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首页> 外文期刊>Molecular and Cellular Biology >Localization of the Rsp5p Ubiquitin-Protein Ligase at Multiple Sites within the Endocytic Pathway
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Localization of the Rsp5p Ubiquitin-Protein Ligase at Multiple Sites within the Endocytic Pathway

机译：Rsp5p泛素蛋白连接酶在内吞途径内的多个位置的定位

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The Saccharomyces cerevisiae RSP5 gene 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 a sla2/end4-1 mutant, 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.
机译：酿酒酵母RSP5 基因编码必需的HECT E3泛素蛋白连接酶。 Rsp5p包含一个N端C2域，在分子中心部分的三个WW域和一个C端催化HECT域。已经鉴定出多种多样的Rsp5p底物和含脊椎动物C2 WW域的HECT E3，包括核蛋白和膜相关蛋白。我们确定了Rsp5p的细胞内定位和定位所必需的决定因素，以便更好地了解Rsp5p活性是如何协调的。使用绿色荧光蛋白融合到Rsp5p和免疫金电子显微镜，我们发现Rsp5p以点状分布在质膜上，对应于可能是内体形成位点以及在周周位点的膜内陷。后者似乎与胞吞中间体相对应，因为在 sla2 / end4-1 突变体中未发现这些结构，并且双免疫金标记证明Rsp5p与内体标记Pep12p和Vps32p共定位。 C2域是本地化的重要决定因素。但是，破坏HECT结构域功能的突变也引起Rsp5p的定位错误，表明酶活性与定位有关。 C2结构域的缺失部分稳定了Fur4p，Fur4p是一种蛋白质，先前被证明会经历Rsp5p和泛素介导的内吞作用。然而，当从蛋白质中删除C2结构域时，Fur4p仍在质膜上泛素化。总之，这些结果表明Rsp5p位于内吞途径内的多个位点，并暗示Rsp5p可能在泛素介导的内吞途径中的多个步骤起作用。

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