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 in serted into the bacterial inner membrane with a single transmembrane a nchor sequence in the NoutCin orientation. The N-terminal sequence imm ediately 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 alter ed, Membrane insertion was analyzed in vivo using the accessibility to externally added protease and in vitro by testing the insertion into inverted Escherichia coil membrane vesicles, In both systems, the orie ntation of the protein was completely reversed for the oppositely char ged mutant coat protein (RD mutant). In addition, we show in vivo that the electrochemical membrane potential is necessary for the transloca tion of both the wild type and the mutant Pf3 coat proteins, suggestin g that membrane insertion is driven by electrophoretic forces.