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

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.