THE INTERACTION OF CALMODULIN WITH CLATHRIN-COATED VESICLES, TRISKELIONS, AND LIGHT-CHAINS - LOCALIZATION OF A BINDING-SITE

The binding of clathrin-coated vesicles, clathrin triskelions, and fre e clathrin light chains to calmodulin-Sepharose was compared, When iso lated from bovine brain, all three components bound to calmodulin-Seph arose in the presence of calcium and could be eluted by its removal, I n contrast, coated vesicles and triskelions isolated from bovine adren al gland did not bind to calmodulin-Sepharose, although the free light chains from adrenal gland bound as effectively as those from brain, A s distinct isoforms of the clathrin light chains are expressed by brai n and adrenal gland, these results implicate the clathrin light chains as the calmodulin-binding component of coated vesicles and triskelion s. Furthermore, the insertion sequences found in the neuron-specific i soforms, although not necessary for the binding of free clathrin light chains to calmodulin, must facilitate the interaction of heavy chain- associated light chains with calmodulin, Recombinant mutants of LCa, w ith deletions spanning the entire sequence, were tested for binding to calmodulin-Sepharose. Those mutants retaining structural integrity, a s assessed by the binding of a panel of monoclonal antibodies, exhibit ed varying amounts of calmodulin binding activity, However, deletion o f the carboxyl-terminal 20 residues abolished calmodulin interaction, Thus, the carboxyl terminus of LCa appears to constitute a calmodulin- binding site, Peptides corresponding to the carboxyl terminus of LCa o r LCb inhibited the interaction of the light chains with calmodulin, s uggesting that this region forms the calmodulin-binding site of both L Ca and LCb, The carboxyl-terminal peptides of LCa and LCb inhibited th e interaction of light chains with calmodulin similar to 10-fold less effectively than a calmodulin-binding peptide derived from smooth musc le myosin light chain kinase, but much more effectively than a calmodu lin-binding peptide derived from adenylate cyclase, This comparison pl aces the clathrin light chain-calmodulin interaction within the physio logical range seen for other calmodulin-binding proteins.