Functional analysis of the promoter of the mitochondrial phosphate carrier human gene: identification of activator and repressor elements and their transcription factors (original) (raw)
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Biogenesis of the mitochondrial phosphate carrier
European Journal of Biochemistry, 1991
The mitochondrial phosphate carrier (Pic) is a member of the family of inner-membrane carrier proteins which are generally synthesized without a cleavable presequence. Surprisingly, the cDNA sequences of bovine and rat P i c suggested the existence of an amino-terminal extension sequence in the precursor of Pic. By expressing P i c in vitro, we found that PIC is indeed synthesized as a larger precursor. This precursor was imported and proteolytically processed by mitochondria, whereby the correct amino-terminus of the mature protein was generated. Import of P i c showed the characteristics of mitochondrial protein uptake, such as dependence on ATP and a membrane potential and involvement of contact sites between mitochondrial outer and inner membranes. The precursor imported in vitro was correctly assembled into the functional form, demonstrating that the authentic import and assembly pathway of P i c was reconstituted when starting with the presequence-carrying precursor. These results are discussed in connection with the recently postulated role of P i c as an import receptor located in the outer membrane.
Journal of Biological Chemistry, 1998
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.
Novel regulatory enhancer in the nuclear gene of the human mitochondrial ATP synthase beta-subunit
The Journal of biological chemistry, 1990
The system coordinating expressions of nuclear coded mitochondrial proteins was investigated by examination of the 5'-flanking region of the human mitochondrial ATP synthase beta-subunit gene. The promoter activity was measured by a transient expression of a chloramphenicol acetyltransferase (CAT) gene connected with various 5'-deletion mutants of the 5'-flanking region. In this experiment, at least two regions enhanced this promoter activity and at least one region repressed it. In one of the enhancing regions, a consensus sequence was found for the genes of other mitochondrial proteins such as those for cytochrome c1 (Suzuki, H., Hosokawa, Y., Nishikimi, M., and Ozawa, T. (1989) J. Biol. Chem. 264, 1368-1374) and the pyruvate dehydrogenase alpha-subunit (Maragos, C., Hutchison, W. M., Hayasaka, K., Brown, G. K., and Dahl, H.-H. M. (1989) J. Biol. Chem. 264, 12294-12298; Ohta, S., Endo, H., Matsuda, K., and Kagawa, Y. (1989) Ann. N. Y. Acad. Sci. 573, 458-460). The char...
FEBS Letters, 1996
Comparison of the sequence of the human mitochondrial phosphate carrier (PiC) gene with cDNA clones characterised from a human heart cDNA library suggested the existence of two isoforms of the PiC, which were generated by alternative splicing of exon IIIA or exon IIIB and which differed in 13 amino acids [Dolce et al. (1994) J. Biol. Chem. 269,10451]. In this work the expression of isoforms A and B of the PiC was investigated in different bovine tissues by Northern blot analysis using two probes that are specific for bovine exon IIIA and exon IIIB, respectively. Isoform A is highly expressed in heart and skeletal muscle. Isoform B is ubiquitously expressed in all tissues that were examined, although at different levels. The tissuespecific expression pattern of the two PiC isoforms is similar to that reported for the isoforms of several mitochondrial proteins required for energy production.
Mitochondrion, 2017
The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is in...
Biochemical and biophysical research communications, 2015
The mitochondrial phosphate carrier (PiC) is a mitochondrial solute carrier protein, which is encoded by SLC25A3 in humans. PiC delivers phosphate, a key substrate of oxidative phosphorylation, across the inner mitochondrial membrane. This transport activity is also relevant for allowing effective mitochondrial calcium handling. Furthermore, PiC has also been described to affect cell survival mechanisms via interactions with cyclophilin D and the viral mitochondrial-localized inhibitor of apoptosis (vMIA). The significance of PiC has been supported by the recent discovery of a fatal human condition associated with PiC mutations. Here, we present first the early studies that lead to the discovery and molecular characterization of the PiC, then discuss the very recently developed mouse models for PiC and pathological mutations in the human SLC25A3 gene.
Phosphate transport in mitochondria: Past accomplishments, present problems, and future challenges
Journal of Bioenergetics and Biomembranes, 1993
The requirement of inorganic phosphate (Pi) for oxidative phosphorylation in eukaryotic cells is fulfilled through specific Pi transport systems. The mitochondrial proton/phosphate symporter (Pi c) is a membrane-embedded protein which translocates Pi from the cytosol into the mitochondrial matrix. Pi c is responsible for the very rapid transport of most of the Pi used in ATP synthesis. During the past five years there have been advances on several fronts. Genomic and cDNA clones for yeast, bovine, rat, and human Pi c have been isolated and sequenced. Functional expression of yeast Pi c in yeast strains deficient in Pi transport and expression in Eseherichia coli of a chimera protein involving Pi c and ATP synthase c~ subunit have been accomplished. Pi c, in contrast to other members of the family of transporters involved in energy metabolism, was demonstrated to have a presequence, which optimizes the import of the precursor protein into mitochondria. Six transmembrane segments appear to be a structural feature shared between Pic and other mitochondrial anion carriers, and recent-site directed mutagenesis studies implicate structure-functional relationships to bacteriorhodopsin. These recent advances on Pic will be assessed in light of a more global interpretation of transport mechanism across the inner mitochondrial membrane.
2012
The GC-box is an important transcriptional regulatory element present in the promoters of many mammalian genes, and is found in most, if not all, oxidative phosphorylation (OXPHOS) promoters. In the present study we examine the effects of three Sp1 family members (Sp2, Sp3, and Sp4) on the adenine nucleotide translocase 2, cytochrome c1, F1-ATPase β-subunit, and the mitochondria transcription factor (mtTFA) promoters in Drosophila SL2 cell line. Sp3, like Sp1, strongly activates transcription all four promoters. SP4 stimulates, moderately, but Sp2 had no effect. In addition, Sp3 can, like Sp1, inhibit transcription from the proximal promoter of the ANT2 gene through binding to the Cbox GC element. By contrast, Sp4 and Sp2 do not repress promoter activity. Furthermore, since Sp4 and Sp2 bind to the Cbox repression element on the ANT2 promoter, but do not repress transcription, inhibition of transcription cannot be explained by steric hindrance of pre-initiation complex assembly. Thes...
… of bioenergetics and …, 1993
Phosphate and oxoglutarate carriers transport phosphate and oxoglutarate across the inner membranes of mitochondria in exchange for OH-and malate, respectively. Both carriers belong to the mitochondrial carrier protein family, characterized by a tripartite structure made up of related sequences about 100 amino acids in length. The results obtained on the topology of the phosphate and oxoglutarate carriers are consistent with the six a-helix model proposed by Saraste and Walker. In both carriers the N-and C-terminal regions are exposed toward the cytosol. In addition, the oxoglutarate cartier has been shown to be a dimer by means of crosslinking studies. The bovine and human genes coding for the oxoglutarate carrier are split into eight and six exons, respectively, and five introns are found in the same position in both genes. The bovine and human phosphate carrier genes have the same organization with nine exons separated by eight introns at exactly the same positions. The phosphate carrier of mammalian mitochondria is synthesized with a cleavable presequence, in contrast to the oxoglutarate Carrier and the other members of the mitochondrial carrier family. The precursor of the phosphate carrier is efficiently imported, proteolytically processed, and correctly assembled in isolated mitoehondria. The presequence-deficient phosphate cartier is imported with an efficiency of about 50% as compared with the precursor of the phosphate carrier and is correctly assembled, demonstrating that the mature portion of the phosphate cartier contains sufficient information for import and assembly into mitochondria.