YND1, a Homologue of GDA1, Encodes Membrane-bound Apyrase Required for Golgi N- andO-Glycosylation in Saccharomyces cerevisiae

The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in thegda1 null mutant caused a significant increase in microsomal membrane-bound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ATP, GDP/GTP, and UDP/UTP and ∼50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The Δynd1 mutant cells were defective in O- and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in Δgda1disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the Δynd1Δgda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells. The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in thegda1 null mutant caused a significant increase in microsomal membrane-bound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ATP, GDP/GTP, and UDP/UTP and ∼50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The Δynd1 mutant cells were defective in O- and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in Δgda1disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the Δynd1Δgda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells. Apyrases (EC 3.6.1.5) are known as enzymes that hydrolyze both di- and triphosphate nucleotides as well as thiamine pyrophosphate. Apyrase family proteins are widely distributed in eukaryotic cells from yeast to mammals (1Plesner L. Int. Rev. Cytol. 1995; 158: 141-214Crossref PubMed Scopus (449) Google Scholar, 2Komoszynski M. Wojtczak A. Biochim. Biophys. Acta. 1996; 1310: 233-241Crossref PubMed Scopus (120) Google Scholar, 3Wang T.-F. Guidotti G. J. Biol. Chem. 1998; 273: 11392-11399Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Different from ATPases that specifically hydrolyze ATP, apyrases generally hydrolyze both ATP and ADP, although the rate of ATP and ADP hydrolysis differs depending on the sources. The main products of ATP hydrolysis by apyrase are AMP and two orthophosphate anions. Apyrases can be divided into ecto-apyrases with the catalytic domain outside the cell and endo-apyrases with the catalytic site inside the cell (2Komoszynski M. Wojtczak A. Biochim. Biophys. Acta. 1996; 1310: 233-241Crossref PubMed Scopus (120) Google Scholar). It has been suggested thatecto-apyrases may participate in neurotransmission (4Edwards F.A. Gibb A.J. FEBS Lett. 1993; 325: 86-89Crossref PubMed Scopus (138) Google Scholar) and blood platelet aggregation and pressure regulation (5Marcus A.J. Safier L.B. FASEB J. 1993; 7: 516-522Crossref PubMed Scopus (241) Google Scholar). However, less is known about the role of endo-apyrases. Although some roles of endo-apyrase have been suggested in protein glycosylation and sugar level control (3Wang T.-F. Guidotti G. J. Biol. Chem. 1998; 273: 11392-11399Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 6Fanta N. Anich M. Mancilla M. Kettlum A.M. Valenzuela M.A. Traverso-Cori A. Arch. Biol. Med. Exp. 1988; 21: 129-133Google Scholar) and in regulation of membrane integrity (7Girolomoni G. Santanionio M.L. Pastore S. Bergstresser P.R. Giannetti A. Cruz P.D. J. Invest. Dermatol. 1993; 100: 282-287Abstract Full Text PDF PubMed Google Scholar), there is no direct evidence for the in vivo function of endo-apyrase (2Komoszynski M. Wojtczak A. Biochim. Biophys. Acta. 1996; 1310: 233-241Crossref PubMed Scopus (120) Google Scholar).Specific oligosaccharide modification of glycoproteins in eukaryotic cells occurs mainly in the lumen of the Golgi apparatus. The substrates for these reactions are nucleotide sugars, which are synthesized in the cytosol. As shown by genetic and biochemical analysis (8Abeijon C. Yanagisawa K. Mandon E.C. Häusler A. Moreman K. Hirschberg C.B. Robbins P.W. J. Cell Biol. 1993; 122: 307-323Crossref PubMed Scopus (158) Google Scholar, 9Capasso J.M. