Interactions of the SecY channel and the SecA ATPase with a translocating polypeptide

A crucial step in the biogenesis of secreted or integral membrane proteins is their passage across cellular membranes. Most of these proteins traverse the membrane via a protein-conducting channel known as Sec61 in eukaryotes, or SecY in prokaryotes. The channel is highly conserved, essential, and present in all forms of life. In bacteria, protein translocation is catalyzed primarily by the interactions between the essential cytosolic ATPase SecA, which binds to SecY and translocates substrates through the channel in an ATP-dependent stepwise manner.;We used an intermediate of translocation to characterize the ATP-dependent SecA conformational changes that result in substrate translocation. Using ATP analogs, we demonstrated that when bound to ATP, SecA strongly interacts with and pushes a substrate into the channel. The substrate is pushed further in the presence of post-hydrolysis mimics of ATP. When SecA is bound to ADP, a second binding site on SecA interacts weakly with the substrate and prevents backsliding. Taken together, our findings suggest that SecA has "two hands" which alternately bind substrates in ATP- and ADP-bound states during translocation.;We demonstrated that during translocation, the two-helix finger of SecA interacted with a translocating substrate, proximal to the entrance to the SecY channel. A tyrosine at the tip of the two-helix finger was important for translocation and mutation of the tyrosine to small polar amino acids abrogated canonical SecA translocation in Gram positive and Gram negative bacteria. Another class of SecA proteins, SecA2, translocates glycosylated proteins and utilizes residues such as serine instead of tyrosine at the tip of the two-helix finger. This indicates that the two-helix finger of SecA is the "first hand," responsible for pushing substrates through SecY. A tyrosine or large hydrophobic amino acid at the tip of the two-helix finger interacts with translocating substrates and exerts the translocation force; canonical SecA substrates are likely excluded from SecA2 due to polar residues at the tip of the two-helix finger. We propose a new model of SecA-dependent translocation in which a tyrosine at the tip of the two-helix finger interacts with a substrate and uses ATP-dependent conformational changes of SecA to drive protein translocation.