Ikoma, Japan—Protein translocation is an essential, nano-scale dynamic process that facilitates the movement of proteins across cellular membranes, enabling them to reach specific locations within the cell or to be transported outside the cell. This process occurs through membrane protein complexes that provide necessary channels for the movement of proteins. In bacteria, a group of proteins forms the SecYEG-SecA complex, which helps cellular proteins translocate across the cytoplasmic membrane. The SecYEG component is a channel through which SecA drives protein translocation, using energy from a molecule called adenosine triphosphate (ATP), also known as the "energy currency" of the cell. Despite many different approaches, observing this process in detail has been quite challenging.
For the first time ever, researchers from Japan directly visualized protein translocation across membranes using high-speed atomic force microscopy (HS-AFM)—an event that had been biochemically predicted but never observed. This pioneering study, published online in Nature Communications on January 8, 2025, was led by Professor Tomoya Tsukazaki from the Nara Institute of Science and Technology (NAIST). The team, including Ms. Yui Kanaoka and Dr. Takayuki Uchihashi from Nagoya University and Dr. Takaharu Mori from Tokyo University of Science; successfully visualized how the SecYEG-SecA complex helps mediate the translocation of unfolded proteins across the bacterial membrane at a molecular level.
The researchers specifically focused on the conformational changes in SecA during the ATP hydrolysis cycle, which is critical to the translocation mechanism. The hydrolysis of ATP serves as the energy source that drives protein transport.
By analyzing the SecYEG-SecA complex dynamics using HS-AFM, the team captured real-time snapshots of SecA transitioning between two distinct conformational states due to the alteration of a region of SecA, PPXD—referred to as the "High" and "Low" states. These changes were linked to the ATP hydrolysis cycle. “ Thirteen years ago, we embarked on this journey to visualize protein translocation across membranes. The challenge of achieving the necessary spatiotemporal resolution pushed the very limits of high-speed AFM. Yet, through relentless dedication, meticulous sample preparation, and patient observation, we are thrilled to have captured these truly groundbreaking images, ” shares Tsukazaki.
Detailed analyses also estimated the rate of protein translocation to be about 2.2 amino acid residues per second, and a single SecYEG-SecA complex was sufficient for successful protein translocation.
“ We not only succeeded in capturing the protein membrane translocation process in real-time—an event previously elusive to visual observation—but also estimated the structural changes of motor proteins and their transport speeds, marking a significant breakthrough. Moving forward, we plan to apply this method to analyze other membrane proteins. This study paves the way for future research in visualizing these dynamics, potentially leading to better comprehension of the molecular mechanisms behind protein translocation, ” concludes Tsukazaki.
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Resource
Title: AFM observation of protein translocation mediated by one unit of SecYEG-SecA complex
Authors: Yui Kanaoka, Takaharu Mori, Wataru Nagaike, Seira Itaya, Yuto Nonaka, Hidetaka Kohga, Takamitsu Haruyama, Yasunori Sugano, Ryoji Miyazaki, Muneyoshi Ichikawa, Takayuki Uchihashi, and Tomoya Tsukazaki
Journal: Nature Communications
DOI: 10.1038/s41467-024-54875-x
Information about the Structural Life Science can be found at the following website: https://bsw3.naist.jp/eng/courses/courses309.html
About Nara Institute of Science and Technology (NAIST)
Established in 1991, Nara Institute of Science and Technology (NAIST) is a national university located in Kansai Science City, Japan. In 2018, NAIST underwent an organizational transformation to promote and continue interdisciplinary research in the fields of biological sciences, materials science, and information science. Known as one of the most prestigious research institutions in Japan, NAIST lays a strong emphasis on integrated research and collaborative co-creation with diverse stakeholders. NAIST envisions conducting cutting-edge research in frontier areas and training students to become tomorrow's leaders in science and technology.
Nature Communications
Imaging analysis
Cells
AFM observation of protein translocation mediated by one unit of SecYEG-SecA complex
8-Jan-2025
The authors declare no competing interests.