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 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.
Phelps
A
A
Boston Biomedical Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Briggs
C
C
Mincone
L
L
Wohlrab
H
H
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D017136:Ion Transport; D008969:Molecular Sequence Data; D016296:Mutagenesis; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D011522:Protons; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D011522:Protons; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine