Institute of Biochemistry, ETH Zürich, Zürich, Switzerland.
Institute of Molecular Health Sciences, ETH Zürich, Zürich, Switzerland.
Elife. 2018 Jun 18;7:e35528. doi: 10.7554/eLife.35528.
In yeast, the glucose-induced degradation-deficient (GID) E3 ligase selectively degrades superfluous gluconeogenic enzymes. Here, we identified all subunits of the mammalian GID/CTLH complex and provide a comprehensive map of its hierarchical organization and step-wise assembly. Biochemical reconstitution demonstrates that the mammalian complex possesses inherent E3 ubiquitin ligase activity, using Ube2H as its cognate E2. Deletions of multiple GID subunits compromise cell proliferation, and this defect is accompanied by deregulation of critical cell cycle markers such as the retinoblastoma (Rb) tumor suppressor, phospho-Histone H3 and Cyclin A. We identify the negative regulator of pro-proliferative genes Hbp1 as a GID/CTLH proteolytic substrate. Indeed, Hbp1 accumulates in cells lacking GID/CTLH activity, and Hbp1 physically interacts and is ubiquitinated in vitro by reconstituted GID/CTLH complexes. Our biochemical and cellular analysis thus demonstrates that the GID/CTLH complex prevents cell cycle exit in G1, at least in part by degrading Hbp1.
在酵母中，葡萄糖诱导的降解缺陷型 (GID) E3 连接酶选择性降解多余的糖异生酶。在这里，我们鉴定了哺乳动物 GID/CTL 复合物的所有亚基，并提供了其层次结构和逐步组装的全面图谱。生化重构成表明，哺乳动物复合物具有内在的 E3 泛素连接酶活性，使用 Ube2H 作为其同源 E2。多个 GID 亚基的缺失会损害细胞增殖，并且这种缺陷伴随着关键细胞周期标志物（如视网膜母细胞瘤 (Rb) 肿瘤抑制因子、磷酸化组蛋白 H3 和细胞周期蛋白 A）的失调。我们确定了促进增殖基因的负调节剂 Hbp1 是 GID/CTL 蛋白酶体的底物。事实上，在缺乏 GID/CTL 活性的细胞中，Hbp1 会积累，并且在体外，重组的 GID/CTL 复合物会与 Hbp1 发生物理相互作用并对其进行泛素化。因此，我们的生化和细胞分析表明，GID/CTL 复合物至少部分通过降解 Hbp1 来防止 G1 期细胞周期退出。
