Kanaoka Yui, Mori Takaharu, Nagaike Wataru, Itaya Seira, Nonaka Yuto, Kohga Hidetaka, Haruyama Takamitsu, Sugano Yasunori, Miyazaki Ryoji, Ichikawa Muneyoshi, Uchihashi Takayuki, Tsukazaki Tomoya
Department of Physics, Graduate School of Science, Nagoya University, Nagoya, Aichi, Japan.
Department of Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, Japan.
Nat Commun. 2025 Jan 8;16(1):225. doi: 10.1038/s41467-024-54875-x.
Protein translocation across cellular membranes is an essential and nano-scale dynamic process. In the bacterial cytoplasmic membrane, the core proteins in this process are a membrane protein complex, SecYEG, corresponding to the eukaryotic Sec61 complex, and a cytoplasmic protein, SecA ATPase. Despite more than three decades of extensive research on Sec proteins, from genetic experiments to cutting-edge single-molecule analyses, no study has visually demonstrated protein translocation. Here, we visualize the translocation, via one unit of a SecYEG-SecA-embedded nanodisc, of an unfolded substrate protein by high-speed atomic force microscopy (HS-AFM). Additionally, the uniform unidirectional distribution of nanodiscs on a mica substrate enables the HS-AFM image data analysis, revealing dynamic structural changes in the polypeptide-crosslinking domain of SecA between wide-open and closed states depending on nucleotides. The nanodisc-AFM approach will allow us to execute detailed analyses of Sec proteins as well as visualize nano-scale events of other membrane proteins.
蛋白质跨细胞膜转运是一个至关重要的纳米级动态过程。在细菌细胞质膜中，该过程的核心蛋白是一种膜蛋白复合物SecYEG（对应于真核生物的Sec61复合物）和一种细胞质蛋白SecA ATP酶。尽管对Sec蛋白进行了三十多年的广泛研究，从基因实验到前沿的单分子分析，但尚无研究能直观地展示蛋白质转运过程。在此，我们通过高速原子力显微镜（HS-AFM），借助嵌入了一个SecYEG-SecA单元的纳米盘，直观呈现了未折叠底物蛋白的转运过程。此外，纳米盘在云母基底上均匀的单向分布使得HS-AFM图像数据分析成为可能，揭示了SecA多肽交联结构域在依赖核苷酸的开放和闭合状态之间的动态结构变化。纳米盘-原子力显微镜方法将使我们能够对Sec蛋白进行详细分析，并直观呈现其他膜蛋白的纳米级事件。
