Stoll KE, Brzovic PS, Davis TN, Klevit RE

The ubiquitin conjugating enzymes Ubc4 and Ubc5 are involved in a variety of ubiquitination pathways in yeast including Rsp5 and Anaphase Promoting Complex (APC)-mediated pathways. We have found the double deletion of ubc4 and ubc5 genes in yeast to be lethal. To investigate the essential pathway disrupted by the ubc4/ubc5 ...

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deletion, several point mutations were inserted in Ubc4. The Ubc4 active site mutation, C86A, and E3 binding mutations A97D/F63A were both unable to rescue the lethal phenotype indicating that an active E3/E2~Ub complex is required for the essential function of Ubc4/5. A mutation that specifically eliminates RING E3-catalyzed isopeptide formation but not HECT E3-transthiolation (N78S-Ubc4) rescued the lethal phenotype. Thus, the essential redundant function performed by Ubc4 and Ubc5 in yeast is with a HECT-type E3, likely the only essential HECT in yeast, Rsp5. Our results also suggest that Ubc1 can weakly replace Ubc4 to transfer mono-Ub with APC, but Ubc4 cannot replace Ubc1 for poly-Ub chain extension on APC substrates. Finally, the backside Ub binding mutant S23R-Ubc4 has no observable effect in yeast. Together, our results are consistent with a model in which Ubc4/5 are 1) the primary E2s for Rsp5 in yeast and 2) act as mono-ubiquitinating E2s in RING E3-catalyzed pathways, in contrast to the processive human orthologue, UbcH5.

Date: Feb. 25, 2011

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