Translocase-Bound SecA Is Largely Shielded from the Phospholipid Acyl Chains†

ABSTRACT: Protein translocation in Escherichia coli is mediated by the SecA ATPase bound to the SecYEG membrane protein complex. SecA translocation ATPase activity as well as protein translocation is dependent on the presence of negatively charged lipids. By using a phospholipid with an acyl chain linked photoactivatable group, the lipid accessibility of SecA bound at the translocase was explored. SecA bound to lipid vesicles containing negatively charged lipids was found to be readily accessible for labeling by the photoactivatable phospholipid. The presence of an excess amount of SecYEG complex resulted in a remarkable reduction in the amount of lipid-accessible SecA irrespective of the nucleotide-bound form of SecA. These data demonstrate that the SecYEG-bound SecA is largely shielded from the phospholipid acyl chains and suggest the presence of two distinct pools of membrane-bound SecA that differ in the degree of lipid association. In Escherichia coli, proteins functioning outside the cytosol are transported across the inner membrane from the site of synthesis to their site of catalysis. Most of the proteins involved in this process, that is, components of the secretion machinery, have been identified by genetic analysis (1, 2)