Members of the Evolutionarily Conserved PMT Family of ProteinO-Mannosyltransferases Form Distinct Protein Complexes among Themselves

Abstract

Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, an essential protein modification. Since PMTs are evolutionarily conserved in fungi but are absent in green plants, the PMT family is a putative target for new antifungal drugs, particularly in fighting the threat of phytopathogenic fungi. The PMT family is phylogenetically classified into PMT1, PMT2, and PMT4 subfamilies, which differ in protein substrate specificity. In the model organism Saccharomyces cerevisiae as well as in many other fungi the PMT family is highly redundant, and only the simultaneous deletion of PMT1/PMT2 and PMT4 subfamily members is lethal. In this study we analyzed the molecular organization of PMT family members in S. cerevisiae. We show that members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p) and that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms. Under certain physiological conditions, however, Pmt1p interacts also with Pmt3p, and Pmt5p with Pmt2p, suggesting a compensatory cooperation that guarantees the maintenance ofO-mannosylation. Unlike the PMT1/PMT2 subfamily members, the single member of the PMT4 subfamily (Pmt4p) acts as a homomeric complex. Using mutational analyses we demonstrate that the same conserved protein domains underlie both heteromeric and homomeric interactions, and we identify an invariant arginine residue of transmembrane domain two as essential for the formation and/or stability of PMT complexes in general. Our data suggest that protein-protein interactions between the PMT family members offer a point of attack to shut down overall proteinO-mannosylation in fungi. Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, an essential protein modification. Since PMTs are evolutionarily conserved in fungi but are absent in green plants, the PMT family is a putative target for new antifungal drugs, particularly in fighting the threat of phytopathogenic fungi. The PMT family is phylogenetically classified into PMT1, PMT2, and PMT4 subfamilies, which differ in protein substrate specificity. In the model organism Saccharomyces cerevisiae as well as in many other fungi the PMT family is highly redundant, and only the simultaneous deletion of PMT1/PMT2 and PMT4 subfamily members is lethal. In this study we analyzed the molecular organization of PMT family members in S. cerevisiae. We show that members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p) and that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms. Under certain physiological conditions, however, Pmt1p interacts also with Pmt3p, and Pmt5p with Pmt2p, suggesting a compensatory cooperation that guarantees the maintenance ofO-mannosylation. Unlike the PMT1/PMT2 subfamily members, the single member of the PMT4 subfamily (Pmt4p) acts as a homomeric complex. Using mutational analyses we demonstrate that the same conserved protein domains underlie both heteromeric and homomeric interactions, and we identify an invariant arginine residue of transmembrane domain two as essential for the formation and/or stability of PMT complexes in general. Our data suggest that protein-protein interactions between the PMT family members offer a point of attack to shut down overall proteinO-mannosylation in fungi. endoplasmic reticulum protein O-mannosyltransferase amino acid blue native polyacrylamide gel electrophoresis dolichyl phosphate-activated mannose hemagglutinin o-phtaldialdehyde N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine l-1-tosylamido-2-phenylethyl chloromethyl ketone Protein O-mannosylation is an evolutionarily conserved protein modification of fundamental importance in many eukaryotes. In yeasts and fungi, the attachment of O-linked mannosyl residues to proteins of the secretory pathway is essential for cell viability (1Gentzsch M. Tanner W. EMBO J. 1996; 15: 5752-5759Google Scholar). In particular it is indispensable for cell wall integrity and normal cellular morphogenesis (2Strahl-Bolsinger S. Gentzsch M. Tanner W. Biochim. Biophys. Acta. 1999; 1426: 297-307Google Scholar, 3Timpel C. Strahl-Bolsinger S. Ziegelbauer K. Ernst J.F. J. Biol. Chem. 1998; 273: 20837-20846Google Scholar, 4Timpel C. Zink S. Strahl-Bolsinger S. Schroppel K. Ernst J.F. J. Bacteriol. 