Negatively charged amino acid residues play an active role in orienting the Sec-independent Pf3 coat protein in the Escherichia coli inner membrane.
The coat protein of Pseudomonas aeruginosa phage Pf3 is transiently inserted into the bacterial inner membrane with a single transmembrane anchor sequence in the N(out)C(in) orientation. The N-terminal sequence immediately flanking the membrane anchor contains one negatively charged residue, whereas the C-terminal hydrophilic segment has two positively charged residues. To investigate how the orientation of this protein is achieved, the three flanking charged amino acid residues were altered. Membrane insertion was analyzed in vivo using the accessibility to externally added protease and in vitro by testing the insertion into inverted Escherichia coli membrane vesicles. In both systems, the orientation of the protein was completely reversed for the oppositely charged mutant coat protein (RD mutant). In addition, we show in vivo that the electrochemical membrane potential is necessary for the translocation of both the wild-type and the mutant Pf3 coat proteins, suggesting that membrane insertion is driven by electrophoretic forces.
Kiefer
D
D
Department of Microbiology, University of Karlsruhe, Germany.
Hu
X
X
Dalbey
R
R
Kuhn
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D002462:Cell Membrane / Q000378:metabolism; D004594:Electrophysiology; D019286:Endopeptidase K / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017105:Pseudomonas Phages / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D036022:Capsid Proteins; C084332:major coat protein, Pseudomonas phage Pf3; D010450:Endopeptidases; D019286:Endopeptidase K