Benli M, Döring F, Robinson D G, Yang X, Gallwitz D
Department of Molecular Genetics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
EMBO J. 1996 Dec 2;15(23):6460-75.
In eukaryotic cells, monomeric GTPases of the Ypt/Rab family function as regulators at defined steps of vesicular transport in exo- and endocytosis. Here we report on the isolation and characterization of two genes (YPT31 and YPT32) of the yeast Saccharomyces cerevisiae which encode members of the Ypt family exhibiting >80% sequence identity. Whereas the disruption of one of the two genes was phenotypically neutral, the disruption of both YPT31 and YPT32 led to lethality. Depletion of wild-type Ypt31p or of a short-lived ubiquitin-Ypt31p in a ypt32 null background led to a massive accumulation of Golgi-like membranes, an inhibition of invertase secretion and defects in vacuolar protein maturation. Similar alterations were observed in a conditional-lethal ypt31-1 mutant at 30 min after shift to the non-permissive temperature. According to subcellular fractionation, a significant part of Ypt31p appeared to be located in Golgi-enriched membrane fractions. In accordance with this, indirect immunofluorescence using affinity-purified anti-Ypt31p antibodies gave a punctate staining similar to that observed with Golgi-located proteins. From the phenotypic alterations observed in ypt31 and ypt32 mutants, it seems likely that the two GTPases are involved in intra-Golgi transport or in the formation of transport vesicles at the most distal Golgi compartment.
在真核细胞中，Ypt/Rab家族的单体GTP酶在胞吐作用和胞吞作用的囊泡运输特定步骤中作为调节因子发挥作用。在此，我们报告了酿酒酵母的两个基因（YPT31和YPT32）的分离与特性，这两个基因编码的Ypt家族成员序列同一性大于80%。虽然两个基因之一的缺失在表型上是中性的，但YPT31和YPT32两者的缺失导致致死性。在ypt32缺失背景下，野生型Ypt31p或短寿命泛素 - Ypt31p的缺失导致类高尔基体膜大量积累、蔗糖酶分泌受抑制以及液泡蛋白成熟缺陷。在条件致死的ypt31 - 1突变体转移到非允许温度30分钟后，也观察到了类似的变化。根据亚细胞分级分离，Ypt31p的很大一部分似乎位于富含高尔基体的膜分级分离物中。与此一致的是，使用亲和纯化的抗Ypt31p抗体进行的间接免疫荧光显示出点状染色，类似于在高尔基体定位蛋白中观察到的染色。从在ypt31和ypt32突变体中观察到的表型变化来看，这两种GTP酶似乎参与了高尔基体内运输或最远端高尔基体区室运输小泡的形成。
