Novel regulatory role of phosphorylated clathrin light chain beta in bovine brain coated vesicles.

The 50-kilodalton (kDa) assembly polypeptide of bovine brain clathrin coated vesicles (CCVs) is phosphorylated in a cyclic nucleotide- and Ca2+-independent manner and is dephosphorylated by a Mg2+-ATP-dependent CCV phosphatase. This report provides evidence for modulation of the phosphorylation reaction of the 50-kDa assembly polypeptide by phosphorylated clathrin light chain beta (pLC beta). In vitro, phosphorylated LC beta inhibits phosphorylation of the 50-kDa polypeptide in CCVs.

Clathrin-coated vesicle subtypes in mammalian brain tissue: detection of polypeptide heterogeneity by immunoprecipitation with monoclonal antibodies.

A panel of monoclonal antibodies (mAbs) was developed to identify polypeptides sorted in subtypes of brain coated vesicles (CVs) and to separate these by immunoprecipitation. The corresponding antigen of some of the mAbs elicited by CV components was present also in synaptosomal plasma membrane, synaptic vesicles, or microsomes. On immunoblots the mAbs reacted with constitutive brain CV proteins, with cargo molecules, and with a novel CV component that interacts with the actin cytoskeleton.

Brain coated vesicle destabilization and phosphorylation of coat proteins.

Two basic polypeptides, bee venom melittin and poly-L-lysine, induced concentration-dependent destabilization of bovine brain coated vesicles. Ultrastructurally the changes observed were aggregation of clathrin coats and segregation of the vesicle membrane, concomitant with the appearance of elongated cisternae of various sizes. Changes in coated vesicle morphology induced by melittin and poly-L-lysine were concurrent with stimulation of phosphate incorporation in proteins of the coat lattice: Mr 33,000 and 100,000.

Phosphorylation characteristics of brain clathrin-coated vesicle endogenous proteins.

Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g.

Mapping two functional domains of clathrin light chains with monoclonal antibodies.

The two forms of clathrin light chains (LCA and LCB) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F.