Glycosylphosphatidylinositol (GPI) Proteins of Saccharomyces cerevisiae Contain Ethanolamine Phosphate Groups on the α1,4-linked Mannose of the GPI Anchor
2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês
10.1074/jbc.m401873200
ISSN1083-351X
AutoresIsabella Imhof, Isabelle Flury, Christine Vionnet, Carole Roubaty, Diane Egger, Andreas Conzelmann,
Tópico(s)Lysosomal Storage Disorders Research
ResumoIn humans and Saccharomyces cerevisiae the free glycosylphosphatidylinositol (GPI) lipid precursor contains several ethanolamine phosphate side chains, but these side chains had been found on the protein-bound GPI anchors only in humans, not yeast. Here we confirm that the ethanolamine phosphate side chain added by Mcd4p to the first mannose is a prerequisite for the addition of the third mannose to the GPI precursor lipid and demonstrate that, contrary to an earlier report, an ethanolamine phosphate can equally be found on the majority of yeast GPI protein anchors. Curiously, the stability of this substituent during preparation of anchors is much greater in gpi7Δ sec18 double mutants than in either single mutant or wild type cells, indicating that the lack of a substituent on the second mannose (caused by the deletion of GPI7) influences the stability of the one on the first mannose. The phosphodiester-linked substituent on the second mannose, probably a further ethanolamine phosphate, is added to GPI lipids by endoplasmic reticulum-derived microsomes in vitro but cannot be detected on GPI proteins of wild type cells and undergoes spontaneous hydrolysis in saline. Genetic manipulations to increase phosphatidylethanolamine levels in gpi7Δ cells by overexpression of PSD1 restore cell growth at 37 °C without restoring the addition of a substituent to Man2. The three putative ethanolamine-phosphate transferases Gpi13p, Gpi7p, and Mcd4p cannot replace each other even when overexpressed. Various models trying to explain how Gpi7p, a plasma membrane protein, directs the addition of ethanolamine phosphate to mannose 2 of the GPI core have been formulated and put to the test. In humans and Saccharomyces cerevisiae the free glycosylphosphatidylinositol (GPI) lipid precursor contains several ethanolamine phosphate side chains, but these side chains had been found on the protein-bound GPI anchors only in humans, not yeast. Here we confirm that the ethanolamine phosphate side chain added by Mcd4p to the first mannose is a prerequisite for the addition of the third mannose to the GPI precursor lipid and demonstrate that, contrary to an earlier report, an ethanolamine phosphate can equally be found on the majority of yeast GPI protein anchors. Curiously, the stability of this substituent during preparation of anchors is much greater in gpi7Δ sec18 double mutants than in either single mutant or wild type cells, indicating that the lack of a substituent on the second mannose (caused by the deletion of GPI7) influences the stability of the one on the first mannose. The phosphodiester-linked substituent on the second mannose, probably a further ethanolamine phosphate, is added to GPI lipids by endoplasmic reticulum-derived microsomes in vitro but cannot be detected on GPI proteins of wild type cells and undergoes spontaneous hydrolysis in saline. Genetic manipulations to increase phosphatidylethanolamine levels in gpi7Δ cells by overexpression of PSD1 restore cell growth at 37 °C without restoring the addition of a substituent to Man2. The three putative ethanolamine-phosphate transferases Gpi13p, Gpi7p, and Mcd4p cannot replace each other even when overexpressed. Various models trying to explain how Gpi7p, a plasma membrane protein, directs the addition of ethanolamine phosphate to mannose 2 of the GPI core have been formulated and put to the test. Many glycoproteins of lower and higher eukaryotes are attached to the plasma membrane by means of a glycosylphosphatidylinositol (GPI) 1The abbreviations used are: GPI, glycosylphosphatidylinositol; CP, complete precursor; Chx, cycloheximide; EtN, ethanolamine; EtN-P, ethanolamine phosphate; Ins, myo-inositol; JBAM, jack bean α-mannosidase; HF, hydrofluoric acid; Man, mannose; MIPC, mannosyl-IPC; M(IP)2C, inositolphosphoryl-MIPC; PE, phosphatidylethanolamine; PI, phosphatidylinositol; WT, wild type; ER, endoplasmic reticulum; PLC, phospholipase C; UPR, unfolded protein response; ConA, concanavalin A. (1McConville M.J. Ferguson M.A. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (803) Google Scholar, 2Takeda J. Kinoshita T. Trends Biochem. Sci. 1995; 20: 367-371Abstract Full Text PDF PubMed Scopus (131) Google Scholar). The carbohydrate structure linking the C-terminal end of GPI proteins to the lipid moiety is identical in GPI anchors from all organisms analyzed so far, namely protein-CO-NH-(CH2)-PO4-6Manα1–2Manα1–6Manα1–4GlcNH2-inositol-PO4-lipid but the GPI anchors from various species differ widely with regard to the side chains attached to this core structure as well as the lipid moieties of the anchor (1McConville M.J. Ferguson M.A. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (803) Google Scholar). This report concerns the ethanolamine phosphate (EtN-P) side chains, which are often present on mannoses 1 and 2 of the core structure (Man1 and Man2 in Fig. 1). Indeed, an EtN-P is invariably found on Man1 of GPI proteins and on the GPI lipids in mammals and in Torpedo californica, but is not found in Trypanosoma brucei, Leishmania major, or Plasmodium falciparum (3McConville M.J. Collidge T.A. Ferguson M.A. Schneider P. J. Biol. 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Chem. 1993; 268: 9998-10002Abstract Full Text PDF PubMed Google Scholar). EtN-P attached to Man1 and Man2, respectively, has also been identified on the complete GPI precursor lipid CP2 of Saccharomyces cerevisiae (11Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar, 12Benachour A. Sipos G. Flury I. Reggiori F. Canivenc-Gansel E. Vionnet C. Conzelmann A. Benghezal M. J. Biol. Chem. 1999; 274: 15251-15261Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 13Imhof I. Canivenc-Gansel E. Meyer U. Conzelmann A. Glycobiology. 2000; 10: 1271-1275Crossref PubMed Scopus (33) Google Scholar) although the identity of the substituent on Man2 has not been formally demonstrated. The presence of these side chains on CP2 came as a surprise in as much as a previous analysis of the pool of the protein-linked GPI anchors of S. cerevisiae had failed to reveal EtN-P or other substituents on Man1 or Man2 (14Fankhauser C. Homans S.W. Thomas-Oates J.E. McConville M.J. Desponds C. Conzelmann A. Ferguson M.A. J. Biol. Chem. 1993; 268: 26365-26374Abstract Full Text PDF PubMed Google Scholar). Candidate EtN-P transferase genes required for the attachment of these substituents have been identified in humans and yeast: PIG-N and MCD4 are involved in the transfer of EtN-P from phosphatidylethanolamine (PE) onto Man1 (13Imhof I. Canivenc-Gansel E. Meyer U. Conzelmann A. Glycobiology. 