The Essential Smp3 Protein Is Required for Addition of the Side-branching Fourth Mannose during Assembly of Yeast Glycosylphosphatidylinositols (2001)

The major glycosylphosphatidylinositols (GPIs) transferred to protein in mammals and trypanosomes contain three mannoses. In Saccharomyces cerevisiae, however, the GPI transferred to protein bears a fourth, α1,2-linked Man on the α1,2-Man that receives the phosphoethanolamine (EthN-P) moiety through which GPIs become linked to protein. We report that temperature-sensitive smp3 mutants accumulate a GPI containing three mannoses and that smp3 is epistatic to thegpi11, gpi13, and gaa1 mutations, which normally result in the accumulation of Man4-GPIs, including the presumed substrate for the yeast GPI transamidase. The Smp3 protein, which is encoded by an essential gene, is therefore required for addition of the fourth Man to yeast GPI precursors. The finding that smp3 prevents the formation of the Man4-GPI that accumulates when addition of EthN-P to Man-3 is blocked in a gpi13 mutant suggests that the presence of the fourth Man is important for transfer of EthN-P to Man-3 of yeast GPIs. The Man3-GPI that accumulates in smp3 is a mixture of two dominant isoforms, one bearing a single EthN-P side branch on Man-1, the other with EthN-P on Man-2, and these isoforms can be placed in separate arms of a branched GPI assembly pathway. Smp3-related proteins are encoded in the genomes ofSchizosaccharomyces pombe, Candida albicans,Drosophila melanogaster, and Homo sapiens and form a subgroup of a family of proteins, the other groups of which are defined by the Pig-B(Gpi10) protein, which adds the third GPI mannose, and by the Alg9 and Alg12 proteins, which act in the dolichol pathway for N-glycosylation. Because Man4-containing GPI precursors are normally formed in yeast and Plasmodium falciparum, whereas addition of a fourth Man during assembly of mammalian GPIs is rare and not required for GPI transfer to protein, Smp3p-dependent addition of a fourth Man represents a target for antifungal and antimalarial drugs. The major glycosylphosphatidylinositols (GPIs) transferred to protein in mammals and trypanosomes contain three mannoses. In Saccharomyces cerevisiae, however, the GPI transferred to protein bears a fourth, α1,2-linked Man on the α1,2-Man that receives the phosphoethanolamine (EthN-P) moiety through which GPIs become linked to protein. We report that temperature-sensitive smp3 mutants accumulate a GPI containing three mannoses and that smp3 is epistatic to thegpi11, gpi13, and gaa1 mutations, which normally result in the accumulation of Man4-GPIs, including the presumed substrate for the yeast GPI transamidase. The Smp3 protein, which is encoded by an essential gene, is therefore required for addition of the fourth Man to yeast GPI precursors. The finding that smp3 prevents the formation of the Man4-GPI that accumulates when addition of EthN-P to Man-3 is blocked in a gpi13 mutant suggests that the presence of the fourth Man is important for transfer of EthN-P to Man-3 of yeast GPIs. The Man3-GPI that accumulates in smp3 is a mixture of two dominant isoforms, one bearing a single EthN-P side branch on Man-1, the other with EthN-P on Man-2, and these isoforms can be placed in separate arms of a branched GPI assembly pathway. Smp3-related proteins are encoded in the genomes ofSchizosaccharomyces pombe, Candida albicans,Drosophila melanogaster, and Homo sapiens and form a subgroup of a family of proteins, the other groups of which are defined by the Pig-B(Gpi10) protein, which adds the third GPI mannose, and by the Alg9 and Alg12 proteins, which act in the dolichol pathway for N-glycosylation. Because Man4-containing GPI precursors are normally formed in yeast and Plasmodium falciparum, whereas addition of a fourth Man during assembly of mammalian GPIs is rare and not required for GPI transfer to protein, Smp3p-dependent addition of a fourth Man represents a target for antifungal and antimalarial drugs. glycosylphosphatidylinositol dolichol phosphate mannose phosphoethanolamine high performance thin layer chromatography jack bean α-mannosidase inositol Glycosylphosphatidylinositols (GPIs)1 with the conserved core structure H2N-CH2-CH2-PO4-6Manα1,2Manα1,6Manα1,4GlcNα1,6Ins phospholipid are made by all eukaryotes. Many GPIs are transferred to the COOH terminus of secretory glycoproteins and serve as membrane anchors, but others remain protein-free (1McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (803) Google Scholar, 2Tiede A. Bastisch I. Schubert J. Orlean P. Schmidt R.E. Biol. Chem. 1999; 380: 503-523Crossref PubMed Scopus (106) Google Scholar, 3McConville M.J. Menon A.K. Mol. Membr. Biol. 2000; 17: 1-16Crossref PubMed Scopus (127) Google Scholar). In Saccharomyces cerevisiae, mannoproteins can be transferred from their GPI anchor to cell wall β-glucan (4Lipke P.N. Ovalle R. J. Bacteriol. 