Mechanism of polyubiquitin chain assembly by an ER-associated ubiquitin ligase

用76个残基的蛋白质泛素（Ub）共价修饰蛋白质，从而改变靶蛋白的稳定性、定位或活性，调节真核细胞功能的几乎所有方面。 该反应需要三种类型的酶的顺序作用;活化酶（E1）在其催化半胱氨酸和泛素中Gly 76的羧基之间形成硫酯键以活化它;缀合酶（E2）从E1接收泛素;泛素连接酶（E3）将泛素分子从E2转移到底物。对于每个真核生物物种，存在两种E1酶，而发现了大约几十种E2酶（表1）和数千种E3连接酶。E3连接酶可以分为两大类，含有HECT结构域（与E6相关蛋白C末端同源）的连接酶和携带催化RING（真正有趣的新基因）结构域的连接酶。 我们已经建立了一个体外泛素化系统，使用纯化的gp 78 c，胞质结构域的ER相关的环连接酶和ube 2g 2。该测定允许我们组装Lys 48连接的多聚泛素链。使用这个系统，我们已经证明，多聚泛素链可以预先组装在催化半胱氨酸的Ube 2g 2被转移到基板之前。在Ube 2g 2及其酵母同源物Ubc 7 p的细胞中检测到活性位点连接的多聚泛素链，但这些链如何组装在E2活性位点上尚不清楚。我们最近发现gp 78通过两个寡聚化位点形成寡聚体，其中一个是gp 78胞质结构域中的疏水片段（gp 78 C）。我们进一步证明，gp 78寡聚体可以使用一种新型相互作用同时与多个Ube 2g 2分子结合，该相互作用主要由与预测的RING结合位点不同的Ube 2g 2表面介导。我们的数据表明，这种gp 78-Ube 2g 2异源寡聚体的形成使多个Ube 2g 2分子紧密接近，允许泛素部分在相邻的Ube 2g 2之间转移，形成活性位点连接的聚泛素链。

Covalent modification of a protein with the 76-residue protein ubiquitin (Ub), thereby changing the stability, localization, or activity of the target protein, regulates almost all aspects of eukaryotic cellular function. This reaction requires the sequential actions of three types of enzymes; an activating enzyme (E1) forms a thioester linkage between its catalytic cysteine and the carboxyl group of Gly76 in ubiquitin to activate it; a conjugating enzyme (E2) that receives ubiquitin from the E1; a ubiquitin ligase (E3) that transfers the ubiquitin molecule from the E2 to a substrate. For every eukaryotic species, there are two E1 enzymes, whereas approximately dozens of E2 enzymes (Table 1) and thousands of E3 ligases are found. The E3 ligases can be classified into two major catagories, the HECT domain (homologous to E6-associated protein C-Terminus)-containing ligases and those carrying a catalytic RING (really interesting new gene) domain. We have established an in vitro ubiquitination system using purified gp78c, a cytosolic domain of an ER-associated RING ligase and ube2g2. The assay allows us to assemble Lys48 linked polyubiquitin chains. Using this system, we have demonstrated that polyubiquitin chains can be preassembled on the catalytic cysteine of Ube2g2 before being transferred to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled on an E2 active site is unclear. We recently discovered that gp78 forms an oligomer via two oligomerization sites, one of which being a hydrophobic segment in the gp78 cytosolic domain (gp78C). We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules using a novel interaction that is primarily mediated by an Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that the formation of such a gp78-Ube2g2 hetero-oligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2 to form active site-linked polyubiquitin chains.