A novel Sec18p/NSF-dependent complex required for Golgi-to-endosome transport in yeast
A novel Sec18p/NSF-dependent complex required for Golgi-to-endosome transport in yeast
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DOI：
10.1091/mbc.8.6.1089
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发表时间：
1997-06-01
影响因子：
3.3
通讯作者：
Emr, SD
中科院分区：
文献类型：
--
作者：
Burd, CG;Peterson, M;Emr, SD
酿酒酵母的液泡蛋白分选（VPS）途径介导蛋白质从高尔基体通过前液泡内体区室定位到液泡。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转运，并导致液泡蛋白的分泌。构建了PEP 12中的温度敏感功能突变（tsf）和显性负突变（其编码内体上的推定SNARE囊泡受体）以及VAC 1（一种与液泡遗传和液泡蛋白分选有关的基因）中的tsf突变，并用于证明Pep 12 p和Vac 1 p是VPS途径的组成部分。Vac 1 p的序列包含两个推定的锌结合环基序，锌指基序，和卷曲螺旋基序。羧基末端RING基序的定点突变强烈影响液泡蛋白分选。Vac 1 p被认为是紧密相关的膜作为单体，并在一个大的抗SDS复合物。通过使用Pep 12 p亲和层析，我们发现Vac 1 p、Vps 45 p（SEC 1家族成员）和Sec 18 p（酵母N-乙基马来酰亚胺敏感因子，NSF）结合Pep 12 p。与该复合物在液泡蛋白分选中的功能作用一致，双pep 12（tsf）vac 1（tsf）和pep 12（tsf）vps 45（tsf）突变体表现出合成的Vps（-）表型，vac 1（tsf）突变体的tsf表型通过VPS 45或PEP 12的过表达而被拯救，在sec 18 -1菌株中显性pep 12等位基因的过表达导致严重的合成生长缺陷，该缺陷通过缺失PEP 12或VAC 1而得到挽救，vac 1 δ细胞的亚细胞分级显示Pep 12 p和Vps 21 p的分级发生了显著变化，Pep 12 p和Vps 21 p是液泡蛋白分选所需的rab家族GT酶。Pep 12 p、Vps 45 p和Vps 21 p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，尽管Vac 1 p的作用表明VPS途径也有新的组分。
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 pep12(tsf)vac1(tsf) and pep12(tsf) vps45(tsf) mutants exhibited synthetic Vps(-) phenotypes, the tsf phenotype of the vac1(tsf) 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.