Effect of different positively charged amino acids, C-terminally of the signal peptidase cleavage site, on the translocation kinetics of a precursor protein in Escherichia coli K-12.
Introduction of positively charged amino acids immediately downstream of the signal sequence in prokaryotic precursor proteins is known to affect the export process. However, it is not clear whether different positively charged amino acids affect the export process similarly. To investigate this, the glutamate at position +2 of outer membrane protein PhoE was substituted by arginine, lysine or histidine. Pulse-chase experiments revealed that the Lys and Arg residues at position +2 caused a reduced processing rate, and that the effect was markedly more severe in the case of the Arg residue. Trypsin accessibility experiments revealed that the accumulated precursors were present in the cytoplasm. Since the degree of the inhibitory effect corresponded to the pKa of the different positively charged amino acids, this suggests that the positively charged residues must be deprotonated during the secretory process.
Struyvé
M
M
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
Bosch
D
D
Visser
J
J
Tommassen
J
J
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D004269:DNA, Bacterial; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D018272:Porins; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005971:Glutamates; D008565:Membrane Proteins; D018272:Porins; D011498:Protein Precursors; D018698:Glutamic Acid; D006639:Histidine; D001120:Arginine; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D008239:Lysine