Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Cell Div. 2011 Mar 31;6:7. doi: 10.1186/1747-1028-6-7.
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.
S73/S97/环基序是 Cdc34 家族 E2 泛素连接酶的标志，这些酶与 SCF E3 泛素连接酶一起促进细胞周期和生长调节相关蛋白的降解。缺乏环基序的 Δ12Cdc34 突变体无法支持生长，这与其无法催化多泛素化有关。然而，缺乏环基序的三重突变体（tm）Cdc34不仅缺乏环基序，还包含 S73K 和 S97D 取代，这些取代在其他 E2 中存在于 K73/D97/no 环基序中，能够支持生长。尽管 tmCdc34 缺乏多泛素化，但它是否支持生长，或者 S73K 和 S97D 取代是否直接或间接纠正了环基序缺失引起的缺陷，目前尚不清楚。
尽管 tmCdc34 在体内和体外产生的多泛素化产物较少，但仍能支持酵母的生存能力，且细胞大小和细胞周期谱正常。Sic1 底物多泛素化的体外缺陷与不能支持生长的 Δ12Cdc34 反应中观察到的缺陷相似。tmCdc34 合成游离泛素的能力仅适度激活，且依赖于底物与 SCFCdc4 的募集。Cka2 激酶对 tmCdc34 中 C 端丝氨酸的磷酸化阻止了游离多泛素链的合成，可能是通过促进它们与底物的结合。然而，tmCDC34 酵母对 Ubp14 C 端泛素水解酶的缺失敏感，并且除 Ubp14 以外的 DUBs 不能有效地将 tmCDC34 酵母提取物中产生的多泛素链解聚，这表明游离链，无论是从头合成的还是从底物中回收的，都具有改变的结构。
催化基序替换会损害 Cdc34 的多泛素化活性，并改变其在体外和体内的调节，但任何基序都可以支持 Cdc34 在酵母生存能力中的功能。因此，S73/S97/环基序介导的强泛素化对于 Cdc34 在酵母生存能力中的作用不是必需的，至少在典型的实验室条件下是这样。
