Abstract
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 (SecA1-826 and SecA10-826, respectively) is the minimum for complementation activity, while with N-terminus deleted by 2-21aa, SecA22-829 is the minimum. Further deletion at the C-terminus of SecA1-826/SecA10-826/SecA22-829 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 SecA22-828 and SecA22-829 could form a dimer. Moreover, the in vitro ATPase and protein translocation activities of SecA22-828 and SecA22-829 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.