Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway.
Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway.
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Rsp5p 泛素蛋白连接酶在内吞途径内多个位点的定位。
DOI：
10.1128/mcb.21.10.3564-3575.2001
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发表时间：
2001
影响因子：
5.3
通讯作者：
Huibregtse,JM
中科院分区：
文献类型：
--
作者：
Wang,G;McCaffery,JM;Wendland,B;Dupré,S;Haguenauer-Tsapis,R;Huibregtse,JM
酿酒酵母RSP 5基因编码HECT E3泛素蛋白连接酶。Rsp 5 p含有一个N-末端C2结构域、三个位于分子中心部分的WW结构域和一个C-末端催化HECT结构域。已经鉴定了Rsp 5 p和脊椎动物C2含WW-domain的HECT E3的不同底物组，包括核和膜相关蛋白。我们确定了Rsp 5 p的细胞内定位和定位所需的决定因素，以便更好地了解Rsp 5 p活动是如何协调的。使用两个绿色荧光蛋白融合Rsp 5 p和免疫金电子显微镜，我们发现，Rsp 5 p分布在一个点状图案在质膜上，对应于膜内陷，这是可能的网站内体形成，以及在泡周网站。后者似乎对应于内吞中间体，因为这些结构在asla 2/end 4 - 1突变体中未观察到，并且双免疫金标记证明Rsp 5 p与内体标记Pep 12 p和Vps 32 p共定位。C2结构域是定位的重要决定因素;然而，破坏HECT结构域功能的突变也引起Rsp 5 p的错误定位，表明酶活性与定位有关。C2结构域的缺失部分地稳定了Fur 4p，Fur 4p是一种先前显示经历Rsp 5 p和泛素介导的内吞作用的蛋白质;然而，当C2结构域从蛋白质中缺失时，Fur 4p仍然在质膜上被泛素化。总之，这些结果表明，Rsp 5 p位于内吞途径内的多个位点，并表明Rsp 5 p可能在泛素介导的内吞途径中的多个步骤发挥作用。
TheSaccharomyces cerevisiae RSP5gene encodes an essential HECT E3 ubiquitin-protein ligase. Rsp5p contains an N-terminal C2 domain, three WW domains in the central portion of the molecule, and a C-terminal catalytic HECT domain. A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-containing HECT E3s have been identified, including both nuclear and membrane-associated proteins. We determined the intracellular localization of Rsp5p and the determinants necessary for localization, in order to better understand how Rsp5p activities are coordinated. Using both green fluorescent protein fusions to Rsp5p and immunogold electron microscopy, we found that Rsp5p was distributed in a punctate pattern at the plasma membrane, corresponding to membrane invaginations that are likely sites of endosome formation, as well as at perivacuolar sites. The latter appeared to correspond to endocytic intermediates, as these structures were not seen in asla2/end4-1mutant, and double-immunogold labeling demonstrated colocalization of Rsp5p with the endosomal markers Pep12p and Vps32p. The C2 domain was an important determinant of localization; however, mutations that disrupted HECT domain function also caused mislocalization of Rsp5p, indicating that enzymatic activity is linked to localization. Deletion of the C2 domain partially stabilized Fur4p, a protein previously shown to undergo Rsp5p- and ubiquitin-mediated endocytosis; however, Fur4p was still ubiquitinated at the plasma membrane when the C2 domain was deleted from the protein. Together, these results indicate that Rsp5p is located at multiple sites within the endocytic pathway and suggest that Rsp5p may function at multiple steps in the ubiquitin-mediated endocytosis pathway.