FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1968271109> ?p ?o ?g. }

• W1968271109 endingPage "15261" @default.
• W1968271109 startingPage "15251" @default.
• W1968271109 abstract "Gpi7 was isolated by screening for mutants defective in the surface expression of glycosylphosphatidylinositol (GPI) proteins. Gpi7 mutants are deficient in YJL062w, herein named GPI7. GPI7 is not essential, but its deletion renders cells hypersensitive to Calcofluor White, indicating cell wall fragility. Several aspects of GPI biosynthesis are disturbed in Δgpi7. The extent of anchor remodeling, i.e. replacement of the primary lipid moiety of GPI anchors by ceramide, is significantly reduced, and the transport of GPI proteins to the Golgi is delayed. Gpi7p is a highly glycosylated integral membrane protein with 9–11 predicted transmembrane domains in the C-terminal part and a large, hydrophilic N-terminal ectodomain. The bulk of Gpi7p is located at the plasma membrane, but a small amount is found in the endoplasmic reticulum. GPI7 has homologues inSaccharomyces cerevisiae, Caenorhabditis elegans, and man, but the precise biochemical function of this protein family is unknown. Based on the analysis of M4, an abnormal GPI lipid accumulating in gpi7, we propose that Gpi7p adds a side chain onto the GPI core structure. Indeed, when compared with complete GPI lipids, M4 lacks a previously unrecognized phosphodiester-linked side chain, possibly an ethanolamine phosphate. Gpi7p contains significant homology with phosphodiesterases suggesting that Gpi7p itself is the transferase adding a side chain to the α1,6-linked mannose of the GPI core structure. Gpi7 was isolated by screening for mutants defective in the surface expression of glycosylphosphatidylinositol (GPI) proteins. Gpi7 mutants are deficient in YJL062w, herein named GPI7. GPI7 is not essential, but its deletion renders cells hypersensitive to Calcofluor White, indicating cell wall fragility. Several aspects of GPI biosynthesis are disturbed in Δgpi7. The extent of anchor remodeling, i.e. replacement of the primary lipid moiety of GPI anchors by ceramide, is significantly reduced, and the transport of GPI proteins to the Golgi is delayed. Gpi7p is a highly glycosylated integral membrane protein with 9–11 predicted transmembrane domains in the C-terminal part and a large, hydrophilic N-terminal ectodomain. The bulk of Gpi7p is located at the plasma membrane, but a small amount is found in the endoplasmic reticulum. GPI7 has homologues inSaccharomyces cerevisiae, Caenorhabditis elegans, and man, but the precise biochemical function of this protein family is unknown. Based on the analysis of M4, an abnormal GPI lipid accumulating in gpi7, we propose that Gpi7p adds a side chain onto the GPI core structure. Indeed, when compared with complete GPI lipids, M4 lacks a previously unrecognized phosphodiester-linked side chain, possibly an ethanolamine phosphate. Gpi7p contains significant homology with phosphodiesterases suggesting that Gpi7p itself is the transferase adding a side chain to the α1,6-linked mannose of the GPI core structure. Glycosylphosphatidylinositol (GPI) 1The abbreviations used are: GPI, glycosylphosphatidylinositol; ASAM, A. satoiα-mannosidase; CP, complete precursor; DAG, diacylglycerol; DHS, dihydrosphingosine; EtN-P, ethanolamine phosphate; GPI-PLD, GPI-specific phospholipase D; Ins, myo-inositol; JBAM, jack bean α-mannosidase; Man, mannose; ORF, open reading frame; pC1 and pC2, protein-derived Ceramides 1and 2; pG1 protein-derivedGlycerophospholipid 1, PI, phosphatidylinositol; PM, plasma membrane; ts, thermosensitive; wt, wild type; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; nt, nucleotide; PCR, polymerase chain reaction; kb, kilobase pair(s); HPLC, high pressure liquid chromatography. 1The abbreviations used are: GPI, glycosylphosphatidylinositol; ASAM, A. satoiα-mannosidase; CP, complete precursor; DAG, diacylglycerol; DHS, dihydrosphingosine; EtN-P, ethanolamine phosphate; GPI-PLD, GPI-specific phospholipase D; Ins, myo-inositol; JBAM, jack bean α-mannosidase; Man, mannose; ORF, open reading frame; pC1 and pC2, protein-derived Ceramides 1and 2; pG1 protein-derivedGlycerophospholipid 1, PI, phosphatidylinositol; PM, plasma membrane; ts, thermosensitive; wt, wild type; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; nt, nucleotide; PCR, polymerase chain reaction; kb, kilobase pair(s); HPLC, high pressure liquid chromatography.