2000; 10: 1271-1275Crossref PubMed Scopus (33) Google Scholar, 15Gaynor E.C. Mondesert G. Grimme S.J. Reed S.I. Orlean P. Emr S.D. Mol. Biol. Cell. 1999; 10: 627-648Crossref PubMed Scopus (112) Google Scholar, 16Hong Y. Maeda Y. Watanabe R. Ohishi K. Mishkind M. Riezman H. Kinoshita T. J. Biol. Chem. 1999; 274: 35099-35106Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), GPI7 in the transfer onto Man2 (12Benachour A. Sipos G. Flury I. Reggiori F. Canivenc-Gansel E. Vionnet C. Conzelmann A. Benghezal M. J. Biol. Chem. 1999; 274: 15251-15261Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and PIG-O and GPI13 in the transfer of EtN-P from PE onto Man3 (17Hong Y. Maeda Y. Watanabe R. Inoue N. Ohishi K. Kinoshita T. J. Biol. Chem. 2000; 275: 20911-20919Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 18Taron C.H. Wiedman J.M. Grimme S.J. Orlean P. Mol. Biol. Cell. 2000; 11: 1611-1630Crossref PubMed Scopus (59) Google Scholar, 19Flury I. Benachour A. Conzelmann A. J. Biol. Chem. 2000; 275: 24458-24465Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 20Menon A.K. Stevens V.L. J. Biol. Chem. 1992; 267: 15277-15280Abstract Full Text PDF PubMed Google Scholar, 21Menon A.K. Eppinger M. Mayor S. Schwarz R.T. EMBO J. 1993; 12: 1907-1914Crossref PubMed Scopus (80) Google Scholar). These genes are homologous to each other, are found throughout the eukaryotic kingdom, possess an N-terminal globular domain facing the lumen of the ER or the extracellular space and multiple transmembrane domains in their C terminus. They are good candidates for the EtN-P transferases themselves, as the N-terminal, globular domains have a distinct homology with verified phosphodiesterases and because the corresponding mutants accumulate GPI lipid intermediates that lack EtN-P residues. A further gene named PIG-F has been implied in the addition of EtN-P side chains, as PIG-F mutants fail to add EtN-P to Man3 in mammalian cells (8Kamitani T. Menon A.K. Hallaq Y. Warren C.D. Yeh E.T. J. Biol. Chem. 1992; 267: 24611-24619Abstract Full Text PDF PubMed Google Scholar, 9Puoti A. Conzelmann A. J. Biol. Chem. 1993; 268: 7215-7224Abstract Full Text PDF PubMed Google Scholar, 22Sugiyama E. DeGasperi R. Urakaze M. Chang H.M. Thomas L.J. Hyman R. Warren C.D. Yeh E.T. J. Biol. Chem. 1991; 266: 12119-12122Abstract Full Text PDF PubMed Google Scholar, 23Inoue N. Kinoshita T. Orii T. Takeda J. J. Biol. Chem. 1993; 268: 6882-6885Abstract Full Text PDF PubMed Google Scholar). GPI11, the yeast homologue of PIG-F, is not required for the addition of EtN-P to Man3 but may be required for the addition of a hydrofluoric acid (HF) labile substituent to Man2 (18Taron C.H. Wiedman J.M. Grimme S.J. Orlean P. Mol. Biol. Cell. 2000; 11: 1611-1630Crossref PubMed Scopus (59) Google Scholar). In yeast, deletion of MCD4, GPI11, or GPI13 is lethal, whereas deletion of GPI7 only compromises cell wall integrity (15Gaynor E.C. Mondesert G. Grimme S.J. Reed S.I. Orlean P. Emr S.D. Mol. Biol. Cell. 1999; 10: 627-648Crossref PubMed Scopus (112) Google Scholar, 18Taron C.H. Wiedman J.M. Grimme S.J. Orlean P. Mol. Biol. Cell. 2000; 11: 1611-1630Crossref PubMed Scopus (59) Google Scholar, 19Flury I. Benachour A. Conzelmann A. J. Biol. Chem. 2000; 275: 24458-24465Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 24Toh-e A. Oguchi T. Genes Genet. Syst. 1998; 73: 365-375Crossref PubMed Scopus (10) Google Scholar). This is not unexpected for GPI13, as in all GPI proteins analyzed it invariably is the EtN-P on Man3 that links the GPI to the protein, and even in mutants that cannot add EtN-P to Man3 or cannot add Man3, the GPI proteins were never found to be attached through an EtN-P on Man1 or Man2. Whereas at present we have a good overall