Identification of a beta-type adaptin in plant clathrin-coated vesicles

Plant clathrin-coated vesicles (CCV), suitably protected against proteolysis, were isolated from zucchini hypocotyls, and screened for the presence of adaptin-like polypeptides using monoclonal antibodies prepared against alpha, beta(beta') and gamma-adaptins of bovine brain. An immunoreactive polypeptide in plant CCV was only detected in the case of the beta(beta')-adaptin antibody. This polypeptide has a molecular mass of 108 kDa in SDS-PAGE, and gives rise to a major cleavage product of 70 kDa after proteolysis with trypsin. Gel filtration of 0.75 M MgCl2-dissociated coat proteins showed that the plant beta(beta')-type adaptin eluted with other polypeptides in a manner similar to the adaptor complexes of brain CCV. Upon subsequent hydroxyapatite chromatography the immunoreactive polypeptide eluted in fractions corresponding to Golgi (HA-I) rather than plasma membrane (HA-II) brain adaptor complexes. In addition, this polypeptide did not shift to a higher molecular mass when subjected to urea-SDS-PAGE. Confirmation of the presence of a beta-type adaptin in plants was provided by dot and Southern blotting experiments using genomic DNA from zucchini hypocotyls and a beta-adaptin cDNA clone from human fibroblasts.