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The proton-translocating ATPase of clathrin-coated vesicles of bovine brain is characterized by ATP specificity. Chloride or bromide serve as co-ions balancing electrogenic proton pumping. ATP-driven proton translocation can be observed in the absence of chloride, provided the membrane potential is collapsed by K+ moving out in the presence of valinomycin. Chloride transport can be observed independent of proton movements in the absence of ATP, provided an inside positive membrane potential is generated by K+ moving from the outside to the inside in the presence of valinomycin.

Clathrin-coated vesicle acidification is mediated by an N-ethylmaleimide-sensitive, vanadate-resistant proton-translocating ATPase. This enzyme is a 530-kDa hetero-oligomer which catalyzes ATP-dependent proton pumping when reconstituted (Xie, X. S., and Stone, D. K. (1986) J. Biol. Chem. 261, 2492-2495). We now report the purification of a second ATPase from bovine brain clathrin-coated vesicles which is inhibited by both N-ethylmaleimide (1 mM) and vanadate (10 microM). Localization of the ATPase to clathrin-coated vesicles was demonstrated by the precipitation of ouabain-resistant, vanadate-sensitive ATPase activity with anti-clathrin antibodies. The enzyme was solubilized with 0.1% polyoxyethylene 9-lauryl ether and has been purified 700-fold to a specific activity of 42 mumol of Pi.mg of protein-1.min-1. A molecular mass of 116 kDa was determined by centrifugation in sucrose gradients prepared in H2O and D2O, by high performance liquid chromatography using gel filtration, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under reducing conditions. The ATPase is unlike any known mammalian E1E2-type ATPase in that it is not inhibited by ouabain or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and it is not activated by Na+, K+, or Ca2+.

We have recently reported the reconstitution of the clathrin-coated vesicle proton-translocating complex with liposomes prepared from ethanol-extracted crude bovine brain lipids. Reconstitution of proton pumping by the isolated proton ATPase has now been achieved with liposomes prepared from pure lipids. Optimal proton pumping, as assessed by ATP-generated acridine orange quenching and 32Pi-ATP exchange, is achieved with liposomes prepared from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol at a weight ratio of 40:26.5:7.5:26, and a lipid-to-protein weight ratio of 200:1. Under such conditions, the extent of decrease of ATP-generated acridine orange quenching is 150-fold greater than that of native clathrin-coated vesicles. Cholesterol is required to stabilize the proteoliposome. Phosphatidylserine, which is most effective in activating ATP-hydrolytic activity of the solubilized enzyme, is not obligatory for reconstitution of proton pumping.

Clathrin-coated vesicles contain a proton translocating ATPase which is insensitive to azide but inhibited by N-ethylmaleimide. The ATP hydrolytic subunit of this proton pump has been solubilized, partially purified, and reconstituted into H+-ATPase-depleted coated vesicle membranes (Xie, X.-S., Stone, D.K., and Racker, E. (1984) J. Biol. Chem. 259, 11676-11678). In this communication we report that the entire proton transporting complex has been solubilized and purified 200-fold. The complex, when reconstituted into brain lipid liposomes, catalyzes azide-resistant, N-ethylmaleimide-sensitive H+ transport manifested as both generation of a pH gradient and an electrical gradient. The complex has an apparent molecular mass of 530 kDa.

A N-ethylmaleimide-sensitive ATPase was extracted and partially purified from clathrin-coated vesicles of bovine brain. During purification the enzyme lost activity which was restored by a purified phospholipid fraction from brain. Phosphatidylserine, but no other commercial phospholipids tested, replaced the brain lipid fraction as activator. Particles depleted of the ATPase exhibited no H+ pump activity when reconstituted with brain phospholipids by the cholate dilution procedure. H+ pump activity was restored by incubating the reconstituted vesicles with the partially purified ATPase.

The clathrin-coated vesicle proton pump is a representative member of the new class of endomembrane proton ATPases that share an inhibitor profile which distinguishes them from classic F1F0 and E1E2-type proton pumps. The coated vesicle proton pump is a large (530 kDa) heteroligomer composed of eight polypeptides with molecular masses of 116, 70, 58, 40, 38, 34, 33 and 17 kDa. The 200-fold purified enzyme catalyses ATP-generated proton pumping when reconstituted in liposomes composed of pure lipids. Subunit function has been determined by partial reaction analysis of subunit and subcomplex activities. The isolated 17 kDa subunit, when co-reconstituted with bacteriorhodopsin, forms a dicyclohexylcarbodiimide-inhibitable proton channel. Selective removal of the 116 kDa subunit transforms the proton ATPase from a Mg2+-activatable to a Ca2+-activatable ATPase. Subsequent dissociation and reconstitution of subunits reveals that the 70, 58, 40 and 33 kDa components are required, in composite, to form a functional ATP-hydrolytic core, and that no single subunit or subcomplex deficient in these subunits can catalyse ATP hydrolysis.

The clathrin-coated vesicle proton-translocating complex is composed of a maximum of eight major polypeptides. Of these potential subunits, only the 17-kDa component, which is a proton pore, has been defined functionally (Sun, S.Z., Xie, X. S., and Stone, D. K. (1987) J. Biol. Chem. 262, 14790-14794). ATPase-and proton-pumping activities of the 200-fold purified proton-translocating complex are supported by Mg2+, whereas Ca2+ will only activate ATP hydrolysis. Like Mg2+-activated ATPase activity, Ca2+-supported ATP hydrolysis is inhibited by N-ethylmaleimide, NO3-, and an inhibitory antibody and is stimulated by Cl- and phosphatidylserine. Thus, Ca2+ prevents coupling of ATPase activity to vectoral proton movement, and Ca2+-activated ATPase activity is a partial reaction useful for analyzing the subunit structure required for ATP hydrolysis. The 530-kDa holoenzyme was dissociated with 3 M urea and subcomplexes, and isolated subunits were partially resolved by glycerol gradient centrifugation. No combination of these components yielded Mg2+-activated ATPase or proton pumping. Ca2+-activated ATP hydrolysis was not catalyzed by a subcomplex containing the 70- and 58-kDa subunits but was restored by recombination of the 70-, 58-, 40-, and 33-kDa polypeptides, indicating that these are subunits of the clathrin-coated vesicle proton pump which are necessary for ATP hydrolysis.

Clathrin-coated vesicles contain a proton translocating ATPase which operates in parallel with a chloride transporter (Xie, X.-S., Stone, D.K., and Racker, E. (1983) J. Biol. Chem. 258, 14834-14838). The polypeptide antibiotic, duramycin, has a dual inhibitory effect on clathrin-coated vesicle acidification. Low amounts of duramycin (5 micrograms/100 micrograms of protein) inhibit by 50% the proton translocation facilitated by chloride translocation. Under these conditions duramycin inhibits also 36Cl uptake when driven by either the electrogenic proton pump or by inward directed K+ movement in the presence of valinomycin. Higher amounts of duramycin (20 micrograms/100 micrograms of protein) are needed to inhibit by 50% the proton pump itself, as evidenced by reduced proton translocation facilitated by an outward potassium movement in the presence of valinomycin. In addition, the amount of duramycin needed to inhibit the proton pump corresponded well with the amount needed to inhibit the ouabain-insensitive, N-ethylmaleimide-sensitive ATPase activity of clathrin-coated vesicles.