STORAGE PROTEIN POLYPEPTIDES IN CLATHRIN COATED VESICLE FRACTIONS FROM DEVELOPING PEA COTYLEDONS ARE NOT DUE TO ENDOMEMBRANE CONTAMINATION

We have isolated clathrin coated vesicles (CCV) from developing pea cotyledons and have purified them extensively. On the basis of particle counting in negatively stained preparations these CCV fractions have less than 10% contamination. The activities of endo- and plasma membrane marker enzymes in CCV fractions have been determined and compared to those for plasma membrane and mixed ER/GApp fractions. According to these measurements contamination of the CCV fractions with smooth surfaced membranes is also less than 10%. Activities of the specific endoplasmic reticulum and Golgi apparatus markers NADPH-cytochrome c-reductase and glucan synthase I were not detected in CCV fractions. Through Western blotting with legumin and vicilin antibodies the relative amounts of prolegumin and provicilin polypeptides in a dilution series of endoplasmic reticulum/Golgi apparatus membranes were compared with that of the CCV fraction. From this it is clear that endomembrane contaminants are not sufficient to account for the presence of storage polypeptides in CCV fractions from pea cotyledons. Storage polypeptides are not detectable in CCV fractions isolated from slow frozen/slow thawed pea cotyledons, whereas previously isolated CCV are hardly affected by such a treatment. Storage polypeptides are also still present in CCV which have been subjected to detergent. These results are discussed in terms of receptor-ligand interaction.