Inorganic polyphosphate is produced and hydrolyzed in F0F1-ATP synthase of mammalian mitochondria

Abstract

Inorganic polyphosphate (polyP) is a polymer present in all living organisms. Although polyP is found to be involved in a variety of functions in cells of higher organisms, the enzyme responsible for polyP production and consumption has not yet been identified. Here, we studied the effect of polyP on mitochondrial respiration, oxidative phosphorylation and activity of F0F1-ATPsynthase. We have found that polyP activates mitochondrial respiration which does not coupled with ATP production (V2) but inhibits ADP-dependent respiration (V3). Moreover, PolyP can stimulate F0F1-ATPase activity in the presence of ATP and, importantly, can be hydrolyzed in this enzyme instead of ATP. Furthermore, PolyP can be produced in mitochondria in the presence of substrates for respiration and phosphate by the F0F1-ATPsynthase. Thus, polyP is an energy molecule in mammalian cells which can be produced and hydrolyzed in the mitochondrial F0F1-ATPsynthase.

Keywords: F0-F1-ATPase; bioenergetics; inorganic polyphosphates; mitochondria.

Figure 1.. Effect of polyP on mitochondrial respiration.

(А) Application of 100, 10 and 5 µM of polyP caused activation of mitochondrial respiration V₂ (V_sub); (B) application of 100 µM of polyP, decreased effect of ADP (V₃), eliminated V₄ state and did not change the effect of CCCP, compare to control; (C) application of 10 µM of polyP activated the V₂ (V_sub) state comparable to 100 µM, (D) application of 5 µM of polyP caused activation of mitochondrial respiration in V₂ (V_sub), respiration rate V₃ state was decreased compared with control. (E) PolyP-induced changes in the respiration rate of isolated mitochondria. The respiration rates in V₂ of control samples was taken as 100%. Application of polyP caused concentration-dependent decrease in RC and P/O ratios compared with control. Respiration rate in % and RC and P/O ratios in arbitrary units, were placed in the center of each bar chart. n = 110 for V₂ in control, n = 55 for control (all other states of respiration), n = 11 for 100 µM of polyP, n = 17 for 10 µM of polyP, n = 12 for 5 µM of polyP. **P < 0.01, NS, not significant.

Figure 2.. Hydrolyzing activity of mitochondrial F₁F₀ ATP-synthase in the presence of inorganic polyphosphates.

(A) ATPase activity of F₁F₀ in the presence of 1 mM of ATP with or without 2 µg/ml oligomycin; (B–E) presence of 1 mM of S, M and LpolyP caused activation of hydrolyzing activity of F₁F₀ in the absence of ATP on 22.9 ± 4.8; 25.5 ± 7 and 29.9 ± 6.2%, respectively, and can be blocked by 2 µg/ml oligomycin (D); (F,G) co-application of ATP and polyP increased the rate of acidulation of the buffer additively to 254 ± 25.6%; 258.2 ± 20.5% and 247.1 ± 14.7 for S polyP, M polyP and L polyP, respectively. **P < 0.01.

Figure 3.. PolyP is a substitute for ATP in a F0F1-ATPase assay.

(A) Short-chain polyP did not increase the ATPase activity, whereas MpolyP (B) and LpolyP (C) at both 5 and 10 mM concentrations, significantly decreased the absorbance at 340 nm overtime, corresponding to an increased consumption of polyP by the F0F1-ATPase (raw traces). 2 µg/ml oligomycin inhibit the effects of MpolyP (B) or LpolyP (C) on the F₀F₁-ATPase activity. (D) Quantification diagram of the rate of ATPase activity, shown as OD/min of the experiments, partially represented on (A C). ***, P < 0.0001.

Figure 4.. Production of polyP in isolated mitochondria.

Measurements of polyP in isolated mitochondria using polyP-DAPI fluorescence. (A) Application of 5 mM succinate in the presence of rotenone (10 µM) induce increase in polyP level, which can be promoted by additional PO₄ and blocked by oligomycin (2 µg/ml). Importantly, the level of polyP in mitochondria was independent of the application of ATP before (B) or after oligomycin (A). (C) The polyP level in mitochondria can be increased by application of substrates of complex I 5 mM glutamate/5 mM malate, inhibited by rotenone (10 µM) and further activated by 5 mM succinate. (D–E) Effects of substrates (D-5 mM glutamate/5 mM malate; E-5 mM succinate) on polyP cold be blocked by oligomycin (2 µg/ml).