Vacuolar H+-translocating Inorganic Pyrophosphatase: Biochemistry and Molecular Biology

Differential sensitivity of membrane‐associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme

“…In other organisms, however, there are membrane-associated PPases which are capable of utilizing the energy contained in the phosphoanhydride bond of PP, for the establishment of transmembrane ionic gradients. Notable examples of energy-transducing, or energy-conse~ing, PPases are the reversible H'-translocating PPase of chromatophores from the purple, non-sulfur bacterium Rhodospirihn rubrum [ 11. the vacuolar H'-translocating PPase of plant cells [2,3] and the membrane-associated PPase of animal and yeast mitochondria [4]. Whereas the capacity of the mitochondrial PPase for ion transport has yet to be demonstrated unequivocally, the enzymes from R rubru~ and plants are PP,-linked ion translocases competent in the primary translocation of H' [5] and H' and/or K' [3,6,7], respectively, and maintenance in this way of an energy buffer alternative to ATP [8].…”

Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana.

“…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)(6)(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, depending on source and preparation (7,8). When account is taken of the large size of the vacuole-it can account for 90-99% of total intracellular volume-the potential bioenergetic impact of the H+-PPase is great.…”

“…The identity of the putative H+-PPase cDNA clones from Arabidopsis is verified by the precise alignment of the deduced amino acid sequence of the protein encoded by AVP-3 with direct internal sequence data acquired from the Mr 64,500-67,000, MgPP,-binding polypeptide of Beta (7,8,11) and N-terminal sequence data obtained from the corresponding polypeptide of Vigna (6).…”

“…Computer searches of both the nucleotide sequence of the cDNA insert of pAVP-3 and the deduced amino acid sequence of the polypeptide encoded by the clone against the GenBank, Genpept, and Swiss-Prot data bases (as of September, 1991) (6)(7)(8)11) and is the only polypeptide of tonoplast vesicles to undergo MgPP1-protectable, free PP1-potentiated labeling by N-[`4C]ethylmaleimide (7,8). In addition, selective purification of the MgPP,-binding subunit of the tonoplast H+-PPase from Vigna and its incorporation into artificial phospholipid bilayers generate proteoliposomes capable of both MgPPi hydrolysis and MgPP,-dependent H+ translocation (C. J. Britten and P.A.R., unpublished results).…”

Common Identity of Substrate Binding Subunit of Vacuolar H+-Translocating Inorganic Pyrophosphatase of Higher Plant Cells

“…H+-translocating PPases have been identified in the energy-coupling membranes of mitochondria, chloroplasts, and phototrophic bacteria (3), but the vacuolar (tonoplast) H+-PPase is the first example of a PPi-energized H+-translocase in a 'nonenergy' coupling membrane (18). In view of the crucial role played by the PPi bond in biological energy transduction, the unique status of PPi as the limiting case of a high-energy phosphate, and the increasing recognition of PPi as a key metabolite in plants (21,27,28), the existence of an energy-conserving H+-PPase on the vacuolar membrane of plant cells is of potentially profound bioenergetic significance.…”