Role of amino-terminal positive charge on signal peptide in staphylokinase export across the cytoplasmic membrane of Escherichia coli.
Staphylokinase mutants having amino acid substitutions within the amino-terminal charged segment of the signal peptide have been produced by in vitro oligonucleotide-directed mutagenesis. When the processing of the gene products was analyzed in Escherichia coli cells, the rate of processing of the mutant staphylokinase precursor decreased as the net charge became more negative. A net positive charge, but not specific amino acid residues, was required on the amino-terminal segment for efficient processing. Staphylokinase precursor having a net negative charge accumulated in the cytoplasm, tending to bind to the cytoplasmic membrane as determined by subcellular fractionation and immunoelectron microscopy. Although a mutant carrying an amino acid substitution in the hydrophobic segment and wild-type staphylokinases had an interfering effect on the processing of other normal secreted proteins, this effect was lost when they also contained charge-altering substitutions in the amino-terminal region. From these results, we concluded that a positive charge on the amino-terminal segment of the staphylokinase signal peptide is required for entrance into the protein export process.
Iino
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T
Takahashi
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Sako
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D000595:Amino Acid Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004563:Electrochemistry; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D006651:Histocytochemistry; D007159:Immunologic Tests; D008666:Metalloendopeptidases; D008854:Microscopy, Electron; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*
D011498:Protein Precursors; D021382:Protein Sorting Signals; D010450:Endopeptidases; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus