The role of the positively charged N-terminus of the signal sequence of E. coli outer membrane protein PhoE in export.
Signal sequences of prokaryotic exported proteins have a dipolar character due to positively charged amino-acid residues at the N-terminus and to a preferentially negatively charged region around the cleavage site. The role of the two lysine residues at the N-terminus of the signal sequence of outer membrane protein PhoE of E. coli-K12 was investigated. Replacement of both of these residues by aspartic acid slightly affected the kinetics of protein translocation in vivo. This export defect, which was observed only when PhoE was overproduced, could not be suppressed by the prlA4 mutation, which has been shown to restore export defects caused by alterations in the hydrophobic core of the signal sequences of various exported proteins. In an in vitro translocation assay, the export defect was more pronounced. Replacement of both lysines by uncharged residues did not disturb the kinetics of protein export in vivo. In the in vitro assay, an extraordinarily efficient processing was detected upon incubation of this precursor with inverted cytoplasmic membrane vesicles. However, this efficient processing was not accompanied by more efficient translocation of the protein. We conclude that the positively charged residues at the N-terminus of the signal sequence are not essential for protein export, but contribute to the efficiency of the process.
Bosch
D
D
Department of Molecular Cell Biology, State University of Utrecht, The Netherlands.
de Boer
P
P
Bitter
W
W
Tommassen
J
J
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001692:Biological Transport; D004252:DNA Mutational Analysis; D004926:Escherichia coli; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D012995:Solubility; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; D021382:Protein Sorting Signals; D014357:Trypsin