Rui-Guang Zhen

Acidic residues necessary for pyrophosphate-energized pumping and inhibition of the vacuolar H+-pyrophosphatase by N,N'-dicyclohexylcarbodiimide.

On the basis of a revised topological model of the vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) derived from the analysis of four published sequences using two structure-predicting programs, TopPred II and MEMSAT, eight acidic amino acid residues located near or within transmembrane α-helices were identified. The codons specifying these amino acids in the cDNA encoding the V-PPase from Arabidopsis thalianawere singly mutated to examine their involvement in pyrophosphate (PPi) hydrolysis and PPi-dependent H+ translocation and the functional significance of the similarities between the sequences encompassing Glu229(227–245) of the V-PPase and theN,N′-dicyclohexylcarbodiimide (DCCD)-binding transmembrane α-helix of the c-subunits of F-ATPases (Nyren, P., Sakai-Nore, Y., and Strid, A. (1993) Plant Cell Physiol. 34, 375–378). Three functional classes were identified after heterologous expression of mutated enzyme in Saccharomyces cerevisiae. Class I (E119Q, E229Q, D573N, E667Q, and E751Q) mutants exhibited PPi hydrolytic and H+ translocation activities and DCCD sensitivities similar to wild type. The one class II mutant obtained (E427Q) was preferentially impaired for H+translocation over PPi hydrolysis but retained sensitivity to DCCD. Class III (E305Q and D504N) mutants exhibited a near complete abolition of both PPi hydrolysis and H+translocation and residual activities with decreased DCCD sensitivity. In none of the mutants was diminished insertion of the V-PPase into the membrane or an increase in the background conductance of the membrane to H+ evident. The decoupled character of E427Q mutants and the enhancement of H+ pumping in E427D mutants by comparison with wild type, in conjunction with the retention of DCCD inhibitability in both E427Q and E427D mutants, implicate a role for Glu427 in DCCD-insensitive H+ translocation by the V-PPase. The proportionate diminution of PPi hydrolytic and H+ translocation activity and conservation of wild type DCCD sensitivity in E229Q mutants refute the notion that Glu229 is the residue whose covalent modification by DCCD is responsible for the abolition of PPi-dependent H+ translocation. Instead, the diminished sensitivity of the residual activities of E305Q and D504N mutants, but not E305D or D504E mutants, to inhibition by DCCD is consistent with the involvement of acidic residues at these positions in inhibitory DCCD binding. The results are discussed with regard to the possible involvement of Glu427 in coupling PPi hydrolysis with transmembrane H+translocation and earlier interpretations of the susceptibility of the V-PPase to inhibition by carbodiimides.

The Molecular and Biochemical Basis of Pyrophosphate-Energized Proton Translocation at the Vacuolar Membrane

Publisher Summary This chapter discusses the molecular and biochemical basis of pyrophosphate–energized proton translocation at the vacuolar membrane. Inorganic pyrophosphatases (PPase) are ubiquitous enzymes that catalyze the hydrolysis of pyrophosphate to orthophosphate. In most organisms, the bulk of PPase activity is soluble and considered to maximize the energy yield from the pyrophosphorylytic cleavage of nucleoside triphosphates, associated with many biosynthetic reactions, by dissipatively hydrolysing PP i . Several lines of inquiry implicate PP i as an important energy source for plant vacuolar function in particular, and plant metabolism in general. Most of the H + phosphohydrolases associated with plant membranes can be distinguished from each other by their inhibitor sensitivities. The F–type H + –ATPases of energy–coupling membranes, the P–type H + –ATPase of plasma membrane and the V–type H + –ATPase of endomembranes are selectively inhibited by azide, orthovanadate and the bafilomycins, respectively. However, strict criteria for the identification of V–PPase activity in uncharacterized membrane fractions, other than through the measurement of azide–, orthovanadate– and bafilomycin–insensitive, K + –stimulated PP i –dependent H + translocation have been lacking. A major complication when attempting to determine the ligand interactions of any PPase, whether soluble or membrane bound, are the multiple ion complexes that PP, is capable of forming in aqueous media.

Aminomethylenediphosphonate: A Potent Type-Specific Inhibitor of Both Plant and Phototrophic Bacterial H+-Pyrophosphatases

