A significant fraction of functional SecA is permanently embedded in the membrane - SecA cycling on and off the membrane is not essential during protein translocation

SecA has been suggested to cycle on and off the cytoplasmic membrane of Escherichia coli during protein translocation, We have reconstituted S-35-SecA onto SecA-depleted membrane vesicles and followed the fate of the membrane-associated S-35-SecA during protein translocation. Some S-35-SecA was released from the membranes in a translocation-independent manner. However, a significant fraction of S-35-SecA remained on the membranes even after incubation with excess SecA. This fraction of S-35-SecA was shown to be integrated into the membrane and was active in protein translocation, indicating that SecA cycling on and off membrane is not required for protein translocation. Proteolysis experiments did not support the model of SecA insertion and deinsertion during protein translocation; instead, a major 48-kDa domain was found persistently embedded in the membrane regardless of translocation status, Thus, in addition to catalyzing ATP hydrolysis, certain domains of SecA probably play an important structural role in the translocation machinery, perhaps forming part of the protein-conducting channels.