SUMO-independent in vivo activity of a SUMO-targeted ubiquitin ligase toward a short-lived transcription factor

Abstract

Many proteins are regulated by ubiquitin-dependent proteolysis. Substrate ubiquitylation can be stimulated by additional post-translational modifications, including small ubiquitin-like modifier (SUMO) conjugation. The recently discovered SUMO-targeted ubiquitin ligases (STUbLs) mediate the latter effect; however, no endogenous substrates of STUbLs that are degraded under normal conditions are known. From a targeted genomic screen, we now identify the yeast STUbL Slx5-Slx8, a heterodimeric RING protein complex, as a key ligase mediating degradation of the MATα2 (α2) repressor. The ubiquitin-conjugating enzyme Ubc4 was found in the same screen. Surprisingly, mutants with severe defects in SUMO-protein conjugation were not impaired for α2 turnover. Unmodified α2 also bound to and was ubiquitylated efficiently by Slx5-Slx8. Nevertheless, when we inactivated four SUMO-interacting motifs (SIMs) in Slx5 that together account for its noncovalent SUMO binding, both in vitro Slx5-Slx8-dependent ubiquitylation and in vivo degradation of α2 were inhibited. These data identify α2 as the first native substrate of the conserved STUbLs, and demonstrate that its STUbL-mediated ubiquitylation does not require SUMO. We suggest that α2, and presumably other proteins, have surface features that mimic SUMO, and therefore can directly recruit STUbLs without prior SUMO conjugation. © 2010 by Cold Spring Harbor Laboratory Press.