The C2 domain of the ubiquitin ligase Rsp5 is required for ubiquitination of the endocytic protein Rvs167 upon change of nitrogen source.

Abstract

Ubiquitination is a key signal for endocytosis of proteins on the plasma membrane. The ubiquitin ligase Rsp5 of Saccharomyces cerevisiae, which contains an amino-terminal membrane-binding C2 domain, three substrate-recognizing tryptophan-tryptophan (WW) domains and a carboxyl-terminal catalytic homologous to the E6-AP carboxyl terminus (HECT) domain, can ubiquitinate plasma membrane proteins directing them for endocytosis. Here, we examined the roles of the C2 domain in endocytosis for the downregulation of the general amino acid permease Gap1, which is one of nitrogen-regulated permeases in S. cerevisiae. First, we constructed several rsp5 mutants producing Rsp5 variants without the C2 domain or with amino acid changes of membrane-binding lysine residues. These mutants showed defects in endocytosis of Gap1 in response to a preferred nitrogen source. Intriguingly, we found that ubiquitination of Gap1 in these mutant cells was highly similar to that in wild-type cells during endocytosis. These results indicate that the C2 domain is essential for endocytosis but not for ubiquitination of substrates such as Gap1. Moreover, genetic and biochemical analyses showed that the endocytic protein Rvs167 was ubiquitinated via Rsp5 and the C2 domain was required for efficient ubiquitination in response to a preferred nitrogen source. Here, we propose a mechanism for the C2 domain-mediated endocytosis of plasma membrane permeases.