1998; 180: 3735-3740Crossref PubMed Google Scholar, 5Fujii T. Shimoi H. Iimura Y. Biochim. Biophys. Acta. 1999; 1427: 133-144Crossref PubMed Scopus (65) Google Scholar). The formation of GPIs is essential for growth of yeasts, Leishmania mexicana, and the bloodstream form of Trypanosoma brucei and for embryonic development in mammals (6Leidich S.D. Drapp D.A. Orlean P. J. Biol. Chem. 1994; 269: 10193-10196Abstract Full Text PDF PubMed Google Scholar, 7Colussi P.A. Orlean P. Yeast. 1997; 13: 139-150Crossref PubMed Scopus (24) Google Scholar, 8Ilgoutz S.C. Zawadzki J.L. Ralton J.E. McConville M.J. EMBO J. 1999; 18: 2746-2755Crossref PubMed Scopus (76) Google Scholar, 9Nagamune K. Nozaki T. Y. K. T. T. R. H. T. A. 2000; PubMed Scopus Google Scholar, K. I. T. T. J. PubMed Google Scholar). GPIs are on an inositol phospholipid in a the of which are in of the A. Bastisch I. Schubert J. Orlean P. Schmidt R.E. Biol. Chem. 1999; 380: 503-523Crossref PubMed Scopus (106) Google Scholar, 3McConville M.J. Menon A.K. Mol. Membr. Biol. 2000; 17: 1-16Crossref PubMed Scopus (127) Google Scholar, Biochem. J. PubMed Scopus Google Scholar, J. T. Biochem. Full Text PDF PubMed Scopus Google Scholar). the core of the GPI can be with side In yeast and the and mannoses can be with phosphoethanolamine (EthN-P) and GPI precursors made in GPI precursors a fourth, α1,2-linked Man on the α1,2-linked Man of the core A. A. EMBO J. 1994; 13: PubMed Scopus Google Scholar, P. A. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). mutants in EthN-P addition to and Man-3 and in GPI transfer to protein accumulate GPIs with mannoses A. I. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Orlean P. Mol. Biol. 2000; PubMed Scopus Google Scholar, P.N. A. J. Biol. PubMed Scopus Google Scholar, H. J. Biol. PubMed Scopus Google Scholar). In in mammalian the contain three mannoses J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. Menon A.K. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, Y. Y. R. K. H. T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google that of be formed J. Biol. Chem. Full Text PDF PubMed Google Scholar, Y. Y. R. K. H. T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. R. K. T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). GPIs in mammals contain three but a fourth, α1,2-linked Man on the GPI anchor of the Ferguson R. T. R. A. PubMed Scopus Google and membrane I. Ferguson M.A.J. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We report that the Smp3 protein, a of a family of phosphate is a for the that adds the fourth Man during yeast GPI the formation of and result in the accumulation of of the fourth mannose is therefore required to the that are the for the yeast GPI transamidase. Because the is and addition of a fourth Man to be of in GPI assembly in yeast is in mammalian addition of the fourth Man represents a target for antifungal drugs. and from mixture and from and jack bean and from by The yeast in are in and the of made for is and as in PubMed Scopus Google Scholar, and the as but of and as in Orlean P. Mol. Biol. 2000; PubMed Scopus Google Scholar, that in to for and to of are The to for and to of are The smp3 K. H. Y. Mol. PubMed Scopus Google to P. Mol. Biol. PubMed Scopus Google to the and The and in a for mutants that the for A. and P. the and in P. Mol. Biol. PubMed Scopus Google to The of by with Orlean P. Mol. Biol. 2000; PubMed Scopus Google Scholar). by with a Orlean P. Mol. Biol. 2000; PubMed Scopus Google and the to and to on with The of from from the of and these and by the of their mutants on the single mutants mutants in a of the and from from the and single the single to on with The presence of the smp3 and gaa1 in these by the on a the mutants the of of not smp3 an J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google to a of of for growth of with and by the Saccharomyces from in which is made by to a containing and of the and of the which the the J. P. PubMed Scopus Google Scholar). in which of is the of the made by to the from and the the and the of P. Mol. Biol. PubMed Scopus Google by by these the to and from the that therefore by of the with the the to the Orlean P. Mol. Biol. 2000; PubMed Scopus Google Scholar). in which is made by to a containing of the and of the which the the which to The from that that in as to for and with of for of GPIs in in in for to and with for as in Orlean P. Mol. Biol. 2000; PubMed Scopus Google In the of by in which of the to for with Orlean P. Mol. Biol. 2000; PubMed Scopus Google Scholar). in with and and and with of in and by by as Orlean P. Mol. Biol. 2000; PubMed Scopus Google Scholar). from of by two of of the of the jack and of EthN-P in Orlean P. Mol. Biol. 2000; PubMed Scopus Google and P. Ferguson M.A.J. PubMed Scopus Google from the Man4-GPI that accumulates in the A. I. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Orlean P. Mol. Biol. 2000; PubMed Scopus Google and a jack bean α-mannosidase by of the with with and and of as S.D. Orlean P. J. Biol. Chem. 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The and a and smp3 mutants a containing the with and which from the and by a major as as a that a in GPI and of in the whereas accumulation by when the these on a and that their GPI is to a in the that the accumulation of the temperature-sensitive smp3 mutants can be by in an a that by a of the of the in for to to become of which with The a with the as and that accumulates as a of the of not in with is to but to by The the and the is with the presence of an to the are of yeast GPI precursors I. P. A. A. 1998; PubMed Scopus Google Scholar, P. Y. M.J. T. H. Biochem. J. 1998; PubMed Scopus Google Scholar, Orlean P. Mol. Biol. 2000; PubMed Scopus Google Scholar, I. A. A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. 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The mutant not accumulate that accumulation is on formation of and the smp3 in the GPI assembly however, is epistatic to and In formation of bears two one of which is on and bears a single EthN-P on and In the accumulation of the a Man4-GPI with three I. P. A. A. 1998; PubMed Scopus Google We that the mutant a growth in to the single the not on with the the formation of the that accumulates in Orlean P. Mol. Biol. 2000; PubMed Scopus Google and in a temperature-sensitive the mutants to formation of in and and and as in smp3 single therefore the formation of the that accumulate in in of three essential proteins that act in the GPI pathway. The finding that smp3 the accumulation of is with the that the prevents the addition of the fourth, mannose to GPIs and to the that is a We by the structure of the of by and and of to by with The from a to that of the to whereas A. to that the of three the of which is in smp3 is therefore in the addition of the fourth, mannose to the GPI The finding that be with the presence of one on the of EthN-P the with and and of two major one as the other as that is a mixture of The of that the GPI from an EthN-P moiety on mannose and therefore that one EthN-P on the Because the GPI that to with the one that of one EthN-P We that of a mixture of two Man3-GPI isoforms, one bearing EthN-P on and one bearing EthN-P on Because of with the of in the in is that the mixture a of a Man3-GPI with a single EthN-P moiety on the presence of be by We the smp3 mutants are in GPI to protein by GPI of a for a GPI in yeast A. H. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). in GPI to in prevents of a form of the protein in the and to remain in a form A. H. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). The two of are by smp3 with and a during which are which is In the the form of the whereas in the the protein a The mutant a in as the mutant S.D. Orlean P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in suggests that is required for transfer of GPIs to protein. to a in protein. The of the and the of on protein a of of of a Man3-GPI be transferred to protein is the these GPIs not the to of that the genomes of pombe, albicans,Drosophila melanogaster, and Homo sapiens proteins The of these protein are The to the and Pig-B(Gpi10) proteins but are to be the and of these pombe, melanogaster, and all of the that the proteins are a separate subgroup of the a of the of of these proteins and to an the of the J. Scholar). that the proteins a separate as the proteins the of and The Smp3 proteins are from other of the family of a on an I. P. A. A. 1998; PubMed Scopus Google that these the in the Smp3-related proteins is in all and Pig-B(Gpi10) in these is to in and a conserved The family of to of protein and as proteins with to one other of these We not the is the of yeast but that the presence of a for a protein is with the that of GPIs that to a fourth Man in of mammalian J. Biol. Chem. Full Text PDF PubMed Google Scholar, Y. Y. R. K. T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the and Smp3 proteins are transfer mannose to GPI to form We therefore these for high in in the other protein. of on a the to a on the accumulation of smp3 mutants not be by of and not to when on a and therefore for one in the that these a high of for their GPIs in The are that the essential Smp3 protein is required for addition of the fourth Man to yeast GPI that in the of transfer of EthN-P to the third GPI Man is not and that is for transfer of GPI precursors to yeast protein. The for the GPI assembly in smp3 mutants and for the that other proteins in the transfer of Man from to is that is the that adds the fourth, Man during assembly of yeast GPIs. that the in smp3 mutants is in the addition of the fourth Man to be for of the of the that adds GPIs to protein, not for formation of GPIs for of the We these The to that the that are normally transferred to protein in yeast all a fourth, mannose GPIs in yeast mannoses J.E. McConville M.J. A. Ferguson M.A.J. J. Biol. Chem. 1993; Full Text PDF PubMed Google and the gaa1 which are in GPI transfer to protein, accumulate Man4-GPIs, the of which is the P.N. A. J. Biol. PubMed Scopus Google Scholar, H. J. Biol. PubMed Scopus Google Scholar). Because smp3 is epistatic to as as and gpi13, which accumulate Man4-GPIs, be required for the formation of all GPI to The the that the a in GPI transfer to protein. the accumulation in these be and of and to protein, to cell growth that the of is to the transfer of to protein, is the presence of the fourth Man is for a GPI to serve as a a for that the fourth mannose is important for transfer of the to Man-3 of the GPI which are blocked in the addition of EthN-P to accumulate a GPI with the structure in Orlean P. Mol. Biol. 2000; PubMed Scopus Google that the GPI by is finding that the formation of in in the that GPI to a fourth We that act on a Man3-GPI a single smp3 mutant be to an accumulation of GPIs bearing two which not the of an is a in the formation of Man4-GPIs, including the presumed for addition of the is that the fourth Man transfer to protein. EthN-P be to Man-3 of in smp3 and in be transferred to protein. the of smp3 mutants with to GPI transfer to protein. of that from a that serve as GPI substrate and that accumulate in the to EthN-P on and be to the of in in of as GPIs with from of the yeast of that are GPIs in of smp3 and of to but the of these are to are however, are of transferred to protein, be to be in however, when to We that the mutant a growth smp3 gaa1 that Smp3p-dependent addition of the fourth Man is for the of GPI for the that bearing two are not the that can be transferred to protein in smp3 and all of the GPI on yeast protein to contain three not be for as by the of smp3 and the of the The of to growth in that the fourth mannose, which is normally on all yeast GPI J.E. McConville M.J. A. Ferguson M.A.J. J. Biol. Chem. 1993; Full Text PDF PubMed Google essential anchor transfer to protein. is that Man is for of mannoproteins the yeast cell wall of a a Man in the GPI and (4Lipke P.N. Ovalle R. J. Bacteriol. 1998; 180: 3735-3740Crossref PubMed Google Scholar, 5Fujii T. Shimoi H. Iimura Y. Biochim. Biophys. Acta. 1999; 1427: 133-144Crossref PubMed Scopus (65) Google Scholar). The fourth however, not form the for all in cell wall proteins, the to β-glucan be through the of a core GPI Man T. Shimoi H. Iimura Y. Biochim. Biophys. Acta. 1999; 1427: 133-144Crossref PubMed Scopus (65) Google Scholar). for the fourth mannose on with the is that the fourth mannose be required for of GPI proteins from the the essential of the protein is is the one by the of a in the addition of the fourth Man to yeast GPI an to form the presumed for EthN-P transfer to in that addition of the fourth Man is for addition of the EthN-P to Man-3 in The in GPI transfer to protein mutants be to the of these and their to and transfer to protein, these mutants accumulate the The a for a accumulation an GPI for growth I. P. A. A. 1998; PubMed Scopus Google Scholar). is an essential protein is required for addition of the third mannose during GPI assembly I. P. A. A. 1998; PubMed Scopus Google Scholar, P. Y. M.J. T. H. Biochem. J. 1998; PubMed Scopus Google Scholar). The major GPIs that can be in yeast mutants can be in a that a branched assembly pathway Orlean P. Mol. Biol. 2000; PubMed Scopus Google and of the two major isoforms of can be placed in one of the The of can be the and in an that is defined by a of from I. P. A. A. 1998; PubMed Scopus Google Scholar, P. Y. M.J. T. H. Biochem. J. 1998; PubMed Scopus Google to that all EthN-P on I. P. A. A. 1998; PubMed Scopus Google Scholar, P. Y. M.J. T. H. Biochem. J. 1998; PubMed Scopus Google Scholar, A. I. