D'Alessio Cecilia, Trombetta Eduardo S, Parodi Armando J
Institute for Biotechnological Research, University of San Martin, CC30, 1650 San Martin, Argentina.
J Biol Chem. 2003 Jun 20;278(25):22379-87. doi: 10.1074/jbc.M300892200. Epub 2003 Apr 9.
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.
真菌、植物和哺乳动物细胞分泌途径中的糖基转移酶产生的核苷二磷酸被转化为单磷酸，以解除对转移酶的抑制，并为反向转运系统提供底物，通过该系统，核苷酸糖从胞质溶胶进入分泌途径腔与核苷单磷酸的排出相偶联。对酵母粟酒裂殖酵母基因组的分析表明，它编码两种具有潜在核苷二磷酸酶活性的酶，即Spgda1p和Spynd1p。对过表达酶的表征表明，Spgda1p是一种GDP酶/UDP酶，而Spynd1p是一种腺苷三磷酸双磷酸酶，因为它能水解核苷三磷酸和二磷酸。亚细胞分级分离表明，这两种活性都定位于高尔基体。单独破坏它们的编码基因不影响细胞活力，但同时破坏这两个基因则是合成致死的。破坏Spgda1+不影响高尔基体N-或O-糖基化，而破坏Spynd1+影响高尔基体N-甘露糖基化，但不影响O-甘露糖基化。虽然在内质网（ER）中未检测到核苷二磷酸酶活性，但UDP-Glc：糖蛋白葡糖基转移酶（GT）介导的N-糖基化在突变体中并未受到严重损害，原因如下：第一，未诱导BiP mRNA表明未发生错误折叠糖蛋白在内质网中的积累；第二，通过将标记的Glc掺入折叠糖蛋白中监测的体内GT依赖性糖基化显示，Spgda1突变体中糖基化部分（35-50%）降低，但在Spynd1突变体中不受影响。结果表明，与迄今为止对真核细胞的假设相反，在粟酒裂殖酵母中，核苷二磷酸酶和糖基转移酶活性可定位于不同的亚细胞区室。初步推测内质网-高尔基体囊泡运输可能参与核苷二磷酸的水解。
