Department of Cell and Molecular Biology, Uppsala University, 752 36 Uppsala, Sweden.
Proc Natl Acad Sci U S A. 2022 Sep 20;119(38):e2204038119. doi: 10.1073/pnas.2204038119. Epub 2022 Sep 12.
Mechanistic details of the signal recognition particle (SRP)-mediated insertion of membrane proteins have been described from decades of in vitro biochemical studies. However, the dynamics of the pathway inside the living cell remain obscure. By combining in vivo single-molecule tracking with numerical modeling and simulated microscopy, we have constructed a quantitative reaction-diffusion model of the SRP cycle. Our results suggest that the SRP-ribosome complex finds its target, the membrane-bound translocon, through a combination of three-dimensional (3D) and 2D diffusional search, together taking on average 750 ms. During this time, the nascent peptide is expected to be elongated only 12 or 13 amino acids, which explains why, in , no translation arrest is needed to prevent incorrect folding of the polypeptide in the cytosol. We also found that a remarkably high proportion (75%) of SRP bindings to ribosomes occur in the cytosol, suggesting that the majority of target ribosomes bind SRP before reaching the membrane. In combination with the average SRP cycling time, 2.2 s, this result further shows that the SRP pathway is capable of targeting all substrate ribosomes to translocons.
信号识别颗粒（SRP）介导的膜蛋白插入的机制细节已经通过几十年的体外生化研究进行了描述。然而，活细胞内途径的动态仍然不清楚。通过将体内单分子追踪与数值建模和模拟显微镜相结合，我们构建了 SRP 循环的定量反应扩散模型。我们的结果表明，SRP-核糖体复合物通过三维（3D）和二维扩散搜索的组合来找到其靶标，即膜结合的转位酶，平均需要 750 毫秒。在此期间，新生肽预计仅延伸 12 或 13 个氨基酸，这解释了为什么在 ，不需要翻译暂停来防止多肽在细胞质中错误折叠。我们还发现，核糖体与 SRP 的结合有很大一部分（75%）发生在细胞质中，这表明大多数靶核糖体在到达膜之前就与 SRP 结合。结合平均 SRP 循环时间 2.2 秒，这一结果进一步表明，SRP 途径能够将所有底物核糖体靶向转位酶。