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Orlean P. Mol. Biol. 2000; PubMed Scopus Google Scholar, I. A. A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The of is a of the Man4-GPI with EthN-P on that accumulates in the mutant Orlean P. Mol. Biol. 2000; PubMed Scopus Google Scholar). for the formation of the two GPIs is that are in an of a branched pathway. to in in addition of the third GPI the mutant accumulates the of I. P. A. A. 1998; PubMed Scopus Google Scholar, P. Y. M.J. T. H. Biochem. J. 1998; PubMed Scopus Google with a not to accumulate that serve as precursors of an as GPI be is that pathway addition of an in which EthN-P is from and one is to Man-2, the addition of EthN-P to of an the GPI not is that is but to of for of We the that the GPIs of the pathway are in GPI assembly the formation of and EthN-P on suggests that addition of EthN-P to is not required for transfer of the α1,2-linked mannose, of with an of EthN-P addition to to accumulation of P. Y. M.J. T. H. Biochem. J. 1998; PubMed Scopus Google Scholar). Because GPI is essential in and (6Leidich S.D. Drapp D.A. Orlean P. J. Biol. Chem. 1994; 269: 10193-10196Abstract Full Text PDF PubMed Google Scholar, 7Colussi P.A. Orlean P. Yeast. 1997; 13: 139-150Crossref PubMed Scopus (24) Google Scholar, 8Ilgoutz S.C. Zawadzki J.L. Ralton J.E. McConville M.J. EMBO J. 1999; 18: 2746-2755Crossref PubMed Scopus (76) Google Scholar, 9Nagamune K. Nozaki T. Y. K. T. T. R. H. T. A. 2000; PubMed Scopus Google be by that mammals and in the of GPI target is the addition of the Man in GPI in not inositol of M.A.J. A. A. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar, A. A. Ferguson M.A.J. EMBO J. 1997; PubMed Scopus Google Scholar). The of a of EthN-P transfer to P. Y. M.J. T. H. Biochem. J. 1998; PubMed Scopus Google Scholar, A. P. Y. H. EMBO J. 1997; PubMed Scopus Google that the in GPI assembly can be in a finding that the for that Smp3p-dependent addition of the fourth mannose to GPI precursors and transfer of the EthN-P to a Man4-GPI are that be by antifungal from the and of the GPI precursors that can be in mammalian is that Man4-GPI precursors are formed and that the EthN-P is to Man-3 of a Man3-GPI J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. Menon A.K. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, Y. Y. R. K. H. T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. R. K. T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). cell in the mammalian of the GPI and accumulate with one to three EthN-P that the major mammalian GPI substrate three mannoses J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, K. Y. J. H. T. Mol. Biol. 2000; PubMed Scopus Google Scholar). is with the that GPIs in mammalian three mannoses (1McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (803) Google Scholar, M.A.J. A. A. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). The in all GPIs a fourth, mannose J.E. McConville M.J. A. Ferguson M.A.J. J. Biol. Chem. 1993; Full Text PDF PubMed Google the presumed GPI are Man4-GPIs, and the of the that of bearing the EthN-P can be The of in mammalian the that addition of a fourth mannose to GPI precursors be in to whereas Smp3p-dependent addition of is essential for therefore represents a target for antifungal and a with and to is encoded in the of the We that the GPI made by P. bearing a fourth mannose P. A. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google the that adds mannose therefore be a target for antimalarial The that the EthN-P to GPIs are The and proteins, which are required for addition of EthN-P to Man-3 of yeast and mammalian GPI Orlean P. Mol. Biol. 2000; PubMed Scopus Google Scholar, I. A. A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. R. K. T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google to in their for GPI EthN-P to a whereas that for a Man4-GPI as for the in in be in the development of antifungal drugs. We for and for and for for from the and of with the of the of and and the