Abstract
While the SecA ATPase drives protein translocation across the bacterial cytoplasmic membrane by interacting with the SecYEG translocon, molecular details of SecA-SecY interaction remain poorly understood. Here, we studied SecY-SecA interaction by using an in vivo site-directed cross-linking technique developed by Schultz and coworkers [Chin, J. W., Martin, A. B., King, D. S., Wang, L., Schultz, P. G. (2002) Proc. Natl. Acad. Sci. USA 99:11020-11024 and Chin, J. W., Schultz, P. G. (2002) ChemBioChem 3:1135-1137]. Benzoyl-phenylalanine introduced into specific SecY positions at the second, fourth, fifth, and sixth cytoplasmic domains allowed UV cross-linking with SecA. Cross-linked products exhibited two distinct electrophoretic mobilities. SecA cross-linking at the most C-terminal cytoplasmic region (C6) was specifically enhanced in the presence of NaN(3), which arrests the ATPase cycle, and this enhancement was canceled by cis placement of some secY mutations that affect SecY-SecA cooperation. In vitro experiments showed directly that SecA approaches C6 when it is engaging in ATP-dependent preprotein translocation. On the basis of these findings, we propose that the C6 tail of SecY interacts with the working form of SecA, whereas C4-C5 loops may offer constitutive SecA-binding sites.