In bacteria, most secretory proteins are translocated across the plasma membrane by the interplay of the SecA ATPase and the SecY channel. How SecA moves a broad range of polypeptide substrates is only poorly understood. Here, we use a combination of bulk biochemistry and single molecule Foerster Resonance Energy Transfer (smFRET) assays to show that SecA moves polypeptides through the SecY-channel by a “push and slide” mechanism. In its ATP-bound state, SecA interacts through a two- helix finger with a subset of amino acids in a substrate, pushing them into the channel. A polypeptide can also passively slide back and forth when SecA is in the predominant ADP-bound state, or when SecA encounters a poorly interacting amino acid in its ATP- bound state. SecA performs multiple rounds of ATP hydrolysis before dissociating from SecY. The proposed “push and slide” mechanism is supported by a mathematical model and explains how SecA moves polypeptides independently of their sequence. This mechanism may also apply to hexameric polypeptide-translocating ATPases.
