Oh Ray S, Bai Xinli, Rommens Johanna M
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8; Program in Genetics & Genomic Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada.
Program in Genetics & Genomic Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada.
J Biol Chem. 2006 Jul 28;281(30):21480-21490. doi: 10.1074/jbc.M601843200. Epub 2006 May 23.
Ubiquitin-conjugating enzyme Ubc6p is a tail-anchored protein that is localized to the cytoplasmic face of the endoplasmic reticulum (ER) membrane and has been implicated in the degradation of many misfolded membrane proteins in yeast. We have undertaken characterization studies of two human homologs, hsUbc6 and hsUbc6e. Both possess tail-anchored protein motifs, display high conservation in their catalytic domains, and are functional ubiquitin-conjugating enzymes as determined by in vitro thiol-ester assay. Both also display induction by the unfolded protein response, a feature of many ER-associated degradation (ERAD) components. Post-translational modification involving phosphorylation of hsUbc6e was observed to be ER-stress-related and dependent on signaling of the PRK-like ER kinase (PERK). The phosphorylation site was mapped to Ser-184, which resides within the uncharacterized region linking the highly conserved catalytic core and the C-terminal transmembrane domain. Phosphorylation of hsUbc6e also did not alter stability, subcellular localization, or interaction with a partner ubiquitin-protein isopeptide ligase. Assays of hsUbc6e(S184D) and hsUbc6e(S184E), which mimic the phosphorylated state, suggest that phosphorylation may reduce capacity for forming ubiquitin-enzyme thiol-esters. The occurrence of two distinct Ubc6p homologs in vertebrates, including one with phosphorylation modification in response to ER stress, emphasizes diversity in function between these Ub-conjugating enzymes during ERAD processes.
泛素结合酶Ubc6p是一种尾锚定蛋白，定位于内质网（ER）膜的细胞质面，参与酵母中许多错误折叠膜蛋白的降解。我们对两种人类同源物hsUbc6和hsUbc6e进行了特性研究。两者都具有尾锚定蛋白基序，其催化结构域具有高度保守性，并且通过体外硫酯测定确定它们是功能性泛素结合酶。两者还表现出由未折叠蛋白反应诱导的特性，这是许多内质网相关降解（ERAD）成分的一个特征。观察到涉及hsUbc6e磷酸化的翻译后修饰与内质网应激相关，并且依赖于蛋白激酶样内质网激酶（PERK）的信号传导。磷酸化位点定位于Ser-184，其位于连接高度保守的催化核心和C末端跨膜结构域的未表征区域内。hsUbc6e的磷酸化也没有改变稳定性、亚细胞定位或与伴侣泛素-蛋白质异肽连接酶的相互作用。对模拟磷酸化状态的hsUbc6e（S184D）和hsUbc6e（S184E）的测定表明，磷酸化可能会降低形成泛素-酶硫酯的能力。脊椎动物中存在两种不同的Ubc6p同源物，其中一种响应内质网应激具有磷酸化修饰，这强调了这些泛素结合酶在ERAD过程中功能的多样性。
