Abstract
Bacterial secretory proteins are translocated post-translationally by the SecA ATPase through the protein-conducting SecY channel in the plasma membrane. During the ATP hydrolysis cycle, SecA undergoes large conformational changes of its two-helix finger and clamp domains, but how these changes result in polypeptide movement is unclear. Here, we use a reconstituted purified system and protease protection assays to show that ATP binding to SecA results in a segment of the translocation substrate being pushed into the channel. This motion is prevented by mutation of conserved residues at the finger's tip. Mutation of SecA's clamp causes backsliding of the substrate in the ATP-bound state. Together, these data support a power stroke model of translocation in which, upon ATP binding, the two-helix finger pushes the substrate into the channel, where it is held by the clamp until nucleotide hydrolysis has occurred.