-anchored proteins represent a subclass of surface proteins found in virtually all eukaryotic organisms (1McConville M.J. Ferguson M.A. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (801) Google Scholar). The genome of Saccharomyces cerevisiae contains more than 70 open reading frames (ORFs) encoding for proteins that, as judged from the deduced primary sequence, can be predicted to be modified by the attachment of a GPI anchor (2Hamada K. Fukuchi S. Arisawa M. Baba M. Kitada K. Mol. Gen. Genet. 1998; 258: 53-59Crossref PubMed Scopus (99) Google Scholar, 3Caro L.H. Tettelin H. Vossen J.H. Ram A.F. van den Ende H. Klis F.M. Yeast. 1997; 13: 1477-1489Crossref PubMed Scopus (281) Google Scholar). In about 25 of them, the presence of an anchor has been confirmed biochemically. A majority of them lose part of the anchor and become covalently attached to the β1,6-glucans of the cell wall (4Lu C.-F. Montijn R.C. Brown J.L. Klis F. Kurjan J. Bussey H. Lipke P.N. J. Cell Biol. 1995; 128: 333-340Crossref PubMed Scopus (188) Google Scholar, 5de-Nobel H. Lipke P.N. Trends Cell Biol. 1994; 4: 42-45Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 6Kollar R. Reinhold B.B. Petrakova E. Yeh H.J. Ashwell G. Drgonova J. Kapteyn J.C. Klis F.M. Cabib E. J. Biol. Chem. 1997; 272: 17762-17775Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). A minority of GPI proteins retain the GPI anchor in an intact form and stay at the plasma membrane (PM). For the biosynthesis of GPI anchors, phosphatidylinositol (PI) is modified by the stepwise addition of sugars and ethanolamine phosphate (EtN-P), thus forming a complete precursor lipid (CP) which subsequently is transferred en bloc by a transamidase onto newly synthesized proteins in the ER (7Takeda J. Kinoshita T. Trends Biochem. Sci. 1995; 4: 367-371Abstract Full Text PDF Scopus (130) Google Scholar, 8Ramalingam S. Maxwell S.E. Medof M.E. Chen R. Gerber L.D. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7528-7533Crossref PubMed Scopus (31) Google Scholar). The identification of genes involved in the biosynthesis of the CP and its subsequent attachment to proteins has been possible through the complementation of mammalian and yeast gpi− mutants, i.e. mutants being deficient in GPI anchoring of membrane proteins (7Takeda J. Kinoshita T. Trends Biochem. Sci. 1995; 4: 367-371Abstract Full Text PDF Scopus (130) Google Scholar, 9Takeda J. Miyata T. Kawagoe K. Iida Y. Endo Y. Fujita T. Takahashi M. Kitani T. Kinoshita T. Cell. 1993; 73: 703-711Abstract Full Text PDF PubMed Scopus (830) Google Scholar, 10Inoue N. Kinoshita T. Orii T. Takeda J. J. Biol. Chem. 1993; 268: 6882-6885Abstract Full Text PDF PubMed Google Scholar, 11Takahashi 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, 12Miyata T. Takeda J. Iida Y. Yamada N. Inoue N. Takahashi M. Maeda K. Kitani T. Kinoshita T. Science. 1993; 259: 1318-1320Crossref PubMed Scopus (424) Google Scholar, 13Kamitani T. Chang H.M. Rollins C. Waneck G.L. Yeh E.T. J. Biol. Chem. 1993; 268: 20733-20736Abstract Full Text PDF PubMed Google Scholar, 14Leidich S.D. Kostova Z. Latek R.R. Costello L.C. Drapp D.A. Gray W. Fassler J.S. Orlean P. J. Biol. Chem. 1995; 270: 13029-13035Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 15Chen R. Udenfriend S. Prince G.M. Maxwell S.E. Ramalingam S. Gerber L.D. Knez J. Medof M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2280-2284Crossref PubMed Scopus (34) Google Scholar, 16Leidich S.D. Orlean P. J. Biol. Chem. 1996; 271: 27829-27837Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Leidich S.D. Drapp D.A. Orlean P. J. Biol. Chem. 1994; 269: 10193-10196Abstract Full Text PDF PubMed Google Scholar, 18Hamburger D. Egerton M. Riezman H. J. Cell Biol. 1995; 129: 629-639Crossref PubMed Scopus (149) Google Scholar, 19Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelmann A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar, 20Yu J. Nagarajan S. Knez J.J. Udenfriend S. Chen R. Medof M.