Mackinnon Andrew L, Paavilainen Ville O, Sharma Ajay, Hegde Ramanujan S, Taunton Jack
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.
Elife. 2014;3:e01483. doi: 10.7554/eLife.01483. Epub 2014 Feb 4.
Membrane protein biogenesis requires the coordinated movement of hydrophobic transmembrane domains (TMD) from the cytosolic vestibule of the Sec61 channel into the lipid bilayer. Molecular insight into TMD integration has been hampered by the difficulty of characterizing intermediates during this intrinsically dynamic process. In this study, we show that cotransin, a substrate-selective Sec61 inhibitor, traps nascent TMDs in the cytosolic vestibule, permitting detailed interrogation of an early pre-integration intermediate. Site-specific crosslinking revealed the pre-integrated TMD docked to Sec61 near the cytosolic tip of the lateral gate. Escape from cotransin-arrest depends not only on cotransin concentration, but also on the biophysical properties of the TMD. Genetic selection of cotransin-resistant cancer cells uncovered multiple mutations clustered near the lumenal plug of Sec61α, thus revealing cotransin's likely site of action. Our results suggest that TMD/lateral gate interactions facilitate TMD transfer into the membrane, a process that is allosterically modulated by cotransin binding to the plug. DOI: http://dx.doi.org/10.7554/eLife.01483.001.
膜蛋白生物合成需要疏水跨膜结构域（TMD）从Sec61通道的胞质前庭协调移动到脂质双层中。由于在这个内在动态过程中难以表征中间体，对TMD整合的分子洞察受到了阻碍。在这项研究中，我们表明，共转运蛋白（一种底物选择性Sec61抑制剂）将新生的TMD捕获在胞质前庭中，从而允许对早期预整合中间体进行详细研究。位点特异性交联揭示了预整合的TMD停靠在侧门胞质末端附近的Sec61上。从共转运蛋白阻滞中逃逸不仅取决于共转运蛋白的浓度，还取决于TMD的生物物理性质。对共转运蛋白抗性癌细胞的遗传筛选发现了多个聚集在Sec61α腔塞附近的突变，从而揭示了共转运蛋白可能的作用位点。我们的结果表明，TMD/侧门相互作用促进TMD转移到膜中，这一过程受到共转运蛋白与塞子结合的变构调节。DOI: http://dx.doi.org/10.7554/eLife.01483.001
