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Negatively charged amino acid residues play an active role in orienting the Sec‐independent Pf3 coat protein in the Escherichia coli inner membrane
Author(s) -
Kiefer Dorothee,
Hu Xintong,
Dalbey Ross,
Kuhn Andreas
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.9.2197
Subject(s) - biology , escherichia coli , amino acid , biophysics , inner membrane , amino acid residue , coat protein , membrane , biochemistry , peptide sequence , gene , rna
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.