Cue1p is an activator of Ubc7p E2 activity in vitro and in vivo
Cue1p is an activator of Ubc7p E2 activity in vitro and in vivo
复制标题
DOI：
10.1074/jbc.m801122200
复制
发表时间：
2008-05-09
影响因子：
4.8
通讯作者：
Hampton, Randolph Y.
中科院分区：
文献类型：
--
作者：
Bazirgan, Omar A.;Hampton, Randolph Y.
Ubc 7 p是一种泛素结合酶（E2），其与内质网（ER）驻留的泛素连接酶（E3）一起起作用以促进内质网相关降解（ERAD）。Ubc 7 p只有在通过Cue 1 p（一种膜锚定的ER蛋白）结合到ER表面时才在ERAD中起作用。Cue 1 p的作用被认为涉及Ubc 7 p在ER表面的被动浓缩。然而，我们的Ubc 7 p的生化研究表明，Cue 1 p可能，此外，刺激Ubc 7 p E2活性。我们已经测试了这个想法，发现它在体外和体内都是正确的。Ubc 7 p结合到Cue 1 p的可溶性结构域显示出强烈增强的体外泛素化活性，在存在和不存在E3的情况下。Cue 1 p还增强了Ubc 7 p在体内的功能，这种激活是从建立的ER锚定作用Cue 1 p分离。最后，我们测试了在体内激活Ubc 7 p的Cue 1 p在一个独立的ER膜和ERAD的测定。嵌合E2连接Ubc 7 p的Cdc 34 p/Ubc 3 p定位域互补的cdc 34 -2 TS表型，和可溶性Cue 1 p结构域的共表达增强互补，这种嵌合Ubc 7 p E2。这些研究揭示了以前未观察到的刺激Ubc 7 p E2活性的Cue 1 p，这是至关重要的完全ERAD和功能独立于众所周知的Cue 1 p锚定功能。此外，它提出了一个以前不受重视的模式，通过Cue 1 p样相互作用的合作伙伴的E2的调节。
Ubc7p is a ubiquitin-conjugating enzyme (E2) that functions with endoplasmic reticulum (ER)-resident ubiquitin ligases (E3s) to promote endoplasmic reticulum-associated degradation (ERAD). Ubc7p only functions in ERAD if bound to the ER surface by Cue1p, a membrane-anchored ER protein. The role of Cue1p was thought to involve passive concentration of Ubc7p at the surface of the ER. However, our biochemical studies of Ubc7p suggested that Cue1p may, in addition, stimulate Ubc7p E2 activity. We have tested this idea and found it to be true both in vitro and in vivo. Ubc7p bound to the soluble domain of Cue1p showed strongly enhanced in vitro ubiquitination activity, both in the presence and absence of E3. Cue1p also enhanced Ubc7p function in vivo, and this activation was separable from the established ER-anchoring role of Cue1p. Finally, we tested in vivo activation of Ubc7p by Cue1p in an assay independent of the ER membrane and ERAD. A chimeric E2 linking Ubc7p to the Cdc34p/Ubc3p localization domain complemented the cdc34-2 TS phenotype, and co-expression of the soluble Cue1p domain enhanced complementation by this chimeric Ubc7p E2. These studies reveal a previously unobserved stimulation of Ubc7p E2 activity by Cue1p that is critical for full ERAD and that functions independently of the well known Cue1p anchoring function. Moreover, it suggests a previously unappreciated mode for regulation of E2s by Cue1p-like interacting partners.