2000; 182: 3063-3071Google Scholar). Impairment ofO-mannosylation also affects the stability, localization, and/or proper function of individual proteins (5Bourdineaud J.P. van der Vaart J.M. Donzeau M. de Sampaio G. Verrips C.T. Lauquin G.J. Mol. Microbiol. 1998; 27: 85-98Google Scholar, 6Buurman E.T. Westwater C. Hube B. Brown A.J. Odds F.C. Gow N.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7670-7675Google Scholar, 7Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Google Scholar, 8Lodder A.L. Lee T.K. Ballester R. Genetics. 1999; 152: 1487-1499Google Scholar, 9Sanders S.L. Gentzsch M. Tanner W. Herskowitz I. J. Cell Biol. 1999; 145: 1177-1188Google Scholar, 10Philip B. Levin D.E. Mol. Cell. Biol. 2001; 21: 271-280Google Scholar). Furthermore, aberrant O-mannosylation can interfere with the retrograde transport of misfolded proteins across the membrane of the endoplasmic reticulum (ER)1 (11Harty C. Strahl S. Romisch K. Mol. Biol. Cell. 2001; 12: 1093-1101Google Scholar).O-mannosylation is not only important in yeast, but also in mammals. It was recently shown that in humans, O-mannosyl glycosylation represents a new pathomechanism for muscular dystrophy and neuronal migration disorders (12Yoshida A. Kobayashi K. Manya H. Taniguchi K. Kano H. Mizuno M. Inazu T. Mitsuhashi H. Takahashi S. Takeuchi M. Herrmann R. Straub V. Talim B. Voit T. Topaloglu H. Toda T. Endo T. Dev. Cell. 2001; 1: 717-724Google Scholar, 13Beltran-Valero De Bernabe D. Currier S. Steinbrecher A. Celli J. Van Beusekom E. Van Der Zwaag B. Kayserili H. Merlini L. Chitayat D. Dobyns W.B. Cormand B. Lehesjoki A.E. Cruces J. Voit T. Walsh C.A. Van Bokhoven H. Brunner H.G. Am. J. Hum. Genet. 2002; 71: 1033-1043Google Scholar). In yeast and fungi, O-mannosylation is initiated in the lumen of the ER by an essential family of proteinO-mannosyltransferases (PMTs). These enzymes catalyze the transfer of mannose from dolichyl phosphate-activated mannose (Dol-P-Man) to serine or threonine residues of secretory proteins (2Strahl-Bolsinger S. Gentzsch M. Tanner W. Biochim. Biophys. Acta. 1999; 1426: 297-307Google Scholar). In Saccharomyces cerevisiae, a total of seven PMT family members (Pmt1–7p) have been identified, which share almost identical hydropathy profiles that predict the PMTs to be integral membrane proteins with multiple transmembrane domains. Pmt1–6 proteins feature an overall protein sequence identity of 57.57. Pmt7p is less conserved. ProteinO-mannosyltransferase activity has been demonstrated for Pmt1–4p and Pmt6p (14Gentzsch M. Tanner W. Glycobiology. 1997; 7: 481-486Google Scholar). Aside from S. cerevisiae PMTs, orthologues are known from many other yeast and fungi, for example,Candida albicans (CaPMT1–2 andCaPMT4–6) and Schizosaccharomyces pombe(SpPMT1, SpPMT3, and SpPMT4) (Refs. 3Timpel C. Strahl-Bolsinger S. Ziegelbauer K. Ernst J.F. J. Biol. Chem. 1998; 273: 20837-20846Google Scholarand 4Timpel C. Zink S. Strahl-Bolsinger S. Schroppel K. Ernst J.F. J. Bacteriol. 2000; 182: 3063-3071Google Scholar). 2T. Willer and S. Strahl, unpublished data.2T. Willer and S. Strahl, unpublished data. Moreover, PMT homologues have been also identified in many multicellular eukaryotes such as Drosophila melanogaster, mouse and, humans (15Martı́n-Blanco E. Garcı́a-Bellido A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6048-6052Google Scholar, 16Jurado L.A. Coloma A. Cruces J. Genomics. 1999; 58: 171-180Google Scholar, 17Willer T. Amselgruber W. Deutzmann R. Strahl S. Glycobiology. 2002; 12: 771-783Google Scholar). Despite their evolutionarily conservation in fungi and throughout the animal kingdom (with the exception of Caenorhabditis elegans), PMTs are not present in green plants.2 This makes the PMT family in fungi especially attractive as a target for the development of new antifungal drugs in order to combat phytopathogenic fungi. The protein O-mannosyltransferases can be divided into three subfamilies: PMT1, PMT2, and PMT4, which include transferases closely related to S. cerevisiae Pmt1p, Pmt2p, and T. Amselgruber W. Deutzmann R. Strahl S. Glycobiology. 