Phosphorylation of the Saccharomyces cerevisiae Snf1 kinase activation loop is determined by the integration of two reaction rates: the rate of phosphorylation by upstream kinases and the rate of dephosphorylation by Glc7. The activities of the Snf1-activating kinases do not appear to be glucose-regulated, since immune complex kinase assays with each of the three Snf1-activating kinases show similar levels of activity when prepared from cells grown in either high or low glucose. In contrast, the dephosphorylation of the Snf1 activation loop was strongly regulated by glucose. When de novo phosphorylation of Snf1 was inhibited, phosphorylation of the Snf1 activation loop was found to be stable in low glucose but rapidly lost upon the addition of glucose. A greater than 10-fold difference in the rates of Snf1 activation loop dephosphorylation was detected. However, the activity of the Glc7-Reg1 phosphatase may not itself be directly regulated by glucose, since the Glc7-Reg1 enzyme was active in low glucose toward another substrate, the transcription factor Mig1. Glucose-mediated regulation of Snf1 activation loop dephosphorylation is controlled by changes in the ability of the Snf1 activation loop to act as a substrate for Glc7.

The Hrd1 ubiquitin ligase plays a role in quality control of two substrates associated with the Sec61 translocon.

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 MATa2 (a2) 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 a2 turnover. Unmodified a2 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 a2 were inhibited. These data identify a2 as the first native substrate of the conserved STUbLs, and demonstrate that its STUbL-mediated ubiquitylation does not require SUMO. We suggest that a2, and presumably other proteins, have surface features that mimic SUMO, and therefore can directly recruit STUbLs without prior SUMO conjugation.[Keywords: Ubiquitin; SUMO; STUbL; proteasome; transcription factor] Supplemental material is available at http://www.genesdev.org.

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Background: Translationally stalled proteins (including those aberrantly translated beyond their stop codons) pose dangers for eukaryotic cells. Results: The ubiquitin ligase Rkr1/Ltn1 targets translationally stalled ER-associated proteins for degradation. Conclusion: Cytosolic and ER-associated translationally stalled proteins are targeted for destruction by related mechanisms. Significance: Mechanisms regulating the degradation of translationally stalled ER-associated proteins may represent therapeutic targets for human disease.