Interactions between SecA and cellular components involved in the translocation of secretory proteins across the cytoplasmic membrane of Escherichia coli were studied by examining changes in the sensitivity of SecA to staphylococcal protease V8. In the presence of ATP, the amino-terminal 95-kDa portion of the SecA molecule became highly resistant to V8 digestion. Adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and ADP were as effective as ATP. For the effect, ATP could be partly replaced by CTP and UTP, but not GTP, as in the case of the protein translocation reaction. In the presence of proOmpA, a presecretory protein, on the other hand, SecA became more sensitive to V8 digestion. The signal peptide region was involved in this effect. The V8-digestion profile in the presence of both proOmpA and ATP or ADP was the same as that in the presence of proOmpA alone. Consistently, proOmpA-induced discharge of ADP or ATP gamma S from SecA was observed by means of flow dialysis. SecA-deprived everted membrane vesicles and an E. coli phospholipid mixture were also effective in making SecA more sensitive to V8 digestion. Among the phospholipids, phosphatidylglycerol and cardiolipin were effective, whereas phosphatidylethanolamine was not. It is suggested that SecA directly interacts with these cellular components and the interactions result in changes in the conformation of SecA. The physiological significance of such interactions in protein secretion is discussed.