INITIAL STEPS IN PROTEIN MEMBRANE INSERTION - BACTERIOPHAGE-M13 PROCOAT PROTEIN BINDS TO THE MEMBRANE-SURFACE BY ELECTROSTATIC INTERACTION

Bacteriophage M13 procoat protein is synthesized on free polysomes prior to its assembly into the inner membrane of Escherichia coli. As an initial step of the membrane insertion pathway, the precursor protein interacts with the cytoplasmic face of the inner membrane. We have used oligonucleotide-directed mutagenesis to study the regions of the procoat protein involved in membrane binding. We find that there is an absolute requirement for positively charged amino acids at both ends of the protein. Replacing these with negatively charged residues resulted in an accumulation of the precursor in the cytoplasm. We propose that the positively charged amino acids are directly involved in membrane binding, possibly directly to the negatively charged phospholipid head groups. This was tested in vitro with artificial liposomes. Whereas wild-type procoat interacted with these liposomes, we found that procoat mutants with negatively charged amino acids at both ends did not bind. Therefore, we conclude that newly synthesized M13 procoat protein binds electrostatically to the negatively charged inner membrane of E. coli.