In vivo assembly of a biological membrane of defined size, shape, and lipid composition

Abstract

At restrictive temperature, mutant ts1 of bacteriophage PM2 makes membrane vesicles inside infected Alteromonas espejiana. A shift from restrictive to permissive temperature resulted in rapid maturation to infectious virions. The membrane vesicles were isolated from cellular membranes by sucrose density gradient centrifugation. Analysis of the unique peak at rho = 1.190 g/cm3 showed spheres of two diameters, 50 nm and 54 nm. The wild-type virus is icosahedral with an average diameter of 60 nm. Gel electrophoresis indicated the absence in the vesicles of the coat and spike proteins. sp27 and sp43, respectively, and the presence of only one viral structural protein, sp6.6. DNA was also present. The lipid in the vesicles was composed of phosphatidylglycerol and phosphatidylethanolamine in a proportion similar to that of the wild-type virus, whose ratio is nearly the inverse of that found in the host membrane. Thus, membrane vesicles made by mutant ts1 resembled the membrane of the wild-type virus in size, shape, and lipid composition, but contained only one of the four structural proteins of the virus. This hydrophobic protein, sp6.6 may be responsible for stimulating membrane morphogenesis.