Efficient in vitro translocation into Escherichia coli membrane vesicles of a protein carrying an uncleavable signal peptide. Characterization of the translocation process.
The translocation into Escherichia coli cytoplasmic membrane vesicles of a protein containing an uncleavable signal peptide was studied. The signal peptide cleavage site of the ompF-lpp chimeric protein, a model secretory protein, was changed from Ala-Ala to Phe-Pro through oligonucleotide-directed site-specific mutagenesis of the ompF-lpp gene on a plasmid. The mutant protein was no longer processed by the signal peptidase. When proteinase K treatment was adopted as a probe for protein translocation into inverted membrane vesicles, the mutant protein exhibited rapid and almost complete translocation, most likely due to the lack of premature cleavage of the signal peptide before the translocation. This result also indicates that cleavage of the signal peptide is not required for translocation of the mature domain of the protein. The establishment of an efficient system made it possible to perform precise and quantitative analysis of the translocation process. The translocation was time-dependent, vesicle-dependent, and required ATP and NADH. Translocation into membrane vesicles was also observed with the uncleavable precursor protein purified by means of immunoaffinity chromatography, although the efficiency was appreciably low. The translocation required only ATP and NADH. Addition of the cytosolic fraction did not enhance the translocation.
Yamane
K
K
Institute of Applied Microbiology, University of Tokyo, Japan.
Matsuyama
S
S
Mizushima
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D019286:Endopeptidase K; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010957:Plasmids; D021382:Protein Sorting Signals / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D013997:Time Factors
D021382:Protein Sorting Signals; D009243:NAD; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; D019286:Endopeptidase K