![]() When expressed from a plasmid in Escherichia coli, it is inserted into the inner membrane with an N outC in orientation. The 44-amino-acid, single membrane-spanning (type III) coat protein of the Pseudomonas aeruginosa phage Pf3 has been studied as a model protein for Sec-independent membrane translocation (Kuhn et al., 1990 Rohrer and Kuhn, 1990 Lee et al., 1992 Cao and Dalbey, 1994). 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. In both systems, the orientation of the protein was completely reversed for the oppositely charged mutant coat protein (RD mutant). 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. To investigate how the orientation of this protein is achieved, the three flanking charged amino acid residues were altered. 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. 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 outC in orientation.
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