PMID:11969418
| Citation |
Kebir, MO and Kendall, DA (2002) SecA specificity for different signal peptides. Biochemistry 41:5573-80 |
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| Abstract |
SecA performs a critical function in the recognition, targeting, and transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. In this study we investigate the substrate specificity of SecA, including the influence of the early mature region of the preprotein on SecA interactions, and the extent to which SecA recognizes targeting signals from different transport pathways. A series of fusion proteins were generated which involved the tandem expression of GST, signal peptide, and the first 30 residues from alkaline phosphatase. These were purified and evaluated for their ability to promote SecA ATPase activity. No significant difference in the stimulation of SecA-lipid ATPase activity between the synthetic wild-type alkaline phosphatase signal peptide and a fusion that also contains the first 30 residues of alkaline phosphatase was observed. The incorporation of sequence motifs in the mature region, which confer SecB dependence in vivo, had no impact on SecA activation in vitro. These results suggest that the early mature region of alkaline phosphatase does not affect the interactions between SecA and the signal peptide. Sec, Tat, and YidC signal peptide fusions were also assayed for their ability to stimulate SecA ATPase activity in vitro and further analyzed in vivo for the Sec dependence of the transport of the corresponding signal peptide mutants of alkaline phosphatase. Our results demonstrate that E. coli Sec signals give the highest level of SecA activation; however, SecA-signal peptide interactions in vitro are not the only arbiter of whether the preprotein utilizes the Sec pathway in vivo. |
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| Keywords |
Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism; Alkaline Phosphatase/genetics; Alkaline Phosphatase/metabolism; Amino Acid Motifs/genetics; Amino Acid Sequence; Bacterial Proteins; Biological Transport/genetics; Capsid/genetics; Capsid/metabolism; Capsid Proteins; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Glutathione Transferase/genetics; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidoreductases, N-Demethylating/genetics; Oxidoreductases, N-Demethylating/metabolism; Protein Precursors/genetics; Protein Precursors/metabolism; Protein Sorting Signals/genetics; Proteolipids/metabolism; Recombinant Fusion Proteins/chemical synthesis; Recombinant Fusion Proteins/metabolism; Signal Transduction/genetics; Substrate Specificity/genetics; beta-Lactamases/genetics; beta-Lactamases/metabolism |
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