Regulation of the RSP5 ubiquitin ligase by an intrinsic ubiquitin-binding site

Abstract

Rsp5 is a homologous to E6AP C terminus (HECT) ubiquitin ligase (E3) that controls many different cellular processes in budding yeast. Although Rsp5 targets a number of different substrates for ubiquitination, the mechanisms that regulate Rsp5 activity remain poorly understood. Here we demonstrate that Rsp5 carries a noncovalent ubiquitin-binding site in its catalytic HECTdomain. The N-terminal lobe of theHECTdomain mediates binding to ubiquitin, and point mutations that disrupt interactions with ubiquitin alter the ability of the Rsp5 HECT domain to assemble polyubiquitin chains in vitro. Point mutations that disrupt ubiquitin binding also result in temperaturesensitive growth defects in yeast, indicating that the Rsp5 ubiquitin-binding site is important for Rsp5 function in vivo. The Nedd4 HECT domain N-lobe also contains ubiquitin-binding activity, suggesting that interactions between the N-lobe and ubiquitin are conserved within the Nedd4 family of ubiquitin ligases. We propose that a subset of HECT E3s are regulated by a conserved ubiquitin-binding site that functions to restrict the length of polyubiquitin chains synthesized by the HECT domain. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.