underlying mechanism remains to be elucidated. The ubiquitous expression of glycosphingolipids suggests that they exert organizing functions in all eukaryotic cells.Animals as well as some plants and fungi generate glycosphingolipids by transferring glucose or galactose to the C1 hydroxyl group of ceramide. These additions can be further decorated by additional sugars and sometimes sulfates to yield hundreds of different glycosphingolipid species (21.Merrill Jr., A.H. Sweeley C.C. Vance D. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam1996: 309-339Google Scholar). In the yeast S. cerevisiae, however, the direct precursor for glycosphingolipid synthesis is not ceramide but inositolphosphorylceramide (IPC, Ref. 22.Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1438: 305-321Crossref PubMed Scopus (129) Google Scholar). IPC is formed by addition of phosphoinositol released from phosphatidylinositol to ceramide, a reaction catalyzed by IPC synthase in a medial compartment of the yeast Golgi (23.Levine T.P. Wiggins C.A. Munro S. Mol. Biol. Cell. 2000; 11: 2267-2281Crossref PubMed Scopus (133) Google Scholar). IPC is then mannosylated to yield mannosyl-IPC (MIPC), which in turn can receive a second phosphoinositol group from phosphatidylinositol to generate the final and by far most abundant sphingolipid, M(IP)2C (22.Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1438: 305-321Crossref PubMed Scopus (129) Google Scholar). MIPC and M(IP)2C synthesis occurs in the lumen of the Golgi (22.Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1438: 305-321Crossref PubMed Scopus (129) Google Scholar, 24.Dean N. Zhang Y.B. Poster J.B. J. Biol. Chem. 1997; 272: 31908-31914Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Whereas IPC is highly enriched in Golgi and vacuolar membranes, the largest amounts of MIPC and M(IP)2C are found in the plasma membrane (25.Hechtberger P. Zinser E. Saf R. Hummel K. Paltauf F. Daum G. Eur. J. Biochem. 1994; 225: 641-649Crossref PubMed Scopus (103) Google Scholar). Hence, the yeast Golgi seems to be a branching point in sphingolipid trafficking from where mannosylated sphingolipids selectively migrate to the cell surface and sphingolipids without the sugar moiety reach the vacuole. However, direct evidence that mannosylated sphingolipids play a role in cargo sorting to the cell surface is lacking.Addressing the biological function of mannosylated sphingolipids in yeast is hampered by the fact that little is known about the enzyme(s) responsible for their synthesis. Three structurally unrelated genes have been implicated in the mannosylation of IPC. The VRG4 gene encodes a nucleotide sugar transporter that mediates GDP-mannose import into the Golgi lumen (24.Dean N. Zhang Y.B. Poster J.B. J. Biol. Chem. 1997; 272: 31908-31914Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Besides being essential for IPC mannosylation, VRG4 also affects N-linked and O-linked glycoprotein modifications (24.Dean N. Zhang Y.B. Poster J.B. J. Biol. Chem. 1997; 272: 31908-31914Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Null mutations in either the CSG1 or CSG2 gene cause a reduction in, but do not completely eliminate MIPC synthesis (26.Beeler T. Gable K. Zhao C. Dunn T. J. Biol. Chem. 1994; 269: 7279-7284Abstract Full Text PDF PubMed Google Scholar, 27.Beeler T.J. Fu D. Rivera J. Monaghan E. Gable K. Dunn T.M. Mol. Gen. Genet. 1997; 255: 570-579Crossref PubMed Scopus (114) Google Scholar). Csg1p is predicted to have a catalytic function since it contains a region of 93 amino acids with homology to the yeast α-1,6-mannosyltransferase, Och1p (27.Beeler T.J. Fu D. Rivera J. Monaghan E. Gable K. Dunn T.M. Mol. Gen. Genet. 1997; 255: 570-579Crossref PubMed Scopus (114) Google Scholar). The function of Csg2p is less obvious. Csg2p contains an EF-Ca2+-binding domain and has been localized to the ER where it may play a role in Ca2+ homeostasis (28.Tanida I. Takita Y. Hasegawa A. Ohya Y. Anraku Y. FEBS Lett. 1996; 379: 38-42Crossref PubMed Scopus (34) Google Scholar). The recent finding that Csg2p forms a complex with Csg1p raises the possibility that IPC mannosyltransferase activity in yeast is regulated by Ca2+ through Csg2p (29.Uemura S. Kihara A. Inokuchi J.I. Igarashi Y. J. Biol. Chem. 2003; 278: 45049-45055Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar).Yeast open reading frame YBR161w, recently renamed CSH1, encodes a protein exhibiting strong similarity to the putative sphingolipid mannosyltransferase, Csg1p (27.Beeler T.J. Fu D. Rivera J. Monaghan E. Gable K. Dunn T.M. Mol. Gen. Genet. 