IMPROVED COATED VESICLE ISOLATION ALLOWS BETTER CHARACTERIZATION OF CLATHRIN POLYPEPTIDES

Postmicrosomal pellets from plant sources are grossly contaminated with ribosomes. Previous purifications of clathrin coated vesicles (CCV) from such subcellular fractions have therefore often involved an RNase treatment. Performed at 30-degrees-C, this step inherently carries with it the dangers of proteolysis. We document here a method for CCV isolation which avoids this. Through the inclusion of suitable antiproteases in the homogenizing and subsequent isolation media, we have also been able to improve the quality of CCV recovered from plant tissues. As a result we have tentatively been able to identify clathrin light chains from zucchini hypocotyl and pea cotyledon CCV. Similar to light chains from bovine brain these polypeptides are heat stable, can be solubilized from neutralized TCA precipitates, bind calcium and clathrin heavy chains. However, in contrast to brain CCV the two light chains of plant CCV are some 10 kDa heavier.