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

Abstract

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