Craighead M W, Bowden S, Watson R, Armstrong J
Imperial Cancer Research Fund, London, England.
Mol Biol Cell. 1993 Oct;4(10):1069-76. doi: 10.1091/mbc.4.10.1069.
The ypt2 gene of the fission yeast Schizosaccharomyces pombe encodes a member of the ypt/rab family of small GTP-binding proteins, related in sequence to Sec4p of Saccharomyces cerevisiae but closer to mammalian rab8. We have introduced a mutation into the gene corresponding to a mutation identified in ypt1, in which a conserved valine residue was altered to asparagine. The mutated ypt2 gene was introduced into the S. pombe genome by gene replacement. The resulting strain was temperature-sensitive for growth. Normal growth was restored by introduction of a plasmid-borne wild-type ypt2 cDNA or by cDNA for rab8 but not by various other rab or ypt sequences. At restrictive temperature the mutant cells accumulated the secretory protein acid phosphatase in a form that appeared to be fully glycosylated and acquired a population of vesicles detectable by electron microscopy. Thus the ypt2 protein, and by inference rab8, appear to function in the last stage of the secretory pathway.
裂殖酵母粟酒裂殖酵母的ypt2基因编码一种小GTP结合蛋白的ypt/rab家族成员，其序列与酿酒酵母的Sec4p相关，但更接近哺乳动物的rab8。我们在该基因中引入了一个与ypt1中鉴定出的突变相对应的突变，其中一个保守的缬氨酸残基被改变为天冬酰胺。通过基因替换将突变的ypt2基因引入粟酒裂殖酵母基因组。所得菌株对生长具有温度敏感性。通过引入质粒携带的野生型ypt2 cDNA或rab8的cDNA可恢复正常生长，但其他各种rab或ypt序列则不能。在限制温度下，突变细胞以似乎完全糖基化的形式积累分泌蛋白酸性磷酸酶，并获得了一群可通过电子显微镜检测到的囊泡。因此，ypt2蛋白，以及由此推断的rab8，似乎在分泌途径的最后阶段发挥作用。