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12580-12585Crossref PubMed Scopus (65) Google Scholar). In our laboratory, a series of recessive gpi− mutants (gpi4 to gpi10) has been obtained by screening for yeast mutants that are unable to display the GPI-anchored α-agglutinin (Sag1p) at the outer surface of the cell wall, although the synthesis and secretion of soluble proteins is normal (21Benghezal M. Lipke P.N. Conzelmann A. J. Cell Biol. 1995; 130: 1333-1344Crossref PubMed Scopus (74) Google Scholar, 22Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar). Here we report on the characterization of gpi7. Four independent gpi7 mutants accumulated M4, an abnormal GPI intermediate that is less hydrophilic than CP2, the precursor accumulating when the transfer of GPIs to proteins is interrupted (18Hamburger D. Egerton M. Riezman H. J. Cell Biol. 1995; 129: 629-639Crossref PubMed Scopus (149) Google Scholar,19Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelmann A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar, 21Benghezal M. Lipke P.N. Conzelmann A. J. Cell Biol. 1995; 130: 1333-1344Crossref PubMed Scopus (74) Google Scholar, 23Sipos G. Puoti A. Conzelmann A. EMBO J. 1994; 13: 2789-2796Crossref PubMed Scopus (67) Google Scholar). Our preliminary characterization of M4 had shown that deacylation by NH3 followed by HF treatment, used to hydrolyze selectively the phosphodiester bonds (Fig. 1), yielded the same Man4-GlcN-inositol fragment as CP2, and we speculated thatgpi7 mutants may be unable to add the EtN-P onto Man3 (Fig. 1) (21Benghezal M. Lipke P.N. Conzelmann A. J. Cell Biol. 1995; 130: 1333-1344Crossref PubMed Scopus (74) Google Scholar). Here we show that this speculation was wrong, that CP2 differs from M4 with regard to a previously unrecognized side chain attached to Man2 (Fig. 1), and that GPI7 is required for the attachment of this side chain. S. cerevisiae strains were FBY11 (MATa ade2-1 ura3-1 leu2-3,112 trp1-1 his3-11,15 gpi8-1), FBY15 (MATα ade2-1 ura3-1 leu2-3,112 trp1-1 his3-11,15 gpi7-1), W303-1B (MATα ade2-1 can1-100 ura3-1 leu2-3, 112 trp1-1 his3-11,15), X2180-1A (MATalys −), FBY122 (MATa ade2-1 ura3-1 leu2-3,112 trp1-1 his3-11,15 gpi8-1 gpi7-1), FBY182 (MATα ade2-1 ura3-1 leu2-3,112 his3-11,15 gpi7::KanMX4), HMSF176 (MATa sec18-1), FBY49 (MATasec18-1 gpi7::KanMX4), C4 (MATaura3-52 leu2-3,112 pmi40), HMSF331 (MATasec53-6), LB2134-3B (MATa mnn9), and YNS3-7A (MATa ura3 his − mnn1 och1::LEU2). Diploid strains were FBY118 (MATa/α ade2-1/ade2-1 ura3-1/ura3-1 leu2-3, 112/leu2-3,112 TRP1/trp1-1 his3-11,15/his3-11,15 LYS/lys −), FBY40 (MATa /α ade2-1/ade2-1 ura3-1/ura3-1 leu2-3,112/leu2-3,112 TRP1/trp1-1 his3-11,15/his3-11,15 LYS/lys − GPI7/gpi7-1), and FBY43 (MATa/α ade2-1/ade2-1 ura3-1/ura3-1 leu2-3,112/leu2-3,112 TRP1/trp1-1 his3-11,15/his3-11,15 LYS/lys − gpi7::KanMX4/gpi7::KanMX4). Maintenance and growth conditions have been described (19Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelmann A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar). The absorbance of dilute cell suspensions was measured in a 1-cm cuvette at 600 nm, and oneA 600 unit of cells corresponds to 1–2 × 107 cells depending on the strain. Escherichia coli strains were HB-101, XL1 blue, and M15 [pREP4] (Qiagen). Materials were obtained from the sources described recently (22Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar). Cysteamine was from Sigma; [3H]dihydrosphingosine was synthesized as described (24Reggiori F. Canivenc-Gansel E. Conzelmann A. EMBO J. 1997; 16: 3506-3518Crossref PubMed Scopus (101) Google Scholar); myriocin was a kind gift of Dr. N. Rao Movva (Novartis, Basel, Switzerland); antibodies to Och1p, alkaline phosphatase, Kex2p, and Wbp1p were kindly donated by Dr. Y. Jigami, National Institute of Bioscience and Human Technology, Ibaraki 305, Japan; Dr. S. Emr, Howard Hughes Medical Institute, University of California, San Diego; Dr. R. Fuller, University of Michigan Medical Center, Ann Arbor, MI; and Dr. M. Aebi, Mikrobiologisches Institut, ETH Zürich, Switzerland, respectively. TheGPI7 gene was cloned by complementation of the ts growth phenotype of