Previous studies showed that certain regions of E. coli SecA can be deleted from its N- and/or C-termini to complement a SecA amber ts mutant. In this study, we determined and characterized the dispensability of both ends of SecA molecules. With N-terminal intact or 9-aa deleted, 826aa (SecA(1-826) and SecA(10-826), respectively) is the minimum for complementation activity, while with N-terminus deleted by 2-21aa, SecA(22-829) is the minimum. Further deletion at the C-terminus of SeCA(1-826)/SeCA(10-828)/SeCA(22-829) abolished the complementation activity in the cells. A hydrophobic amino acid is required for the 826th residue in the minimal-length SecAs. Chemical crosslinking and gel filtration result showed that both purified SecA(22-828) and SecA(22-829) could form a dimer. Moreover, the in vitro ATPase and protein translocation activities of SecA(22-828) and SecA(22-829) were similar, though lower than wild-type SecA. The active mutants had more truncated SecA in soluble than membrane-bound form, but was more stably embedded in membranes. In contrast, the inactive mutants tended to have truncated SecA more membrane-bound than soluble form, and were more loosely bound and easily chased out. Thus, the loss of complementation appears to be related to their altered subcellular localization and stability in the membranes. This study defines the substantial regions of N- and C-termini of SecA that may be deleted without losing complementation activity. (C) 2014 Elsevier Inc. All rights reserved.