1997; 255: 570-579Crossref PubMed Scopus (114) Google Scholar, 29.Uemura S. Kihara A. Inokuchi J.I. Igarashi Y. J. Biol. Chem. 2003; 278: 45049-45055Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Here we report that Csh1p is functionally homologous to Csg1p and provide evidence that Csg1p and Csh1p function as two independent sphingolipid mannosyltransferases. Loss of Csg1p and Csh1p had no effect on the delivery of vacuolar proteins or on the packaging of cell surface components into distinct classes of secretory vesicles. From these results, we conclude that the organization of the various post-Golgi delivery pathways in yeast does not depend on production of mannosylated sphingolipids.EXPERIMENTAL PROCEDURESStrains and Plasmids—Unless indicated otherwise, yeast strains were grown at 28 °C to mid-logarithmic phase (0.5–1.0 OD600) in synthetic dextrose (S.D.) medium or in yeast extract-peptone-dextrose (YEPD) medium. Yeast transformations were carried out as described (30.Elble R. BioTechniques. 1992; 13: 18-20PubMed Google Scholar). The yeast mutants Δpep12Δvam3, Δanp1, Δmnn10 and Δvan1 were all derived from the strain SEY6210 (MATα ura3-52 his3 Δ200 leu2-3-112 trpl-Δ901 suc2-Δ9 lys2-801) and have been described elsewhere (31.Holthuis J.C.M. Nichols B.J. Pelham H.R.B. Mol. Biol. Cell. 1998; 9: 3383-3397Crossref PubMed Scopus (95) Google Scholar, 32.Jungmann J. Rayner J.C. Munro S. J. Biol. Chem. 1999; 274: 6579-6585Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). All other gene deletion phenotypes were characterized in the strain EHY227 (MATα sec6-4 TPI1::SUC2::TRP1 ura3-52 his3 Δ200 leu2-3-112 trp1-1). For the deletion of CSG1, CSH1, and IPT1 genes, 450–550 base pair fragments of the promoter and ORF 3′-end of each gene were amplified by PCR from yeast genomic DNA. The gene promoters and ORF ends were cloned into NotI/EcoRI and SpeI/MluI sites located on either site of a loxP-HIS3-loxP cassette that was ligated into the EcoRI/SpeI sites of a pBluescript KS– vector (Stratagene, La Jolla, CA; the loxP-HIS3-loxP plasmid was a gift of T. Levine, University College London, UK). Gene deletion constructs were linearized with NotI and MluI and transformed into EHY227 to generate Δcsg1 (JHY075), Δcsh1 (JHY088), and Δipt1 (JHY079) strains. Double deletions were performed sequentially in EHY227 by repeated use of the loxP-HIS3-loxP cassette and subsequent removal of the HIS3 marker by excisive recombination using Cre recombinase (33.Sauer B. Mol. Cell Biol. 1987; 7: 2087-2096Crossref PubMed Scopus (359) Google Scholar), yielding the Δcsg1Δcsh1 strain (JHY090). In each case, the correct integration or excision event was confirmed by PCR.Aur1p was tagged at its C terminus with three copies of the hemagglutinin (HA) epitope using the PCR knock-in approach (34.Wach A. Brachat A. Alberti-Segui C. Rebischung C. Philippsen P. Yeast. 1997; 13: 1065-1075Crossref PubMed Scopus (507) Google Scholar) and plasmid p3xHAt-HIS5 (S. Munro, MRC-LMB, Cambridge, UK). Pma1p was tagged at its N terminus with one copy of the HA epitope using integration plasmid pRS305Δ51 as described (35.Ziman M. Chuang J.S. Schekman R.W. Mol. Biol. Cell. 1996; 7: 1909-1919Crossref PubMed Scopus (118) Google Scholar). Vam3p was tagged at its N terminus with three copies of the HA epitope using integration plasmid pRS405(HA)3VAM3 (B. Nichols, MRC-LMB, Cambridge, UK). Expression plasmids encoding Myc-tagged invertase, Myc-tagged Mnt1p and GFP-tagged Sed5p have been described previously (23.Levine T.P. Wiggins C.A. Munro S. Mol. Biol. Cell. 2000; 11: 2267-2281Crossref PubMed Scopus (133) Google Scholar).Promoter regions (650 bp) and open reading frames of CSG1 and CSH1 were PCR-amplified from yeast genomic DNA and subsequently ligated into single copy vector pRS413 (CEN, HIS3) or multicopy vector pRS425 (2μ, LEU2) (36.