FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2122116013> ?p ?o ?g. }

• W2122116013 endingPage "20919" @default.
• W2122116013 startingPage "20911" @default.
• W2122116013 abstract "Many eukaryotic proteins are anchored by glycosylphosphatidylinositol (GPI) to the cell surface membrane. The GPI anchor is linked to proteins by an amide bond formed between the carboxyl terminus and phosphoethanolamine attached to the third mannose. Here, we report the roles of two mammalian genes involved in transfer of phosphoethanolamine to the third mannose in GPI. We cloned a mouse gene termed Pig-o that encodes a 1101-amino acid PIG-O protein bearing regions conserved in various phosphodiesterases.Pig-o knockout F9 embryonal carcinoma cells expressed very little GPI-anchored proteins and accumulated the same major GPI intermediate as the mouse class F mutant cell, which is defective in transferring phosphoethanolamine to the third mannose due to mutantPig-f gene. PIG-O and PIG-F proteins associate with each other, and the stability of PIG-O was dependent upon PIG-F. However, the class F cell is completely deficient in the surface expression of GPI-anchored proteins. A minor GPI intermediate seen inPig-o knockout but not class F cells had more than three mannoses with phosphoethanolamines on the first and third mannoses, suggesting that this GPI may account for the low expression of GPI-anchored proteins. Therefore, mammalian cells have redundant activities in transferring phosphoethanolamine to the third mannose, both of which require PIG-F. Many eukaryotic proteins are anchored by glycosylphosphatidylinositol (GPI) to the cell surface membrane. The GPI anchor is linked to proteins by an amide bond formed between the carboxyl terminus and phosphoethanolamine attached to the third mannose. Here, we report the roles of two mammalian genes involved in transfer of phosphoethanolamine to the third mannose in GPI. We cloned a mouse gene termed Pig-o that encodes a 1101-amino acid PIG-O protein bearing regions conserved in various phosphodiesterases.Pig-o knockout F9 embryonal carcinoma cells expressed very little GPI-anchored proteins and accumulated the same major GPI intermediate as the mouse class F mutant cell, which is defective in transferring phosphoethanolamine to the third mannose due to mutantPig-f gene. PIG-O and PIG-F proteins associate with each other, and the stability of PIG-O was dependent upon PIG-F. However, the class F cell is completely deficient in the surface expression of GPI-anchored proteins. A minor GPI intermediate seen inPig-o knockout but not class F cells had more than three mannoses with phosphoethanolamines on the first and third mannoses, suggesting that this GPI may account for the low expression of GPI-anchored proteins. Therefore, mammalian cells have redundant activities in transferring phosphoethanolamine to the third mannose, both of which require PIG-F. glycosylphosphatidylinositol aldehyde dehydrogenase Chinese hamster ovary endoplasmic reticulum phosphoethanolamine glutathione S-transferase kilobase pair(s) mannose polymerase chain reaction 1-chloro-3-tosylamido-7-amino-2-heptanone Many proteins on the eukaryotic cell surface are anchored by glycosylphosphatidylinositol (GPI)1 (1.McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (797) Google Scholar, 2.Kinoshita T. Ohishi K. Takeda J. J. Biochem. 1997; 122: 251-257Crossref PubMed Scopus (122) Google Scholar, 3.Schultz C. Gilson P. Oxley D. Youl J. Bacic A. Trends Plant Sci. 1998; 3: 426-431Abstract Full Text Full Text PDF Scopus (150) Google Scholar, 4.Kapteyn J.C. van den Ende H. Klis F.M. Biochim. Biophys. Acta. 1999; 1426: 373-383Crossref PubMed Scopus (317) Google Scholar, 5.Tiede A. Bastisch I. Schubert J. Orlean P. Schmidt R.E. Biol. Chem. 1999; 380: 503-523Crossref PubMed Scopus (105) Google Scholar). The common backbone, EtNP-6Manα-1,2Manα-1,6Manα-1,4GlcNα-1,6myo-inositol-P-lipid (where EtNP, Man, GlcN, and P are phosphoethanolamine, mannose, glucosamine, and phosphate, respectively), is assembled in the endoplasmic reticulum (ER) by the sequential additions of sugar and EtNP components to phosphatidylinositol (6.Englund P.T. Annu. Rev. Biochem. 1993; 62: 121-138Crossref PubMed Google Scholar, 7.Stevens V.L. Biochem. J. 1995; 310: 361-370Crossref PubMed Scopus (102) Google Scholar). This core is conserved in all eukaryotes but modified by various side chains in different organisms. In the yeast Saccharomyces cerevisiae (8.Sutterlin 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, 9.Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar) and in mammalian cells (10.Hirose S. Prince G.M. Sevlever D. Ravi L. Rosenberry T.L. Ueda E. Medof M.E. J. Biol. Chem. 1992; 267: 16968-16974Abstract Full Text PDF PubMed Google Scholar), the first mannose (Man1) is modified by EtNP at position 2. The second mannose (Man2) of mammalian and yeast GPIs can also be modified by EtNP at position 6 (11.Ueda E. Sevlever D. Prince G.M. Rosenberry T.L. Hirose S. Medof M.E. J. Biol. Chem. 1993; 268: 9998-10002Abstract Full Text PDF PubMed Google Scholar, 12.Benachour 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 (109) Google Scholar). EtNP on the third mannose (Man3) is transferred from phosphatidylethanolamine (13.Menon A.K. Stevens V.L. J. Biol. Chem. 1992; 267: 15277-15280Abstract Full Text PDF PubMed Google Scholar, 14.Menon A.K. Eppinger M. Mayor S. Schwarz R.T. EMBO J. 1993; 12: 1907-1914Crossref PubMed Scopus (80) Google Scholar), whereas donors for EtNP on Man1 and Man2 have not been clarified.In yeast, two homologous gene products involved in side chain modification of mannose residues were characterized. Gpi7p is involved in the addition of a side chain, probably EtNP, to Man2, which is not essential for growth (12.Benachour 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 (109) Google Scholar). Mcd4p, which is essential for growth, is probably involved in EtNP transfer to Man1 (15.Gaynor E.C. Mondesert G. Grimme S.J. Reed S.I. Orlean P. Emr S.D. Mol. Biol. Cell. 1999; 10: 627-648Crossref PubMed Scopus (111) Google Scholar, 16.Hong 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 (117) Google Scholar). Mouse F9 cells defective in Pig-n, a mouse homologue of MCD4, accumulate GPI precursors without EtNP on Man1 (16.Hong 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 (117) Google Scholar). YW3548 (17.Sutterlin C. Horvath A. Gerold P. Schwarz R.T. Wang Y. Dreyfuss M. Riezman H. EMBO J. 1997; 16: 6374-6383Crossref PubMed Scopus (86) Google Scholar) inhibits the addition of EtNP to Man1 in both yeast and mammalian cells by inhibiting Mcd4p and Pig-n (8.Sutterlin 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, 16.Hong 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 (117) Google Scholar), indicating that yeast Mcd4p is involved in transferring EtNP to Man1. Mcd4p and Gpi7p are large proteins of about 120 kDa having an amino-terminal lumenal domain and multiple transmembrane domains in the carboxyl-terminal portion. They have regions conserved in phosphodiesterases and nucleotide pyrophosphatases within the amino-terminal lumenal domain (12.Benachour 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 (109) Google Scholar, 15.Gaynor E.C. Mondesert G. Grimme S.J. Reed S.I. Orlean P. Emr S.D. Mol. Biol. Cell. 1999; 10: 627-648Crossref PubMed Scopus (111) Google Scholar). It is likely that they are involved in transfer of EtNP to mannoses, although their enzyme activities remain to be demonstrated.In the yeast genome, there is a third gene, YLL031c, homologous toMCD4 and GPI7 (12.Benachour 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 (109) Google Scholar, 15.Gaynor E.C. Mondesert G. Grimme S.J. Reed S.I. Orlean P. Emr S.D. Mol. Biol. Cell. 1999; 10: 627-648Crossref PubMed Scopus (111) Google Scholar). Although YLL031c has not been shown to be involved in GPI biosynthesis, its disruption in yeast caused a phenotype similar to those of other GPI anchoring mutants; namely, germinated spores had lethal growth defect after one to two generations with heterogeneous bud sizes (18.Zhang N. Ismail T. Wu J. Woodwark K.C. Gardner D.C.J. Walmsley R.M. Oliver S.G. Yeast. 