title: The essential Ubc4/Ubc5 function in yeast is HECT E3-dependent, and RING E3-dependent pathways require only monoubiquitin transfer by Ubc4.
abstract: The ubiquitin (Ub)-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 deletion, several point mutations were inserted in Ubc4. The Ubc4 active site mutation C86A and the E3-binding mutations A97D and 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/Ubc5. 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 and Ubc5 are 1) the primary E2s for Rsp5 in yeast and 2) act as monoubiquitinating E2s in RING E3-catalyzed pathways, in contrast to the processive human ortholog UbcH5.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Kate E
authors.initials: KE
authors.lastname: Stoll
authors.forename: Peter S
authors.initials: PS
authors.lastname: Brzovic
authors.forename: Trisha N
authors.initials: TN
authors.lastname: Davis
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Klevit
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D020134:Catalytic Domain / Q000235:genetics; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism* / Q000502:physiology*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C489270:UBC5 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; C063109:ubiquitin-conjugating enzyme UBC4; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; C469287:RSP5 protein, S cerevisiae