Electron microscopic visualization of asymmetric precursor translocation intermediates: SecA functions as a dimer

Abstract

SecA, the ATPase of Sec translocase, mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. Here we report the structures of Escherichia coli Sec intermediates during preprotein translocation as visualized by electron microscopy to probe the oligomeric states of SecA during this process. We found that the translocase holoenzyme is symmetrically assembled by SecA and SecYEG on proteoliposomes, whereas the translocation intermediate 31 (I(31)) becomes asymmetric because of the presence of preprotein. Moreover, SecA is a dimer in these two translocation complexes. This work also shows surface topological changes in the components of translocation intermediates by immunogold labeling. The channel entry for preprotein translocation was found at the center of the I(31) structures. Our results indicate that the presence of preprotein introduces asymmetry into translocation intermediates, while SecA remains dimeric during the translocation process.