The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways
The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways
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DOI：
10.1091/mbc.11.10.3365
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发表时间：
2000-10-01
影响因子：
3.3
通讯作者：
Hochstrasser, M
中科院分区：
文献类型：
--
作者：
Amerik, AY;Nowak, J;Hochstrasser, M
酿酒酵母DOA4基因编码一种脱泛素酶，该酶是快速降解泛素-蛋白酶体途径底物所必需的。遗传和生化数据都表明，Doa4通过促进泛素从泛素化的中间产物循环到蛋白酶体而在这一途径中发挥作用。在这里，我们描述了12个自发的doa4-1突变的基因外抑制基因的分离；这些基因涉及7个不同的基因，其中6个基因被克隆。令人惊讶的是，所有克隆的DID(Doa4非依赖性降解)基因都编码空泡蛋白分类(VPS)途径的组成部分。具体地说，它们都是E类VPs因子，它们在晚期内体/泡前室成熟成多囊体并随后与液泡融合的过程中发挥作用。六个DID蛋白中有四个在结构上相关，这表明功能上存在重叠。在野生型和几株VPS中，Doa4-绿色荧光蛋白呈胞质/核分布。然而，在缺乏Vps4/Did6 ATPase的细胞中，像其他几个VPs因子一样，很大一部分Doa4-绿色荧光蛋白集中在靠近液泡的晚期内吞体样E类隔室。这些结果表明，蛋白质去泛素化和膜内蛋白转运之间存在一种意想不到的联系，Doa4在内体晚期/泡前隔室作用，从泛素化的膜蛋白中回收泛素，进入液泡。
The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin-proteasome pathway substrates. Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome. Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned. Surprisingly, all of the cloned DID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway. Ln particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Four of the six Did proteins are structurally related, suggesting an overlap in function. In wild-type and several vps strains, Doa4-green fluorescent protein displays a cytoplasmic/nuclear distribution. However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4-green fluorescent protein, like several other Vps factors, concentrates at the late endosome-like class E. compartment adjacent to the vacuole. These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.