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Abstract
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.
Highlights
From the Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, 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
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
It should be noted that a more than 3-fold increase, compared to the wild-type, in the initial rate of translocation was achieved with the (Lys)., mutant. These results suggest that the rate of translocation is proportional to the number of the positive charge and that a +2 charge is by no means the best in terms of the rate of translocation
Summary
The E. coli strains used were Q13Lpp- for preparation of cytosolic fraction S150-2, K003 (Lpp- AuncB-C-TnlO). For preparation of membrane vesicles [14], and JM83 [15] for DNA manipulation. In uitro transcription of genes coding for uncleavable OmpF-Lpp and its mutant forms was performed with SP6 RNA polymerase as described [16]. The translation reaction was carried out in the presence of Trana5S-label as described previously [9]. The translocation reaction was carried out using purified inverted membrane vesicles at 37 “C after the addition of 5 mM ATP and 5. The amount of protein in the OmpF-Lpp band on a gel was determined densitometrically with a Shimadzu CS-930 chromate-scanner. The efficiency of translocation was expressed as the ratio of the intensities of protein bands on fluorograms before and after the proteinase K treatment
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