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7051-7055Crossref PubMed Scopus (108) Google Scholar), nucleotide sugars are imported from the cytosol to the Golgi lumen by highly specific transporters, which may also antiport the corresponding nucleoside monophosphate from the Golgi lumen to the cytosol. NDPase hydrolyzes nucleoside diphosphate to nucleoside monophosphate, which is a putative antiporter of nucleotide sugars. This allows the entry of additional nucleotide sugars from the cytosol to the Golgi lumen. Thereby, the NDPase is considered to play a critical role in the translocation of these nucleotide sugars from the cytosol into the lumen (10Hirschberg C.B. Clapham D.E. Ehrlich B.E. Transporter of Nucleotides and Nucleotide Derivatives in the Endoplasmic Reticulum and Golgi Apparatus. Rockefeller University Press, New York1996: 105-120Google Scholar).In Saccharomyces cerevisiae, mannosylation of N- and O-linked oligosaccharides is mainly regulated by a GDPase that converts GDP to GMP. GDA1, the gene for GDPase, has been cloned and found to encode a type II membrane protein (8Abeijon C. Yanagisawa K. Mandon E.C. Häusler A. Moreman K. Hirschberg C.B. Robbins P.W. J. Cell Biol. 1993; 122: 307-323Crossref PubMed Scopus (158) Google Scholar). Gda1p shows a high activity toward GDP, but a low activity toward UDP and no activity toward ATP and ADP (11Yanagisawa K. Resnick D. Abeijon C. Robbins P.W. Hirschberg C.B. J. Biol. Chem. 1990; 265: 19351-19355Abstract Full Text PDF PubMed Google Scholar). Deletion of GDA1results in a marked reduction in Golgi mannosylation of proteins and lipids in vivo (8Abeijon C. Yanagisawa K. Mandon E.C. Häusler A. Moreman K. Hirschberg C.B. Robbins P.W. J. Cell Biol. 1993; 122: 307-323Crossref PubMed Scopus (158) Google Scholar) and causes a 4-fold lower rate of GDP-mannose entry into Golgi vesicles in dolichol-P-Man synthase-deficient cells (12Berninsone P. Miret J.J. Hirschberg C.B. J. Biol. Chem. 1994; 269: 207-211Abstract Full Text PDF PubMed Google Scholar). Nevertheless, in Δgda1cells, invertase is still heavily glycosylated, and in addition, a significant amount of GMP, as well as very low residual nonspecific nucleoside phosphatase activity unrelated to Gda1p, was detected in the Golgi membranes (8Abeijon C. Yanagisawa K. Mandon E.C. Häusler A. Moreman K. Hirschberg C.B. Robbins P.W. J. Cell Biol. 1993; 122: 307-323Crossref PubMed Scopus (158) Google Scholar, 12Berninsone P. Miret J.J. Hirschberg C.B. J. Biol. Chem. 1994; 269: 207-211Abstract Full Text PDF PubMed Google Scholar). These observations suggested the importance of residual GDPase activity in the Golgi lumen of the Δgda1mutant. Another possibility could be a direct supply of GMP from GDP-mannose by mannosyl-phosphate transferase. The MNN4 gene is known to encode a positive regulator of mannosyl phosphorylation (13Odani T. Shinmma Y. Wang X.-H. Jigami Y. FEBS Lett. 1997; 420: 186-190Crossref PubMed Scopus (71) Google Scholar, 14Odani T. Shimma Y. Tanaka A. Jigami Y. Glycobiology. 1996; 6: 805-810Crossref PubMed Scopus (65) Google Scholar), but the analysis of mannosylation of invertase in Δgda1Δmnn4 double disruptant cells showed no effect on mannosylation when compared with that in Δgda1 alone. This result strongly indicated that the remaining GMP did not arise from mannosyl phosphorylation in the Golgi lumen of Δgda1 cells and prompted us to investigate other GDPases involved in protein glycosylation. At that time, it was already known that theSaccharomyces Genome Database contains at least one homologue of Gda1p that possess ∼20% amino acid identity to Gda1p and that also belongs to the apyrase superfamily, as determined by sequence alignment (15Handa M. Guidotti G. Biochem. Biophys. Res. Commun. 