Membranes markers in highly purified clathrin-coated vesicles from Cucurbita hypocotyls

The two plasma-membrane (PM) markers: 1,3-β-glucan synthase and naphthylphthalamic-acid-binding capacity are localized in two peaks of activity on isopycnic Ficoll/D2O gradients prepared from a zucchini (Cucurbita pepo L.) hypocotyl post-microsomal fraction. The denser peak overlaps with the major distribution of clathrin and represents a region of the gradient enriched in coated vesicles (cv). Further purification of the pooled cv-fractions has shown that these PM marker activities are not borne by the cv, but are instead carried by smooth membrane fragments also present in these fractions. As judged from the results of Western blotting with polyclonal antibodies prepared against the 100-kilodalton (kDa) subunit of a PM H(+)-ATPase and the 70-kDa subunit of a tonoplastic H(+)-ATPase, these contaminants are of both PM and endomembrane origin. The PM contaminants however, differ from phase-partitioning- and free-flow-electrophoresis-purified PM prepared from microsomal fractions of zucchini hypocotyls in terms of their bouyant density in Ficoll/D2O gradients. Moreover, they do not appear to be present as sealed, outside-out, vesicles. Highly purified cv fractions from zucchini hypocotyls cross-react with subunit antibodies from both vacuolar and PM H (+)-ATPases. These results are discussed in terms of recent findings on cv ATPases from bovine brain.