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). A second version of these constructs was prepared, but then with 3 copies of the HA epitope fused to the C termini of CSG1 and CSH1, using PCR.Lipid Analysis—Exponentially grown cells (0.5 OD600) were inoculated in 5 ml of S.D. medium containing 10 μCi myo-[3H]inositol (16 Ci/mmol; ICN Biomedicals, Eschwege) and grown for 16 h at 30 °C. Cells were harvested by centrifugation, washed twice with 10 mm NaN3 and lipids extracted by bead bashing in H2O/methanol/chloroform (5: 16:16). The organic extracts were dried and subjected to butyl alcohol/water partitioning. Lipids recovered from the butyl alcohol phase were deacylated by mild base treatment using 0.2 n NaOH in methanol. After neutralizing with 1 m acetic acid, lipids were extracted with chloroform and separated by TLC using chloroform/methanol/4.2 m NH3 (9:7:3). The TLC plate was dipped in 0.4% 2,5-diphenyloxazol dissolved in 2-methylnaphthalene supplemented with 10% xylene (37.Bonner W.M. Stedman J.D. Anal. Biochem. 1978; 89: 247-256Crossref PubMed Scopus (121) Google Scholar) and 3H-labeled lipids detected by fluorography using Kodak X-Omat S films exposed at –80 °C. Alternatively, 3H-labeled lipids were detected by exposure to BAS-TR2040 imaging screens (Fuji, Japan) and read out on a BIO-RAD Personal Molecular Imager (BioRad, Hercules, CA).Analysis of IPC Mannosyltransferase Activity in Cell Extracts—Exponentially grown Δcsg1Δcsh1 cells (2.5 OD600) were inoculated in 50 ml S.D. medium containing 100 μCi myo-[3H]inositol and then grown for 16 h at 30 °C. Cells were harvested by centrifugation, washed twice with 10 mm NaN3, and lysed by bead bashing in lysis buffer (50 mm Hepes, pH 7.2, 10 mm MnCl2, 1 mm NEM) in the presence of fresh protease inhibitors. After removal of unbroken cells (500 × g, 10 min), membranes were collected (100,000 × g, 60 min) and solubilized in 1 ml of lysis buffer containing 1% and fresh protease inhibitors. After incubation for 60 min at room temperature, the extract was centrifuged (100,000 × g, 60 min), and 50-μl aliquots were stored at –80 °C. In addition, 400 OD600 of non-radiolabeled, exponentially-grown wild-type or Δcsg1Δcsh1 cells transformed with multicopy CSG1, CSH1, or control plasmids were lysed by bead bashing in 4 ml of ice-cold lysis buffer containing fresh protease inhibitors. Upon removal of unbroken cells, total membranes were collected, resuspended in 1 ml of ice-cold lysis buffer containing 1% Triton X-100, and rotated at 4 °C for 60 min.For IPC mannosyltransferase assays, 50 μl of radiolabeled extract was mixed with 150 μl of unlabeled extract and then preincubated with 10 mm GDP-mannose (Sigma-Aldrich) for 10 min at 30 °C. Reactions were diluted 10-fold in lysis buffer and then incubated for 2 h at 30 °C. Reactions were stopped by adding 6.4 ml of chloroform:methanol (1:2.2). Lipids were extracted, deacylated, and separated by TLC as above.Antibodies and Immunoblotting—Peptides corresponding to C-terminal regions of Csg1p and Csh1p (Fig. 1) were synthesized and then coupled to a carrier before immunization of rabbits. The resulting antisera were affinity-purified against peptides coupled to NHS-activated Sepharose 4 Fast Flow according to instructions of the manufacturer (Amersham Biosciences). Affinity-purified antibodies were used at a dilution of 1:1000 for immunoblot analysis and at 1:250 for immunofluorescence microscopy. Rabbit polyclonal antibodies to CPY, Gos1p, Pep12p, Tlg1p, and Tlg2p were described previously (38.Holthuis J.C.M. Nichols B.J. Dhruvakumar S. Pelham H.R. EMBO J. 1998; 17: 113-126Crossref PubMed Scopus (216) Google Scholar). Rabbit polyclonal antibodies to Sso2p were provided by S. Keränen, (Biotechnology and Food Research, Espoo, Finland) and to Gas1p by H. Riezman (Sciences II, Geneve, Switzerland). The Myc epitope was detected with mouse monoclonal antibody 9E10 or with rabbit polyclonal antibodies (Santa Cruz Biotechnology) and the HA epitope with rat monoclonal antibody 3F10, mouse monoclonal antibody 12CA5 (Roche Applied Science) or rabbit polyclonal antibodies (Santa Cruz Biotechnology). For immunoblotting, all antibody incubations were carried out in phosphate-buffered