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A study investigated the steady-state interaction of the Hsc70 protein with clathrin baskets at pH6 and quantitatively determine the function of a 100-kDa cofactor and DnaJ homolog called auxilin in this interaction. it was found that clathrin baskets activated the Hsc70 adenosine triphosphatase activity 100-fold with a dissociation constant of 0.2 microns in the presence, but not absence, of auxilin. Experimental methods and results are presented.

The multimeric clathrin assembly proteins AP-1 and AP-2 with molecular masses of approximately 270 kDa and the monomeric clathrin assembly proteins AP180 and auxilin with molecular masses of approximately 90 kDa catalyze the assembly of clathrin into artificial clathrin baskets under physiological conditions. We have now identified a much smaller approximately 20-kDa clathrin assembly protein in 0.5 M Tris, pH 7.0, extracts of bovine-brain coated vesicles and purified it to near homogeneity. A polyclonal antibody against this protein did not cross-react with any of the other assembly proteins, and sequencing data suggest that this new protein is similar or identical to myelin basic protein (MBP). At a molar ratio of 3 molecules per clathrin triskelion, MBP catalyzes polymerization of clathrin into artificial baskets that appear structurally similar to the baskets assembled by the other assembly proteins. In addition, like the other baskets, the clathrin-MBP baskets are uncoated by hsp70. MBP represents a significant fraction of the total assembly protein activity present in 0.5 M Tris, pH 7.0, extracts of coated vesicles. It is not clear if it acts as an assembly protein in vivo, but because it is well characterized and easily available, MBP will be a useful protein to investigate the mechanism of clathrin assembly and disassembly in vitro.

Clathrin-coated vesicles transport selected integral membrane proteins from the cell surface and the trans-Golgi network to the endosomal system. Before fusing with their target the vesicles must be stripped of their coats. This process is effected by the chaperone protein hsp70c together with a 100K cofactor which we here identify as the coat protein auxilin. Auxilin binds with high affinity to assembled clathrin lattices and, in the presence of ATP, recruits hsp70c. Dissociation of the lattice does not depend as previously supposed on clathrin light chains or on the amino-terminal domain of the heavy chain. The presence of a J-domain at its carboxy terminus now defines auxilin as a member of the DnaJ protein family. In conjunction with hsp70, DnaJ proteins catalyse protein folding, protein transport across membranes and the selective disruption of protein-protein interactions. We show that deletion of the J-domain of auxilin results in the loss of cofactor activity.

We have determined the amino acid sequences of the essential light chains (ELC) and regulatory light chains (RLC) of myosin from two species of clam, Mercenaria mercenaria and Macrocallista nimbosa, using protein chemistry methods. The N-termini of all four proteins were blocked, and sequencing was carried out on various chemically and enzymatically produced peptide fragments. Cleavage of either Mercenaria RLC (MRLC) or Macrocallista RLC (VLC) at its 3 Arg yielded four peptides, three of which could not be sequenced directly, due to an N-terminal blocking group and 2 Arg-Gln bonds in these proteins. The fourth peptide was partially and specifically cleaved at an unusually reactive residue, Met-64, which is invariant in all known RLC sequences. A comparison of all available molluscan ELC and RLC sequences was carried out in search of clues to functionally important features of these proteins in muscles which are regulated by a Ca(2+)-sensitive myosin. By analogy with other RLCs, VRLC and MRLC may be phosphorylated at Ser-11 by an endogenous kinase. All myosin light chains, like troponin C and calmodulin, contain four homologous regions, I to IV, each of which contains a twelve-residue potential Ca(2+)-binding loop flanked on either side by a pair of helices. All RLCs, including those from Ca(2+)-insensitive myosins, contain a divalent cation-binding site in region I. Clam and other molluscan ELCs contain a single Ca(2+)-binding site in region III. This site is present only in the ELCs of myosins that are regulated by direct binding of Ca2+. The ELC site III is likely to play a key role in the regulation of molluscan muscle contraction.

The amino acid sequence of the scallop myosin essential light chain (SELC) was determined from analysis of the intact, S-carboxymethylated protein and peptides produced by cleavage at its four methionine residues by cyanogen bromide digestion and at its six arginine residues by citraconylation and tryptic digestion. SELC contains 156 amino acid residues, including three cysteines, four tyrosines, one tryptophan, two histidines, and an unblocked amino-terminal proline. The protein has a calculated Mr of 17,616. SELC is an acidic protein, with a net charge of 18- at physiological pH. Comparative analysis reveals four homologous domains (I-IV), which arose by reduplication of a gene for a small, ancestral calcium binding protein. Each domain has a helix-loop-helix structure, with all the ligands for calcium binding located within a 12-residue segment that spans the loop and the first turn of the following helix. Potential calcium binding sequences were found in the ancestral sites III (residues 94-105) and IV (residues 132-143). Mutations in critical positions in domains I and II seem to preclude the possibility of calcium binding in the amino-terminal half of SELC. An unexpected third potential calcium binding segment (at residues 119-130, predicted to be in helical conformation) was found in domain IV. A reactive thiol group (Cys-78) that is involved in binding of regulatory light chains was tentatively located in an extended "linker region", which connects the two halves of the molecule.