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 μM, and the PPi concentration needed for one-half maximal activity of the maize (Zea mays) root tonoplast H+-pyrophosphatase is 30 μM. 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 → 2Pi (reaction 2) H+cyt → H+vac (reactions 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 (PP i) hydrolyzed. A proton gradient (ΔpH) can be built up in tonoplast vesicles using PFP as a PPi-regenerating system. The Δ 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 Δ pH attained by the pyrophosphatase coupled to the PFP reaction can be maintained by PFP activities far below those found in higher plants tissues.