In vitro kinetic analysis of the role of the positive charge at the amino-terminal region of signal peptides in translocation of secretory protein across the cytoplasmic membrane in Escherichia coli.
By using an in vitro system for the translocation of secretory proteins in Escherichia coli, detailed and quantitative studies were performed as to the function of the positively charged amino acid residues at the amino terminus of the signal peptide. Uncleavable OmpF-Lpp, a model secretory protein carrying an uncleavable signal peptide, and mutant proteins derived from it were used as translocation substrates. When the positive charge, +2 (LysArg) for the wild-type, was changed to 0, -1, or -2, little or no translocation was observed. The number of the positive charge was altered by introducing different numbers of Lys or Arg residues into the amino terminus. The rate of translocation was roughly proportional to this number, irrespective of whether the charged amino acid residues were Lys or Arg. When the amino-terminal LysArg was replaced by His residues, translocation took place more efficiently at pH 6.5 than pH 8.0, whereas that of the wild-type was about the same as the two pH values. We conclude that the signal peptide requires a positive charge at its amino-terminal region to function in the translocation reaction and that the rate of translocation is roughly proportional to the number of the positively charged group, irrespective of the amino acid species that donates the charge. Evidence suggesting that the positive charge is involved in the binding of precursor proteins to the membrane surface to initiate translocation is also presented.
Sasaki
S
S
Institute of Applied Microbiology, University of Tokyo, Japan.
Matsuyama
S
S
Mizushima
S
S
D000595:Amino Acid Sequence; D001120:Arginine; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004563:Electrochemistry; D004926:Escherichia coli / Q000378:metabolism*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D006639:Histidine; D001120:Arginine; D008239:Lysine