Burd C G, Peterson M, Cowles C R, Emr S D
Division of Cellular and Molecular Medicine, University of California, San Diego, School of Medicine, La Jolla 92093-0668, USA.
Mol Biol Cell. 1997 Jun;8(6):1089-104. doi: 10.1091/mbc.8.6.1089.
The vacuolar protein-sorting (VPS) pathway of Saccharomyces cerevisiae mediates localization of proteins from the trans-Golgi to the vacuole via a prevacuolar endosome compartment. Mutations in class D vacuolar protein-sorting (vps) genes affect vesicle-mediated Golgi-to-endosome transport and result in secretion of vacuolar proteins. Temperature-sensitive-for-function (tsf) and dominant negative mutations in PEP12, encoding a putative SNARE vesicle receptor on the endosome, and tsf mutations in VAC1, a gene implicated in vacuole inheritance and vacuolar protein sorting, were constructed and used to demonstrate that Pep12p and Vac1p are components of the VPS pathway. The sequence of Vac1p contains two putative zinc-binding RING motifs, a zinc finger motif, and a coiled-coil motif. Site-directed mutations in the carboxyl-terminal RING motif strongly affected vacuolar protein sorting. Vac1p was found to be tightly associated with membranes as a monomer and in a large SDS-resistant complex. By using Pep12p affinity chromatography, we found that Vac1p, Vps45p (SEC1 family member), and Sec18p (yeast N-ethyl maleimide-sensitive factor, NSF) bind Pep12p. Consistent with a functional role for this complex in vacuolar protein sorting, double pep12tsfvac1tsf and pep12tsf vps45tsf mutants exhibited synthetic Vps- phenotypes, the tsf phenotype of the vac1tsf mutant was rescued by overexpression of VPS45 or PEP12, overexpression of a dominant pep12 allele in a sec18-1 strain resulted in a severe synthetic growth defect that was rescued by deletion of PEP12 or VAC1, and subcellular fractionation of vac1 delta cells revealed a striking change in the fractionation of Pep12p and Vps21p, a rab family GTPase required for vacuolar protein sorting. The functions of Pep12p, Vps45p, and Vps21p indicate that key aspects of Golgi-to-endosome trafficking are similar to other vesicle-mediated transport steps, although the role of Vac1p suggests that there are also novel components of the VPS pathway.
酿酒酵母的液泡蛋白分选（VPS）途径通过前液泡内体区室介导蛋白质从反式高尔基体到液泡的定位。D类液泡蛋白分选（vps）基因的突变影响囊泡介导的高尔基体到内体的运输，并导致液泡蛋白的分泌。构建了编码内体上假定的SNARE囊泡受体的PEP12中的功能温度敏感（tsf）和显性负突变，以及与液泡遗传和液泡蛋白分选有关的基因VAC1中的tsf突变，并用于证明Pep12p和Vac1p是VPS途径的组成部分。Vac1p的序列包含两个假定的锌结合RING基序、一个锌指基序和一个卷曲螺旋基序。羧基末端RING基序中的定点突变强烈影响液泡蛋白分选。发现Vac1p作为单体与膜紧密结合，并存在于一个大的抗SDS复合物中。通过使用Pep12p亲和色谱法，我们发现Vac1p、Vps45p（SEC1家族成员）和Sec18p（酵母N-乙基马来酰亚胺敏感因子，NSF）与Pep12p结合。与该复合物在液泡蛋白分选中的功能作用一致，双pep12tsfvac1tsf和pep12tsf vps45tsf突变体表现出合成的Vps-表型，vac1tsf突变体的tsf表型通过VPS45或PEP12的过表达得以挽救，在sec18-1菌株中过表达显性pep12等位基因导致严重的合成生长缺陷，通过缺失PEP12或VAC1得以挽救，vac1δ细胞的亚细胞分级分离显示Pep12p和Vps21p（液泡蛋白分选所需的rab家族GTP酶）的分级分离发生了显著变化。Pep12p、Vps45p和Vps21p的功能表明，高尔基体到内体运输的关键方面与其他囊泡介导的运输步骤相似，但Vac1p的作用表明VPS途径中也存在新的组分。