picture of the major stages of the GPI biosynthesis pathway, many questions concerning the sequence of events and their importance for the biosynthetic process remain to be elucidated. One particular difficulty is to know which GPI lipid is attached to nascent proteins by the GPI transamidase complex in the ER. Even though there is a wealth of partial structures of lipids accumulating in mutants that are unable to synthesize or to attach GPI lipids to proteins, or of lipids that are synthesized by microsomes in vitro, it is doubtful that all these lipids are normal intermediates of the pathway. Thus, it cannot be excluded a priori that under physiological conditions certain EtN-Ps are added to GPI anchors only after the GPI lipid has been added to proteins or the protein has left the ER. Here we describe our attempts to address a few of the several unresolved questions about the biosynthetic route and the role of the EtN-P residues on Man1 and Man2 in yeast. Strains, Media, and Materials—Saccharomyces strains are listed in Table I. Cells were grown on rich medium (YPD) or minimal media SDaaUA or SGaaUA, containing 2% glucose (D) or galactose (G) at 30 °C and amino acids (designated with aa), uracil (U), and adenine (A) but without inositol (Ins) (25Sherman F. Methods Enzymol. 2002; 350: 3-41Crossref PubMed Scopus (983) Google Scholar). Chemicals, radiochemicals, and inhibitors were from sources described (11Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar). PI-specific PLC (PI-PLC) from Bacillus cereus was from ICN Biomedicals Inc. (number 195685) (Aurora, OH) or Roche Molecular Biochemicals (number 1-143-069) (Rotkreuz, Switzerland); GPI-PLD purified from bovine serum was the kind gift of Dr. U. Brodbeck. Pentoxifylline, dipyramidole, and ethaverine were from Sigma, 3-isobutyl-1-methylxanthine and papaverine were from Fluka, Buchs, Switzerland. Pronase for anchor preparation was from Sigma, catalog number P-5147, or Roche Molecular Biochemicals, nuclease-free, catalog number 165-921. Concanavalin (ConA)-Sepharose was from Amersham Biosciences (number 17-0440-01).Table IS. cerevisiae strainsStrainGenotypeSourceX2180-1AMATa lys--canrRandy SchekmanSF 226-1CMATa sec12-4Randy SchekmanHMSF176MATa sec18-1Randy SchekmanFBY182MATα ade2-1 ura3-1 leu2-3,112 his3-11,15 gpi7::KanMX4Ref. 12Benachour A. Sipos G. Flury I. Reggiori F. Canivenc-Gansel E. Vionnet C. Conzelmann A. Benghezal M. J. Biol. Chem. 1999; 274: 15251-15261Abstract Full Text Full Text PDF PubMed Scopus (110) Google ScholarFBY15MATα ade2-1 ura3-1 leu2-3,112 trp1-1 his3-11,15 gpi7-1Ref. 12Benachour A. Sipos G. Flury I. Reggiori F. Canivenc-Gansel E. Vionnet C. Conzelmann A. Benghezal M. J. Biol. Chem. 1999; 274: 15251-15261Abstract Full Text Full Text PDF PubMed Scopus (110) Google ScholarFBY49MATa sec18-1 gpi7::KanMX4Ref. 12Benachour A. Sipos G. Flury I. Reggiori F. Canivenc-Gansel E. Vionnet C. Conzelmann A. Benghezal M. J. Biol. Chem. 1999; 274: 15251-15261Abstract Full Text Full Text PDF PubMed Scopus (110) Google ScholarFBY91gpi8-1 sec18-1 leu2-3,112This studyFBY115MATα ade2-1 ura3-1 trp1-1 lys-gpi7-1FBY122MATa ura3-1, ade2-1, his3-11,15, trp1-1, leu2-3,112 gpi8-1 gpi7-1Ref. 49Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelmann A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google ScholarYAT2626MATa leu2, his3, trp1, ura3, ade2, can1, gpi7::LEU2Ref. 33Toh-e A. Oguchi T. Genes Genet. Syst. 2002; 77: 309-322Crossref PubMed Scopus (13) Google ScholarZY400MATa ade2-101 leu2-3,112 ura3-52 suc2-9 mnn9::URA3 gal2 pep4::CATClint Ballou17A-H42MATa trp1-289 ura 3-52 leu2Ref. 38Packeiser A.N. Urakov V.N. Polyakova Y.A. Shimanova N.I. Shcherbukhin V.D. Smirnov V.N. Ter-Avanesyan M.D. Yeast. 