Protease protection assays show polypeptide movement into the SecY channel by power-strokes of the SecA ATPase

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 back-sliding 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.