The deubiquitinating enzyme Ubp2 modulates Rsp5-dependent Lys 63-linked polyubiquitin conjugates in Saccharomyces cerevisiae

Abstract

The functions of Lys63-linked polyubiquitin chains are poorly understood, as are the enzymes that specifically generate Lys 63-linked conjugates. Rsp5 is a HECT (homologous to E6AP C terminus) ubiquitin ligase involved in numerous processes, and an associated deubiquitinating enzyme, Ubp2, modulates its activity. Adramatic increase in Lys63-linked conjugates was observed in ubp2Δ cells. The formation of these was Rsp5-dependent, and ubp2Δ phenotypes could be suppressed by prevention of formation of Lys63 conjugates. Cell wall integrity was impaired in rsp5-1 cells and in cells defective in Lys 63-polyubiquitination, as assayed by calcofluor white sensitivity, and ubp2Δ and rup1Δ mutants suppressed the calcofluor white sensitivity of rsp5-1. A large fraction of the Lys63 conjugates in ubp2Δ cells bound to Rsp5, and a proteomics approach was used to identify Rsp5 substrates subject to Ubp2 regulation. Two closely related proteins, Csr2 and Ecm21, were among the identified proteins. Both were efficiently Lys 63-polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. Together, these results indicate that Ubp2 modulates Lys63-polyubiquitination of Rsp5 substrates in vivo, including ubiquitination of two newly identified Rsp5 substrates. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.