1999; 15: 1287-1296Crossref PubMed Scopus (9) Google Scholar). Therefore, it is likely that YLL031cp is involved in EtNP transfer to Man3. Man1 is modified at position 2, whereas Man2 and Man3 are modified at position 6. Consistent with these different positions of modification, Gpi7p and YLL031cp share an amino acid identity (38%) that is higher than that of Gpi7p and Mcd4p (24%) and that of YLL031cp and Mcd4p (21%) (12.Benachour 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 (109) Google Scholar,16.Hong 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 (117) Google Scholar).Mammalian GPI biosynthesis mutant cells of complementation class F are defective in EtNP transfer to Man3 (10.Hirose S. Prince G.M. Sevlever D. Ravi L. Rosenberry T.L. Ueda E. Medof M.E. J. Biol. Chem. 1992; 267: 16968-16974Abstract Full Text PDF PubMed Google Scholar, 19.Sugiyama E. DeGasperi R. Urakaze M. Chang H.M. Thomas L.J. Hyman R. Warren C.D. Yeh E.T.H. J. Biol. Chem. 1991; 266: 12119-12122Abstract Full Text PDF PubMed Google Scholar, 20.Puoti A. Conzelmann A. J. Biol. Chem. 1993; 268: 7215-7224Abstract Full Text PDF PubMed Google Scholar), for which mutation inPig-f is responsible (21.Inoue N. Kinoshita T. Orii T. Takeda J. J. Biol. Chem. 1993; 268: 6882-6885Abstract Full Text PDF PubMed Google Scholar, 22.Ohishi K. Kurimoto Y. Inoue N. Endo Y. Takeda J. Kinoshita T. Genomics. 1996; 34: 340-346Crossref PubMed Scopus (19) Google Scholar). However, PIG-F protein does not have structural similarity to YLL031cp, and yeast has a PIG-F homologue, YDR302wp. Here, we report molecular cloning of a mouse YLL031c homologue termed Pig-o (phosphatidylinositol glycan, class O) and show that PIG-O and PIG-F are involved in the addition of EtNP to Man3. We also show that mammalian cells have another mechanism of adding EtNP to Man3 that requires PIG-F but not PIG-O.DISCUSSIONIn the present study, we cloned and characterized Pig-oand analyzed the roles of PIG-O and PIG-F in GPI biosynthesis. The first conclusion of this study is that PIG-O and PIG-F act together in transferring EtNP to Man3. This is based on two results: 1)Pig-o knockout cells and class F cells defective in PIG-F accumulated the same major GPI intermediate, H6, which bears three mannoses lacking EtNP on Man3; and 2) PIG-O and PIG-F formed a protein complex in the ER, and the stability of the former was dependent upon the latter. The second conclusion is that although this mechanism of EtNP transfer to Man3 is predominant, there must be a second mechanism that also requires PIG-F but not PIG-O. This conclusion is supported by two observations: 1) Pig-o knockout cells express a low level of GPI-anchored proteins, whereas class F cells are completely deficient in the surface expression of GPI-anchored protein; and 2)Pig-o knockout but not class F cells generated a minor GPI with more than three mannoses that has EtNPs on Man1 and Man3.Association of PIG-O and PIG-FBoth PIG-O and PIG-F were found exclusively in the ER, where the GPI anchor is synthesized, and they specifically associated with each other. PIG-F, which is a very hydrophobic protein, may associate with the carboxyl-terminal hydrophobic region of PIG-O.The expression of PIG-O was dependent upon PIG-F, i.e. the level of PIG-O expression was at least three times higher in the presence than in the absence of PIG-F. However, the expression of PIG-F was not affected by a lack of PIG-O. Presumably, most PIG-O was associated with PIG-F, whereas some PIG-F may exist free from PIG-O. These results suggest that PIG-O acts together with PIG-F in the ER and that PIG-F may have at least one more partner that is involved in the second mechanism of transferring EtNP to Man3 (see below for further discussion).Common and Different Phenotypes of Pig-o Knockout and Class F Mutant CellsPig-o knockout and class F cells share common defective phenotypes but they have some differences too. Both cells are deficient in the surface