Franco Zanotti

Structural and functional characterization of Fof1-ATP synthase on the extracellular surface of rat hepatocytes

Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-F(o)F(1)-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F(1) (alpha/beta and gamma) and F(o) (F(o)I-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF(1) is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H(+) and are inhibited by F(1) and F(o)-targeting inhibitors. We hypothesise that ecto-F(o)F(1)-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.

Age-dependent changes in the mitochondrial F0F1 ATP synthase

The age dependence of ATP hydrolase activity and oligomycin sensitive passive proton conduction in sonicated submitochondrial particles of rat brain and rat heart has been investigated. The results show an increase of Vmax of the ATP hydrolase activity and decrease of oligomycin sensitive passive proton conduction with increase of the age of rats from 3 to 6 months. Decrease of ATPase activity and increase of oligomycin sensitive proton conduction occur with further aging to 24 months. Immunoblot analysis shows that both the F(1) and F(0) contents of mitochondria vary with the age of rats, the former exhibiting relatively larger changes.

3,5-diiodo-L-thyronine upregulates rat-liver mitochondrial FoF1-ATP synthase by GA-binding protein/nuclear respiratory factor-2

Besides triiodothyronine (T3), 3,5-diiodo-L-thyronine (T2) has been reported to affect mitochondrial bioenergetic parameters. T2 effects have been considered as independent of protein synthesis. Here, we investigated the effect of in vivo chronic T2 administration to hypothyroid rats on liver mitochondrial F(o)F(1)-ATP synthase activity and expression. T2 increased state 4 and state 3 oxygen consumption and raised ATP synthesis and hydrolysis, which were reduced in hypothyroid rats. Immunoblotting analysis showed that T2 up-regulated the expression of several subunits (alpha, beta, F(o)I-PVP and OSCP) of the ATP synthase. The observed increase of beta-subunit mRNA accumulation suggested a T2-mediated nuclear effect. Then, the molecular basis underlying T2 effects was investigated. Our results support the notion that the beta-subunit of ATP synthase is indirectly regulated by T2 through, at least in part, the activation of the transcription factor GA-binding protein/nuclear respiratory factor-2. These findings provide new insights into the T2 role on bioenergetic mechanisms.

New aspects of the mechanism of inorganic phosphate and dicarboxylate transport in mitochondria

1. Introduction It is generally accepted that the transport of inor- ganic phosphate in mitochondria is mediated by two distinct translocating systems localized in the inner membrane [l-7]. The first mediates the exchange- diffusion of Pi with OH-(phosphate carrier) and the second that of Pi with dicarboxylates (dicarboxyla- te carrier). The observation that Pi -Pi exchange is inhibited when both N-ethylmaleimide (NEM) and butylmalonate (BM) are present [4,7], together with the finding that dicarboxylate-dicarboxylate or Pi- dicarboxylate exchanges are not inhibited by NEM [8,9], are considered as main evidence that the two carriers are well distinct functional entities. We have however, found that butymalonate interfers with the mechanism of action of NEM since it removes or pre- vents in intact mitochondria the extrabinding of Pi in- duced by NEM [lo]. These findings have prompted us to reinvestigate the effect of NEM and BM on the transport of Pi and dicarboxylic acids in rat-liver mito- chondria. The results reported in this paper indicate that the mitochondrial membrane contains only one transporting system which, depending on the prevailing conditions, mediates either Pi-OH-or Pi-dicarboxy- late exchange. 2. Methods Rat-liver mitochondria were loaded with Pi, malate or succinate by preincubation for 10 min at 4” C in the presence of 250 mM sucrose, 5 I.cg/ml oligomycin, 0.34 m 200 mM sucrose; 20 mM Tris-HCI; oligomycin; rotenone and antimycin at the concentrations indicated above. The pH was ad- justed to 7.2. At the time specified in the legends nige- kin, butylmalonate, N-ethylmaleimide and the coun- ter-anions were added. Incubation was carried out at 4°C directly in small centrifuge tubes in a total vol of 1 ml and mitochondria separated from the suspen- ding medium by rapid centrifugation at 20 000 g. Before incubation Pi was determined chemically [ 1 l] and malate enzymatically [ 121. Their specific activity was calculated by measuring the corresponding total radioactivity of 12% HC104 extracts of loaded mito- chondria. Pi, malate and [14C] succinate content of mitochondrial pellet was corrected for that present in the sucrose space. 3. Results The sensitivity of the Pi carrier to NEM and BM was studied by following the efflux of P, from mito- chondria induced by the addition of nigericin [6]. Fig. 1 shows that NEM at 200 PM fully inhibited the Pi-OH-exchange whilst BM, even at a very high con- centration as 5 mM (not shown), had no effect at all. However when these two compounds were added to- gether different results, according to the sequence of additions and NEM concentration, were obtained. BM, added after NEM, increased the inhibition of Pi efflux

Effect of the ATPase inhibitor protein IF1 on H+ translocation in the mitochondrial ATP synthase complex

The H(+) F(o)F(1)-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F(1) moiety of the complex protrudes at the inner side of the membrane, the F(o) sector spans the membrane reaching the outer side. The IF(1) component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F(o)F(1)-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF(1) (residue 42-58) to the F(1)-alpha/beta subunits. We have analyzed the effect of native purified IF(1) the IF(1)-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K(+)] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F(o)F(1) complex. The results show that IF(1), and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F(1) to the F(o) side and in the opposite direction. Cross-linking of IF(1) to F(1)-alpha/beta subunits inhibits the ATP-driven H(+) translocation but enhances H(+) conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F(o)F(1) complex.

