The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP receptor targeting pathway, whereas the heptameric Sec complex has been proposed to mediate ribosome-independent post-translational translocation of proteins with less hydrophobic signal sequences that escape recognition by the SRP. However, multiple reports have proposed that the Sec complex may function cotranslationally and be involved in translocation or integration of SRP-dependent protein translocation substrates. To provide insight into these conflicting views, we induced expression of the tobacco etch virus protease to achieve rapid inactivation of the Sec complex by protease-mediated cleavage within the cytoplasmic domain of the Sec63 protein. Protein translocation assays conducted after tobacco etch virus protease induction revealed a complete block in translocation of two well-characterized substrates of the Sec complex, carboxypeptidase Y (CPY) and Gas1p, when the protease cleavage sites were located at structural domain boundaries in Sec63. However, integration of SRP-dependent membrane protein substrates was not detectably impacted. Moreover, redirecting CPY to the cotranslational pathway by increasing the hydrophobicity of the signal sequence rendered translocation of CPY insensitive to inactivation of the Sec complex. We conclude that the Sec complex is primarily responsible for the translocation of yeast secretome proteins with marginally hydrophobic signal sequences.

中文翻译：

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酵母 Sec 复合物的快速灭活选择性地阻止了翻译后易位蛋白质的运输。

酵母内质网具有三个不同的蛋白质易位通道。与翻译核糖体结合的异源三聚体 Sec61 和 Ssh1 复合物通过信号识别粒子 (SRP) 和 SRP 受体靶向途径介导靶向内质网的蛋白质的共翻译易位，而七聚体 Sec 复合物已被提议介导核糖体独立后-具有较少疏水性信号序列的蛋白质的翻译易位，从而逃避 SRP 的识别。然而，多项报告提出 Sec 复合物可能协同翻译并参与 SRP 依赖性蛋白质易位底物的易位或整合。为了深入了解这些相互矛盾的观点，我们诱导烟草蚀刻病毒蛋白酶的表达，以通过蛋白酶介导的 Sec63 蛋白细胞质域内的裂解实现 Sec 复合物的快速灭活。在烟草蚀刻病毒蛋白酶诱导后进行的蛋白质易位分析显示，当蛋白酶切割位点位于 Sec63 中的结构域边界时，Sec 复合体的两个充分表征的底物、羧肽酶 Y (CPY) 和 Gas1p 的易位完全阻断。然而，SRP 依赖性膜蛋白底物的整合并未受到可检测的影响。此外，通过增加信号序列的疏水性将 CPY 重定向到共翻译途径，使得 CPY 的易位对 Sec 复合物的失活不敏感。