THE MAJOR COAT PROTEIN OF FILAMENTOUS BACTERIOPHAGE-F1 SPECIFICALLY PAIRS IN THE BACTERIAL CYTOPLASMIC MEMBRANE

Filamentous bacteriophage are long, thin single-stranded DNA viruses t hat infect male strains of Escherichia coli without killing the host. Each phage contains approximately 2700 copies of the major coat protei n, pVIII, which exists as a 5.2 kDa cytoplasmic membrane protein prior to incorporation into phage. Studies from a number of groups analyzin g the behavior of wild-type and mutant pVIII in detergents suggested t hat pVIII might pair under these conditions. In order to test whether pVIII molecules pair in vivo in the cytoplasmic membrane, four plasmid -encoded pVIII variants were constructed in which specific residues in the transmembrane region were mutated to cysteine in an attempt to st abilize any pair via disulfide bridges. Variants A35C and I39C were un able to complement phage with an amber mutation in gene VIII. The I39C variant was unable to be packaged into phage particles even though it was inserted into the membrane. In the case of A35C, the inability to complement was not due to a packaging defect because the variant prot ein could be packaged into phage in the presence of wild-type pVIII. W estern blot analysis of cytoplasmic membrane samples revealed that the A35C variant formed stable disulfide dimers in vivo. Expression of A3 5C interfered with wild-type phage infection, indicating that the asse mbly :machinery may recognize the disulfide dimers in some nonproducti ve way. The results indicate that pVIII may specifically pair along a particular face in the cytoplasmic membrane prior to assembly; however , these pairs must be able to be separated in order for normal assembl y to occur. (C) 1998 Academic Press Limited.