Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences, Stockholm University, Svante Arrhenius väg 16C, SE-10691 Stockholm, Sweden.
Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences, Stockholm University, Svante Arrhenius väg 16C, SE-10691 Stockholm, Sweden.
J Mol Biol. 2020 Dec 4;432(24):166694. doi: 10.1016/j.jmb.2020.10.028. Epub 2020 Oct 31.
Many unanswered questions remain in understanding the biosynthesis of the peptide hormone insulin. Here we elucidate new aspects in the mechanism of co-translational translocation initiation of pre-proinsulin in the endoplasmic reticulum. We utilize a translational arrest peptide derived from the x-box-binding protein (Xbp1) to induce ribosomal stalling and generate translocation intermediates. We find that the insulin signal sequence is rather weakly gating and requires the assistance of auxiliary translocon components to initiate translocation. Probing the translational intermediates with chemical crosslinking, we identified an early interaction with the translocon-associated protein (TRAP) complex. The TRAPβ subunit interacts with pre-proinsulin before the peptide enters the Sec61 translocon channel in a signal sequence-dependent manner. We describe the substrate sequence determinants that are recognized by TRAP on the cytosolic site of the membrane to facilitate substrate-specific opening of the Sec61 translocon channel. Our findings support the hypothesis that the TRAP-dependence is in part determined by the content of glycine and proline residues mainly within the signal sequence.
在理解多肽激素胰岛素的生物合成过程中，仍有许多悬而未决的问题。在这里，我们阐明了内质网中前胰岛素原共翻译易位起始机制的新方面。我们利用来自 X 盒结合蛋白 (Xbp1) 的翻译阻断肽诱导核糖体停滞并产生易位中间产物。我们发现胰岛素信号序列的门控作用相当弱，需要辅助易位蛋白复合物的协助才能起始易位。通过化学交联探测翻译中间产物，我们鉴定出与易位相关蛋白 (TRAP) 复合物的早期相互作用。TRAPβ 亚基在信号序列依赖性方式下，在肽进入 Sec61 易位通道之前与前胰岛素原相互作用。我们描述了 TRAP 在膜的胞质侧识别的底物序列决定因素，以促进 Sec61 易位通道的底物特异性开放。我们的发现支持这样的假设，即 TRAP 的依赖性部分取决于信号序列中主要的甘氨酸和脯氨酸残基的含量。