the gpi7-1/gpi8-1 double mutant as described (19Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelmann A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar). The three plasmids complementing gpi7-1 contained a 4.3-kb common DNA restriction fragment that was partially sequenced by the dideoxy sequencing method (25Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52610) Google Scholar). The complementing insertSphI/SspI of 3.6 kb was cloned into theSphI/SmaI-digested YEp352 multicopy vector (26Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1993; 2: 163-167Crossref Scopus (1080) Google Scholar) or YCplac33 single copy vector (27Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2515) Google Scholar) to generate pBF41 (Fig. 3C) and pBF43, respectively. One step disruption of GPI7 was done as described (28Wach A. Brachat A. Pöhlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2235) Google Scholar). Briefly, the 1.5-kb long KanMX4 module was PCR-amplified by using pFA6a-KanMX4 as template and the following two adapter primers: GPI7-forwards (5′-CTTCACCAAGTTAGCAAGATGAACTTGAAGCAGTTCACGTGCCtcgatgaattcgagctc-3′) with 17 nucleotides (nt) of homology to the pFA6a-KanMX4 multiple cloning site (in lowercase) and 43 nt of homology toGPI7 (in uppercase) starting 18 nt upstream of the start codon (bold); GPI7-backwards (5′-ATCAAGAGCGCAAAGGAGGGCCAATTCAGGTAACCAGCCATTCAcgtacgctgcaggtcgac-3′) with 18 nt of homology to the pFA6a-KanMX4 multiple cloning site (lowercase) and 44 nt of homology to the ORF ofGPI7 in the region immediately upstream of the stop codon. This PCR DNA fragment was used to transform the diploid strain FBY118, homozygous for GPI7, and FBY40, a heterozygousgpi7-1/GPI7 strain. The correct targeting of the PCR-made KanMX4 module into the GPI7 locus in geneticin-resistant clones was verified by PCR with whole yeast cells using primers GPI7-plus (5′-GTTCATCTACCACGCAC-3′) starting 36 nt upstream of the ATG, GPI7-minus (5′-GACCCAAGTAATGCAGG-3′) starting 631 base pairs downstream of the ATG and the K2 primer of the KanMX4 module (5′-GTATTGATGTTGGACG-3′). Plasmid pBF41 (Fig. 3C) was digested withBstYI and EcoRV to generate a 633-base pair fragment of GPI7. This fragment was inserted into the multiple cloning site of the bacterial expression vector pQE-30 (Qiagen) digested with BamHI/SmaI thus generating the plasmid pBF402. This plasmid was used to transform the E. coli strain M15[pREP4]. Expression of the recombinant protein was induced with isopropyl-1-thio-β-d-galactopyranoside and purified on a nickel-nitrilotriacetic acid-agarose column (Qiagen) under denaturing conditions according to the manufacturer's instructions. A polyclonal antiserum was raised against this Gpi7p fragment by repeated intramuscular injections of 100 μg of recombinant protein into a rabbit. Ten mg of the recombinant protein were coupled to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) according to the manufacturer's instructions, and antiserum was affinity purified as described (29Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 541-552Google Scholar). The nature of the association of Gpi7p with the membrane was determined following a previously described protocol (30Feldheim D. Schekman R. J. Cell Biol. 1994; 126: 935-943Crossref PubMed Scopus (81) Google Scholar) except that the EDTA concentration in buffer G was increased from 2 to 20 mm. Gpi7p protease sensitivity was examined by proteinase K digestion of microsomes essentially as described (31Baxter B.K. James P. Evans T. Craig E.A. Mol. Cell. Biol. 1996; 16: 6444-6456Crossref PubMed Scopus (67) Google Scholar). Briefly, 100 A 600 of washed W303 cells were resuspended in 1 ml of lysis buffer (20 mm HEPES, pH 7.5, 500 mm sucrose, 3 mm magnesium acetate, 20 mm EDTA, 1 mm dithiothreitol) and were lysed by agitation with glass beads at 4 °C. The homogenate was centrifuged for 5 min at 600 × g to remove unbroken cells, and the supernatant was centrifuged for 15 min at 13,000 × g. The membrane pellet was resuspended in 240 μl of the same lysis buffer and split into 6 aliquots of equal size. Aliquots of microsomes were incubated for 20 min on ice with or without 0.5% Triton X-100 and