1996; 218: 916-923Crossref PubMed Scopus (273) Google Scholar).Here we report that the residual GDPase activity in the Δgda1 mutant results from this homologue of Gda1p. It turned out to be a typical apyrase that can hydrolyze not only nucleoside diphosphates, as Gda1p does, but also nucleoside triphosphates. We also demonstrate that additional expression of this yeast apyrase can complement the lack of GDPase activity of Gda1p and plays an important role in controlling the protein glycosylation in Golgi compartments. For the above reason, we named the GDA1homologue YND1 (yeast nucleosidediphosphatase) to indicate a possible redundancy between the YND1 and GDA1 genes. In this paper, we present the first direct evidence for in vivo endo-apyrase function in protein glycosylation and suggest the involvement of yeast apyrase in cell wall integrity. Apyrases (EC 3.6.1.5) are known as enzymes that hydrolyze both di- and triphosphate nucleotides as well as thiamine pyrophosphate. Apyrase family proteins are widely distributed in eukaryotic cells from yeast to mammals (1Plesner L. Int. Rev. Cytol. 1995; 158: 141-214Crossref PubMed Scopus (449) Google Scholar, 2Komoszynski M. Wojtczak A. Biochim. Biophys. Acta. 1996; 1310: 233-241Crossref PubMed Scopus (120) Google Scholar, 3Wang T.-F. Guidotti G. J. Biol. Chem. 1998; 273: 11392-11399Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Different from ATPases that specifically hydrolyze ATP, apyrases generally hydrolyze both ATP and ADP, although the rate of ATP and ADP hydrolysis differs depending on the sources. The main products of ATP hydrolysis by apyrase are AMP and two orthophosphate anions. Apyrases can be divided into ecto-apyrases with the catalytic domain outside the cell and endo-apyrases with the catalytic site inside the cell (2Komoszynski M. Wojtczak A. Biochim. Biophys. Acta. 1996; 1310: 233-241Crossref PubMed Scopus (120) Google Scholar). It has been suggested thatecto-apyrases may participate in neurotransmission (4Edwards F.A. Gibb A.J. FEBS Lett. 1993; 325: 86-89Crossref PubMed Scopus (138) Google Scholar) and blood platelet aggregation and pressure regulation (5Marcus A.J. Safier L.B. FASEB J. 1993; 7: 516-522Crossref PubMed Scopus (241) Google Scholar). However, less is known about the role of endo-apyrases. Although some roles of endo-apyrase have been suggested in protein glycosylation and sugar level control (3Wang T.-F. Guidotti G. J. Biol. Chem. 1998; 273: 11392-11399Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 6Fanta N. Anich M. Mancilla M. Kettlum A.M. Valenzuela M.A. Traverso-Cori A. Arch. Biol. Med. Exp. 1988; 21: 129-133Google Scholar) and in regulation of membrane integrity (7Girolomoni G. Santanionio M.L. Pastore S. Bergstresser P.R. Giannetti A. Cruz P.D. J. Invest. Dermatol. 1993; 100: 282-287Abstract Full Text PDF PubMed Google Scholar), there is no direct evidence for the in vivo function of endo-apyrase (2Komoszynski M. Wojtczak A. Biochim. Biophys. Acta. 1996; 1310: 233-241Crossref PubMed Scopus (120) Google Scholar). Specific oligosaccharide modification of glycoproteins in eukaryotic cells occurs mainly in the lumen of the Golgi apparatus. The substrates for these reactions are nucleotide sugars, which are synthesized in the cytosol. As shown by genetic and biochemical analysis (8Abeijon C. Yanagisawa K. Mandon E.C. Häusler A. Moreman K. Hirschberg C.B. Robbins P.W. J. Cell Biol. 1993; 122: 307-323Crossref PubMed Scopus (158) Google Scholar, 9Capasso J.M. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7051-7055Crossref PubMed Scopus (108) Google Scholar), nucleotide sugars are imported from the cytosol to the Golgi lumen by highly specific transporters, which may also antiport the corresponding nucleoside monophosphate from the Golgi lumen to the cytosol. NDPase hydrolyzes nucleoside diphosphate to nucleoside monophosphate, which is a putative antiporter of nucleotide sugars. This allows the entry of additional nucleotide sugars from the cytosol to the Golgi lumen. Thereby, the NDPase is considered to play a critical role in the translocation of these nucleotide sugars from the cytosol into the lumen (10Hirschberg C.B. Clapham D.E. Ehrlich B.E. Transporter of Nucleotides and Nucleotide Derivatives in the Endoplasmic Reticulum and Golgi Apparatus. Rockefeller University Press, New York1996: 105-120Google Scholar). In Saccharomyces cerevisiae, mannosylation of N- and O-linked oligosaccharides is mainly