expression of GPI-anchored proteins. However, low levels of Thy-1 and ScaI remained on thePig-o knockout F9 cells. In contrast, class F cells are completely deficient in the surface Thy-1 expression. Therefore, PIG-O is involved in but not essential for GPI-anchoring of proteins, whereas PIG-F is essential for it. Both Pig-o knockout and class F cells did not generate a mature GPI, H8, but accumulated a GPI intermediate, H6. H6 has three mannoses and EtNP modification on Man1 but lacks EtNP on Man3 (10.Hirose S. Prince G.M. Sevlever D. Ravi L. Rosenberry T.L. Ueda E. Medof M.E. J. Biol. Chem. 1992; 267: 16968-16974Abstract Full Text PDF PubMed Google Scholar). Therefore, both cells are defective in the transfer of EtNP to Man3 (see Fig. 7 for the pathway). Class F cells generated one minor GPI, termed F-1, whereas Pig-o knockout cells generated two minor GPIs, termed KO-1 and KO-2. We concluded that F-1 and KO-1 are the same GPI (see below for discussion), but KO-2 was seen only in Pig-o knockout cells.Possible Structures of GPIs, KO-1/F-1 and KO-2KO-1 and F-1 had similar mobilities on TLC, slightly slower than that of H7. H7 bears three mannoses with EtNPs on Man1 and Man3. After the treatment with α-mannosidase, KO-1 and F-1 behaved similarly and became a GPI migrating slightly more slowly than H6. H6 bears three mannoses with EtNP on Man1. KO-1 must have EtNP on Man1 because its generation was inhibited by YW3548/BE49385A. Although we did not analyze the number of mannose residues in KO-1 and F-1, we speculate based on these results that they have three or four mannoses with EtNP on Man1 and Man2 (Fig.7).KO-2 migrated more slowly than H7, and after α-mannosidase treatment, its product behaved similarly to H7 on TLC. KO-2 has EtNP on Man1 because its generation was inhibited by YW3548/BE49385A. KO-2, therefore, has more than three, most likely four, mannoses with EtNP on Man1 and Man3.Two Mechanisms of Transfer of EtNP to Man3 in Mammalian CellsThe complex of PIG-O and PIG-F should be responsible for the addition of EtNP to Man3 in H6 to generate H7 and subsequently H8. It is very likely that PIG-O bears a catalytic site because three family members, Mcd4p/Pig-n, Gpi7p, and PIG-O, correspond to EtNP additions to Man1, Man2, and Man3, respectively, and because they have regions with homology to various phosphodiesterases and nucleotide pyrophosphatases. Consistent with the idea that these regions are involved in the catalytic site, the temperature-sensitive mutant allele of MCD4 had a mutation within one of these regions (15.Gaynor E.C. Mondesert G. Grimme S.J. Reed S.I. Orlean P. Emr S.D. Mol. Biol. Cell. 1999; 10: 627-648Crossref PubMed Scopus (111) Google Scholar). These regions are within the lumenal domains, suggesting that transfer of EtNP to Man3 occurs on the lumenal side of the ER.PIG-F is required for the stable expression of PIG-O. It is unlikely that this is the only role of PIG-F because PIG-O expressed in the absence of PIG-F did not cause the surface Thy-1 expression in class F cells. PIG-F may play some role in the addition of EtNP to Man3.In the absence of PIG-O, GPI-anchored proteins were expressed at low levels. Because KO-2 may have Man3 with EtNP on it, it seems possible that KO-2 accounts for the residual GPI-anchoring in Pig-oknockout cells. Generation of KO-2 should require PIG-F because it was not seen in class F cells. Consistent with the notion that KO-2 is competent for attachment to proteins, class F cells are completely deficient in the surface expression of GPI-anchored proteins. Because PIG-F may not be a catalytic component, it would act together with a catalytic component other than PIG-O to generate KO-2. Because Gpi7p is responsible for addition of EtNP to Man2 at position 6 in yeast and EtNP on Man3 is also at position 6, a mammalian homologue of Gpi7p seems to be a candidate of the second partner for PIG-F. Human and mouse sequences homologous to Gpi7p are found in the GenBankTM data base. They should be cloned and characterized to determine whether they represent