Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis
Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis
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DOI：
10.1091/mbc.12.2.421
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发表时间：
2001-02-01
影响因子：
3.3
通讯作者：
Hicke, L
中科院分区：
文献类型：
--
作者：
Dunn, R;Hicke, L
酵母Rsp 5 p及其哺乳动物同源物Nedd 4是质膜蛋白的泛素依赖性内吞作用所需的hect结构域泛素-蛋白连接酶（E3）。由于泛素化足以诱导内化，因此E3介导的泛素化是质膜蛋白内吞的关键调节事件。Rsp 5 p是一种必需的多结构域蛋白，含有一个氨基末端C2结构域、三个WW蛋白-蛋白相互作用结构域和一个携带E3活性的羧基末端hect结构域。在这项研究中，我们证明，Rsp 5 p是外周相关的膜，并提供证据表明，Rsp 5 p的功能作为一个多聚体蛋白复合物的一部分。我们定义了Rsp 5 p及其结构域在酵母α-因子受体Ste 2 p的泛素依赖性内化中的功能。温度敏感的rsp 5突变体不能泛素化或内化Ste 2 p在nonpermissive温度。整个C2结构域的缺失对a因子内化没有影响;然而，三个WW结构域中的任何一个的点突变都损害了受体泛素化和内化。这些观察结果表明，WW结构域在选择磷酸化蛋白作为内吞货物的重要调控事件中发挥作用。此外，在C2和WW 1结构域的突变有更严重的缺陷，运输液相标记物的液泡比受体内化，这表明Rsp 5 p功能在多个步骤的内吞途径。
Yeast Rsp5p and its mammalian homologue, Nedd4, are hect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins. Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex. We define the function of Rsp5p and its domains in the ubiquitin-dependent internalization of the yeast a-factor receptor, Ste2p. Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on a-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization. These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo. In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway.