Expression in Escherichia coli, functional characterization, and tissue distribution of isoforms A and B of the phosphate carrier from bovine mitochondria.
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 (Pi/Pi antiport and Pi/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.
Fiermonte
G
G
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy.
Dolce
V
V
Palmieri
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001149:Arsenates / Q000494:pharmacology; D001483:Base Sequence; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D007536:Isomerism; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001149:Arsenates; D002352:Carrier Proteins; D018076:DNA, Complementary; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D011994:Recombinant Proteins; C025657:arsenic acid