1999; 15: 1485-1501Crossref PubMed Scopus (21) Google Scholar521-17A-H42MATa trp1-289 ura 3-52 leu2 ssu21 (=mcd4ts)Ref. 38Packeiser A.N. Urakov V.N. Polyakova Y.A. Shimanova N.I. Shcherbukhin V.D. Smirnov V.N. Ter-Avanesyan M.D. Yeast. 1999; 15: 1485-1501Crossref PubMed Scopus (21) Google ScholarFBY413MATa leu2-Δ1 trp1-Δ63 his3-Δ200 ura3-52Ref. 19Flury I. Benachour A. Conzelmann A. J. Biol. Chem. 2000; 275: 24458-24465Abstract Full Text Full Text PDF PubMed Scopus (57) Google ScholarFBY1102MATa leu2-Δ1 trp1-Δ63 his3-Δ200 ura3-52 gpi13::HIS3-GAL1UAS-GPI13Ref. 19Flury I. Benachour A. Conzelmann A. J. Biol. Chem. 2000; 275: 24458-24465Abstract Full Text Full Text PDF PubMed Scopus (57) Google ScholarFBY1104MATa leu2-Δ1 trp1-Δ63 his3-Δ200 ura3-52 mcd4::HIS3 GAL1UAS-MCD4 YNR019w::kanMX4Ref. 19Flury I. Benachour A. Conzelmann A. J. Biol. Chem. 2000; 275: 24458-24465Abstract Full Text Full Text PDF PubMed Scopus (57) Google ScholarC4MATa leu2-3,112 ura3-52 pmi 40Ref. 30Sipos G. Puoti A. Conzelmann A. EMBO J. 1994; 13: 2789-2796Crossref PubMed Scopus (67) Google ScholarFBY166gpi8::kanMX ura3-1::URA3-GAL1UAS-GPI8 ade2-1 his3-11,15 leu2-3,112 trp1-1 lys+Ref. 50Meyer U. Benghezal M. Imhof I. Conzelmann A. Biochemistry. 2000; 39: 3461-3471Crossref PubMed Scopus (71) Google ScholarFBY182MATα ade2-1 ura3-1 leu2-3,112 his3-11,15 gpi7::KanMX4Ref. 12Benachour A. Sipos G. Flury I. Reggiori F. Canivenc-Gansel E. Vionnet C. Conzelmann A. Benghezal M. J. Biol. Chem. 1999; 274: 15251-15261Abstract Full Text Full Text PDF PubMed Scopus (110) Google ScholarFBY631MATa ade2-1, his3-11,15, leu2-3,112, trp1-1, gpi10-1This studyCWH4MATα ura3-52 lys2 gpi1Frans Klis Open table in a new tab Preparation of Radiolabeled GPI Protein Anchor Peptides and Anchor Peptide Head Groups—Exponentially growing cells were labeled with myo-[2-3H]inositol in Ins-free SDaaUA as described (26Guillas I. Pfefferli M. Conzelmann A. Methods Enzymol. 2000; 312: 506-515Crossref PubMed Google Scholar). Washed cells were broken with glass beads in chloroform/methanol (1:1), and proteins were delipidated in chloroform/methanol/water (10:10:3), and then Lester solvent (ethanol/water/diethyl ether/pyridine/concentrated NH4OH (15:15:5:1:0.018)) at 37 °C for 15 min as described (26Guillas I. Pfefferli M. Conzelmann A. Methods Enzymol. 2000; 312: 506-515Crossref PubMed Google Scholar, 27Hanson B.A. Lester R.L. J. Lipid Res. 1980; 21: 309-315Abstract Full Text PDF PubMed Google Scholar). In some experiments (Table IV) the latter solvent was replaced by chloroform, methanol, 1.5 mm triethylamine (10:10:3) or proteins were extracted without preliminary organic solvent extraction (Table V) by just boiling cells in sample buffer, or by breaking cells with glass beads in TPIN buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 30 μg/ml leupeptin, pepstatin, and antipain) or by incubation for 5 min at room temperature in 100 mm NaOH as described (28Kushnirov V.V. Yeast. 2000; 16: 857-860Crossref PubMed Scopus (674) Google Scholar). Proteins were solubilized by boiling in sample buffer K (60 mm Tris-HCl, pH 6.8, 5% glycerol, 2% SDS, 4% 2-mercaptoethanol, 0.0025% bromphenol blue). Anchor peptides were then prepared essentially as outlined in Fig. 3 and described before (26Guillas I. Pfefferli M. Conzelmann A. Methods Enzymol. 