a functional homologue of Gpi7p and act with PIG-F in transferring EtNP to Man3.Note Added in ProofTwo papers describing functions of yeast orthologues of PIG-F and PIG-O were recently published (Taron, C. H., Wiedman, J. M., Grimme, S. J., and Orlean, P. (2000) Mol. Biol. Cell 11, 1611–1630; Flury, I., Benachour, A., and Conzelmann, A. (May 22, 2000) J. Biol. Chem.10.1074/jbc.M003844200). Many proteins on the eukaryotic cell surface are anchored by glycosylphosphatidylinositol (GPI)1 (1.McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (797) Google Scholar, 2.Kinoshita T. Ohishi K. Takeda J. J. Biochem. 1997; 122: 251-257Crossref PubMed Scopus (122) Google Scholar, 3.Schultz C. Gilson P. Oxley D. Youl J. Bacic A. Trends Plant Sci. 1998; 3: 426-431Abstract Full Text Full Text PDF Scopus (150) Google Scholar, 4.Kapteyn J.C. van den Ende H. Klis F.M. Biochim. Biophys. Acta. 1999; 1426: 373-383Crossref PubMed Scopus (317) Google Scholar, 5.Tiede A. Bastisch I. Schubert J. Orlean P. Schmidt R.E. Biol. Chem. 1999; 380: 503-523Crossref PubMed Scopus (105) Google Scholar). The common backbone, EtNP-6Manα-1,2Manα-1,6Manα-1,4GlcNα-1,6myo-inositol-P-lipid (where EtNP, Man, GlcN, and P are phosphoethanolamine, mannose, glucosamine, and phosphate, respectively), is assembled in the endoplasmic reticulum (ER) by the sequential additions of sugar and EtNP components to phosphatidylinositol (6.Englund P.T. Annu. Rev. Biochem. 1993; 62: 121-138Crossref PubMed Google Scholar, 7.Stevens V.L. Biochem. J. 1995; 310: 361-370Crossref PubMed Scopus (102) Google Scholar). This core is conserved in all eukaryotes but modified by various side chains in different organisms. In the yeast Saccharomyces cerevisiae (8.Sutterlin 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, 9.Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar) and in mammalian cells (10.Hirose S. Prince G.M. Sevlever D. Ravi L. Rosenberry T.L. Ueda E. Medof M.E. J. Biol. Chem. 1992; 267: 16968-16974Abstract Full Text PDF PubMed Google Scholar), the first mannose (Man1) is modified by EtNP at position 2. The second mannose (Man2) of mammalian and yeast GPIs can also be modified by EtNP at position 6 (11.Ueda E. Sevlever D. Prince G.M. Rosenberry T.L. Hirose S. Medof M.E. J. Biol. Chem. 1993; 268: 9998-10002Abstract Full Text PDF PubMed Google Scholar, 12.Benachour 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 (109) Google Scholar). EtNP on the third mannose (Man3) is transferred from phosphatidylethanolamine (13.Menon A.K. Stevens V.L. J. Biol. Chem. 1992; 267: 15277-15280Abstract Full Text PDF PubMed Google Scholar, 14.Menon A.K. Eppinger M. Mayor S. Schwarz R.T. EMBO J. 1993; 12: 1907-1914Crossref PubMed Scopus (80) Google Scholar), whereas donors for EtNP on Man1 and Man2 have not been clarified. In yeast, two homologous gene products involved in side chain modification of mannose residues were characterized. Gpi7p is involved in the addition of a side chain, probably EtNP, to Man2, which is not essential for growth (12.Benachour 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 (109) Google Scholar). Mcd4p, which is essential for growth, is probably involved in EtNP transfer to Man1 (15.Gaynor E.C. Mondesert G. Grimme S.J. Reed S.I. Orlean P. Emr S.D. Mol. Biol. Cell. 1999; 10: 627-648Crossref PubMed Scopus (111) Google Scholar, 16.Hong 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 (117) Google Scholar). Mouse F9 cells defective in Pig-n, a mouse homologue of MCD4, accumulate GPI precursors without EtNP on Man1 (16.Hong 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 (117) Google Scholar). YW3548 (17.Sutterlin C. Horvath A. Gerold P. Schwarz R.T. Wang Y. Dreyfuss M. Riezman H. EMBO J. 1997; 16: 6374-6383Crossref PubMed Scopus (86) Google Scholar) inhibits the addition of EtNP to Man1 in both yeast and mammalian cells by inhibiting Mcd4p and Pig-n (8.Sutterlin 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, 16.Hong 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 (117) Google Scholar), indicating that yeast Mcd4p is involved in transferring EtNP to Man1. Mcd4p and Gpi7p are large proteins of about 120 kDa having an amino-terminal lumenal domain and multiple transmembrane domains in the carboxyl-terminal portion. They have regions conserved in phosphodiesterases and nucleotide pyrophosphatases within the amino-terminal lumenal domain (12.Benachour 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 (109) Google Scholar, 15.Gaynor E.C. Mondesert G. Grimme S.J. Reed S.I. Orlean P. Emr S.D. Mol. Biol. Cell. 1999; 10: 627-648Crossref PubMed Scopus (111) Google Scholar). It is likely that they are involved in transfer of EtNP to mannoses, although their enzyme activities remain to be demonstrated. In the yeast genome, there is a third gene, YLL031c, homologous toMCD4 and GPI7 (12.Benachour 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 (109) Google Scholar, 15.Gaynor E.C. Mondesert G. Grimme S.J. Reed S.I. Orlean P. Emr S.D. Mol. Biol. Cell. 1999; 10: 627-648Crossref PubMed Scopus (111) Google Scholar). Although YLL031c has not been shown to be involved in GPI biosynthesis, its disruption in yeast caused a phenotype similar to those of other GPI anchoring mutants; namely, germinated spores had lethal growth defect after one to two generations with heterogeneous bud sizes (18.Zhang N. Ismail T. Wu J. Woodwark K.C. Gardner D.C.J. Walmsley R.M. Oliver S.G. Yeast. 1999; 15: 1287-1296Crossref PubMed Scopus (9) Google Scholar). Therefore, it is likely that YLL031cp is involved in EtNP transfer to Man3. Man1 is modified at position 2, whereas Man2 and Man3 are modified at position 6. Consistent with these different positions of modification, Gpi7p and YLL031cp share an amino acid identity (38%) that is higher than that of Gpi7p and Mcd4p (24%) and that of YLL031cp and Mcd4p (21%) (12.Benachour 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 (109) Google Scholar,16.Hong 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 (117) Google Scholar). Mammalian GPI biosynthesis mutant cells of complementation class F are defective in EtNP transfer to Man3 (10.Hirose S. Prince G.M. Sevlever D. Ravi L. Rosenberry T.L. Ueda E. Medof M.E. J. Biol. Chem. 1992; 267: 16968-16974Abstract Full Text PDF PubMed Google Scholar, 19.Sugiyama E. DeGasperi R. Urakaze M. Chang H.M. Thomas L.J. Hyman R. Warren C.D. Yeh E.T.H. J. Biol. Chem. 1991; 266: 12119-12122Abstract Full Text PDF PubMed Google Scholar, 20.Puoti A. Conzelmann A. J. Biol. Chem. 1993; 268: 7215-7224Abstract Full Text PDF PubMed Google Scholar), for which mutation inPig-f is responsible (21.Inoue N. Kinoshita T. Orii T. Takeda J. J. Biol. Chem. 1993; 268: 6882-6885Abstract Full Text PDF PubMed Google Scholar, 22.Ohishi K. Kurimoto Y. Inoue N. Endo Y. Takeda J. Kinoshita T. Genomics. 1996; 34: 340-346Crossref PubMed Scopus (19) Google Scholar). However, PIG-F protein does not have structural similarity to YLL031cp, and yeast has a PIG-F homologue, YDR302wp. Here, we report molecular cloning of a mouse YLL031c homologue termed Pig-o (phosphatidylinositol glycan, class O) and show that PIG-O and PIG-F are involved in the addition of EtNP to Man3. We also show that mammalian cells have another mechanism of adding EtNP to Man3 that requires PIG-F but not PIG-O. DISCUSSIONIn the present study, we cloned and characterized Pig-oand analyzed the roles of PIG-O and PIG-F in GPI biosynthesis. The first conclusion of this study is that PIG-O and PIG-F act together in transferring EtNP to Man3. This is based on two results: 1)Pig-o knockout cells and class F cells defective in PIG-F accumulated the same major GPI intermediate, H6, which bears three mannoses lacking EtNP on Man3; and 2) PIG-O and PIG-F formed a protein complex in the ER, and the stability of the former was dependent upon the latter. The second conclusion is that although this mechanism of EtNP transfer to Man3 is predominant, there must be a second mechanism that also requires PIG-F but not PIG-O. This conclusion is supported by two observations: 1) Pig-o knockout cells express a low level of GPI-anchored