SecA functions in vivo as a discrete anti‐parallel dimer to promote protein transport

• Published in: Molecular microbiology 2017-02, Vol.103 (3), p.439-451
• Main Authors: Banerjee, Tithi, Lindenthal, Christine, Oliver, Donald
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphatases - physiology; Bacterial proteins; Bacterial Proteins - metabolism; Bacterial Proteins - physiology; Cell Membrane - metabolism; Dimerization; Hydrolysis; Ligands; Membrane Transport Proteins - metabolism; Microbiology; Protein Binding; Protein Structure, Quaternary; Protein Transport - physiology; SEC Translocation Channels - metabolism; SEC Translocation Channels - physiology
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.13567

Summary SecA ATPase motor protein plays a central role in bacterial protein transport by binding substrate proteins and the SecY channel complex and utilizing its ATPase activity to drive protein translocation across the plasma membrane. SecA has been shown to exist in a dynamic monomer–dimer equilibrium modulated by translocation ligands, and multiple structural forms of the dimer have been crystallized. Since the structural form of the dimer remains a controversial and unresolved question, we addressed this matter by engineering ρ‐benzoylphenylalanine along dimer interfaces corresponding to the five different SecA X‐ray structures and assessing their in vivo photo‐crosslinking pattern. A discrete anti‐parallel 1M6N‐like dimer was the dominant if not exclusive dimer found in vivo, whether SecA was cytosolic or in lipid or SecYEG‐bound states. SecA bound to a stable translocation intermediate was crosslinked in vivo to a second SecA protomer at its 1M6N interface, suggesting that this specific dimer likely promotes active protein translocation. Taken together, our studies strengthen models that posit, at least in part, a SecA dimer‐driven translocation mechanism. SecA ATPase facilitates protein transport through the integral membrane SecY channel complex. The physiological form of the various SecA dimers that have been crystallized was explored by engineering a site‐specific crosslinker into potential dimer interfaces and performing in vivo photo‐crosslinking. The results indicate that a single discrete dimer species is present within the cell, and this species was also captured during arrested protein transport through the SecY channel utilizing a SecA‐OmpA‐GFP trimeric protein.