The loop-less tmCdc34 E2 mutant defective in polyubiquitination in vitro and in vivo supports yeast growth in a manner dependent on Ubp14 and Cka2

tmCdc34 supports yeast viability with normal cell size and cell cycle profile despite producing fewer polyubiquitin conjugates in vivo and in vitro. The in vitro defect in Sic1 substrate polyubiquitination is similar to the defect observed in reactions with Δ12Cdc34 that cannot support growth. The synthesis of free polyubiquitin by tmCdc34 is activated only modestly and in a manner dependent on substrate recruitment to SCFCdc4. Phosphorylation of C-terminal serines in tmCdc34 by Cka2 kinase prevents the synthesis of free polyubiquitin chains, likely by promoting their attachment to substrate. Nevertheless, tmCDC34 yeast are sensitive to loss of the Ubp14 C-terminal ubiquitin hydrolase and DUBs other than Ubp14 inefficiently disassemble polyubiquitin chains produced in tmCDC34 yeast extracts, suggesting that the free chains, either synthesized de novo or recycled from substrates, have an altered structure.The catalytic motif replacement compromises polyubiquitination activity of Cdc34 and alters its regulation in vitro and in vivo, but either motif can support Cdc34 function in yeast viability. Robust polyubiquitination mediated by the S73/S97/loop motif is thus not necessary for Cdc34 role in yeast viability, at least under typical laboratory conditions.The covalent attachment of ubiquitin to other proteins often serves as the signal for their degradation by the 26 S proteasome [1]. Protein ubiquitination depends on a cascade of ubiquitin transfer reactions that begins with the formation of a high-energy thiolester between the C-terminus of ubiquitin and the catalytic site cysteine of the E1 ubiquitin-activating enzyme. The activated ubiquitin is transesterified to the active site cysteine of one of many E2 ubiquitin-conjugating enzymes and then conjugated to specific substrates in a manner dependent upon specific E3 ubiquitin ligases. While the term "ubiquitin ligase" implies that all E3s are enzymes, only the HECT-type E3s contain a catalytic site cysteine that di