2000; 312: 506-515Crossref PubMed Google Scholar) except that the number of delipidation and washing steps was reduced to obtain quantitative recovery rather than full delipidation of anchor peptides and the ConA-Sepharose buffer was 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 1 mm each of CaCl2, MgCl2, MnCl2, phenylmethylsulfonyl fluoride, and benzamidine. Soluble head groups were obtained from purified radiolabeled anchor peptides through limiting methanolic NH3 deacylation (29Roberts W.L. Myher J.J. Kuksis A. Low M.G. Rosenberry T.L. J. Biol. Chem. 1988; 263: 18766-18775Abstract Full Text PDF PubMed Google Scholar) followed by PI-PLC treatment, for which the peptides were dissolved in 20 mm Tris-HCl, pH 7.5, 0.2 mm EDTA, 20% 1-propanol or by GPI-PLD treatment in 50 mm Tris-HCl, pH 7.4, 10 mm NaCl, 20% propanol, 2.6 mm CaCl2. Incubations with PI-PLC or GPI-PLD were for 16 h at 37 °C. Lipids were removed by butanol extraction.Table IVX2180–1A and sec18–1 gpi7Δ cells do not contain trans-acting factors influencing the stability of EtN-P groups during the extraction of GPI anchor head groupsAcetolysis/JBAM/HF/NAcAcetolysis/HF/NAcAcetolysis/HF/NAc/JBAMSubstitution on Man1%X2180-1A*/X2180-1A14.291.0015.6X2180-1A*/X2180-1A + PDI + TEA14.286.6016.4X2180-1A*/sec18-1 gpi7Δ13.590.3015.0sec18-1 gpi7Δ*/X2180-1A + PDI90.492.35097.95sec18-1gpi7Δ*/sec18-1gpi7Δ91.396.3094.85sec18-1 gpi7Δ*/sec18-1gpi7Δ + PDI91.695.0096.45 Open table in a new tab Table VOmission of protein delipidation by organic solvents during the isolation of GPI anchors increases the stability of ethanolaminephosphate on Man1Strains/extraction conditionsAcetolysis/JBAM/HF/NAcAcetolysis/HF/NAcAcetolysis/HF/NAc/JBAMSubstitution on Man1%X2180-1A (wt) boil in SDS55.891.2051.5sec18-1 gpi7 Δ boil in SDS93.694.5098.1X2180-1A (wt) break with glass beads in Tris buffer, then boil in SDS65.491.5071.5X2180-1A, treated with 0.1 m NaOH for 5 min, boil in SDS64.590.6071.2 Open table in a new tab Testing Enzymes for Contaminating Phosphodiesterase Activity Removing EtN-P Side Chains—Radiolabeled CP lipids were generated by metabolic labeling of pmi40 with [3H]mannose (30Sipos G. Puoti A. Conzelmann A. EMBO J. 1994; 13: 2789-2796Crossref PubMed Scopus (67) Google Scholar) or sec18 gpi8 double mutants with myo-[2-3H]Ins. CP lipids were purified by preparative TLC and then incubated under exactly the same conditions as used for preparing GPI anchor peptides. In some experiments we added a mixture of the phosphodiesterase inhibitors pentoxifylline, dipyramidole, ethaverine, 3-isobutyl-1-methylxanthine and papaverine at 1, 0.25, 0.1, 0.1, and 0.25 mm final concentrations, respectively. To test for a phosphodiesterase activity that would remove EtN-P, CaCl2 in GPI-PLD buffer was replaced by 10 mm EDTA so that the GPI-PLD itself was inactive (31Davitz M.A. Hom J. Schenkman S. J. Biol. Chem. 1989; 264: 13760-13764Abstract Full Text PDF PubMed Google Scholar). After incubation the potential degradation of CP lipids and the appearance of less polar lipids was assessed by TLC in solvent 1 followed by radioscanning/fluorography. Analysis of Head Groups—Liberated head groups were subjected to acetolysis, JBAM and HF treatments, N-acetylation using methods listed in Ref. 11Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar, and paper chromatography in solvent methyl ethyl ketone/pyridine/water (20:12:11) (9Puoti A. Conzelmann A. J. Biol. Chem. 1993; 268: 7215-7224Abstract Full Text PDF PubMed Google Scholar). myo-[14C]Ins was added to each sample as an internal standard before paper chromatography allowing for exact positioning of Man-GlcNAc-Ins and GlcNAc-Ins peaks. Lipid Analysis—Lipids were