The energy-dependent transport of solutes across the vacuolar membrane (tonoplast) of plant cells is driven by two H' pumps: a vacuolar ("V-type") H+-ATPase (EC 3.6.1.3) and a HW-translocating (pyrophosphate-energized) inorganic pyrophosphatase (H+-PPase; EC 3.6.1.1). The H+-PPase, like the V-type H+-ATPase, is abundant and ubiquitous in the vacuolar membranes of plant cells, and both enzymes make a substantial contribution to the transtonoplast HW-electrochemical potential difference. Here, we report the cloning and sequence of cDNAs encoding the tonoplast H+-PPase of Arabidopsis thaiana. The protein predicted from the nucleotide sequence of the cDNAs is constituted of 770 amino acids and has a molecular weight of 80,800. It is a highly hydrophobic integral membrane protein, and the structure derived from hydrophilicity plots contains at least 13 transmembrane spans. Since the tonoplast H+-PPase appears to be constituted of one polypeptide species and genomic Southern analyses indicate that the gene encoding the M, 80,800 polypeptide is present in only a single copy in the genome of Arabidopsis, it is suggested that the H+-PPase has been cloned in its entirety. The lack of sequence identities between the tonoplast H+-PPase and any other characterized H+ pump or PP-dependent enzyme implies a different evolutionary origin for this translocase.The chemiosmotic hypothesis (1) contends that membranebound H+ pumps constitute the primary transducers by means of which living cells interconvert light, chemical, and electrical energy. Through the establishment and maintenance of transmembrane electrochemical gradients, H+ pumps energize the transport of other solutes or, in the special case of the energy-coupling membranes of mitochondria, chloroplasts, and bacteria, transduce the H+ electrochemical gradient generated by membrane-linked anisotropic redox reactions to the synthesis of ATP (1). Given the multitude of biological reactions energized by ATP, primary H+ translocation and the interconversions of ATP have come to be recognized as the principal generators of usable energy in the cell. Intriguing, therefore, is the fact that the vacuolar membrane (tonoplast) of plant cells contains not only a H+-ATPase (EC 3.6.1.3) (2, 3) but also an inorganic pyrophosphate-energized H+-pyrophophatase (H+-PPase; EC 3.6.1.1) (2). Both enzymes catalyze inward electrogenic H+ translocation (from cytosol to vacuole lumen), but the H+-PPase is unusual in its exclusive use of PPj as energy source (4).The tonoplast H+-PPase appears to be important for plant cell function: it is widespread, active, and abundant. The enzyme is ubiquitous in the vacuolar membranes of plant cells (2) and capable of establishing a H+ gradient of similar, and often greater, magnitude than the H+-ATPase on the same membrane (2, 5-7). The Mr 64,500-73,000 substrate (MgPPi)-binding subunit of the H+-PPase constitutes between 1% (8) and 10%1o (6, 7) of total vacuolar membrane protein and the purified enzyme has a turnover number of between 50 and 100 s-1, ...

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There have been conflicting reports in the literature concerning the polypeptide composition of the vacuolar H+-translocating inorganic pyrophosphatase (tonoplast H+-PPase) of plant cells. The major subunit(s) of the enzyme have been attributed to polypeptides of relative molecular weight (Mr) 64,500 (Beta vulgaris), 67,000 (Beta vulgaris), 73,000 (Vigna radiata), and 37,000 to 45,000 (Zea mays). Here, we reconcile these differences to show, through the combined application of independent purification, affinitylabeling, sequencing, and immunological procedures, that the major polypeptide associated with the H+-PPase from all of these organisms, and Arabidopsis thaliana, corresponds to the same moiety. The principal polypeptide components of the H+-PPase purified from Beta and Vigna by independent procedures have similar apparent subunit masses when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under

An H+-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophoresis. Overall, a 150-fold purification of the PPase was obtained, from the tonoplast fraction, through anion exchange chromatography of the detergent-solubilized membranes followed by ammonium sulfate precipitation and gel filtration chromatography. The purified polypeptide showed no cross-reactivity with antibodies raised against the 67 kilodalton subunit of the tonoplast ATPase.The existence of two distinct H+-translocating pumps, an ATPase and a PPase,2 both potentially contributing to the electrochemical gradient across the vacuolar membrane, has now been well documented. The enzymes have been shown to be separable by chromatography after detergent-solubilization of tonoplast vesicles isolated from red beet (15) and from the CAM plant Kalanchoe daigremontia (3). Both proton pumps caused accumulation of neutral red in individual vacuoles of Nitella (17), and in a patch-clamp study of sugar beet vacuoles, Hedrich and Kurkdjian (4) demonstrated the presence of the two electrogenic pumps by measuring membrane potentials induced by ATP and/or PPi in the same vacuole.As a first step toward a more complete molecular analysis of the protein, it was necessary to purify the PPase and elucidate its polypeptide composition. A recent study which reported the purification of the PPase from microsomal fractions of corn seedlings (12) could not, however, be applied to our system of purified tonoplast from red beet, and we have therefore developed an alternative purification scheme. ' Supported by the Natural Sciences and Engineering Research Council of Canada and the Fonds pour la Formation de Chercheurs et l'Aide a la Recherche of Quebec.2 Abbreviations: PPase, pyrophosphatase; BTP, bis-tris propane; Chaps, 3-[(3-cholamidopropyl)dimethyl-ammonio]-l-propanesulfonate; EB, elution buffer; RB, running buffer; SM, suspension medium. MATERIALS AND METHODS Tonoplast PreparationFresh red beets (Beta vulgaris L.) were bought commercially, kept at 4°C and used within 24 h.Tonoplast vesicles were prepared as described (14) with some modifications. Peeled and diced beets (350 g) were homogenized at 4°C in a blender with two 30-s bursts in 350 mL homogenization buffer containing 10 mM glycerophosphate, 0.65 M ethanolamine (adjusted to pH 8.0 with concentrated H2SO4), 0.28 M choline chloride, 25 mM K-metabisulfite, 3 mm BTP-EDTA, 0.2% BSA (fraction V, essentially fatty acid-free), 10% (w/v) insoluble PVP, 5 mm DTT, and 1 mM PMSF in 70 mm BTP-Mes (pH 8.0). The homogenate was filtered through cheesecloth and centrifuged at 80,000g for 30 min in a Beckman Type 45 Ti rotor. The pellets were resuspended with a Dounce homogenizer in a small volume of SM containing 10% (w/v) glycerol, 5 mm BTP-Mes (pH 8.0), 1 mm BTP-EDTA, and 5 mm DTT, then mad...

The functional sizes of the vacuolar H(+)-ATPase (V-ATPase; EC 3.6.1.34) and H(+)-pyrophosphatase (PPase; EC 3.6.1.1) from vacuolar membranes of red beet (Beta vulgaris L.) were estimated by radiation inactivation, both for substrate hydrolysis and for H+ transport. For the V-ATPase, the radiation-inactivation size for H+ transport was 446 (403-497) kDa and that for ATP hydrolysis was 394 (359-435) kDa. The low values of both of these estimates suggest that not all subunits which may co-purify with V-ATPases are required for either hydrolysis or transport. For the PPase, the radiation-inactivation size for hydrolysis was 91 (82-103) kDa, suggesting that the minimum functional unit for hydrolysis is the 81 kDa monomer. In contrast to the V-ATPase, the PPase gave a radiation-inactivation size for transport which was 3-4-fold larger than that for hydrolysis (two estimates for transport gave 307 and 350 kDa), indicating that a single catalytic subunit is insufficient for transport activity.