Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are retro-translocated into the cytosol and degraded by the ubiquitin-proteasome system, a pathway termed luminal ER-associated protein degradation (ERAD-L). Retro-translocation is mediated by a conserved protein complex, consisting of the ubiquitin ligase Hrd1 and four associated proteins (Der1, Usa1, Hrd3, and Yos9). Photo-crosslinking experiments provided preliminary evidence for the polypeptide path through the membrane, but did not reveal specific interactions between amino acids in the substrate and Hrd1 complex. Here, we have used site-specific disulfide crosslinking to map the interactions of a glycosylated model substrate with the Hrd1 complex in live S. cerevisiae cells. Together with available electron cryo-microscopy (cryo-EM) structures, the results show that the substrate interacts on the luminal side with both a groove in Hrd3 and the lectin domain of Yos9, and inserts a loop into the membrane, with one side of the loop interacting with the lateral gate of Der1, and the other with the lateral gate of Hrd1. Our disulfide crosslinking experiments also show that two Hrd1 molecules can interact through their lateral gates, and that Hrd1 auto-ubiquitination is required for the disassembly of these Hrd1 dimers. Taken together, these data define the path of a polypeptide through the ER membrane and suggest that auto-ubiquitination of inactive Hrd1 dimers is required to generate active Hrd1 monomers.

内质网 (ER) 腔中错误折叠的蛋白质被逆向转运到胞质溶胶中并被泛素-蛋白酶体系统降解，该途径称为腔内 ER 相关蛋白降解 (ERAD-L)。逆向易位由保守的蛋白质复合物介导，该复合物由泛素连接酶 Hrd1 和四种相关蛋白（Der1、Usa1、Hrd3 和 Yos9）组成。光交联实验为多肽通过膜的路径提供了初步证据，但没有揭示底物中氨基酸与 Hrd1 复合物之间的特定相互作用。在这里，我们使用位点特异性二硫键交联来绘制糖基化模型底物与活酵母中 Hrd1 复合物的相互作用。细胞。连同可用的电子冷冻显微镜 (cryo-EM) 结构，结果表明，底物在腔侧与 Hrd3 中的凹槽和 Yos9 的凝集素结构域相互作用，并将一个环插入膜中，一侧为环与 Der1 的侧门相互作用，另一个与 Hrd1 的侧门相互作用。我们的二硫键交联实验还表明，两个 Hrd1 分子可以通过它们的侧门相互作用，并且这些 Hrd1 二聚体的分解需要 Hrd1 自动泛素化。总之，这些数据定义了多肽通过 ER 膜的路径，并表明非活性 Hrd1 二聚体的自泛素化是产生活性 Hrd1 单体所必需的。