extracted from labeled cells using CHCl3/CH3OH/H2O (10:10:3), desalted by butanol/water partitioning, and analyzed by TLC on Silica Gel 60 plates using the same solvent (solvent 1) followed by fluorography. Biosynthesis of GPI Lipids in Vitro—For GPI biosynthesis in microsomes in vitro we followed a previously used protocol (11Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar). Briefly, spheroplasts were generated by incubation for 60 min at 37 °C in buffer A (10 mm azide, 1.4 m sorbitol, 50 mm K2HPO4, pH 7.5, 40 mm 2-mercaptoethanol) using Zymolyase (0.2 mg/ml) or Quantazyme (3 units/1 A600 unit of cells), spheroplasts were washed 2 times in the same buffer but without 2-mercaptoethanol, were broken by forcing them through a 0.4-mm needle using a syringe, the cell lysate was centrifuged at 4 °C at 3000 × g for 5 min, and then 75,000 × g for 60 min. Pellets P3 and P75 were resuspended in 0.8 m sorbitol, 10 mm triethanolamine, pH 7.2, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 2 μg/ml pepstatin, 2 μg/ml chymostatin, 2 μg/ml antipain and could be frozen at that stage. For the standard assay the two pellets were pooled, diluted into 100 mm Tris-HCl, pH 7.5, 3 mm MgCl2, 0.5 mm MnCl2, 1 mm EGTA, 1 mm ATP, 1 mm CoA, 1 mm GDP-mannose, 20 μg/ml tunicamycine, and 50 μg/ml nikkomycin and incubated in a final volume of 100 μl with 3–6 μCi of UDP-[3H]GlcNAc for 60–90 min at 30 °C. Plasmids and Plasmid Construction—A pUPRE expressing lacZ from an artificial promoter containing UPRE was obtained from Dr. Ralph Menzel, Berlin (32Menzel R. Vogel F. Kargel E. Schunck W.H. Yeast. 1997; 13: 1211-1229Crossref PubMed Scopus (43) Google Scholar). For Fig. 10A we used multicopy TOp2141, TOp662, or YEp24 containing PSD1, ECM33, or no insert that all were contributed by Dr. Akio Toh-e (33Toh-e A. Oguchi T. Genes Genet. Syst. 2002; 77: 309-322Crossref PubMed Scopus (13) Google Scholar). pBF111 containing GPI7 with a deletion of amino acids 192–321 was constructed by opening pBF43 (YCplac33 containing GPI7 behind its own promoter) with SmaI and BssHII. The gap was filled with the EcoRV-BssHII fragment excised from the same plasmid, pBF43. pBF112 containing GPI7 with a deletion of amino acids 54–191 was generated by opening pBF43 with PflMI, blunting with Klenow polymerase, and then cutting with BssHII. The thus generated gap was filled with a SmaI-BssHII fragment from pBF43. pBF113 was constructed by amplifying the N-terminal part of Gpi7p by PCR using primers 5′-agacgttcaacaaattgatatcgt-3′ and 5′-cgcgACGCGTACCGGTcaaaagaggataattataatttgt-3′ having restriction sites MluI and AgeI (uppercase). The last 39 amino acids of Wbp1p (including its transmembrane domain and the KKXX motif), the stop codon plus the transcription terminator were amplified with primers 5′-cgcgACGCGTACCGGTtcttgggtttatattagcgccattt-3′ and 5′-cgcggaattcgagctcGGTACCccttaatacaaactgcaaaagagttt-3′. The two PCR fragments were cut with MluI and EcoRV and MluI and KpnI, respectively, and ligated into pBF43 opened with EcoRV and KpnI. pBF114 was constructed by amplifying nucleotides 409–1197 of GPI7 with primers 5′-tggCTGCAGcagttcatccaacata-3′ and 5′-gGTTTAAACGGTACCttacaattcatcgtgtgcagacttggttaacgtttcttga-3′. The PCR fragment was cut with PstI and KpnI and inserted into pBF43 opened with the same two enzymes. To obtain multicopy plasmids, the XhoI-KpnI fragments of pBF111, pBF112, pBF113, pBF114, and pBF43 were transferred into YEplac195, which was opened with SalI (same overhang as XhoI) and KpnI, thus yielding vectors pBF116, pBF117, pBF118, pBF119, and pBF120, respectively. In