Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis

Yeast Rsp5p and its mammalian homologue, Nedd4, are hect domain ubiquitin-p rotein ligases (E3s) required for the ubiquitin-dependent endocytosis of pl asma membrane proteins. Because ubiquitination is sufficient to induce inte rnalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein c ontaining an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In t his study, we demonstrate that Rsp5p is peripherally associated with membra nes and provide evidence that Rsp5p functions as part of a multimeric prote in complex. We define the function of Rsp5p and its domains in the ubiquiti n-dependent internalization of the yeast a-factor receptor, Ste2p. Temperat ure-sensitive rsp5 mutants were unable to ubiquitinate or to internalize St e2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on a-factor internalization; however, point mutations in any of t he three WW domains impaired both receptor ubiquitination and internalizati on. These observations indicate that the WW domains play a role in the impo rtant regulatory event of selecting phosphorylated proteins as endocytic ca rgo. In addition, mutations in the C2 and WW1 domains had more severe defec ts on transport of fluid-phase markers to the vacuole than on receptor inte rnalization, suggesting that Rsp5p functions at multiple steps in the endoc ytic pathway.