Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane
PRECURSOR PROTEINS
Molecular Sequence Data
03 medical and health sciences
Adenosine Triphosphate
Bacterial Proteins
Species Specificity
BINDING
Escherichia coli
Amino Acid Sequence
Protein Precursors
Phospholipids
Adenosine Triphosphatases
0303 health sciences
Binding Sites
SecA Proteins
Escherichia coli Proteins
Cell Membrane
RECOGNITION
Membrane Transport Proteins
COLI PLASMA-MEMBRANE
Biological Transport
GENE
EXPORT
COMPONENT
PHOSPHOLIPIDS
RESOLUTION
ESCHERICHIA-COLI
SEC Translocation Channels
Bacillus subtilis
DOI:
10.1111/j.1365-2958.1993.tb01200.x
Publication Date:
2006-10-28T01:18:22Z
AUTHORS (7)
ABSTRACT
SummarySecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton‐motive‐force‐driven translocation across the cytoplasmic membrane. A putative ATP‐binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A‐type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine‐rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site‐directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild‐type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (kcat/km) of the K106N SecA for lipid‐stimulated ATP hydrolysis was only 1% of that of the wild‐type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild‐type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid mono‐layer, in contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton‐motive‐force‐dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino‐terminal domain of SecA is part of the catalytic ATP‐binding site. This site may be involved in the ATP‐driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.
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