Jedd G, Richardson C, Litt R, Segev N
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637, USA.
J Cell Biol. 1995 Nov;131(3):583-90. doi: 10.1083/jcb.131.3.583.
Small GTPases of the rab family are involved in the regulation of vesicular transport. The restricted distribution of each of these proteins in mammalian cells has led to the suggestion that different rab proteins act at different steps of transport (Pryer, N. K., L. J. Wuestehube, and R. Sheckman. 1992. Annu Rev. Biochem. 61:471-516; Zerial, M., and H. Stenmark. 1993. Curr. Opin. Cell Biol. 5:613-620). However, in this report we show that the Ypt1-GTPase, a member of the rab family, is essential for more than one step of the yeast secretory pathway. We determined the secretory defect conferred by a novel ypt1 mutation by comparing the processing of several transported glycoproteins in wild-type and mutant cells. The ypt1-A136D mutant has a change in an amino acid that is conserved among rab GTPases. This mutation leads to a rapid and tight secretory block upon a shift to the restrictive temperature, and allows for the identification of the specific steps in the secretory pathway that directly require Ypt1 protein (Ypt1p). The ypt1-A136D mutant exhibits tight blocks in two secretory steps, ER to cis-Golgi and cis- to medial-Golgi, but later steps are unaffected. Thus, it is unlikely that Ypt1p functions as the sole determinant of fusion specificity. Our results are more consistent with a role for Ypt1/rab proteins in determining the directionality or fidelity of protein sorting.
Rab家族的小GTP酶参与囊泡运输的调控。这些蛋白质在哺乳动物细胞中的分布受限，这表明不同的Rab蛋白在运输的不同步骤发挥作用（Pryer, N. K., L. J. Wuestehube, and R. Sheckman. 1992. 《生物化学年度评论》61:471 - 516；Zerial, M., and H. Stenmark. 1993. 《细胞生物学当前观点》5:613 - 620）。然而，在本报告中我们表明，Rab家族成员Ypt1 - GTP酶对于酵母分泌途径的多个步骤至关重要。我们通过比较野生型和突变体细胞中几种运输的糖蛋白的加工过程，确定了一种新型ypt1突变导致的分泌缺陷。ypt1 - A136D突变体的一个氨基酸发生了变化，该氨基酸在Rab GTP酶中保守。这种突变在转移到限制温度时导致快速且紧密的分泌阻断，并允许鉴定分泌途径中直接需要Ypt1蛋白（Ypt1p）的特定步骤。ypt1 - A136D突变体在两个分泌步骤中表现出紧密的阻断，即从内质网到顺式高尔基体以及从顺式到中间高尔基体，但后续步骤不受影响。因此，Ypt1p不太可能作为融合特异性的唯一决定因素。我们的结果更符合Ypt1/Rab蛋白在确定蛋白质分选的方向性或保真度方面的作用。
