A regulatory mechanism for Rsp5, a multifunctional ubiquitin ligase in Saccharomyces cerevisiae: characterization of its interaction with a deubiquitinating enzyme

HECT E3 ubiquitin ligases are widely distributed from yeast to human cells and play important roles in many physiological processes. Rsp5, an essential HECT E3 ligase in Saccharomyces cerevisiae, is involved in many biological processes, including transcriptional activation, endocytic trafficking, mitochondrial inheritance, and RNA export pathways. Although Rsp5 has been shown to regulate multiple pathways targeting multiple substrates, mechanisms for regulating the biochemical activity of Rsp5 are largely uncharacterized (121, 199). To gain further insight into the regulation of this enzyme, I identified proteins that copurified with epitope-tagged Rsp5. Ubp2, a deubiquitinating enzyme, was a prominent copurifying protein. Rup1, a previously uncharacterized UBA domain protein, was required for binding of Rsp5 to Ubp2 both in vitro and in vivo. Biochemical and genetic evidence are consistent with a model that Ubp2and Rup1 antagonizes Rsp5-catalyzed substrate ubiquitination. In vivo and in vitro experiments showed that Rsp5 and Ubp2 display strong preferences for assembly and disassembly of K63-linked polyubiquitination, respectively. A large fraction of the K63 conjugates in ubp2∆ cells bound to Rsp5, and a proteomics approach was therefore used to identify Rsp5 substrates subject to Ubp2 regulation. Two proteins implicated in cell wall integrity, Csr2 and Ecm21, were identified and both proteins were efficiently K63- polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. I have also shown that cell wall integrity is impaired in rsp5-1 cells and this can be rescued by either ubp2∆ or rup1∆ mutation, suggesting that the Ubp2/Rup1 complex negatively regulates Rsp5-mediated cell wall homeostasis. Together, these data represent a novel regulatory mechanism for Rsp5 and suggest that similar mechanisms might be utilized by its mammalian homologues. Furthermore, this work provides a basis for studying the mechanism for differential polyubiquitin chain type synthesis by HECT E3 ligases.