The suitability of different pyrophosphate (PPi) analogs as inhibitors of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) of tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata was investigated. Five 1,1-diphosphonates and imidodiphosphate were tested for their effects on substrate hydrolysis by the V-PPase at a substrate concentration corresponding to the Km of the enzyme. The order of inhibitory potency (apparent inhibition constants, Kiapp values, [mu]M, in parentheses) of the compounds examined was aminomethylenediphosphonate (1.8) > hydroxymethylenediphosphonate (5.7) [almost equal to] ethane-1-hydroxy-1,1-diphosphonate (6.5) > imidodiphosphate (12) > methylenediphosphonate (68) >> dichloromethylenediphosphonate (>500). The specificity of three of these compounds, aminomethylenediphosphonate, imidodiphosphate, and methylenediphosphonate, was determined by comparing their effects on the V-PPase and vacuolar H+-ATPase from Vigna, plasma membrane H+-ATPase from Beta vulgaris, H+-PPi synthase of chromatophores prepared from Rhodospirillum rubrum, soluble PPase from Saccharomyces cerevisiae, alkaline phosphatase from bovine intestinal mucosa, and nonspecific monophosphoesterase from Vigna at a PPi concentration equivalent to 10 times the Km of the V-PPase. Although all three PPi analogs inhibited the plant V-PPase and bacterial H+-PPi synthase with qualitatively similar kinetics, whether substrate hydrolysis or PPi-dependent H+-translocation was measured, neither the vacuolar H+-ATPase nor plasma membrane H+-ATPase nor any of the non-V-PPase-related PPi hydrolases were markedly inhibited under these conditions. It is concluded that 1, 1-diphosphonates, in general, and aminomethylenediphosphonate, in particular, are potent type-specific inhibitors of the V-PPase and its putative bacterial homolog, the H+-PPi synthase of Rhodospirillum.

1-Chloro-2,4-Dinitrobenzene-Elicited Increase in Vacuolar Glutathione-S-Conjugate Transport Activity

Unlike most other characterized organic solute transport in plants, uptake of the model compound S-(2,4-dinitrophenyl)glutathione (DNP-GS) and related glutathione-S-conjugated by vacuolar membranes is directly energized by MgATP. Here we show that exogenous application of the DNP-GS precursor 1-chloro-2,4-dinitrobenzene (CDNB) to seedlings of Vigna radiata (mung bean) increases the capacity of vacuolar membrane vesicles isolated from hypocotyls for MgATP-dependent DNP-GS transport in vitro. Our findings are 4-fold: (a) Pretreatment of seedlings with CDNB causes a progressive increase in MgATP-dependent DNP-GS uptake by vacuolar membrane vesicles, whereas the same range of CDNB concentrations causes only marginal stimulation when the compound benoxacor [4-(dichloroacetyl)-3,4-dihydro-3-methyl-2H-1,4-benzoxazine] is included in the pretreatment solution. (b) Increased DNP-GS uptake is accompanied by a proportionate and selective increase in Vmax(DNP-GS) but not in Km(DNP-GS) or Km(MgATP). (c) CDNB-enhanced DNP-GS uptake is not accompanied by a change in the density profile or sidedness of the vacuolar membrane fraction. (d) Basal and CDNB-enhanced DNP-GS uptake are indistinguishable in terms of their inhibitor profiles. On the basis of these findings, it is inferred that pretreatment with CDNB increases the amount or recruitment of functional transporter into the vacuolar membrane and that agents such as benoxacor antagonize the effects otherwise seen with CDNB in this sytem.

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

1,1‐Diphosphonate analogs of pyrophosphate, containing an amino or a hydroxyl group on the bridge carbon atom, are potent inhibitors of the H+‐translocating pyrophosphatases of chromatophores prepared from the bacterium Rhodospirillum rubrum and vacuolar membrane vesicles prepared from the plant Vigna radiata. The inhibition constant for aminomethylenediphosphonate, which binds competitively with respect to substrate, is below 2 μM. Rat liver mitochondrial pyrophosphatase is two orders of magnitude less sensitive to this compound but extremely sensitive to imidodiphosphate. By contrast, fluoride is highly effective only against the mitochondrial pyrophosphatase. It is concluded that the mitochondrial pyrophosphatase and the H+‐pyrophosphatases of chromatophores and vacuolar membranes belong to two different classes of enzyme.

Molecular Dissection of Vacuolar H+-Pyrophosphatase

It is now established that inorganic pyrophosphate (PPi) is a major energy source for electrogenic H+-translocation across the vacuolar membrane (tonoplast) of plant cells (Rea et al 1992a; Rea and Poole 1993). The enzyme responsible, the vacuolar H+-translocating pyrophosphatase (V-PPase; EC 3.6.1.1), which is an abundant membrane constituent, comprising 1–10% of total vacuolar membrane protein, exclusively utilizes PPi as substrate (Rea and Poole 1986) and, in parallel with the V-ATPase (EC 3.6.1.3), generates an inside-acid, inside-positive transtonoplast H+-electrochemical potential difference. The H+ gradient so generated serves to energize a wide range of ΔpH and/or Δψ-coupled transport processes, including the vacuolar accumulation of low molecular weight solutes (sugars, amino acids, carboxylic acids, mineral ions) and the lumenal localization of storage proteins and lysosomal-type hydrolases. Because the plant vacuole is the largest intracellular organelle known, frequently accounting for 40 –99% of total intracellular volume, and directly participates in many fundamental physiological processes such as cytosolic pH stasis, “excretion” of metabolically perturbing agents (e.g. xenobiotics and alkaloids), sequestration of regulatory Ca2+, turgor regulation and nutrient storage and retrieval, any meaningful account of plant cell metabolism is contingent on an understanding of the functional characteristics and organization of the primary energizers responsible for the establishment of the requisite transmembrane ion gradients: the V-PPase and V-ATPase.