Nucleoside Diphosphatase and Glycosyltransferase Activities Can Localize to Different Subcellular Compartments in Schizosaccharomyces pombe

Abstract

Nucleoside diphosphates generated by glycosyltransferases in the fungal, plant, and mammalian cell secretory pathways are converted into monophosphates to relieve inhibition of the transferring enzymes and provide substrates for antiport transport systems by which the entrance of nucleotide sugars from the cytosol into the secretory pathway lumen is coupled to the exit of nucleoside monophosphates. Analysis of the yeast Schizosaccharomyces pombe genome revealed that it encodes two enzymes with potential nucleoside diphosphatase activity, Spgda1p and Spynd1p. Characterization of the overexpressed enzymes showed that Spgda1p is a GDPase/UDPase, whereas Spynd1p is an apyrase because it hydrolyzed both nucleoside tri and diphosphates. Subcellular fractionation showed that both activities localize to the Golgi. Individual disruption of their encoding genes did not affect cell viability, but disruption of both genes was synthetically lethal. Disruption of Spgda1 ؉ did not affect Golgi N-or O-glycosylation, whereas disruption of Spynd1 ؉ affected Golgi N-mannosylation but not O-mannosylation. Although no nucleoside diphosphatase activity was detected in the endoplasmic reticulum (ER), N-glycosylation mediated by the UDP-Glc:glycoprotein glucosyltransferase (GT) was not severely impaired in mutants because first, no ER accumulation of misfolded glycoproteins occurred as revealed by the absence of induction of BiP mRNA, and second, in vivo GT-dependent glucosylation monitored by incorporation of labeled Glc into folding glycoproteins showed a partial (35-50%) decrease in Spgda1 but was not affected in Spynd1 mutants. Results show that, contrary to what has been assumed to date for eukaryotic cells, in S. pombe nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments. It is tentatively suggested that ER-Golgi vesicle transport might be involved in nucleoside diphosphate hydrolysis.Almost all nucleotide sugar-dependent glycosyltransferases in the secretory pathway generate nucleoside diphosphates that are converted into monophosphates to relieve inhibition of the transferring enzymes and provide substrates for antiport transport systems by which the entrance of nucleotide sugars from the cytosol into the lumen of the secretory pathway is coupled to the exit of nucleoside monophosphates (1). Several secretory pathway nucleoside diphosphatases have been described already. There are two enzymes displaying such enzymatic activity in Saccharomyces cerevisiae, a GDPase/UDPase and an apyrase (denominated Gda1p and Ynd1p, respectively) (2-4). Apyrases differ from nucleoside diphosphatases, as the former are able to degrade not only nucleoside diphosphates but also triphosphates. Both enzymatic activities are localized to the Golgi apparatus. Irrespective of their cellular origin, all enzymes able to hydrolyze nucleoside diphosphates (nucleoside diphosphatases proper and apyrases) share four highly similar sequences that have been called apyrase conserved regions. Analysis of th...

ire-1-dependent Transcriptional Up-regulation of a Lumenal Uridine Diphosphatase from Caenorhabditis elegans

“…The only apyrase of S. cerevisiae and S. pombe, Ynd1p, occurs in the yeast GA and participates in the glycosylation process (11)(12)(13). The ATPase activity of ScYnd1p is normally down-regulated by the binding of its cytoplasmic C terminus to Vma13p, a subunit of the vacuolar H ϩ -ATPase (49).…”

“…3A). The other ORF, C33H5.14, whose gene product is NTP-1, also has five ACR domains but is more closely related to yeast and human apyrases, Ynd1p and ENTPD5 (9,11,12,13), than to UDA-1 (Fig. 3C).…”

“…Loss of ScGDA1 function results in a drastic reduction of GDP-mannose-dependent (as opposed to lipid-linked mannose) mannosylation of proteins and lipids (10). The ynd1gda1 double mutant is synthetically lethal in S. pombe but not in S. cerevisiae, although the latter cells grow poorly and show severe cell-wall defects (11,12).…”

“…This unconventional mechanism for dealing with ER-generated, lumenal UDP by the S. pombe Golgi ENTPDase was hypothesized to be the result of antero transport of ER-derived vesicles containing UDP to the GA (11).…”

“…Saccharomyces cerevisiae and Schizosaccharomyces pombe each have two GA enzymes that degrade nucleoside diphosphates: ScGda1p (10) and SpGda1p (11), which are solely nucleotide diphosphatases and have preferred activity toward GDP and UDP; ScYnd1p (12,13) and SpYnd1p (11) are apyrases and use nucleoside di-and tri-phosphates as substrates. Loss of ScGDA1 function results in a drastic reduction of GDP-mannose-dependent (as opposed to lipid-linked mannose) mannosylation of proteins and lipids (10).…”