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

Abstract

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, ...

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

“…SDS gels and sequences that align with each other and the deduced sequence of Arabidopsis H+-PPase cDNA clone pAVP-3 (23). The deduced N terminus of pAVP, in turn, aligns with the direct sequence data derived from the N terminus of the corresponding subunit of the enzyme from Vigna (11).…”

“…The open reading frame of the cDNA insert of pAVP-3 encodes a 770-amino acid polypeptide with a predicted Mr of 80,800 (molecular mass, 81 kD) (23). The corresponding immunoreactive polypeptide of Arabidopsis microsomes, on the other hand, has an apparent MA of 66,800 on SDS gels (Fig.…”

“…Rea, in preparation). In conjunction with the amino acid sequence data deduced from pAVP-3, which show that the MgPPi-binding polypeptide of the tonoplast H+-PPase of Arabidopsis is extremely hydrophobic and contains at least 13 transmembrane spans, in addition to hydrophilic domains containing potential MgPPi-binding motifs (23), these findings strongly suggest that subunits in addition to the polypeptide encoded by AVP-3 need not be invoked to account for the capacity of the tonoplast H+-PPase for PPi-energized transtonoplast HW-translocation.…”

“…The N-terminal sequence data for the enzyme from Vigna were taken from Maeshima and Yoshida (11). cDNA clone pAVP-3, encoding the H+-PPase from Arabidopsis, was isolated by immunoscreens of a XZAP expression library with antibody affinity purified against the Mr 66,000 subunit of the enzyme from Vigna (23 complex. This component alone copurifies with PPase activity during detergent solubilization and chromatography and is the only polypeptide of tonoplast vesicles to undergo MgPPiprotectable, free PPi-potentiated labeling by ["4C]NEM.…”

Oxygen exchange reactions catalyzed by vacuolar H+‐translocating pyrophosphatase Evidence for reversible formation of enzyme‐bound pyrophosphate

“…Protein purification [3][4][5][6], covalent modification [3,5], molecular cloning [7][8][9] and heterologous expression [9] have demonstrated the abundance, high catalytic activity, novelty and structural simplicity of the V-PPase, and patch clamp analyses implicate the pump in the primary translocation of both H ÷ and K ÷ into the vacuole [10]. However, despite these advances, very little is understood of how the one polypeptide species constituting the V-PPase accomplishes catalysis.…”