The isolation and enrichment of coated vesicles from suspension-cultured carrot cells

Summary A method has been developed to isolate and purify coated vesicles from suspension cultured carrot (Daucus carota L.) cells. It incorporates features of centrifugation methods (sucrose step gradient; Ficoll/D₂O gradient) previously employed in the isolation of coated vesicles from mammalian brain tissue. Most important is the treatment of the crude coated vesicle fraction (postmicrosomal supernatant) with ribonuclease to remove ribosomes which are a serious source of contamination in such fractions. The fraction finally obtained is contaminated to the extent of 30% of total observed particles in negatively stained preparations with naked vesicles whose diameter are smaller than those of the coated vesicles. These vesicles are interpreted as being coated vesicles which have been stripped of their coats. SDS-PAGE of coated vesicle fractions purified by this method reveal significant differences in the polypeptide patterns obtained from plant and animal systems.