Membrane assembly from purified components. I. Isolated M13 procoat does not require ribosomes or soluble proteins for processing by membranes.
The coat protein of coliphage M13 is an integral protein of the host-cell cytoplasmic membrane prior to its assembly into virions. It is initially synthesized as procoat, a soluble precursor with a 23 amino acid leader sequence at its amino terminus. 35S-labeled procoat accumulates during an in vitro translation reaction that contains 35S-methionine and RNA from M13-infected cells. Radiochemically pure procoat has been isolated from in vitro translation reactions by extraction into an organic solvent and gel filtration through Sephadex LH-60. Radiochemically pure procoat can be used as substrate in rapid and quantitative assays for leader peptidase and for leader peptide hydrolase, an enzyme that degrades the leader peptide after its release from procoat. Procoat solubility, digestion by leader peptidase and processing by membranes are affected by the presence of Mg2+ ion. Isolated procoat is soluble in water at low ionic strength and mildly alkaline pH as well as in detergent solutions. It is cleaved to coat protein by purified E. coli leader peptidase and by inverted E. coli inner-membrane vesicles. These properties of the purified procoat mirror those of the procoat in crude extracts. This suggests that there are no other soluble components that are necessary for the assembly of procoat into the membrane and its conversion to coat; specifically, it provides powerful evidence that protein synthesis is not involved.
D002462:Cell Membrane / Q000378:metabolism; D003090:Coliphages / Q000032:analysis; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000502:physiology; D012697:Serine Endopeptidases; D012995:Solubility; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D008565:Membrane Proteins; D011498:Protein Precursors; D014764:Viral Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase