| Abstract
| - The homodimeric mitochondrial phosphate transport protein (PTP),which has six transmembrane helices per subunit, catalyzes inorganic phosphate transport in anelectroneutral and pH gradient-dependent manner across the inner membrane. We have replaced theGlu, Asp, and His residues of theyeast PTP to assess their role in the transport mechanism. Mutantswith physiologically relevant transportactivity were identified by their ability to rescue the PTP null mutantyeast from glycerol medium. Fiveresidues appear critical for transport: His-32 in helix A, Glu-126and -137 in helix C, and Asp-39 and-236 at the matrix ends of helices A and E. These mutant PTPs areexpressed at near normal levels inyeast. This yeast PTP and the mutants were expressed inEscherichia coli as inclusion bodies,solubilized,purified, and reconstituted. Their transport activities correlatewell with the physiological assays. Noneof the transport inactivating mutations appear to be due to majorprotein conformation changes as assayedby the efficiency of PTP incorporation into liposomes. Only theGlu95Gln (cytosolic helices B andC-connecting segment), Glu163Gln and Glu164Gln (matrix helices C andD-connecting segment), andGlu126Asp (helix C) show a near 70% decrease in liposome incorporationefficiency. In addition, mutationsat either end of helix D increase phosphate transport 2-fold. Wewould like to suggest that Glu-126,His-32, and Glu-137 (similar to Asp-96, Lys-216, and Asp-85 ofbacteriorhodopsin) form a protoncotransport pathway that is coupled in an as yet undefined manner(possibly via His-32) to a phosphatetransport pathway, which may include helix D.
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