Synthesis, assembly into the cytoplasmic membrane, and proteolytic processing of the precursor of coliphage M13 coat protein.

The major coat protein (gene 8 product) of coliphage M13 is an integral protein of the host cell cytoplasmic membrane prior to virus assembly. It is synthesized as a precursor, termed procoat, with an extra 23 NH2terminal residues. We have studied the synthesis, assembly, and processing of procoat protein by amino acid pulse-labeling E. coli which are infected by either M13 or by M13 with amber mutations. Pulse-labeled procoat is found in the soluble fraction and little radioactivity is incorporated into membrane-bound coat protein by short pulse labeling. For wild type M13-infected cells, the soluble procoat sediments at about 5 S in a sucrose gradient. Pulselabeled procoat rapidly chases from the soluble fraction. For amber 7 M13-infected cells, the pulse-labeled procoat is soluble and sediments at about 5 s, as in the wild type virus infections. During chase, procoat is lost from the soluble fraction and appears in the membrane fraction at short chase times as coat protein begins to appear. The appearance of coat protein during “chase” parallels the loss of procoat and is not affected by the addition of the protein synthesis inhibitor puromycin at the beginning of the chase. These data for wild type and amber 7 M13 infections strongly suggest that soluble procoat is the precursor of membrane-bound procoat, which is then proteolytically cleaved to yield coat protein. For amber 5 M13-infected cells, procoat is found in the soluble fraction with a sedimentation coefficient of 1.2 S, which is that expected for the monomeric protein. A portion of the procoat is found in the membrane; even after long chase times, only one-half of the membrane-bound procoat is converted to coat protein. The reasons for these alterations in procoat metabolism in M13 amber 5-infected cells are not known.