proteins, whereas class F cells are completely deficient in the surface expression of GPI-anchored protein; and 2)Pig-o knockout but not class F cells generated a minor GPI with more than three mannoses that has EtNPs on Man1 and Man3.Association of PIG-O and PIG-FBoth PIG-O and PIG-F were found exclusively in the ER, where the GPI anchor is synthesized, and they specifically associated with each other. PIG-F, which is a very hydrophobic protein, may associate with the carboxyl-terminal hydrophobic region of PIG-O.The expression of PIG-O was dependent upon PIG-F, i.e. the level of PIG-O expression was at least three times higher in the presence than in the absence of PIG-F. However, the expression of PIG-F was not affected by a lack of PIG-O. Presumably, most PIG-O was associated with PIG-F, whereas some PIG-F may exist free from PIG-O. These results suggest that PIG-O acts together with PIG-F in the ER and that PIG-F may have at least one more partner that is involved in the second mechanism of transferring EtNP to Man3 (see below for further discussion).Common and Different Phenotypes of Pig-o Knockout and Class F Mutant CellsPig-o knockout and class F cells share common defective phenotypes but they have some differences too. Both cells are deficient in the surface expression of GPI-anchored proteins. However, low levels of Thy-1 and ScaI remained on thePig-o knockout F9 cells. In contrast, class F cells are completely deficient in the surface Thy-1 expression. Therefore, PIG-O is involved in but not essential for GPI-anchoring of proteins, whereas PIG-F is essential for it. Both Pig-o knockout and class F cells did not generate a mature GPI, H8, but accumulated a GPI intermediate, H6. H6 has three mannoses and EtNP modification on Man1 but lacks EtNP on Man3 (10.Hirose S. Prince G.M. Sevlever D. Ravi L. Rosenberry T.L. Ueda E. Medof M.E. J. Biol. Chem. 1992; 267: 16968-16974Abstract Full Text PDF PubMed Google Scholar). Therefore, both cells are defective in the transfer of EtNP to Man3 (see Fig. 7 for the pathway). Class F cells generated one minor GPI, termed F-1, whereas Pig-o knockout cells generated two minor GPIs, termed KO-1 and KO-2. We concluded that F-1 and KO-1 are the same GPI (see below for discussion), but KO-2 was seen only in Pig-o knockout cells.Possible Structures of GPIs, KO-1/F-1 and KO-2KO-1 and F-1 had similar mobilities on TLC, slightly slower than that of H7. H7 bears three mannoses with EtNPs on Man1 and Man3. After the treatment with α-mannosidase, KO-1 and F-1 behaved similarly and became a GPI migrating slightly more slowly than H6. H6 bears three mannoses with EtNP on Man1. KO-1 must have EtNP on Man1 because its generation was inhibited by YW3548/BE49385A. Although we did not analyze the number of mannose residues in KO-1 and F-1, we speculate based on these results that they have three or four mannoses with EtNP on Man1 and Man2 (Fig.7).KO-2 migrated more slowly than H7, and after α-mannosidase treatment, its product behaved similarly to H7 on TLC. KO-2 has EtNP on Man1 because its generation wa" @default.
• W2122116013 created "2016-06-24" @default.
• W2122116013 creator A5005083287 @default.
• W2122116013 creator A5041156218 @default.
• W2122116013 creator A5051611788 @default.
• W2122116013 creator A5072623856 @default.
• W2122116013 creator A5082394458 @default.
• W2122116013 creator A5091406841 @default.
• W2122116013 date "2000-07-01" @default.
• W2122116013 modified "2023-09-30" @default.
• W2122116013 title "Requirement of PIG-F and PIG-O for Transferring Phosphoethanolamine to the Third Mannose in Glycosylphosphatidylinositol" @default.
• W2122116013 cites W1485988308 @default.
• W2122116013 cites W1492544808 @default.
• W2122116013 cites W1503098738 @default.
• W2122116013 cites W1561571702 @default.
• W2122116013 cites W1562203178 @default.
• W2122116013 cites W1583769399 @default.
• W2122116013 cites W1824901304 @default.
• W2122116013 cites W1891938237 @default.
• W2122116013 cites W1944304173 @default.
• W2122116013 cites W1968271109 @default.