2002; 12: 771-783Google Scholar, V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). of the PMT1 and PMT2 show and from PMT4 subfamily PMT family members share three conserved sequence show between PMT1/PMT2 and PMT4 subfamily members V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). the PMT1/PMT2 and PMT4 protein in S.L. Gentzsch M. Tanner W. Herskowitz I. J. Cell Biol. 1999; 145: 1177-1188Google Scholar, M. Tanner W. Glycobiology. 1997; 7: 481-486Google Scholar). in fungi the PMT1/PMT2 subfamily is highly redundant, the PMT4 subfamily has only T. Amselgruber W. Deutzmann R. Strahl S. Glycobiology. 2002; 12: 771-783Google Scholar, V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). the PMT family members, from S. cerevisiae has been Pmt1p is an integral ER membrane with seven domains S. A. J. Biol. Chem. 1999; Scholar). the the the lumen of the domains that are between transmembrane and two and transmembrane and are the ER lumen and are essential for Pmt1p activity V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar, S. A. J. Biol. Chem. 1999; Scholar). The of invariant amino acid residues in that are in the and/or of protein and/or V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). of PMTs from highly conserved present in V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; which are also in and function is Sci. 2000; Scholar). Pmt1p a heteromeric with in and this formation is essential for activity V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar, M. T. Tanner W. Scholar). and of Pmt1p are in Pmt1p-Pmt2p interactions V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). Pmt1p, is known the molecular organization of the of the O-mannosylation In the present study we analyzed the molecular assembly of the PMT family in We demonstrate that formation is of for members of the PMT family in S. cerevisiae. members of the PMT1 subfamily heteromeric complexes with members of the PMT2 subfamily in acts as a homomeric complex. Despite the between PMT1/PMT2 and PMT4 we show that the same and residues protein-protein The S. cerevisiae are in I. and the of D. A. with the yeast M. A. S. A. J. Biol. Chem. 1999; V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; M. Gentzsch M. Bussey H. Tanner W. J. Biol. Chem. V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; and the for J. T. Scholar). and in by sequence are Mol. Cell. Biol. M. Gentzsch M. Bussey H. Tanner W. J. Biol. Chem. M. Gentzsch M. Bussey H. Tanner W. J. Biol. Chem. T. Gentzsch M. Tanner W. T. Gentzsch M. Tanner W. V. T. Gentzsch M. Tanner W. 1997; V. T. Gentzsch M. Tanner W. 1997; M. Gentzsch M. Bussey H. Tanner W. J. Biol. Chem. M. Tanner W. EMBO J. 1996; 15: 5752-5759Google The of O-mannosylation in of S. of unpublished in a new was of the PMT2 by a M. Gentzsch M. Bussey H. Tanner W. J. Biol. Chem. into with the same the a was and into with and in total of of the hemagglutinin to the of PMT2 by R. to and Scholar). that in sequence was by with the and the other of U. with the and The into and The was into of the was into with sequence of the was In the of this we a between the we and the yeast data we a from the yeast and the and into and These analyses that in to the data three pairs to is in The in the amino acid sequence of of of of the sequence was by with The The sequence was by a with a from E. J. J. Cell. 1996; that the yeast membrane R. and the yeast with and in The PMT4 and to was from S. cerevisiae and The was with and into into the of the the In the of the to the of amino of was and the and arginine residue of was for a by the and to of by sequence of the of was into to of with the same the a was and into in acid of was to by the and to of by sequence of the was into a of V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; with the a was and into to with proteins of and the to of S. cerevisiae Pmt3p, to of to of and to of The by S. cerevisiae as and for for PMT4, for The with the sequence by into a The proteins in E. The proteins from and into by to with the protein J. Biol. Chem. Scholar). as V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). as V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). of and was of from of the yeast was with of to protein for to protein as E. D. Scholar). with of for with of chloromethyl and and with of in of and proteins from as in