AFM observation of protein translocation mediated by one unit of SecYEG-SecA complex
Yui Kanaoka, Takaharu Mori, Wataru Nagaike, Seira Itaya, Yuto Nonaka, Hidetaka Kohga, Takamitsu Haruyama, Yasunori Sugano, Ryoji Miyazaki, Muneyoshi Ichikawa, Takayuki Uchihashi, Tomoya Tsukazaki
Abstract
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. Protein translocation across cellular membranes is an essential and nano-scale dynamic process. Here, the authors visualize protein translocation via the SecYEG-SecA complex in a nanodisc by high-speed atomic force microscopy (HS-AFM), shedding light on the structural changes of SecA in this process.