Ubp2 regulates Rsp5 ubiquitination activity in vivo and in vitro.

The yeast HECT-family E3 ubiquitin ligase Rsp5 has been implicated in diverse cell functions. Previously, we and others [1], [2] reported the physical and functional interaction of Rsp5 with the deubiquitinating enzyme Ubp2, and the ubiquitin associated (UBA) domain-containing cofactor Rup1. To investigate the mechanism and significance of the Rsp5-Rup1-Ubp2 complex, we examined Rsp5 ubiquitination status in the presence or absence of these cofactors. We found that, similar to its mammalian homologues, Rsp5 is auto-ubiquitinated in vivo. Association with a substrate or Rup1 increased Rsp5 self-ubiquitination, whereas Ubp2 efficiently deubiquitinates Rsp5 in vivo and in vitro. The data reported here imply an auto-modulatory mechanism of Rsp5 regulation common to other E3 ligases.