Isolation of Clathrin Coated Vesicles

Plant clathrin-coated vesicles (CCVs) were isolated from cucumber and zucchini hypocotyls (Depta et al., 1991; Holstein et al., 1994). CCV components were protected against proteolysis using homogenization media composed of 0.1 M MES (pH 6.4), 1 mM EGTA, 3 mM EDTA, 0.5 mM MgCl2, a mixture of proteinase inhibitors and 2% (w/v) fatty-acid-free BSA (Holstein et al., 1994). The crude CCV fraction (40 000-120 000 g pellet) was further purified by cen-trifugation in Ficoll/sucrose according to Campbell et al., (1983) and then by isopycnic centrifugation in a sucrose density gradient using a vertical rotor (160 000 g, 2.5 h, Depta et al., 1991). CCV-enriched fractions (collected at 40-45% sucrose) were removed, pooled and pelleted. CCV fractions were stored at - 80 °C for further use. Immunoblotting was performed using monoclonal antibodies against mammalian adaptins and clathrin. Confirmation of the presence of a P-type adaptin in plants was provided by dot and Southern blotting experiments using genomic DNA from zucchini hypocotyls and a P-adaptin cDNA clone from human fibroblasts (Holstein et al., 1994).

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