PDF(Pubmed)
Abstract：
The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N-terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro-alpha-factor. The unbinding parameters including off-rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko-Hummer-Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off-rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor.
摘要：
Sec61转位子允许分泌前蛋白在多肽生物合成过程中从胞质溶胶转位到内质网腔。这些蛋白质具有在转位上对接的N-末端信号肽(SP)。SP突变可以消除易位并引起疾病，建议对这种SP/Sec61相互作用发挥重要作用。然而,这种结合的详细生物物理表征仍然缺失。这里,光学镊力谱用于表征Sec61与pre-α因子SP之间解离过程的动力学参数。通过将破裂力数据拟合到Dudko-Hummer-Szabo模型，获得了包括解离速率常数和到过渡状态的距离在内的去束缚参数。有趣的是,易位抑制剂mycolactone增加解离速率并加速SP/Sec61解离，同时也削弱了互动。尽管在SP中含有单点突变的易位缺陷突变体消除了SP/Sec61结合的特异性，导致不稳定的相互作用。总之,我们定量表征了信号肽和转位子之间的解离过程，以及如何通过易位抑制剂改变解结合参数。
