Transport of the VSV-encoded glycoprotein (G protein) between successive compartments of the Golgi has been reconstituted in a cell-free system and is measured, in a rapid and sensitive new assay, by the coupled incorporation of 3H-N-acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transported during mixed incubations from the "donor" compartment in Golgi from VSV-infected CHO clone 15B cells (missing a key Golgi GlcNAc transferase) to the next, successive "acceptor" compartment (containing the GlcNAc transferase) in Golgi from wild-type CHO cells. Golgi fractions used in this assay have been extensively purified, and account for all of the donor and acceptor activity in the cells. Together with several other lines of evidence, this indicates that the cell-free system is highly specific, measuring only transport between sequential compartments in the Golgi stack. Transport in vitro is almost as efficient as in the cell, and requires ATP and the cytosol fraction in addition to protein components on the cytoplasmic surface of the Golgi membranes.

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 .

Cell-free fusion between endocytic vesicles has been obtained in a sensitive assay based on the avidin-biotin binding reaction. Chinese hamster ovary cells are allowed to endocytose either avidin-linked j3-galactosidase or biotinylated IgG. Postnuclear supernatant extracts prepared from these cells are incubated at 37°C in the presence of an ATP-regenerating system. Fusion between vesicles from the two extracts permits the avidin-biotin association to occur, so that the amount of avidin-enzyme-biotinylated IgG complex produced is proportional to the amount of fusion between vesicles. The amount of complex formed is measured, after detergent lysis of the vesicles, by an ELISA technique, using a fluorogenic substrate for fi-galactosidase. The fusion process requires ATP hydrolysis and specific cytosolic proteins. The vesicles that fuse appear to be endocytic vesicles populated by the endocytosed proteins within 5 min of their internalization at 37°C. Ionophores and weak bases do not inhibit fusion, suggesting that a pH gradient across the vesicle membrane is not crucial for the fusion process.Eukaryotic cells internalize macromolecules and fluid by the process of endocytosis (1, 2). Central to this process are the steps offorming an endocytic vesicle, transporting the vesicle to its specific target site, and fusing this vesicle with its target compartment. Presently, our understanding ofthe function of cell proteins in mediating endocytic processes is limited to the role of receptor proteins in receptor-mediated uptake (3, 4) and to the metabolism of clathrin-coated structures (5-8).One approach for identifying other proteins that mediate endocytic events is to devise cell-free systems that faithfully reconstruct the processes seen in vivo and then dissect out the factors critical for their operation in the cell-free milieu. This functional approach has the advantage of not only identifying such factors but providing a basis for uncovering their mechanistic role in endocytosis. This paper describes a cell-free system that reconstitutes endocytic vesicle fusion. Vesicles isolated from cells that have endocytosed avidin-p-galactosidase (Av-/3-Gal) conjugates are incubated at 37°C, in the presence of ATP and cytosolic proteins, with vesicles from cells that have internalized biotinylated IgG molecules. Biotinylated insulin is added to scavenge any Av-p8-Gal released by vesicle breakage. When vesicles from the two fractions fuse, the avidin-biotin association occurs (Fig. 1, step a). The amount of Av-P-Gal-IgG complex formed is thus a measure of the extent of fusion. This complex is assayed by an ELISA technique (Fig. 1, steps b and c), which binds the IgG to a solid phase and measures its entrained B-galactosidase with a fluorescent substrate.Previous systems designed to detect endocytic vesicle fusion (9, 10) have relied on developing the signal representing fusion events within the fused vesicle products. Since the environmental conditions within the fused vesicles cannot be /)a b~KK c known with certainty,...

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.