Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane

• DOI: 10.1111/j.1365-2958.1993.tb01200.x
• Publication Date: 2006-10-28T01:18:22Z
• Authors: J. van der Wolk; M. Klose; E. Breukink; R. A. Demel; B. de Kruijff; R. Freudl; A. J. M. Driessen
• Keywords: Walker motifs; Translocase; Wild type

Summary SecA is the precursor protein binding subunit of bacterial translocase, which consists SecY/E as integral membrane domain. an ATPase, and couples hydrolysis ATP to release bound proteins allow their proton‐motive‐force‐driven translocation across cytoplasmic membrane. A putative ATP‐binding motif can be predicted from amino acid sequence with homology consensus Walker A‐type motif. The role this domain not known. lysine residue at position 106 end glycine‐rich loop in Bacillus subtilis was replaced by asparagine through site‐directed mutagenesis (K106N SecA). similar replacement introduced adjacent 101 (K101N Wild‐type mutant were expressed a high level purified homogeneity. catalytic efficacy ( k cat / m ) K106N for lipid‐stimulated only 1% that wild‐type K101N SecA. retained ability bind ATP, but its ATPase activity stimulated proteins. Mutant affinity Escherichia coli inner vesicles insert into phospholipid mono‐layer, contrast wild type, insertion prevented ATP. blocks proton‐motive‐force‐dependent chase intermediate fully translocated proOmpA. It concluded GKT amino‐terminal part site. This site may involved ATP‐driven recycling function allows association proteins, bilayer.