Summary: Fractionation on sucrose density gradients of spheroplast lysates from NCYC 366 yielded a fraction with a peak density of 1.05 g ml, intermediate between that of membranes and intracellular low-density vesicles. Electron microscopy showed the fraction to consist of membranes associated with intracellular vesicles. Evidence for the presence of plasma membranes in the association fraction was obtained by using I-labelled spheroplasts. The fraction was free from detectable amounts of cytochromes and DNA. Fractionation on sucrose density gradients of incubation mixtures containing isolated crude plasma-membranes and vesicles gave rise to a visible intermediate-density band, which electron microscopy showed to contain membranes associated with vesicles. Evidence for the presence of plasma membranes in the intermediate-density band came from incubating mixtures containing I-labelled crude plasma-membranes, and evidence for the presence of vesicles came from using I-labelled vesicles. Supplementing incubation mixtures with calcium chloride sharply increased the size of the intermediate-density band. Cycloheximide and methylbenzimidazol-2-yl-carbamate had no effect on its formation. Purified plasma-membranes failed to form an intermediate-density band when incubated with vesicles. Supplementing these incubation mixtures with calcium chloride did not produce an intermediate-density band, but caused extensive association of vesicles with plasma membranes that pelleted in gradients. Isolated vesicles were not osmotically active; their polyphosphate content was 1.61 μmol orthophosphate equivalent (mg vesicle protein). They had diameters in the range 0.45-0.65 μm, as measured on electron micrographs of thin sections. The data reported provide further evidence for a role for intracellular low-density vesicles in envelope growth in


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