Phosphate transport in rat-liver mitochondria.

Vesicles have been prepared by mild sonication of a purified inner membrane fraction from rat liver mitochondria. Such vesicles demonstrate an energy-dependent net concentrative uptake of inorganic phosphate in the absence of added Na+, Ca’+, or ionophores. Phosphate uptake can be supported by a variety of substrates including /?-hydroxybutyrate, succinate, and ascorbate + tetramethylphenylenediamine. These inner membrane vesicles have a membrane orientation opposite to that of intact mitochondria as shown by six different types of experiments: negative staining with phosphotungstic acid under hypotonic conditions reveals 90 A F1-ATPase particles only on the membrane periphery; an ATPase inhibitor peptide too large to permeate the inner membrane inhibits ATPase activity of the vesicles by greater than 90%; atractyloside, which prevents ATP from reaching the F1-ATPase in intact mitochondria (i.e. by inhibiting exchange of external ATP for internal ADP), has no effect on the ATPase activity of vesicles preloaded with ADP; hydroxyl ion ejection and uptake of lipophilic anions take place when respiration is supported by succinate; and finally, p-chloromercuribenzoate and mersalyl almost completely inhibit /3-hydroxybutyrate dehydrogenase activity under conditions where these agents are shown not to cross the inner mitochondrial membrane. Phosphate uptake by these inverted inner membrane vesicles is inhibited by uncoupling agents, by p-chloromercuribenzoate and mersalyl, and by the membrane-permeable sulfhydryl reagent N-ethylmaleimide. In contrast, both n-butyl malonate, a specific inhibitor of the phosphate/dicarboxylate exchange system, and atractyloside, a specific inhibitor of the adenine nucleotide exchange system fail to inhibit phosphate uptake. Results presented here show that energy-dependent phosphate movement can take place from the matrix surface to the cytoplasmic surface of the mitochondrial inner membrane of rat liver. This activity may be the expression of an electrogenically driven uniport process and represent either a different mode of action of the phosphate/hydroxyl antiporter, which is known to transport phosphate into mitochondria, or a different system for phosphate transport.