Functional properties of the reconstituted phosphate carrier from bovine heart mitochondria: Evidence for asymmetric orientation and characterization of three different transport modes

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The two isoforms of the mammalian mitochondrial phosphate carrier (PiC), A and B, differing in the sequence near the N terminus, arise from alternative splicing of a primary transcript of the PiC gene (Dolce, V., Iacobazzi, V., Palmieri, F., and Walker, J. E. (1994) J. Biol. Chem. 269, 10451-10460). To date, the PiC isoforms A and B have not been studied at the protein level. To explore the tissue-distribution and the potential functional differences between the two isoforms, polyclonal site-directed antibodies specific for PiC-A and PiC-B were raised, and the two bovine isoforms were obtained by expression in Escherichia coli and reconstituted into phospholipid vesicles. Western blot analysis demonstrated that isoform A is present in high amounts in heart, skeletal muscle, and diaphragm mitochondria, whereas isoform B is present in the mitochondria of all tissues examined. Heart and liver bovine mitochondria contained 69 and 0 pmol of PiC-A/mg of protein, and 10 and 8 pmol of PiC-B/mg of protein, respectively. In the reconstituted system the pure recombinant isoforms A and B both catalyzed the two known modes of transport (P i /P i antiport and P i /H ؉ symport) and exhibited similar properties of substrate specificity and inhibitor sensitivity. However, they strongly differed in their kinetic parameters. The transport affinities of isoform B for phosphate and arsenate were found to be 3-fold lower than those of isoform A. Furthermore, the maximum transport rate of isoform B is about 3-fold higher than that of isoform A. These results support the hypothesis that the sequence divergence between PiC-A and PiC-B may have functional significance in determining the affinity and the translocation rate of the substrate through the PiC molecule.The transport of inorganic phosphate across the inner mitochondrial membrane into the matrix compartment is essential for the oxidative phosphorylation of ADP to ATP. This transport is catalyzed by the phosphate carrier (PiC) 1 which has been purified from different sources and reconstituted into liposomes in an active form (1-4). In the reconstituted system the native bovine PiC protein catalyzes the P i /H ϩ symport as well as the P i /P i exchange, which functions by a sequential mechanism (5). The primary structure of the mature PiC is made up of three tandemly related domains about 100 amino acids in length (6). These repetitive elements are related to those found in the other well characterized members of the mitochondrial carrier family (see Refs. 7-10 for reviews) (11,12). They are also found in a number of other proteins of known sequence but of unknown function, which therefore belong to the same protein superfamily (7-10). By examination of the transmembrane topography of the PiC in the inner mitochondrial membrane, it has been proposed that both the N-and C-terminal regions of the PiC protrude toward the cytosol and that the polypeptide chain spans the membrane six times (13). Only one gene for the PiC has been detected in man and cow (14), of which the human one has...

Section: Constants Of Recombinant Pic-a and Pic-b Isoformsmentioning

confidence: 39%

Section: Construction Of Expression Plasmids Coding For Pic Isoforms mentioning

confidence: 99%

Section: Construction Of Expression Plasmids Coding For Pic Isoforms mentioning

confidence: 99%

See 1 more Smart Citation

Expression in Escherichia coli, Functional Characterization, and Tissue Distribution of Isoforms A and B of the Phosphate Carrier from Bovine Mitochondria

Fiermonte¹,

Dolce²,

Palmieri

1998

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125

“…Its mechanism is of the simultaneous (sequential) type, involving a ternary complex in transport catalysis that requires the binding of two ligands at the same time (Stappen and Krä mer, 1994). An additional property that places the PIC into this functional family is its ability to switch to uniport (efflux) activity after chemical modification with some mercurial reagents (Stappen and Krä mer, 1993). It was previously shown that the aspartate/glutamate carrier (Dierks et al, 1990a), the ADP/ATP carrier (Dierks et al, 1990b), and the carnitine carrier (Indiveri et al, 1991) can reversibly be converted by mercurial reagents from coupled antiport to uncoupled uniport, a function that comprises both carrier-like and channel-like properties.…”

mentioning

confidence: 99%

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

Schroers

Krämer

Wohlrab

1997

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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.

“…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.…”

mentioning

confidence: 99%

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

Engstová¹,

Žáčková²,

Růžička³

et al. 2001

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