The Reversible Antiport-Uniport Conversion of the Phosphate Carrier from Yeast Mitochondria Depends on the Presence of a Single Cysteine

Abstract

Wild type and mutant phosphate carriers (PIC) fromSaccharomyces 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 (P i ؊ / P i ؊ antiport and P i ؊ net transport, i.e. P i ؊ /OH ؊ antiport) and uncoupled uniport (mercuric chloride-induced P i ؊ 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.

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

“…The study of mitochondrial anion carriers has shown that limited modifications can convert carriers into channels. These modifications include manipulation of the ionic composition of the medium in the case of the ADP\ATP carrier [91], chemical modification in the case of the phosphate carrier [92], or site-directed mutagenesis in the case of UCP1 [93]. Following such modifications, a loss of specificity of the transport was noted, and sometimes the channel exhibited certain pore-like properties and allowed large molecules to go through.…”

α-Isopropylmalate, a Leucine Biosynthesis Intermediate in Yeast, Is Transported by the Mitochondrial Oxalacetate Carrier

“…In forward exchange kinetic measurements, the initial transport rate was calculated from the radioactivity taken up by the proteoliposomes after 1 min in the initial linear range of substrate uptake. In backward exchange experiments, the decrease in radioactivity inside the liposomes was fitted to the equation: ␣ ϭ 100 (1 Ϫ e Ϫkt ), where ␣ is the percentage of isotopic equilibrium (20). The rates were expressed as apparent velocities, i.e.…”

Co-precipitation of Phosphate and Iron Limits Mitochondrial Phosphate Availability in Saccharomyces cerevisiae Lacking the Yeast Frataxin Homologue (YFH1)

“…Inorganic phosphate, which is vital for oxidative phosphorylation, is transported to the mitochondrial matrix by two partly redundant carriers, Mir1 and Pic2 (22,23). Interestingly, Mir1 is also involved in protein import into mitochondria (24), and Pic2 can reversibly change its transport mode (antiport-uniport) according to the redox conditions (25). More generally, phosphate is involved in the homeostasis of several cations in different cellular compartments (26).…”