Coughlan Christina M, Walker Jennifer L, Cochran Jared C, Wittrup K Dane, Brodsky Jeffrey L
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
J Biol Chem. 2004 Apr 9;279(15):15289-97. doi: 10.1074/jbc.M309673200. Epub 2004 Jan 26.
The rate-limiting step in protein secretion is folding, which occurs in the endoplasmic reticulum (ER) lumen, and almost all secreted proteins contain disulfide bonds that form in the ER and stabilize the native state. Secreted proteins unable to fold may aggregate or they may be subject to ER-associated protein degradation. To examine the fate of aberrant forms of a well characterized, disulfide-bonded secreted protein, we expressed bovine pancreatic trypsin inhibitor in yeast. Bovine pancreatic trypsin inhibitor is a single domain, 58-amino acid polypeptide containing three disulfide bonds, and yeast cells secrete the wild type protein. In contrast, the Y35L mutant, which folds rapidly but is unstable, remains soluble and is not secreted. Surprisingly, the proteolysis of Y35L is unaffected in yeast containing mutations in genes encoding factors required for ER-associated protein degradation and is stable if artificially retained in the ER. Rather, Y35L is diverted from the Golgi to the vacuole and degraded. Because only the mutant protein is quantitatively proteolyzed these data suggest that a post-ER quality control check-point diverts unstable proteins to the vacuole for degradation.
蛋白质分泌的限速步骤是折叠，这一过程发生在内质网（ER）腔中，几乎所有分泌蛋白都含有在内质网中形成并稳定天然状态的二硫键。无法折叠的分泌蛋白可能会聚集，或者可能会经历内质网相关蛋白降解。为了研究一种特征明确的、含二硫键的分泌蛋白异常形式的命运，我们在酵母中表达了牛胰蛋白酶抑制剂。牛胰蛋白酶抑制剂是一个单结构域、含58个氨基酸的多肽，含有三个二硫键，酵母细胞可分泌野生型蛋白。相比之下，Y35L突变体虽然折叠迅速但不稳定，它仍保持可溶状态且不被分泌。令人惊讶的是，Y35L的蛋白水解在编码内质网相关蛋白降解所需因子的基因发生突变的酵母中不受影响，并且如果人工保留在内质网中则是稳定的。相反，Y35L从高尔基体转向液泡并被降解。由于只有突变蛋白被定量蛋白水解，这些数据表明内质网后质量控制检查点会将不稳定蛋白转向液泡进行降解。