proteinase K. Digestions were stopped by addition of phenylmethylsulfonyl fluoride (final concentration 4 mm, added from a 200 mm stock in ethanol) and kept on ice for an additional 10 min before being boiled in sample buffer (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar). The subcellular localization of Gpi7p was determined essentially as described (33Gaynor E.C. Emr S.D. J. Cell Biol. 1997; 136: 789-802Crossref PubMed Scopus (164) Google Scholar). Briefly, 500 A 600 units of mid-log phase W303-1B cells were broken by agitation with glass beads in 200 mm sorbitol, 25 mm PIPES, pH 6.8, 50 mm KCl, 5 mm NaCl, 10 mm EDTA, 10 mm NaN3/NaF, 1 mmphenylmethylsulfonyl fluoride, leupeptin, pepstatin, and antipain, each at 30 μg/ml). After removal of the unbroken cells the homogenate was centrifuged for 10 min at 8,000 × g at 4 °C to generate pellet P8 and supernatant S8. S8 was divided in two and either precipitated by the addition of trichloroacetic acid to 10% or centrifuged at 100,000 × g for 1 h to generate pellet P100. For zymolyase treatment the cells were washed and resuspended at 50 A 600 units/ml in zymolyase buffer (1.2 m sorbitol, 50 mmK2HPO4 pH 7.5, 40 mm2-mercaptoethanol, 20 mm EDTA, 10 mmNaN3, 10 mm NaF) containing zymolyase 20T. After a 40-min incubation at 30 °C, cells were placed on a cushion of 1.5 m sorbitol, 50 mmK2HPO4, pH 7.5, 20 mm EDTA, 10 mm NaN3, 10 mm NaF and were centrifuged. For Western blotting, all the samples were denatured during 5 min at 95 °C in reducing sample buffer and run on a 6, 7.5, or 10% SDS-PAGE for detection of antigens, respectively (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar). Western blotting was carried out with anti-Wbp1p, anti-alkaline phosphatase, anti-Gas1p, or anti-Kex2p antisera or with affinity purified anti-Gpi7p or anti-Och1p antibodies, always using the chemiluminescence ECL kit from Amersham Pharmacia Biotech, Buckinghamshire, UK. Previously described procedures were used to label cells with [2-3H]Man (23Sipos G. Puoti A. Conzelmann A. EMBO J. 1994; 13: 2789-2796Crossref PubMed Scopus (67) Google Scholar), [4,5-3H]DHS, or [2-3H]Ins (24Reggiori F. Canivenc-Gansel E. Conzelmann A. EMBO J. 1997; 16: 3506-3518Crossref PubMed Scopus (101) Google Scholar) and to label microsomes with UDP-[3H]GlcNAc (22Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar). Delipidated protein extracts for SDS-PAGE and lipid extracts were made as described (24Reggiori F. Canivenc-Gansel E. Conzelmann A. EMBO J. 1997; 16: 3506-3518Crossref PubMed Scopus (101) Google Scholar). Lipid extracts were analyzed by ascending TLC using 0.2-mm thick silica gel 60 plates with the solvent 1 (chloroform/methanol, 0.25% KCl in water, 55:45:10, v/v) or solvent 2 (chloroform/methanol/water, 10:10:3, v/v). Radioactivity was detected and quantitated by one- and two-dimensional radioscanning (LB 2842; Berthold AG, Regensdorf, Switzerland). TLC plates were sprayed with EN3HANCE and exposed to film (X-Omat; Eastman Kodak Co.) at −80 °C. Lipid extracts were deacylated with NaOH (34Puoti A. Desponds C. Conzelmann A. J. Cell Biol. 1991; 113: 515-525Crossref PubMed Scopus (77) Google Scholar) and treated with JBAM (35Ralton J.E. Milne K.G. Güther M.L.S. Field R.A. Ferguson M.A.J. J. Biol. Chem. 1993; 268: 24183-24189Abstract Full Text PDF PubMed Google Scholar) as described. For GPI-PLD treatment lipid extracts were dissolved in 20 mm Tris-HCl, pH 7.4, 0.1 mm CaCl2, 20% 1-propanol. Incubations were for 12 h at 37 °C. All treated lipid extracts were desalted by partitioning between n-butyl alcohol and an aqueous solution of 0.1 mm EDTA, 5 mm Tris-HCl, pH 7.5, and back extraction of the butanol phase with water before TLC (23Sipos G. Puoti A. Conzelmann A. EMBO J. 1994; 13: 2789-2796Crossref PubMed Scopus (67) Google Scholar). Lipids were treated with methanolic NH3 to remove the acyl group on Ins (36Roberts 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) and cleaved using nitrous acid (37Güther M.L. Masterson W.J. Ferguson M.A. J. Biol. Chem. 1994; 269: 18694-18701Abstract Full Text PDF PubMed Google Scholar) as described. Lipids were purified by preparative TLC on 0.2-mm thick Silica Gel 60 plates (Merck, Germany) in