Proton transport in maize tonoplasts supported by fructose-1,6-bisphosphate cleavage. Pyrophosphate-dependent phosphofructokinase as a pyrophosphate-regenerating system

Abstract

The energy derived from pyrophosphate (PPi) hydrolysis is used to pump protons across the tonoplast membrane, thus forming a proton gradient. In a plant's cytosol, the concentration of PPi varies between 10 and 800 microm, and the PPi concentration needed for one-half maximal activity of the maize (Zea mays) root tonoplast H+-pyrophosphatase is 30 microm. In this report, we show that the H+-pyrophosphatase of maize root vacuoles is able to hydrolyze PPi (Reaction 2) formed by Reaction 1, which is catalyzed by PPi-dependent phosphofructokinase (PFP): Fructose-1,6-bisphosphate (F1,6BP) + Pi <--> PPi +Fructose-6-phosphate (F6 P) (reaction 1) PPi --> 2 Pi (reaction 2) H+cyt --> H+vac (reaction 3) F1,6BP + H+cyt <--> H+vac + F6P + Pi (reaction 4) During the steady state, one-half of the inorganic phosphate released (Reaction 4) is ultimately derived from F1,6BP, whereas PFP continuously regenerates the pyrophosphate (PPi) hydrolyzed. A proton gradient (DeltapH) can be built up in tonoplast vesicles using PFP as a PPi-regenerating system. The Delta pH formed by the H+-pyrophosphatase can be dissipated by addition of 20 mm F6P, which drives Reaction 1 to the left and decreases the PPi available for the H+-pyrophosphatase. The maximal Delta pH attained by the pyrophosphatase coupled to the PFP reaction can be maintained by PFP activities far below those found in higher plants tissues.

Figure 1.

Cleavage of F1,6BP and Mg²⁺ dependence. The F1,6BP cleavage was dependent on MgCl₂ (A) and F1,6BP (B) concentrations. The MgCl₂ dependence was measured in a medium containing: 50 mm MOPS-Tris buffer (pH 7.0), 60 milliunits mL^–1 mung bean (Vigna radiata) PFP, 0.1 mg mL^–1 maize vacuolar microsomal protein, 100 mm KCl, 2 μm F2,6BP, and 10 mm F1,6BP (A). When the F1,6BP dependence was measured, the MgCl₂ concentration was fixed at 0.6 mm(B). The reaction was performed at 30°C and started by addition of 0.1 mm P_i. Aliquots of 0.8 mL were removed, and the reaction was stopped by the addition of 0.2 mL of 50% (w/v) trichloroacetic acid. The figure shows a representative experiment. Similar results were obtained with three different vesicle preparations.

Figure 2.

Membrane proton gradient supported by PFP, F1,6BP, and P_i. The assay conditions were identical to those described in Figure 1B, except that the 9-amino-6-chloro-2-methoxyacridine (ACMA; 3 μm) was present and F1,6BP, F2,6BP, and P_i concentrations are listed below for each panel. At the end of gradient formation, 1 μm carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) was added to dissipate the H⁺ gradient in A and B. In C, the addition of PFP activator, F2,6BP, enhanced the proton gradient formation, and the addition of 20 mm F6P dissipated it. The arrows indicate the sequential addition of: A, 25 milliunits of PFP, 2 μm F2,6BP, 2 mm P_i, and 0.6 mm F1,6BP; B, 30 μm P_i was included in the reaction medium before the sequential addition of: 25 milliunits of PFP; 4 μm F2,6BP; 0.6 mm F1,6BP, and 2 mm P_i; C, 10 milliunits of PFP, 2 mm P_i, 0.1 mm F1,6BP, 4 μm F2,6BP, and 20 mm F6P. The figure shows a representative experiment. Similar results were obtained in three different vesicle preparations.

Figure 3.

PP_i saturation kinetics of maize root H⁺-PPase. The assay medium composition was 50 mm MOPS-Tris (pH 7.0), 100 mm KCl, 0.6 mm MgCl₂, 3 μm FCCP, and 0.030 mg mL^–1 maize vesicle protein. The PP_i concentrations are shown on the abscissa. The reaction was started by addition of vesicles. The amount of PP_i hydrolyzed never exceeded 5% of the total PP_i added to the medium. The kinetic parameters shown in the figure were calculated from a nonlinear regression analysis applied to the Hill equation using the program ENZFITTER. The specific activities shown are means ± se of at least four independent preparations.

Figure 4.

PFP dependence for the formation of the membrane proton gradient. The reaction was carried out in medium containing 10 mm MOPS-Tris (pH 7.0), 3 μm ACMA, 1 mm MgCl₂, 100 mm KCl, 2 μm F2,6BP, 2 mm P_i, 0.1 mg mL^–1 maize vacuolar microsomal protein, and 0.6 mm F1,6BP. The initial rate of H⁺ pumping was taken in the linear phase of fluorescence quenching of ACMA and was expressed as percentage ΔF per minute after the addition of 1, 3, 8, and 18 milliunits of PFP to the cuvette (2 mL) in the absence (○) or in the presence (•) of 12 mm NaF or 2 μm FCCP (Δ). The figure shows a representative experiment. Similar results were obtained in three different vesicle preparations. The dotted line represents the maximal rate of H⁺ pumping measured when 0.3 mm PP_i was added to the reaction mixture in the absence of the PP_i-PFP regenerating system.

Figure 5.

Free-energy changes (ΔG) associated with the conversion of F1,6BP to build up a H⁺ gradient in tonoplast vesicles or with conversion to pyruvate by the glycolytic pathway. After Glc or Fru phosphorylation, the F1,6BP formed can be diverted to PP_i synthesis by PP_i-PFP (ΔG₁), and the PP_i is hydrolyzed by H⁺-PPase to build up a proton gradient of 2 units of pH in the tonoplast vesicles (ΔG₂). Alternatively, the F1,6BP is converted into triose-P by aldolase (ΔG₃). The triose-P is converted by glycolytic enzymes to pyruvate (ΔG₄). ΔG₁ and ΔG₂ were calculated as shown in Table II (condition A) using concentrations in the plant cell cytosol. The ΔG₃ and ΔG₄ were calculated from glycolytic reactions described by Kubota and Ashihara (1990).