Quality control of a transcriptional regulator by SUMO-targeted degradation

Abstract

MOT1 encodes an essential ATPase that functions as a general transcriptional regulator in vivo by modulating TATA-Binding Protein (TBP) DNA-binding activity. To better understand the role(s) of MOT1 in vivo, we selected for genomic suppressors of a mot1 temperature-sensitive (Ts-) mutation. This selection identified mutations in SPT15 (TBP) and BUR6, both of which are clearly linked with MOT1 at the functional level. The vast majority of the suppressor mutations, however, unexpectedly occurred in six genes that encode known components of the SUMO pathway and in two other genes with unknown functions, SLX5 and SLX8. The results presented in Chapter I, including extensive synthetic lethality observed between slx5Delta and slx8Delta and SUMO pathway mutations, as well as increased SUMOylation levels in slx5Delta and slx8Delta strains, suggest that SLX5 and SLX8 are new components or regulators of the SUMO pathway.;Following the publication of these findings, it was reported recently that Slx5 and Slx8 are heterodimeric RING-domain-containing proteins that possess SUMO-targeted ubiquitin ligase (STUbL) activity in vitro. Slx5-Slx8 and its orthologs were proposed to target SUMO conjugates for ubiquitin-mediated proteolysis, but the only in vivo substrate identified to date was the mammalian PML protein, and the physiological importance of SUMO-targeted ubiquitylation remained largely unknown. To address these issues, I set out to identify the SUMOylated substrate that is responsible for suppression of the mot1-301 phenotypes. The results presented in Chapter II demonstrated that Mot1 is SUMOylated in vivo and that disrupting the Slx5-Slx8 pathway by mutation of the target lysines in Mot1, by deletion of SLX5 or the ubiquitin E2 UBC4, or by inhibition of the proteosome, suppresses mot1-301 mutant phenotypes and increases the stability of the Mot1-301 protein. The Mot1-301 mutant protein is targeted for proteolysis by SUMOylation to a much greater extent than wild-type Mot1, suggesting a quality control mechanism. In support of this idea, growth of yeast in the presence of the arginine analog canavanine results in increased SUMOylation and Slx5-Slx8-mediated degradation of wild-type Mot1. These results therefore demonstrated that Mot1 is an in vivo STUbL target in yeast and suggest a role for SUMO-targeted degradation in protein quality control.