solvent 2. Radioactive spots were localized by radioscanning, scraped, and eluted with solvent 2. A second run on TLC was done to obtain radiochemically pure M0, M4, and CP2. Soluble head groups were obtained from lipids through GPI-PLD treatment done as above, followed by limiting methanolic NH3deacylation (36Roberts 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). Non-hydrolyzed GPIs were removed by butanol extraction (23Sipos G. Puoti A. Conzelmann A. EMBO J. 1994; 13: 2789-2796Crossref PubMed Scopus (67) Google Scholar). The water-soluble head groups were treated with JBAM (0.5 units) or ASAM (5 microunits) as described (38Puoti A. Conzelmann A. J. Biol. Chem. 1992; 267: 22673-22680Abstract Full Text PDF PubMed Google Scholar). HF dephosphorylation was done as described (39Puoti A. Conzelmann A. J. Biol. Chem. 1993; 268: 7215-7224Abstract Full Text PDF PubMed Google Scholar). The generated fragments were analyzed by paper chromatography in methylethyl ketone/pyridine/H2O (20:12:11) as described (39Puoti A. Conzelmann A. J. Biol. Chem. 1993; 268: 7215-7224Abstract Full Text PDF PubMed Google Scholar). Before paper chromatography the products were N-acetylated and desalted over mixed-bed ion exchange resin AG-501-X8 (Bio-Rad) unless indicated otherwise (34Puoti A. Desponds C. Conzelmann A. J. Cell Biol. 1991; 113: 515-525Crossref PubMed Scopus (77) Google Scholar). Acetolysis was done as described (40Schneider P. Ferguson M.A. McConville M.J. Mehlert A. Homans S.W. Bordier C. J. Biol. Chem. 1990; 265: 16955-16964Abstract Full Text PDF PubMed Google Scholar). Radiolabeled Manx-GlcNAc-[3H]Ins (x = 1, 2, 3, 4) chromatography standards (Figs. 5 and6, standards 1–4) were generated through fragmentation of [3H]Man-labeled head groups of CP2, isolated frompmi40 by acetolysis then HF, HF then ASAM, JBAM then HF, and HF treatments, respectively. The GlcNAc-[3H]Ins standard (Figs. 5 and 6, standard 0) was generated by HF treatment of the [3H]Ins-labeled head group of M0, obtained fromsec53 cells. All standards were N-acetylated. Dionex HPLC analysis of non-dephosphorylated head groups was done exactly as described (23Sipos G. Puoti A. Conzelmann A. EMBO J. 1994; 13: 2789-2796Crossref PubMed Scopus (67) Google Scholar). Anchor peptides were prepared from labeled proteins as described (23Sipos G. Puoti A. Conzelmann A. EMBO J. 1994; 13: 2789-2796Crossref PubMed Scopus (67) Google Scholar).Figure 6M4 of Δgpi7 contains an HF-sensitive substituent on Man1 but lacks an HF-sensitive substituent on Man2. CP2 and M4 head groups were obtained from [3H]Man labeled pmi40 and [3H]Ins labeled Δgpi7, respectively. A and B, head groups of M4 were subjected to acetolysis and then either treated with JBAM (B) or left untreated (A). Finally all products were dephosphorylated with HF,N-acetylated, and analyzed by paper chromatography.C and D, head groups were treated for 12 h with HF, desalted, treated with JBAM, treated with HF for 60 h,N-acetylated, and finally analyzed by paper chromatography. Standards 0–4 are Manx-GlcNAc-Ins (x = 0, 1, 2, 3, 4). Free [3H]Man ran out of the paper shown in C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For limiting HF treatment, aliquots of radiolabeled head groups derived from CP2 and M4 and prepared as above were dephosphorylated with 50 μl of 48% aqueous HF at 0 °C as described (38Puoti A. Conzelmann A. J. Biol. Chem. 1992; 267: 22673-22680Abstract Full Text PDF PubMed Google Scholar) for 0–28 h. After neutralization with saturated LiOH, samples were desalted by gel filtration through an 8-ml Sephadex G-10 (Amersham Pharmacia Biotech) column. Samples were then dried in the Speed-Vac and treated with JBAM prior to complete HF dephosphorylation (60 h, 0 °C). Samples were neutralized again with LiOH and N-acetylated. Aliquots were dried and then directly applied to Whatman paper No. 1M and analyzed by descending chromatography as described above. As reported before (19Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelmann A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar) and shown in Fig. 2, wild type (wt) cells do not contain polar GPIs (lane 1), gpi8-1 accumulates CP2 as the most polar GPI