regulated by a GDPase that converts GDP to GMP. GDA1, the gene for GDPase, has been cloned and found to encode a type II membrane protein (8Abeijon C. Yanagisawa K. Mandon E.C. Häusler A. Moreman K. Hirschberg C.B. Robbins P.W. J. Cell Biol. 1993; 122: 307-323Crossref PubMed Scopus (158) Google Scholar). Gda1p shows a high activity toward GDP, but a low activity toward UDP and no activity toward ATP and ADP (11Yanagisawa K. Resnick D. Abeijon C. Robbins P.W. Hirschberg C.B. J. Biol. Chem. 1990; 265: 19351-19355Abstract Full Text PDF PubMed Google Scholar). Deletion of GDA1results in a marked reduction in Golgi mannosylation of proteins and lipids in vivo (8Abeijon C. Yanagisawa K. Mandon E.C. Häusler A. Moreman K. Hirschberg C.B. Robbins P.W. J. Cell Biol. 1993; 122: 307-323Crossref PubMed Scopus (158) Google Scholar) and causes a 4-fold lower rate of GDP-mannose entry into Golgi vesicles in dolichol-P-Man synthase-deficient cells (12Berninsone P. Miret J.J. Hirschberg C.B. J. Biol. Chem. 1994; 269: 207-211Abstract Full Text PDF PubMed Google Scholar). Nevertheless, in Δgda1cells, invertase is still heavily glycosylated, and in addition, a significant amount of GMP, as well as very low residual nonspecific nucleoside phosphatase activity unrelated to Gda1p, was detected in the Golgi membranes (8Abeijon C. Yanagisawa K. Mandon E.C. Häusler A. Moreman K. Hirschberg C.B. Robbins P.W. J. Cell Biol. 1993; 122: 307-323Crossref PubMed Scopus (158) Google Scholar, 12Berninsone P. Miret J.J. Hirschberg C.B. J. Biol. Chem. 1994; 269: 207-211Abstract Full Text PDF PubMed Google Scholar). These observations suggested the importance of residual GDPase activity in the Golgi lumen of the Δgda1mutant. Another possibility could be a direct supply of GMP from GDP-mannose by mannosyl-phosphate transferase. The MNN4 gene is known to encode a positive regulator of mannosyl phosphorylation (13Odani T. Shinmma Y. Wang X.-H. Jigami Y. FEBS Lett. 1997; 420: 186-190Crossref PubMed Scopus (71) Google Scholar, 14Odani T. Shimma Y. Tanaka A. Jigami Y. Glycobiology. 1996; 6: 805-810Crossref PubMed Scopus (65) Google Scholar), but the analysis of mannosylation of invertase in Δgda1Δmnn4 double disruptant cells showed no effect on mannosylation when compared with that in Δgda1 alone. This result strongly indicated that the remaining GMP did not arise from mannosyl phosphorylation in the Golgi lumen of Δgda1 cells and prompted us to investigate other GDPases involved in protein glycosylation. At that time, it was already known that theSaccharomyces Genome Database contains at least one homologue of Gda1p that possess ∼20% amino acid identity to Gda1p and that also belongs to the apyrase superfamily, as determined by sequence alignment (15Handa M. Guidotti G. Biochem. Biophys. Res. Commun. 1996; 218: 916-923Crossref PubMed Scopus (273) Google Scholar). Here we report that the residual GDPase activity in the Δgda1 mutant results from this homologue of Gda1p. It turned out to be a typical apyrase that can hydrolyze not only nucleoside diphosphates, as Gda1p does, but also nucleoside triphosphates. We also demonstrate that additional expression of this yeast apyrase can complement the lack of GDPase activity of Gda1p and plays an important role in controlling the protein glycosylation in Golgi compartments. For the above reason, we named the GDA1homologue YND1 (yeast nucleosidediphosphatase) to indicate a possible redundancy between the YND1 and GDA1 genes. In this paper, we present the first direct evidence for in vivo endo-apyrase function in protein glycosylation and suggest the involvement of yeast apyrase in cell wall integrity. We thank Claudia Abeijon for yeast strains, Ken-Ichi Nakayama for help with chitinase sugar chain analysis, S. K. Roy for help with preparation of intact Golgi vesicles, and Joe Horecka for invaluable advice. We are indebted to Mami Kainuma, Yumi Maeda, and Takehiko Yoko-o for long fruitful discussions and to Neta Dean for critical reading of the manuscript.