Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway

The Saccharomyces cerevisiae RSP5 gene encodes an essential HECT E3 ubiquit in-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 do main. A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-co ntaining 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 under stand how Rsp5p activities are coordinated. Using both green fluorescent pr otein fusions to Rsp5p and immunogold electron microscopy, me found that Rs p5p was distributed in a punctate pattern at the plasma membrane, correspon ding to membrane invaginations that are likely sites of endosome formation, os well as at perivacuolar sites, The latter appeared to correspond to end ocytic intermediates, as these structures were not seen in a sla2/end4-1 mu tant, and double-immunogold labeling demonstrated colocalization of Rsp5p w ith 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 a ctivity is linked to localization, Deletion of the C2 domain partially stab ilized Fur lp, a protein previously shown to undergo Rsp5p- and ubiquitin-m ediated endocytosis; however, Fur4p was still ubiquitinated at the plasma m embrane when the C2 domain was deleted from the protein. Together, these re sults indicate that Rsp5p is located at multiple sites within the endocytic pathway and suggest that Rsp5p mag function at multiple steps in the ubiqu itin-mediated endocytosis pathway.