The protease-protected 30 kDa domain of SecA is largely inaccessible to the membrane lipid phase

SecA binds to the inner membrane of Escherichia coli through low affinity lipid interactions or with high affinity at SecYEG, the integral domain of preprotein translocase. Upon addition of preprotein and nucleotide, a 30 kDa domain of SecYEG-bound SecA is protected from proteolysis via membrane insertion, Such protection could result from some combination of insertion into the lipid phase, into a proteinaceous environment or across the membrane, To assess the exposure of SecYEG-bound SecA to membrane lipids, a radiolabeled, photoactivatable and lipid-partitioning crosslinker, 3-trifluoromethyl-3-(m-[I-125]iodophenyl) diazirine benzoic acid ester, was incorporated into inner membrane vesicles, The 30 kDa domain of SecYEG-bound SecA, inserted into the membrane in response to translocation ligands, is 18-fold less labeled than SecY, which is labeled effectively. In contrast, incorporation of the purified 30 kDa SecA fragment into crosslinker-containing detergent micelles or addition of detergent to crosslinker-containing membranes bearing the protease-protected SecA domain readily allows for labeling of this domain, We propose that the protease-inaccessible 30 kDa SecA domain is shielded from the fatty acyl membrane phase by membrane-spanning SecYEG helices and/or is largely exposed to the periplasm.