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Abstract：
Endoplasmic reticulum-associated degradation (ERAD) plays a critical role in the destruction of terminally misfolded proteins at the secretory pathway. The system also regulates expression levels of several proteins such as Pca1p, a cadmium exporter in yeast. To gain better insight into the mechanisms underlying ERAD of Pca1p and other polytopic proteins by the proteasome in the cytosol, our study determined the roles for the molecular factors of ERAD in dislodging Pca1p from the endoplasmic reticulum (ER). Inactivation of the 20S proteasome leads to accumulation of ubiquitinated Pca1p in the ER membrane, suggesting a role for the proteasome in extraction of Pca1p from the ER. Pca1p formed a complex with the proteasome at the membrane in a Doa10p E3 ligase-dependent manner. Cdc48p is required for recruiting the proteasome to Pca1p. Although the Ufd2p E4 ubiquitin chain extension enzyme is involved in efficient degradation of Pca1p, Ufd2p-deficient cells did not affect the formation of a complex between Pca1p and the proteasome. Two other polytopic membrane proteins undergoing ERAD, Ste6*p and Hmg2p, also displayed the same outcomes observed for Pca1p. However, poly-ubiquitinated Cpy1*p, a luminal ERAD substrate, was detected in the cytosol independent of proteolytic activities of the proteasome. These results indicate that extraction and degradation of polytopic membrane proteins at the ER is a coupled event. This mechanism would relieve the cost of exposed hydrophobic domains in the cytosol during ERAD.
摘要：
内质网相关降解（ERAD）在分泌途径的末端错误折叠蛋白的破坏中起关键作用。该系统还调节几种蛋白质的表达水平，如Pca1p，酵母中的镉出口国。为了更好地了解Pca1p和其他多表位蛋白通过胞质溶胶中的蛋白酶体ERAD的潜在机制，我们的研究确定了ERAD分子因子在将Pca1p从内质网（ER）中移出中的作用。20S蛋白酶体的失活导致泛素化Pca1p在ER膜中的积累，提示蛋白酶体在从ER提取Pca1p中的作用。Pca1p以Doa10pE3连接酶依赖性方式与膜上的蛋白酶体形成复合物。Cdc48p是将蛋白酶体募集到Pca1p所必需的。尽管Ufd2pE4泛素链延长酶参与Pca1p的高效降解，Ufd2p缺陷细胞不影响Pca1p和蛋白酶体之间复合物的形成。另外两种正在进行ERAD的多位膜蛋白，Ste6*p和Hmg2p，也显示了与Pca1p相同的结果。然而,聚泛素化Cpy1*p，管腔ERAD基质，在细胞质中检测到，与蛋白酶体的蛋白水解活性无关。这些结果表明，在ER处提取和降解多位膜蛋白是偶联事件。该机制将减轻ERAD期间胞质溶胶中暴露的疏水结构域的成本。