• W2122116013 cites W1974998411 @default.
• W2122116013 cites W1975304761 @default.
• W2122116013 cites W1980314703 @default.
• W2122116013 cites W1988627101 @default.
• W2122116013 cites W1990476575 @default.
• W2122116013 cites W2000144601 @default.
• W2122116013 cites W2003322429 @default.
• W2122116013 cites W2012116939 @default.
• W2122116013 cites W2017501248 @default.
• W2122116013 cites W2031977328 @default.
• W2122116013 cites W2055043387 @default.
• W2122116013 cites W2056799965 @default.
• W2122116013 cites W2072538271 @default.
• W2122116013 cites W2099694432 @default.
• W2122116013 cites W2106882534 @default.
• W2122116013 cites W2107175187 @default.
• W2122116013 cites W2110764616 @default.
• W2122116013 cites W2117919289 @default.
• W2122116013 cites W2124676316 @default.
• W2122116013 cites W2129597030 @default.
• W2122116013 cites W2132358136 @default.
• W2122116013 cites W2133308882 @default.
• W2122116013 cites W2135197366 @default.
• W2122116013 cites W2146488895 @default.
• W2122116013 cites W2147611281 @default.
• W2122116013 cites W2156852013 @default.
• W2122116013 cites W2164321363 @default.
• W2122116013 cites W2175428614 @default.
• W2122116013 cites W2328105636 @default.
• W2122116013 cites W4322384960 @default.
• W2122116013 doi "https://doi.org/10.1074/jbc.m001913200" @default.
• W2122116013 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10781593" @default.
• W2122116013 hasPublicationYear "2000" @default.
• W2122116013 type Work @default.
• W2122116013 sameAs 2122116013 @default.
• W2122116013 citedByCount "78" @default.
• W2122116013 countsByYear W21221160132012 @default.
• W2122116013 countsByYear W21221160132013 @default.
• W2122116013 countsByYear W21221160132014 @default.
• W2122116013 countsByYear W21221160132015 @default.
• W2122116013 countsByYear W21221160132016 @default.
• W2122116013 countsByYear W21221160132017 @default.
• W2122116013 countsByYear W21221160132018 @default.
• W2122116013 countsByYear W21221160132020 @default.
• W2122116013 countsByYear W21221160132021 @default.
• W2122116013 countsByYear W21221160132022 @default.
• W2122116013 countsByYear W21221160132023 @default.
• W2122116013 crossrefType "journal-article" @default.
• W2122116013 hasAuthorship W2122116013A5005083287 @default.
• W2122116013 hasAuthorship W2122116013A5041156218 @default.
• W2122116013 hasAuthorship W2122116013A5051611788 @default.
• W2122116013 hasAuthorship W2122116013A5072623856 @default.
• W2122116013 hasAuthorship W2122116013A5082394458 @default.
• W2122116013 hasAuthorship W2122116013A5091406841 @default.
• W2122116013 hasBestOaLocation W21221160131 @default.
• W2122116013 hasConcept C134018914 @default.
• W2122116013 hasConcept C185592680 @default.
• W2122116013 hasConcept C2775887612 @default.
• W2122116013 hasConcept C2778815084 @default.
• W2122116013 hasConcept C55493867 @default.
• W2122116013 hasConcept C86803240 @default.
• W2122116013 hasConceptScore W2122116013C134018914 @default.
• W2122116013 hasConceptScore W2122116013C185592680 @default.
• W2122116013 hasConceptScore W2122116013C2775887612 @default.
• W2122116013 hasConceptScore W2122116013C2778815084 @default.
• W2122116013 hasConceptScore W2122116013C55493867 @default.
• W2122116013 hasConceptScore W2122116013C86803240 @default.
• W2122116013 hasIssue "27" @default.
• W2122116013 hasLocation W21221160131 @default.
• W2122116013 hasOpenAccess W2122116013 @default.
• W2122116013 hasPrimaryLocation W21221160131 @default.
• W2122116013 hasRelatedWork W1031544844 @default.
• W2122116013 hasRelatedWork W1521550497 @default.
• W2122116013 hasRelatedWork W2004382209 @default.
• W2122116013 hasRelatedWork W2029983943 @default.
• W2122116013 hasRelatedWork W2042048723 @default.
• W2122116013 hasRelatedWork W2071868532 @default.

• next