MITOCHONDRIAL PHOSPHATE-TRANSPORT PROTEIN - REPLACEMENTS OF GLUTAMIC,ASPARTIC, AND HISTIDINE-RESIDUES AFFECT TRANSPORT AND PROTEIN CONFORMATION AND POINT TO A COUPLED PROTON TRANSPORT PATH

The homodimeric mitochondrial phosphate transport protein (PTP), which has six transmembrane helices per subunit, catalyzes inorganic phosph ate transport in an electroneutral and pH gradient-dependent manner ac ross the inner membrane. We have replaced the Glu, Asp, and His residu es of the yeast PTP to assess their role in the transport mechanism. M utants with physiologically relevant transport activity were identifie d by their ability to rescue the PTP null mutant yeast from glycerol m edium. Five residues appear critical for transport: His-32 in helix A, Glu-126 and -137 in helix C, and Asp-39 and -236 at the matrix ends o f helices A and E. These mutant PTPs are expressed at near normal leve ls in yeast, This yeast PTP and the mutants were expressed in Escheric hia coli as inclusion bodies, solubilized, purified, and reconstituted . Their transport activities correlate well with the physiological ass ays. None of the transport inactivating mutations appear to be due to major protein conformation changes as assayed by the efficiency of PTP incorporation into liposomes. Only the Glu95Gln (cytosolic helices B and C-connecting segment), Glu163Gln and Glu164Gln (matrix helices C a nd D-connecting segment), and Glu126Asp (helix C) show a near 70% decr ease in liposome incorporation efficiency. In addition, mutations at e ither end of helix D increase phosphate transport 2-fold. We would lik e to suggest that Glu-126, His-32, and Glu-137 (similar to Asp-96, Lys -216, and Asp-85 of bacteriorhodopsin) form a proton cotransport pathw ay that is coupled in an as yet undefined manner (possibly via His-32) to a phosphate transport pathway, which may include helix D.