lipid (lane 8), and gpi7-1 and the gpi7-1/gpi8-1 double mutant accumulate M4 (lanes 4 and 6), thus demonstrating that gpi7-1 is epistatic to gpi8-1 and suggesting that, during GPI biosynthesis, Gpi7p may act before Gpi8p. Although the originalgpi7 mutants and the unrelated gpi8-1 mutant were not significantly temperature-sensitive (ts) for growth, the growth of the gpi7-1/gpi8-1 double mutant was strongly temperature-dependent. Transfection of a genomic library into this double mutant allowed the isolation of clones containing complementing plasmids (19Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelmann A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar). These clones were labeled withmyo-[3H]inositol ([3H]Ins) at 37 °C, and the lipids were extracted and analyzed by TLC. Upon transfection some gpi7-1/gpi8-1 indeed had regained the ability to make CP2 (Fig. 2, lanes 6 and 7) and showed the same lipid profile as gpi8-1 (Fig. 2, lane 8). All these clones harbored plasmids containing YJL062w as the only complete ORF. Transfection of a multicopy vector containing YJL062w under its own promoter (pBF41, Fig. 3C) into gpi7-1almost completely cured the accumulation of M4 (Fig. 2, lane 5). As expected, the accumulation of CP2 by gpi8-1 was not abolished by the overexpression of YJL062w (Fig. 2, lanes 8 and 9). YJL062w predicts an 830-amino acid membrane protein with an N-terminal signal sequence for insertion into the ER, 5 potential N-glycosylation sites, and about 9–11 putative transmembrane domains (Fig. 3, A and B). YJL062w was deleted and replaced by the selectable marker KanMX4. On rich medium the deletants grew about as rapidly as wt cells at all temperatures. Thus, YJL062w is not an essential gene. We were unable to sporulate ΔYJL062/ΔYJL062 diploids indicating that YJL062 is required for sporulation. However, ΔYJL062/YJL062 heterozygotes sporulated readily and ΔYJL062 spores germinated normally. In accordance with previous results on gpi7 mutants (21Benghezal M. Lipke P.N. Conzelmann A. J. Cell Biol. 1995; 130: 1333-1344Crossref PubMed Scopus (74) Google Scholar), growth of ΔYJL062 (= Δgpi7, see below) on plates at 37 °C was severely inhibited by 0.5 mg/ml Calcofluor White. ΔYJL062 accumulated M4 at even higher levels than gpi7-1,and this accumulation was almost completely suppressed by the transfection of pBF41 (Fig. 2, lanes 2 and 3). Residual accumulation of M4 may be due to some cells that lost the complementing plasmid. Transfection of YJL062w under its own promoter on a single copy vector (plasmid pBF43) was sufficient to suppress the accumulation of M4 in a homozygous ΔYJL062/ΔYJL062 diploid (Fig. 2,lanes 11 and 12). As can be seen in Fig. 2,gpi7-1, ΔYJL062, ΔYJL062/ΔYJL062, andgpi8-1 mutants also show minor amounts of the GlcNα1,6(acyl→)Ins-P-DAG GPI intermediate M0, the accumulation of which is believed to reflect a build up of GPI intermediates throughout the biosynthetic pathway (Fig. 2, lanes 2, 4, 6, 8, and11). (It should be noted that some intermediates of intermediate size are obscured on TLC by PI and inositol phosphoceramide (41Sipos G. Reggiori F. Vionnet C. Conzelmann A. EMBO J. 1997; 16: 3494-3505Crossref PubMed Scopus (83) Google Scholar).) As expected, expression of YJL062w abolishes the accumulation of M0 in gpi7-1 and ΔYJL062 (Fig. 2,lanes 3, 5, and 12) but not ingpi7-1/gpi8-1 nor gpi8-1 (lanes 7 and9), since in the latter the GPI biosynthesis remains blocked. To evaluate if the mutation in gpi7-1 is genetically linked to YJL062w, YJL062w was disrupted in a heterozygousgpi7-1/GPI7 diploid. Correct replacement of one YJL062w locus was verified by PCR in two independent geneticin-resistant transformants. The verified deletants were sporulated, and a total of 26" @default.
• W1968271109 created "2016-06-24" @default.
• W1968271109 creator A5000591351 @default.
• W1968271109 creator A5004778008 @default.
• W1968271109 creator A5043642645 @default.
• W1968271109 creator A5052363755 @default.
• W1968271109 creator A5058055140 @default.
• W1968271109 creator A5060891801 @default.
• W1968271109 creator A5064468023 @default.
• W1968271109 creator A5074499137 @default.
• W1968271109 date "1999-05-01" @default.
