No abstract

Uncoating ATPase, an abundant 70,000-mol-wt polypeptide mediating the ATPdependent dissociation of clathrin from coated vesicles and empty clathrin cages, has been purified to virtual homogeneity from calf brain cytosol . Uncoating protein is present in cells in amounts roughly stoichiometric with clathrin . This enzyme is isolated as a mixture of monomers and dimers, both forms being active . ATP can support protein-facilitated dissociation of clathrin at micromolar levels; all other ribotriphosphates as well as deoxy-ATP are inactive . The clathrin that is released from cages consists of trimers (triskelions) in a stoichiometric complex with uncoating ATPase . These complexes with clathrin have little tendency to self-associate at neutral pH, and at acidic pH they interfere with the assembly of free clathrin . The possible existence and function of these complexes as clathrin carriers in cells would explain why uncoating protein is made in quantities equivalent to clathrin .

A cycle of clathrin assembly and disassembly drives the formation of coated vesicles, intermediates in intracellular protein transport. The heavy chain of clathrin is needed for assembly, but the function of the clathrin light chains has remained obscure. An enzyme has now been purified which uses ATP hydrolysis to power the release of clathrin from coated vesicles, presumably recycling the coat protein for repeated rounds of vesicle budding. This 'uncoating ATPase' requires clathrin light chains for its action.

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles . The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity . Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages . The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly . The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein . Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released . This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release .An important step in interorganelle transport is the removal ofthe clathrin coat from freshly budded coated vesicles. This facilitates the eventual fusion of the contained vesicle with its target membrane and also liberates the clathrin for use in further rounds of coated vesicle budding (1,5,15,17,24). The accompanying paper (18) described the purification of a protein capable of removing clathrin from coated vesicles. The uncoating protein will also dissociate artificial cages made only of clathrin and its light chains . Disassembly requires micromolar levels of ATP; moreover, nonhydrolyzable analogues of ATP show little ability to support this enzymatic uncoating (14,18) . These results suggest that nucleotide hydrolysis is needed to power disassembly of clathrin.Any enzyme that efficiently couples ATP hydrolysis to another process will hydrolyze ATP only when all of the substrates needed for the reaction are present. Therefore, we have investigated whether the uncoating protein possesses an ATPase activity that is manifest only in the presence of its other substrate, clathrin cages.