V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; of by for The was by for to the proteins for from of of with of with of and in of to in C.A. S. A. in Scholar). total of and V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; a in of proteins by for The was by for of polyacrylamide to in was from a to to a of native polyacrylamide gel electrophoresis was a polyacrylamide from with a as H. G. Scholar). was to both polyacrylamide gel and The also G. This was electrophoresis for by was for yeast from a and with of The cell was in of as V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). The membrane was in of and for in of o-phtaldialdehyde was to of to of membrane and in the for The was with by or and to S. Tanner W. J. and S. R. L. M. EMBO J. 12: a of and M. T. Tanner W. a of and a of a of The and and complexes by the O-mannosyltransferase activity was as (14Gentzsch M. Tanner W. Glycobiology. 1997; 7: 481-486Google Scholar). that underlie the of we formation is a feature of the yeast PMT family We that S. cerevisiae molecular Pmt5p and Pmt6p in yeast as shown by analyses and from the deletion that the are highly and We also of and These to PMT complexes by and PMT we a cerevisiae Pmt1p V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). was in a deletion and from and was from with The and an of the and analyzed by and the with to Pmt1p to Pmt6p the of the the same was Pmt1p the and shown M. T. Tanner W. we that is the of Pmt1p Moreover, a for be in the and The of was but this was highly In or Pmt6p not be of from and deletion that interacts with of and the of Pmt1p-Pmt2p and complexes a of was in and as that to Pmt1p but not Pmt3p, or Pmt6p that Pmt1p interacts with of In in the a for Pmt5p be The of Pmt5p was however, the was not and highly Furthermore, of from a deletion that interacts with Pmt5p of Pmt1p not data that in yeast Pmt1p and a protein complex. In we that Pmt1p also interacts with Pmt3p, and that interacts with we the of which is the of was from of and In the a of Pmt1p, but not Pmt2p, or Pmt6p was present Pmt5p was highly with the and that interacts with the between and Pmt5p was of other proteins and and data not data show that and Pmt5p-Pmt3p complexes are present in S. cerevisiae. however, Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms. The and complexes which we in are less with Pmt1p-Pmt2p or Pmt5p-Pmt3p that the complexes are not and to that Pmt1p-Pmt2p and Pmt5p-Pmt3p represent the predominant PMT complexes in yeast, we H. G. that native protein from and polyacrylamide to native PMT complexes complexes by with of complexes that in from yeast two protein with an of and by The highly represents the The in of in in S. A. J. Biol. Chem. 1999; is to an migration of Pmt1p by the of the an to protein and/or the to which is in H. G. Scholar). The formation of Pmt1p was by and Pmt1p not In to Pmt1p, protein complexes with an molecular of be The of Pmt1p to molecular complexes to in which is highly to of PMT complexes Protein complexes with molecular of also by and not that in yeast in complexes are only present in with that in a but not in deletion the of the complexes by shown in and this is we in the of a of protein complexes with an of be the of of the These complexes not only by but also by not In complexes not These data that Pmt1p interacts with and however, represents the also that protein complexes almost only in the of Pmt5p not with an heteromeric with The of and complexes in yeast the complexes are is that complexes are in the of their to for a of this we analyzed and the of and are of from a that the of both and Pmt5p is with in the is The of Pmt1p is not suggesting that Pmt1p is not These that in the of the and complexes data we analyzed formation by Under the we in yeast only of a protein with an of be which is by and as well as not In complexes be in of the formation of and complexes in we in O-mannosyltransferase activity and we O-mannosyltransferase activity of and complexes (Refs. M. Tanner W. Glycobiology. 