• W1968271109 modified "2023-09-30" @default.
• W1968271109 title "Deletion of GPI7, a Yeast Gene Required for Addition of a Side Chain to the Glycosylphosphatidylinositol (GPI) Core Structure, Affects GPI Protein Transport, Remodeling, and Cell Wall Integrity" @default.
• W1968271109 cites W1485814238 @default.
• W1968271109 cites W1485988308 @default.
• W1968271109 cites W1492544808 @default.
• W1968271109 cites W1508389393 @default.
• W1968271109 cites W1543999675 @default.
• W1968271109 cites W1561571702 @default.
• W1968271109 cites W1561649218 @default.
• W1968271109 cites W1562203178 @default.
• W1968271109 cites W1564262044 @default.
• W1968271109 cites W1571485829 @default.
• W1968271109 cites W1601138755 @default.
• W1968271109 cites W1602150101 @default.
• W1968271109 cites W1626163495 @default.
• W1968271109 cites W1767192041 @default.
• W1968271109 cites W1884967233 @default.
• W1968271109 cites W1891938237 @default.
• W1968271109 cites W1892543101 @default.
• W1968271109 cites W1927235811 @default.
• W1968271109 cites W1944304173 @default.
• W1968271109 cites W1960975851 @default.
• W1968271109 cites W1967390330 @default.
• W1968271109 cites W1972565685 @default.
• W1968271109 cites W1980969996 @default.
• W1968271109 cites W1987433777 @default.
• W1968271109 cites W1996677209 @default.
• W1968271109 cites W2006267913 @default.
• W1968271109 cites W2024537054 @default.
• W1968271109 cites W2029152767 @default.
• W1968271109 cites W2051441654 @default.
• W1968271109 cites W2051722675 @default.
• W1968271109 cites W2053255035 @default.
• W1968271109 cites W2054323447 @default.
• W1968271109 cites W2061188947 @default.
• W1968271109 cites W2063287165 @default.
• W1968271109 cites W2063397347 @default.
• W1968271109 cites W2065698593 @default.
• W1968271109 cites W2067607091 @default.
• W1968271109 cites W2068496808 @default.
• W1968271109 cites W2080429285 @default.
• W1968271109 cites W2081978633 @default.
• W1968271109 cites W2096179261 @default.
• W1968271109 cites W2100057937 @default.
• W1968271109 cites W2100285359 @default.
• W1968271109 cites W2100837269 @default.
• W1968271109 cites W2103831114 @default.
• W1968271109 cites W211139468 @default.
• W1968271109 cites W2114388625 @default.
• W1968271109 cites W2115073295 @default.
• W1968271109 cites W2127217613 @default.
• W1968271109 cites W2129371499 @default.
• W1968271109 cites W2135197366 @default.
• W1968271109 cites W2135556297 @default.
• W1968271109 cites W2138270253 @default.
• W1968271109 cites W2145999632 @default.
• W1968271109 cites W2146222671 @default.
• W1968271109 cites W2147276807 @default.
• W1968271109 cites W2148417653 @default.
• W1968271109 cites W2154786726 @default.
• W1968271109 cites W2158714788 @default.
• W1968271109 cites W2159703228 @default.
• W1968271109 cites W246661795 @default.
• W1968271109 cites W334144690 @default.
• W1968271109 cites W77949942 @default.
• W1968271109 doi "https://doi.org/10.1074/jbc.274.21.15251" @default.
• W1968271109 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10329735" @default.
• W1968271109 hasPublicationYear "1999" @default.
• W1968271109 type Work @default.
• W1968271109 sameAs 1968271109 @default.
• W1968271109 citedByCount "120" @default.
• W1968271109 countsByYear W19682711092012 @default.
• W1968271109 countsByYear W19682711092013 @default.
• W1968271109 countsByYear W19682711092014 @default.
• W1968271109 countsByYear W19682711092015 @default.
• W1968271109 countsByYear W19682711092016 @default.
• W1968271109 countsByYear W19682711092017 @default.
• W1968271109 countsByYear W19682711092018 @default.
• W1968271109 countsByYear W19682711092019 @default.
• W1968271109 countsByYear W19682711092020 @default.
• W1968271109 countsByYear W19682711092021 @default.
• W1968271109 countsByYear W19682711092022 @default.
• W1968271109 crossrefType "journal-article" @default.
• W1968271109 hasAuthorship W1968271109A5000591351 @default.
• W1968271109 hasAuthorship W1968271109A5004778008 @default.
• W1968271109 hasAuthorship W1968271109A5043642645 @default.
• W1968271109 hasAuthorship W1968271109A5052363755 @default.

• next