Characterizzation and functional analysis of the promoter of the human mitochondrial phosphate carrier gene

INTRODUCTION: The phosphate carrier (PiC) belongs to the mitochondrial carrier protein family. Its physiological role is to catalyze the transport of inorganic phosphate into the mitochondria. Only one PiC gene in man and cow, that gives rise to two alternatively spliced isoforms (A and B), has been detected (1). The recombinant, reconstituted isoforms exhibit similar substrate specificity and inhibitor sensitivity but differ in their kinetic parameters and tissue distribution (2). Until now nothing was known about the transcriptional control of the PiC gene. We have analyzed the promoter of the human PiC gene and identified an activation domain and an inhibition domain. MATERIALS AND METHODS: Transient transfection of Hela cells, EMSA and DNA footprinting were performed as described in ref. 3. Silencer proteins were purified by affinity chromatography and identified by mass spectrometry as described (3). RESULTS AND CONCLUSIONS: Through deletion analysis of the 5’ flanking regulatory region of the human PiC (-1213/-25) and transient transfection we have identified two domains in the promoter of the human PiC gene: an activation domain (-223/-25) and an inhibition domain (-1017/-814). The most effective promoter activity in transfected HeLa cells corresponded to the region containing putative binding sites for Sp1 (-163/-142) and CREB (-138/-116). These DNA sequences were active in gel shift assays in the presence of HeLa cell nuclear extracts or recombinant Sp1 and CREB, respectively. The CREB element is important for basal expression and for induced expression through the cAMP/protein kinase pathway. In fact, upon stimulation with foskolin, we observed an increase in the level of both PiC mRNA and PiC protein. Both footprinting and transfection of deletion constructs of the inhibition region (-1017/-814) showed that PiC silencer activity extends over 25 nts (-943/-919), which specifically binds two proteins present in HeLa cell nuclear extracts. These transcription factors were purified by DNA affinity, analyzed by mass spectrometry and identified as p54nrb/NonO and PSF. These findings may provide insight into the control of PiC gene expression in different cell types and under different growth conditions. 1. Dolce V., et al. (1994) J. Biol. Chem. 269, 10451-10460. 2. Fiermonte G., et al. (1998) J. Biol. Chem. 273, 22782-22787. 3. Iacobazzi V., et al. (2005) Biochem. J. 391, 613-621.