saline containing 5% dried milk and 0.5% Tween-20. After incubation with peroxidase-conjugated secondary antibodies (Bio-Rad), blots were developed using a chemiluminescent substrate kit (Pierce). Chemiluminescent bands were quantified using a GS-710 calibrating imaging densitometer (BioRad) with QuantityOne software.Immunofluorescence Microscopy—Exponentially grown cells were fixed and mounted on glass-slides as described previously (38.Holthuis J.C.M. Nichols B.J. Dhruvakumar S. Pelham H.R. EMBO J. 1998; 17: 113-126Crossref PubMed Scopus (216) Google Scholar). All antibody incubations were performed in phosphate-buffered saline supplemented with 2% dried milk and 0.1% saponin for 2 h at room temperature. Primary polyclonal antibodies to Csg1p, Csh1p, and the rat monoclonal 9F10 to HA were used at a dilution of 1:400, 1:150, and 1:250 respectively. Fluorescein- or Cy3-conjugated secondary antibodies (Amersham Biosciences) were used at a dilution of 1:100. Fluorescence microscopy and image acquisition were carried out using a Leica DMRA microscope (Leitz, Wetzlar, Germany) equipped with a cooled CCD camera (KX85, Apogee Instruments Inc., Tucson, AZ) driven by Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).Fractionation of Secretory Vesicles—Exponentially grown sec6-4 cells expressing HA-tagged Pma1p (2.0 OD600) were inoculated into YEPD medium (500-ml culture per gradient) and then grown for 14–16 h at 25 °C to 0.7 OD600/ml. Next, cells were collected (500 × g, 5 min) resuspended in 250 ml of YEPD and then shifted to 38 °C for 60 min to induce the sec6–4 secretory block. Spheroplasting, cell lysis, and collection of membrane pellet enriched in secretory vesicles (SVs) were performed essentially as described (7.Harsay E. Bretscher A. J. Cell Biol. 1995; 131: 297-310Crossref PubMed Scopus (206) Google Scholar) except that SVs were collected on a 60% Nycodenz cushion in lysis buffer. SVs were resuspended in 1.5 ml of lysis buffer adjusted to 30% Nycodenz and then loaded at the bottom of a 11-ml linear 16–26% Nycodenz/0.8 M sorbitol gradient. Following centrifugation at 100,000 × gav for 16 h min at 4 °C in a Beckman SW41Ti rotor, 0.6-ml fractions were collected from the top of the gradient. Fraction densities were determined by reading refractive indices on a Bausch and Lomb refractometer. Equal amounts per fraction were subjected to immunoblotting and analyzed for ATPase and invertase enzyme activity as described (7.Harsay E. Bretscher A. J. Cell Biol. 1995; 131: 297-310Crossref PubMed Scopus (206) Google Scholar).Immunoisolation of Secretory Vesicles—Immunoisolations of Pma1p-HA-containing SVs were performed using magnetic Dynabeads protein G (Dynal Biotech GmbH, Hamburg, Germany) loaded with mouse anti-HA (12CA5) or anti-Myc (9E10) monoclonal antibodies. Beads were incubated with antibodies for 40 min at room temperature and antibodies bound quantified by SDS-PAGE. Anti-HA beads contained 0.35 μgof 12CA5/μl of bead-slurry and control beads contained 0.1 μg of 9E10/μl bead-slurry. For immunoisolation of Pma1p-containing vesicles, a 300-μl reaction was prepared in lysis buffer containing 126 μl Dynabeads slurry, 5 mg/ml bovine serum albumin, and 15 μl membranes from Nycodenz gradient PM-ATPase peak fractions obtained by fractionating membranes derived from 1 g of cells. The reactions were rotated gently at 4 °C for 2 h. Supernatants were subjected to centrifugation (100,000 × g, 1 h, 4 °C), and membrane pellets were resuspended in 100 μl of SDS sample buffer. Beads were washed twice for 30 min in 1 ml of bovine serum albumin-containing lysis buffer, twice in lysis buffer, and resuspended in 75 μl of SDS sample buffer. Bound and unbound membranes were analyzed by immunoblotting.RESULTSCSH1 Encodes a Novel Putative IPC Mannosyltransferase— Comparative sequence analysis revealed that Csh1p is 67% identical to Csg1p and has the same predicted protein topology, namely a putative N-terminal signal sequence and two potential transmembrane segments localized to the C-terminal half of the protein (Fig. 1). Between the signal sequence and first transmembrane segment there is a region of 93 residues sharing 29% identity with the luminal portion (residues 