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.