The reversible antiport-uniport conversion of the phosphate carrier from yeast mitochondria depends on the presence of a single cysteine.
Wild type and mutant phosphate carriers (PIC) from Saccharomyces cerevisiae mitochondria were expressed in Escherichia coli as inclusion bodies, solubilized, purified, and optimally reconstituted into liposomal membranes. This PIC can function as coupled antiport (Pi-/Pi- antiport and Pi- net transport, i.e. Pi-/OH- antiport) and uncoupled uniport (mercuric chloride-induced Pi- efflux). The basic kinetic properties of these three transport modes were analyzed. The kinetic properties closely resemble those of the reconstituted PIC from beef heart mitochondria. A competitive inhibitor of phosphate transport by the PIC, phosphonoformic acid, was used to establish functional overlap between the the physiological transport modes and the induced efflux mode. Replacement mutants were used to relate the reversible switch from antiport to uniport to a specific residue of the carrier. There are only three cysteines in the yeast PIC. They are at positions 28, 134, and 300 and were replaced by serine, both individually and in combinations. Cysteine 300 near the C-terminal loop and cysteine 134 located within the third transmembrane segment are accessible to bulky hydrophilic reagents from the cytosolic side, whereas cysteine 28 within the first transmembrane segment is not. None of the three cysteines is relevant to the two antiport modes. Cysteine 134 was identified to be the major target of bulky SH reagents, that lead to complete inactivation of the physiological transport modes. The reversible conversion between coupled antiport and uncoupled uniport of the PIC depends on the presence of one single cysteine (cysteine 28) in the PIC monomer, i.e. two cysteines in the functionally active dimer. The consequences of this result with respect to a functional model of the carrier protein are discussed.
Schroers
A
A
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Krämer
R
R
Wohlrab
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D002352:Carrier Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D011994:Recombinant Proteins; D003545:Cysteine