Characterization of the minimal length of functional SecA in Escherichia coli

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.

Establishment of serine protease htrA mutants in Helicobacter pylori is associated with secA mutations

“…Na et al . 45 demonstrated that the C-terminal 70–75 amino acids can be deleted from SecA without losing complementation activity in E . coli .…”

“…coli . These included C-terminal protein truncation 45 or cysteine to serine substitutions 48 . Hence, by analogy, we can hypothesize that the secA suppressor mutations of Δ htrA or htrA S221A in H .…”

SecA – a multidomain and multitask bacterial export protein

“…The indispensability of the SecA function for cell viability, as well as the importance of the Sec secretion-dependent virulence factors for pathogenicity of numerous bacterial pathogens, make this protein a good candidate for a therapeutic target (for example Na et al, 2015).…”

“…2). The amino acid residues 12-830 of SecA are considered as a catalytic core of this protein, which is essential for bacterial viability and maintaining the translocation process (Or et al, 2005;Na et al, 2015).…”

SecA inhibitors as potential antimicrobial agents: differential actions on SecA-only and SecA-SecYEG protein-conducting channels

“…It is important to note that the roles of SecA and ATP hydrolysis in bacterial growth are not well understood. For example, a truncation of SecA by deletion of short N-terminal or C-terminal reduces both translocation ATPase and even protein translocation, but does not significantly affect the growth of E. coli (Floyd et al 2014;Na et al 2015). The rationales for these assays and summaries of structures of various SecA inhibitors have been described (Segers and Anne 2011;Rao et al 2014;Chaudhary et al 2015a).…”