pBF121, Gpi7p contains an additional 6 amino acids (RSKKHQ) at its C terminus. pBF121 was obtained by amplifying the C terminus of Gpi7p with primers 5′-acttGCGCGCgttttcttccaa-3′ (uppercase is the RS of BssHII) and 5′-TCTATGTTAGTTTGTTTTTTTCGACCTatcaagagcgcaaaggaggg-3′ and amplifying the transcription terminator of GPI7 with primers 5′-AGGTCGAAAAAAACAAACTAAcatagaattgttcacgtggtctaaa-3′ (sequence encoding RSKKHQ in uppercase) and 5′-agtgagtacatGGTACCaccttattat-3′. The two PCR fragments were used as templates for crossed PCR and the final product was cut with BssHII and KpnI and then ligated into pBF43 that was opened in the same way. The multicopy version pBF122 was obtained as described above. Addition of Ethanolamine Phosphate to Man1 Is a Prerequisite for the Addition of Man3 by Gpi10p—During GPI biosynthesis, Man3 is transferred from dolicholphosphomannose to Man2 by PIG-B in mammals and its homologue GPI10 in yeast (11Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar, 34Takahashi M. Inoue N. Ohishi K. Maeda Y. Nakamura N. Endo Y. Fujita T. Takeda J. Kinoshita T. EMBO J. 1996; 15: 4254-4261Crossref PubMed Scopus (108) Google Scholar, 35Sutterlin C. Escribano M.V. Gerold P. Maeda Y. Mazon M.J. Kinoshita T. Schwarz R.T. Riezman H. Biochem. J. 1998; 332: 153-159Crossref PubMed Scopus (78) Google Scholar). Mammalian and yeast mutants in these genes accumulate Man-(EtN-P→)Man-GlcN-(acyl→)PI. When treated with the fungal inhibitor YW3548, yeast cells accumulate Man-Man-GlcN-(acyl→)PI and YW3548 has therefore been postulated to be an inhibitor of the EtN-P transferase PIG-N/MCD4 (35Sutterlin C. Escribano M.V. Gerold P. Maeda Y. Mazon M.J. Kinoshita T. Schwarz R.T. Riezman H. Biochem. J. 1998; 332: 153-159Crossref PubMed Scopus (78) Google Scholar, 36Sutterlin 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) than of Gpi10p, as initially proposed. This notion was confirmed by the finding that overexpression of MCD4 made yeast cells resistant to YW3548, whereas overexpression of GPI10 had a much smaller, albeit significant effect, especially at low inhibitor concentrations (16Hong Y. Maeda Y. Watanabe R. Ohishi K. Mishkind M. Riezman H. Kinoshita T. J. Biol. Chem. 1999; 274: 35099-35106Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 35Sutterlin C. Escribano M.V. Gerold P. Maeda Y. Mazon M.J. Kinoshita T. Schwarz R.T. Riezman H. Biochem. J. 1998; 332: 153-159Crossref PubMed Scopus (78) Google Scholar). The implication is that Gpi10p cannot add onto Man-Man-GlcN-(acyl→)PI and requires a substrate that contains EtN-P on Man1. This hypothesis, however, was difficult to reconcile with the occurrence of major GPI lipids accumulating in mutants such as smp3 (lipid 3-1-2), gpi11 (lipid 11-2), and gpi13 (part of lipid a) as these lipids contain 3 or 4 mannoses but lack EtN-P on Man1. In this context it was of interest to know the structure of the intermediates accumulating in mcd4 mutants. Several mcd4 mutants had previously been labeled with [3H]inositol but accumulated only very small amounts of Ins-labeled GPI lipids that could not be structurally analyzed (15Gaynor E.C. Mondesert G. Grimme S.J. Reed S.I. Orlean P. Emr S.D. Mol. Biol. Cell. 1999; 10: 627-648Crossref PubMed Scopus (112) Google Scholar, 19Flury I. Benachour A. Conzelmann A. J. Biol. Chem. 2000; 275: 24458-24465Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 37Storey M.K. Wu W.I. Voelker D.R. Biochim. Biophys. Acta. 2001; 1532: 234-247Crossref PubMed Scopus (19) Google Scholar). We therefo
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