Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Department of Cellular Biochemistry, University Medical Centre Göttingen, Göttingen, Germany.
Nat Cell Biol. 2020 Mar;22(3):274-281. doi: 10.1038/s41556-020-0473-4. Epub 2020 Feb 24.
During endoplasmic-reticulum-associated protein degradation (ERAD), misfolded proteins are polyubiquitinated, extracted from the ER membrane and degraded by the proteasome. In a process called retrotranslocation, misfolded luminal proteins first need to traverse the ER membrane before ubiquitination can occur in the cytosol. It was suggested that the membrane-embedded ubiquitin ligase Hrd1 forms a retrotranslocation pore regulated by cycles of auto- and deubiquitination. However, the mechanism by which auto-ubiquitination affects Hrd1 and allows polypeptides to cross the membrane and whether Hrd1 forms a membrane-spanning pore remained unknown. Here, using purified Hrd1 incorporated into different model membranes, we show that Hrd1 auto-ubiquitination leads to the opening of a pore. Substrate binding increases the pore size and its activity, whereas deubiquitination closes the pore and renders it unresponsive to substrate. We identify two binding sites for misfolded proteins in Hrd1, a low-affinity luminal site and a high-affinity cytoplasmic site formed following auto-ubiquitination of specific lysine residues in Hrd1's RING domain. We propose that the affinity difference between the luminal and cytoplasmic binding sites provides the initial driving force for substrate movement through Hrd1.
在内质网相关蛋白降解（ERAD）过程中，错误折叠的蛋白被多泛素化，从内质网膜中提取出来，并被蛋白酶体降解。在一个称为逆向转运的过程中，错误折叠的腔蛋白首先需要穿过内质网膜，然后才能在细胞质中发生泛素化。有人提出，膜嵌入的泛素连接酶 Hrd1 形成一个逆向转运孔，由自动和去泛素化的循环调节。然而，自动泛素化如何影响 Hrd1 并允许多肽穿过膜，以及 Hrd1 是否形成一个跨膜孔仍然未知。在这里，我们使用纯化的 Hrd1 整合到不同的模型膜中，表明 Hrd1 的自动泛素化导致孔的打开。底物结合增加了孔的大小和活性，而去泛素化则关闭了孔，并使其对底物无反应。我们确定了 Hrd1 中两个错误折叠蛋白的结合位点，一个是低亲和力的腔位，一个是高亲和力的细胞质位，这是在 Hrd1 的 RING 结构域中特定赖氨酸残基的自动泛素化之后形成的。我们提出，腔和细胞质结合位点之间的亲和力差异为底物通过 Hrd1 的运动提供了初始驱动力。
