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.
Highlights
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]
No evidence was provided, it was assumed that mammalian cell endoplasmic reticulum (ER) nucleoside diphosphatases were required for hydrolysis of UDP generated by the UDP-Glc:glycoprotein glucosyltransferase (GT), an enzyme involved in the quality control of glycoprotein folding [11]
Like S. cerevisiae Gda1p and Ynd1p, both S. pombe enzymes localized to the Golgi, as shown by sucrose gradient centrifugation of postnuclear supernatants
Summary
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]. No evidence was provided, it was assumed that mammalian cell ER nucleoside diphosphatases were required for hydrolysis of UDP generated by the UDP-Glc:glycoprotein glucosyltransferase (GT), an enzyme involved in the quality control of glycoprotein folding [11] The presence of nucleoside diphosphatase and nucleotide sugar-dependent glycosyltransferase activities in the mammalian cell ER and Golgi compartments and the exclusive presence of the former activity in the S. cerevisiae Golgi, that is, in the only secretory pathway compartment in which nucleoside diphosphates are known to occur in this yeast, supported the notion that the presence of glycosyltransferase-generated nucleoside diphosphates in a subcellular compartment necessarily implied the presence of an enzyme able to hydrolyze them in the same compartment. On the characterization of S. pombe nucleoside diphosphatases we made the unexpected observation that this yeast, like S. cerevisiae, only expresses two Golgi-located nucleoside diphosphatase activities, demonstrating that, contrary to what has been assumed so far, nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments
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