1997; 7: 481-486Google and T. Gentzsch M. Tanner W. Scholar, shown in O-mannosyltransferase activity is in and with of an of in and not and in which and complexes are in O-mannosyltransferase activity is by and with O-mannosyltransferase activity of and deletion from yeast of membrane proteins in the in the transfer of from to the of three are in a new from yeast of membrane proteins in the in the transfer of from to the of three are In data show that members of the PMT1 subfamily (Pmt1p and Pmt5p) complexes with members of the PMT2 subfamily (Pmt2p and and that Pmt1p-Pmt2p and Pmt5p-Pmt3p pairs are the predominant forms. Under and complexes can be to We not interactions between members of the PMT1 or PMT2 subfamily and the only member of the PMT4 In with other proteins be was from from a yeast not the of which not by the of other PMT family member not We to complexes to interactions or is with other an of was by of the to the of of the of a by that this is for was in a S. cerevisiae and with and of was to protein and which differ in molecular by a with shown in with and that is present in homomeric complexes in was in a deletion and was of between and and analyzed by as that in the of with an of which is in with a of of complexes with an molecular of be with the formation of data suggest that homomeric however, the with other molecular be homomeric of and was from in and and In was with and of was to protein of of the and of from with of protein was with or and and proteins identified by in to the other PMT family members homomeric we underlie homomeric and heteromeric PMT formation and/or We that a endoplasmic of Pmt1p is for activity but not for Pmt1p-Pmt2p formation V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar, also the same be the for we the of and the deletion in and yeast formation was by that in the of complexes with an of and can be in in to Furthermore, and are complexes in from to data we that is to interact with as well as with is in a it not the that this is essential for activity it not Our data show that domain of to in the same as of S. cerevisiae Pmt1p, amino acid residue in transmembrane domain two the is essential for the formation of heteromeric Pmt1p-Pmt2p complexes V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar, also In of Pmt1p for in a of activity V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar, also is for Pmt1p-Pmt2p complexes and as well activity are that a amino acid that is important for the of this arginine residue is highly conserved between PMT family members, we this residue also affects homomeric We of of in with The protein was and in a yeast a and a and of with that in yeast and the protein show an identical and are not to the of the that is essential for activity in In of a and, or In to which almost to and with a for in data show that underlie the formation of heteromeric complexes between members of the PMT1 and PMT2 and homomeric In yeast the PMT1 and PMT2 but not the PMT4 subfamily are highly In this study we demonstrate that the formation of protein complexes is a feature of PMT family members in We in members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and shown in Pmt1p-Pmt2p and Pmt5p-Pmt3p are the predominant complexes between PMT1 and PMT2 subfamily members in S. cerevisiae Under certain conditions, however, Pmt1p can interact with Pmt3p, and Pmt5p with Pmt2p, This can for of the is as is the in In the of the PMT4 subfamily homomeric complexes we that the same conserved protein domains both heteromeric and homomeric the PMT family in S. cerevisiae, Pmt1p is closely related to Pmt5p identity and and to identity and In of this of the proteins Pmt1p and as well as and have by This is by the that Pmt1p and Pmt5p are to other the of and 1997; that and a protein that from an of the yeast a of the to protein complexes between individual members of the PMT1 and the PMT2 subfamily and in S. cerevisiae. This also and complexes can be only certain physiological of suggest that Pmt1p-Pmt2p and complexes the same protein Pmt1p-Pmt2p complexes represent the predominant of in from to have shown that the simultaneous deletion of PMT1 in the of of in O-mannosyltransferase with yeast (1Gentzsch M. Tanner W. EMBO J. 1996; 15: 5752-5759Google Scholar, T. Gentzsch M. Tanner W. Scholar). In in O-mannosylation of the same protein is in and such as the cell wall proteins and (14Gentzsch M. Tanner W. Glycobiology. 