Interaction of mitochondrial phosphate carrier with fatty acids and hydrophobic phosphate analogs

Mitochondrial transporters, in particular uncoupling proteins and the ADP/A TP carrier, are known to mediate uniport of anionic fatty acids (FAs), allo wing FA cycling which is completed by the passive movement of FAs across th e membrane in their protonated form. This study investigated the ability of the mitochondrial phosphate carrier to catalyze such a mechanism and, furt hermore, how this putative activity is related to the previously observed H gCl2-induced uniport mode. The yeast mitochondrial phosphate carrier was ex pressed in Escherichia coli and then reconstituted into lipid vesicles. The FA-induced H+ uniport or Cl- uniport were monitored fluorometrically after HgCl2 addition. These transport activities were further characterized by t esting various inhibitors of the two different transport modes. The phospha te carrier was found to mediate FA cycling, which led to H+ efflux in prote oliposomes. This activity was insensitive to ATP, mersalyl or N-ethylmaleim ide and was inhibited by methylenediphosphonate and iminodi(methylenephosph onate), which are new inhibitors of mitochondrial phosphate transport. Also , the HgCl2 induced Cl- uniport mediated by the reconstituted yeast PIC, wa s found to be inhibited by these reagents. Both methylenediphosphonate and iminoai(methylenephosphonate) blocked unidirectional Cl- uptake , whereas C l- efflux was inhibited by iminodi(methylenephosphonate) and phosphonoformi c acid only. These results suggest that a hydrophobic domain, interacting w ith FAs, exists in the mitochondrial phosphate carrier, which is distinct f rom the phosphate transport pathway. This domain allows for FA anion unipor t via the phosphate carrier and consequently, FA cycling that should lead t o uncoupling in mitochondria. This might be considered as a side function o f this carrier. (C) 2000 Elsevier Science Ltd. All rights reserved.