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Volume 271, Number 48, Issue of November 29, 1996 pp. 30980-30985
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Reconstitution of ATPase Activity from Individual Subunits of the Clathrin-coated Vesicle Proton Pump
THE REQUIREMENT AND EFFECT OF THREE SMALL SUBUNITS

(Received for publication, May 23, 1996, and in revised form, September 19, 1996)

From the Division of Molecular Transport, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9121

The vacuolar-type proton pump of clathrin-coated vesicles is composed of two general domains, a peripheral, catalytic sector (V_C) and a transmembranous proton channel (V_B). In its native form, the enzyme can hydrolyze both MgATP and CaATP, whereas V_C, when separated from V_B, loses its MgATPase activity and switches to a state that can hydrolyze only CaATP. Further dissociation of V_C results in subcomplexes that are depleted of one or more subunits and lack ATPase activity altogether. Reconstitution of recombinant subunits to these biochemically prepared subcomplexes has demonstrated the necessity of polypeptides of 70, 58, 40, and 33 kDa (subunits A, B, C, and E, respectively) for CaATPase activity of the V_C complex. The current studies demonstrate that mixtures of these four recombinant subunits cannot support CaATPase activity in the absence of a biochemically prepared subcomplex. Investigation of the other components required for ATPase activity has led to the identification of three additional polypeptides present in preparations of V_C, with apparent molecular masses of 15, 14, and 10 kDa. Each of these proteins was found to activate ATPase activity of mixtures of subunits A, B, C, and E. In addition, ATPase reconstituted from these individual subunits hydrolyses ATP, not only in the presence of Ca²⁺ but also in the presence of Mg²⁺. Investigation of the individual properties of these three subunits revealed that the 10-kDa polypeptide is subunit F, as determined by immunoblot analysis. This subunit had no effect on MgATPase activity of V_C but stimulated CaATPase activity 6-fold in the presence of subunit D. Under optimal conditions the 14-kDa component resulted in a 10-fold stimulation and the 15-kDa component a 20-fold stimulation of MgATPase activity; based on this observation, the 14- and 15-kDa polypeptides were named subunits G and H, respectively. In addition, proton pumping activity was reconstituted through the reassembly of subunits A-H with V_B and SFD, a previously described pump component composed of polypeptides of 50 and 57 kDa (Xie, X.-S, Crider, B.P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809-25815). Together, these experiments completely define the catalytic center of the vacuolar proton pump of clathrin-coated vesicles.

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