PMID:11179425

Citation	Dunn, R and Hicke, L (2001) Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis. Mol. Biol. Cell 12:421-35
Abstract	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 alpha-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 alpha-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.
Links	PubMed PMC30953
Keywords	Amino Acid Sequence; Binding Sites; Catalytic Domain; Cell Membrane/metabolism; Endocytosis/physiology; Endosomal Sorting Complexes Required for Transport; Isoquinolines/metabolism; Ligases/genetics; Ligases/metabolism; Membrane Proteins/genetics; Membrane Proteins/metabolism; Molecular Sequence Data; Mutation; Peptides/metabolism; Protein Structure, Tertiary; Receptors, Mating Factor; Receptors, Peptide/metabolism; Saccharomyces cerevisiae/genetics; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins; Transcription Factors; Ubiquitin-Protein Ligase Complexes; Ubiquitin-Protein Ligases; Ubiquitins/metabolism; Vacuoles/metabolism
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