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首页> 外文期刊>Cell Division >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
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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

机译：无环tmCdc34 E2突变体在体内和体外的多聚泛素化缺陷，以依赖于Ubp14和Cka2的方式支持酵母生长

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Background The S73/S97/loop motif is a hallmark of the Cdc34 family of E2 ubiquitin-conjugating enzymes that together with the SCF E3 ubiquitin ligases promote degradation of proteins involved in cell cycle and growth regulation. The inability of the loop-less Δ12Cdc34 mutant to support growth was linked to its inability to catalyze polyubiquitination. However, the loop-less t riple m utant (tm) Cdc34, which not only lacks the loop but also contains the S73K and S97D substitutions typical of the K73/D97o loop motif present in other E2s, supports growth. Whether tmCdc34 supports growth despite defective polyubiquitination, or the S73K and S97D substitutions, directly or indirectly, correct the defect caused by the loop absence, are unknown. Results 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. Conclusions 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.
机译：背景S73 / S97 /环基序是E2泛素结合酶的Cdc34家族的标志，它与SCF E3泛素连接酶一起促进了参与细胞周期和生长调节的蛋白质的降解。无环的Δ12 Cdc34突变体不能支持生长与它不能催化多聚泛素化有关。但是，无环三态突变体（tm）Cdc34不仅缺少环，而且还包含其他E2中存在的K73 / D97 /无环基序典型的S73K和S97D取代。尚不清楚尽管多泛素化存在缺陷， tm Cdc34是否支持生长，还是直接或间接地通过S73K和S97D替代来纠正由环缺失引起的缺陷。结果 tm Cdc34支持酵母活力，具有正常的细胞大小和细胞周期谱，尽管体内和体外产生的泛素结合物较少。 Sic1底物多聚泛素的体外缺陷类似于在与不能支持生长的Δ12 Cdc34反应中观察到的缺陷。 tm Cdc34合成游离多聚泛素仅适度激活，其方式取决于底物募集到SCF Cdc4 的方式。 Cka2激酶使 tm Cdc34中C端丝氨酸的磷酸化阻止了游离多聚泛素链的合成，这可能是由于促进了它们与底物的附着。尽管如此， tm CDC34酵母菌对Ubp14 C端泛素水解酶的丢失很敏感，除了Ubp14以外的DUBs不能有效地拆卸 tm CDC34酵母提取物中产生的多聚泛素链，这表明从头合成或从底物回收的自由链具有改变的结构。结论催化基序替换损害了Cdc34的多聚泛素化活性，并改变了其在体内和体外的调控，但任一基序均可支持Cdc34在酵母生存能力中的功能。因此，至少在典型的实验室条件下，由S73 / S97 /环基序介导的强健的多聚泛素化对于Cdc34在酵母生存力中的作用不是必需的。

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