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.
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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DOI：
10.1186/1747-1028-6-7
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发表时间：
2011-03-31
期刊：
影响因子：
2.3
通讯作者：
Skowyra D
中科院分区：
文献类型：
--
作者：
Lass A;Cocklin R;Scaglione KM;Skowyra M;Korolev S;Goebl M;Skowyra D
S73/S97/loop基序是CDC34家族E2泛素结合酶的标志，它与SCF E3泛素连接酶一起促进参与细胞周期和生长调节的蛋白质的降解。无环Δ12Cdc34突变体无法支持生长与其无法催化多泛素化有关。然而，无环三重突变体(Tm)CDC34不仅缺少环，还包含其他E2中存在的K73/D97/无环基序中典型的S73K和S97D替换，支持生长。尽管有缺陷的多泛素化，tmCDc34是否支持生长，或者S73K和S97D替换是否直接或间接地纠正了环缺失造成的缺陷，目前尚不清楚。尽管在体内和体外产生的多泛素结合物较少，但tmCDC34支持具有正常细胞大小和细胞周期特征的酵母活性。SIC1底物多泛素化的体外缺陷类似于与Δ12Cdc34反应中观察到的缺陷，不能支持生长。由tmCdc34合成的游离泛素仅被温和地激活，并且依赖于底物对SCFCdc4的募集。Cka2激酶使tmCDC34的C-末端丝氨酸磷酸化，可能通过促进多聚泛素链与底物的结合来阻止其合成。然而，tmCDC34酵母对Ubp14 C末端泛素水解酶的丢失和Ubp14以外的DUBS不能有效地分解tmCDC34酵母提取物中产生的多泛素链敏感，这表明无论是从头合成的自由链还是从底物中回收的自由链，都具有改变的结构。催化基序替换影响了CDc34的多泛素化活性，并改变了其在体外和体内的调节，但任一基序都可以支持CDc34在酵母活性中的功能。因此，至少在典型的实验室条件下，由S73/S97/LOOP基序介导的强大的多泛素化对于CDC34在酵母活性中的作用是不必要的。
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 triple mutant (tm) Cdc34, which not only lacks the loop but also contains the S73K and S97D substitutions typical of the K73/D97/no 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. 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.
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影响因子：
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作者：
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通讯作者：
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