96–197) of the yeast α-1,6-mannosyltransferase, Och1p (39.Nakayama K. Nagasu T. Shimma Y. Kuromitsu J. Jigami Y. EMBO J. 1992; 11: 2511-2519Crossref PubMed Scopus (242) Google Scholar). This region contains a conserved DXD motif that occurs in a wide range of glycosyltransferase families and likely forms part of a catalytic site (40.Wiggins C.A. Munro S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7945-7950Crossref PubMed Scopus (315) Google Scholar).Csg1p is required for accumulation of mannosylated sphingolipids in yeast and its similarity to Och1p suggests that the protein serves as an IPC mannosyltransferase (27.Beeler T.J. Fu D. Rivera J. Monaghan E. Gable K. Dunn T.M. Mol. Gen. Genet. 1997; 255: 570-579Crossref PubMed Scopus (114) Google Scholar). However, loss of Csg1p is not sufficient to abolish IPC mannosylation (see also below), raising the possibility that Csh1p represents an alternative IPC mannosyltransferase that functions independently of Csg1p. To investigate this possibility, we constructed yeast strains in which the ORFs of CSG1, CSH1, or both were removed. TLC analysis of alkaline-treated lipid extracts prepared from myo-[3H]inositol-labeled cells showed that, compared with the wild-type strain, the Δcsg1 mutant produced greatly reduced levels of the mannosylated sphingolipids MIPC and M(IP)2C, and accumulated IPC-C and IPC-D (Fig. 2A, lanes 1 and 2; note that IPC-C contains a monohydroxylated C26 fatty acid whereas the C26 fatty acid in IPC-D is dihydroxylated; (41.Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Unlike Δcsg1 cells, the Δcsh1 mutant produced IPC and mannosylated IPC species at ratios similar to those in wild-type cells (Fig. 2A, lanes 1 and 3). In the Δcsg1Δcsh1 double mutant, however, production of MIPC and M(IP)2C was completely abolished (Fig. 2A, lane 4). These results are consistent with those reported in a recent study (29.Uemura S. Kihara A. Inokuchi J.I. Igarashi Y. J. Biol. Chem. 2003; 278: 45049-45055Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and indicate that Csg1p and Csh1p have redundant functions in IPC mannosylation.Fig. 2Csg1p and Csh1p have redundant functions in sphingolipid mannosylation. A, deletion of CSG1 and CSH1 abolishes IPC mannosylation. Yeast cells were labeled overnight with myo-[3H]inositol and the lipid extracts either deacylated by mild alkaline hydrolysis with NaOH (+) or control incubated (–). Lipids were extracted, separated by TLC, and then visualized by autoradiography as described under "Experimental Procedures." Lane 1, wild-type; lane 2, Δcsg1; lane 3, Δcsh1; lane 4, Δcsg1Δcsh1; lanes 5 and 6, Δipt1. Note that IPT1 is known to have an essential function in M(IP)2C synthesis. B, analysis of IPC mannosyltransferase activity in Triton X-100 extracts derived from wild-type and Δcsg1Δcsh1 cells. Extracts prepared from myo-[3H]inositol-labeled Δcsg1Δcsh1 cells were either control-incubated (lane 1) or mixed with extracts of unlabeled wild-type cells (lanes 2 and 3), Δcsg1Δcsh1 cells (lane 4), or Δcsg1Δcsh1 cells transformed with a multicopy vector containing CSG1 (lane 5) or CSH1 (lane 6). Incubations were performed in the presence (+) or absence (–) of 1 mm GDP-mannose as described under "Experimental Procedures." Lipids were extracted, deacylated, and then separated by TLC before autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The block in MIPC and M(IP)2C synthesis observed in Δcsg1Δcsh1 cells can be explained by a complete loss of IPC mannosyltransferase activity, but may also be due to a defective delivery of IPC or GDP-mannose to the transferase-containing compartment. To explore these possibilities, we analyzed the IPC mannosyltransferase activity in detergent extracts derived from wild-type and Δcsg1Δcsh1 cells. To this end, Triton X-100 extracts prepared from myo-[3H]inositol-labeled Δcsg1Δcsh1 cells were mixed with extracts from unlabeled wild-type or mutant cells, and then incubated in the presence or absence of externally added GDP-mannose. When extracts from inositol-labeled Δcsg1Δcsh1 cells were incubated with unlabeled wild-type cell extracts, radioactive IPC was converted to MIPC and M(IP)2C
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