3,5-Diiodo-L-Thyronine Administration To Hypothyroid Rats Rapidly Enhances Fatty Acid Oxidation Rate and Bioenergetic Parameters in Liver Cells

Growing evidence shows that, among triiodothyronine derivatives, 3,5 diiodo-L-thyronine (T2) plays an important role in energy metabolism and fat storage. In the present study, short-term effects of T2 administration to hypothyroid rats on fatty acid oxidation rate and bioenergetic parameters were investigated. Within 1 h following T2 injection, state 3 and state 4 respiration rates, which were reduced in hypothyroid mitochondria, were noticeably increased particularly in succinate- with respect to glutamate/malate-energized mitochondria. Maximal respiratory activity, observed when glutamate/malate/succinate were simultaneously present in the respiratory medium, was significantly stimulated by T2 treatment. A T2-induced increase in respiratory rates was also observed when palmitoyl-CoA or L-palmitoylcarnitine were used as substrates. No significant change in respiratory control index and ADP/O ratio was observed. The activities of the mitochondrial respiratory chain complexes, especially Complex II, were increased in T2-treated rats. In the latter, Complex V activities, assayed in both ATP synthesis and hydrolysis direction, were enhanced. The rate of fatty acid oxidation, followed by conversion of [14C]palmitate to CO2 and ketone bodies, was higher in hepatocytes isolated from T2-treated rats. This increase occurs in parallel with the raise in the activity of carnitine palmitoyltransferase-I, the rate limiting enzyme of fatty acid β-oxidation, assayed in situ in digitonin-permeabilized hepatocytes. Overall, these results indicate that T2 rapidly increases the ability of mitochondria to import and oxidize fatty acids. An emerging idea in the literature is the ability of T2 to reduce adiposity and dyslipidemia and to prevent the development in liver steatosis. The results of the present study, showing a rapid T2-induced increase in the ability of mitochondria to import and oxidize fatty acids, may contribute to understand the biochemical mechanisms of T2-metabolic effects.

The γ subunit of F1 and the PVP protein of Fo (FoI) are components of the gate of the mitochondrial FoF1 H+-ATP synthase

The γ subunit of the F1 moiety of the bovine mitochondrial H+‐ATP synthase is shown to function as a component of the gate. Addition of purified γ subunit to Fo‐liposomes inhibits transmembrane proton conduction. This inhibition can be removed by the bifunctional thiol reagent diamide. Immunoblot analysis shows that the diamide effect is likely due to disulphide bridging of the γ subunit with the PVP protein of the Fo sector.

Mitochondrial F0F1 H+-ATP synthase. Characterization of F0 components involved in H+ translocation.

The membrane F0, sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F1‐depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin‐sensitive proton conduction, or recombined with purified F1 to give the oligomycin‐sensitive F0F1‐ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDS‐polyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F0 polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD‐binding protein, the nuclear encoded protein [(1987) J. Mol. Biol. 197, 89–100], characterized as an intrinsic component of F0, (F0I, PVP protein [(1967) J. Biol. Chem. 242, 2547–2551]) is involved in H+ translocation and the sensitivity of this process to the F0 inhibitors, DCCD and oligomycin.

Regulatory role of the ATPase inhibitor protein on proton conduction by mitochondrial H+-ATPase complex

This study shows that the natural inhibitor protein of mitochondrial H+‐ATPase complex (IF1) inhibits, in addition to the catalytic activity, the proton conductivity of the complex. The inhibition of ATPase activity by IF1 is less effective in the purified F1 than in submitochondrial particles where F1 is bound to F0. No inhibition of H+ conductivity by F0 is observed in F1‐depleted particles

3,5-diiodo-L-thyronine increases FoF1-ATP synthase activity and cardiolipin level in liver mitochondria of hypothyroid rats.

Short-term effects of 3,5-L-diiodothyronine (T2) administration to hypothyroid rats on FoF1-ATP synthase activity were investigated in liver mitochondria. One hour after T2 injection, state 4 and state 3 respiration rates were noticeably stimulated in mitochondria subsequently isolated. FoF1-ATP synthase activity, which was reduced in mitochondria from hypothyroid rats as compared to mitochondria from euthyroid rats, was significantly increased by T2 administration in both the ATP-synthesis and hydrolysis direction. No change in β-subunit mRNA accumulation and protein amount of the α-β subunit of FoF1-ATP synthase was found, ruling out a T2 genomic effect. In T2-treated rats, changes in the composition of mitochondrial phospholipids were observed, cardiolipin (CL) showing the greatest alteration. In mitochondria isolated from hypothyroid rats the decrease in the amount of CL was accompanied by an increase in the level of peroxidised CL. T2 administration to hypothyroid rats enhanced the level of CL and decreased the amount of peroxidised CL in subsequently isolated mitochondria, tending to restore the CL value to the euthyroid level. Minor T2-induced changes in mitochondrial fatty acid composition were detected. Overall, the enhanced FoF1-ATP synthase activity observed following injection of T2 to hypothyroid rats may be ascribed, at least in part, to an increased level of mitochondrial CL associated with decreased peroxidation of CL.