Mitochondrial Phosphate Transport Protein. Replacements of Glutamic, Aspartic, and Histidine Residues Affect Transport and Protein Conformation and Point to a Coupled Proton Transport Path

Abstract

The homodimeric mitochondrial phosphate transport protein (PTP), which has six transmembrane helices per subunit, catalyzes inorganic phosphate transport in an electroneutral and pH gradient-dependent manner across the inner membrane. We have replaced the Glu, Asp, and His residues of the yeast PTP to assess their role in the transport mechanism. Mutants with physiologically relevant transport activity were identified by their ability to rescue the PTP null mutant yeast from glycerol medium. 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 of helices A and E. These mutant PTPs are expressed at near normal levels in yeast. This yeast PTP and the mutants were expressed in Escherichia coli as inclusion bodies, solubilized, purified, and reconstituted. Their transport activities correlate well with the physiological assays. 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 and D-connecting segment), and Glu126Asp (helix C) show a near 70% decrease in liposome incorporation efficiency. In addition, mutations at either end of helix D increase phosphate transport 2-fold. We would like 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 pathway that is coupled in an as yet undefined manner (possibly via His-32) to a phosphate transport pathway, which may include helix D.

The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP

“…It has been suggested that UCP1 was derived from an anion\proton symporter [39]. The membrane potential of respiring mitochondria opposes the net accumulation of anions in this organelle, and therefore proton co-transport would ensure electroneutrality, as with the phosphate carrier [97]. The binding of a nucleotide to UCP1 induces a conformational change that results in the marked stability of the interaction of the nucleotide with UCP1, and is consistent with the hypothesis of an aborted transport of nucleotide.…”

“…In theory, proton transport by UCP1 contradicts its relationship with anion carriers. However, co-transport of a proton by the mitochondrial phosphate carrier ensures electroneutrality [97], or makes the exchange sensitive to the electrochemical gradient across the inner membrane in case of the aspartate\glutamate carrier [98]. Therefore proton pathways existed in mitochondrial carriers before the appearance of UCP1 in mammals.…”

Natural and Azido Fatty Acids Inhibit Phosphate Transport and Activate Fatty Acid Anion Uniport Mediated by the Mitochondrial Phosphate Carrier

“…We found that AzNPP i together with diphosphonates belongs to nontransportable P i analogs. Thus, a specific gate of PIC does not allow sulfate and tungstate to pass (7), and, on the other hand, it is able to accommodate the methyl group and part of the phosphonomethyl and phosphonomethylimino groups, or a phenyl attached to the phosphate. Wohlrab et al (7) hypothesized that some acid residues of yeast PIC, such as Glu 163 , Glu 164 , Glu 192 , and Glu 196 might ensure specificity for phosphate and exclude interaction with sulfate.…”

“…Thus, a specific gate of PIC does not allow sulfate and tungstate to pass (7), and, on the other hand, it is able to accommodate the methyl group and part of the phosphonomethyl and phosphonomethylimino groups, or a phenyl attached to the phosphate. Wohlrab et al (7) hypothesized that some acid residues of yeast PIC, such as Glu 163 , Glu 164 , Glu 192 , and Glu 196 might ensure specificity for phosphate and exclude interaction with sulfate. Interestingly, glutamate Glu 190 on UCP1 conveys the pH dependence of nucleotide di-and triphosphates binding to this protein (44).…”

“…It was thought to mediate a stoichiometric P i *H ϩ symport (7,13), but alternatively, a P i / OH Ϫ antiport has been proposed to be the most plausible mode (13,14). In addition to monovalent phosphate (15), arsenate and divalent monofluorophosphate (16) are also translocated.…”