The yeast Npi1/Rsp5 ubiquitin ligase lacking its N-terminal C-2 domain is competent for ubiquitination but not for subsequent endocytosis of the Gap1permease

The yeast ubiquitin ligase Npi1/Rsp5 and its mammalian homologue Nedd4 are involved in ubiquitination of various cell surface proteins, these being su bsequently internalized by endocytosis and degraded in the vacuole/lysosome . Both enzymes consist of an N-terminal C-2 domain, three to four successiv e WW(P) domains, and a C-terminal catalytic domain (HECT) containing a high ly conserved cysteine residue involved in ubiquitin thioester formation, In this study, we show that the conserved cysteine of the HECT domain is requ ired for yeast cell viability and for ubiquitination and subsequent endocyt osis of the Gap1 permease. In contrast, the C-2 domain of Npi1/Rsp5 is not essential to cell viability. Its deletion impairs internalization of Gap1, without detectably affecting ubiquitination of the permease. This suggests that Npi1/Rsp5 participates, via its C2 domain, in endocytosis of ubiquitin ated permeases, (C) 1999 Academic Press.