1997; 7: 481-486Google Scholar, V. T. Gentzsch M. Tanner W. 1997; Scholar). In in O-mannosylation is not in or (1Gentzsch M. Tanner W. EMBO J. 1996; 15: 5752-5759Google Scholar, M. Tanner W. Glycobiology. 1997; 7: 481-486Google Scholar, V. T. Gentzsch M. Tanner W. 1997; Scholar). deletion of in a in from to a by with the in activity in (1Gentzsch M. Tanner W. EMBO J. 1996; 15: 5752-5759Google Scholar). In analyses of the O-mannosylation of in and Gentzsch and Tanner (1Gentzsch M. Tanner W. EMBO J. 1996; 15: 5752-5759Google that in by Pmt1p and as well as Pmt3p, the same proteins as Pmt1p and Pmt2p, but into in the of Pmt1p and The that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes is by the that of PMT1, PMT2, but not PMT4 and is in to cell that the of misfolded proteins in the ER L. Cell. 2000; Scholar). In Pmt1p-Pmt2p and Pmt5p-Pmt3p are to the same of substrate Pmt5p-Pmt3p complexes for substrate and, only a in yeast In the of Pmt1p and Pmt2p, however, Pmt5p-Pmt3p for O-mannosylation in be to the and which we only in in and or physiological such as in The of in O-mannosyltransferase activity in that and as and feature substrate to Our data suggest a compensatory cooperation between PMT1/PMT2 subfamily members, which in S. cerevisiae only the simultaneous deletion of PMT subfamily members in a and cell (1Gentzsch M. Tanner W. EMBO J. 1996; 15: 5752-5759Google Scholar). This is by the that the of a not in the formation of complexes the of Gentzsch M. T. Tanner W. that of PMT2 a of in O-mannosyltransferase activity in yeast and in deletion compensatory cooperation between the members of the PMT1/PMT2 subfamily in S. cerevisiae is also by the that in the yeast S. only member of PMT subfamily is the deletion of the single PMT2 subfamily member is In S. cerevisiae Pmt6p an overall sequence identity of with and of with In the of and that Pmt6p interacts with with not and from other in that Pmt6p interacts with other not with not identify we analyzed of the or the are with was that S. R. L. M. EMBO J. 12: S. R. J. Cell Biol. R. R. I. S. L. M. EMBO J. or R. J. Cell Biol. interact with Pmt6p not are to the of Pmt6p as antifungal it is that in S. cerevisiae and in C. albicans only deletion of in with PMT1/PMT2 subfamily members (1Gentzsch M. Tanner W. EMBO J. 1996; 15: 5752-5759Google Scholar, J.F. 2001; Scholar). to protein O-mannosylation both PMT1/PMT2 and PMT4 subfamily members be in from PMT1/PMT2 subfamily members, such as substrate and conserved sequence S.L. Gentzsch M. Tanner W. Herskowitz I. J. Cell Biol. 1999; 145: 1177-1188Google Scholar, M. Tanner W. Glycobiology. 1997; 7: 481-486Google Scholar, V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). In homomeric protein complexes as demonstrated in this study In of it is of particular importance that the of the and/or stability of PMT complexes in both PMT1/PMT2 and PMT4 Our mutational analyses that which is highly conserved between PMT family members, is for complexes and activity and the in Pmt1p affects Pmt1p-Pmt2p complexes and in of activity V. T. M. Strahl-Bolsinger S. J. Biol. Chem. 2000; Scholar). PMT complexes offer a point of attack to proteinO-mannosylation in fungi. are a of protein O-mannosyltransferases or heteromeric protein is that complexes an of a of feature of proteins is that O-linked are in Sci. 15: Scholar). are to important for protein a O-mannosylation proteins are in the lumen of the ER G. R. 71: C. Strahl S. Romisch K. Mol. Biol. Cell. 2001; 12: 1093-1101Google Scholar). and S. Strahl, unpublished data. the which be by function has been for other such as J. Biol. Chem. 1997; or the complexes and J. S. EMBO J. 1998; Scholar, J. S. J. Biol. Chem. 1999; Scholar, J. S. J. Biol. Chem. 2002; Scholar). Furthermore, members of the PMT1 and PMT2 subfamily the same protein substrate (14Gentzsch M. Tanner W. Glycobiology. 1997; 7: 481-486Google Scholar, V. T. Gentzsch M. Tanner W. 1997; serine and threonine residues and the same Since to are known that are this to be this is by the that is by but other PMT family member (11Harty C. Strahl S. Romisch K. Mol. Biol. Cell. 2001; 12: 1093-1101Google Scholar). the of PMT complexes it be important to their substrate and of PMTs specificity. We C. M. and S. for and C. J. T. and H. for with the of and We are to L. J. and U. for and We W. Tanner for many and for the of the