INTRODUCTION: The phosphate carrier (PiC) is a nuclear encoded protein that belongs to the mitochondrial carrier protein family. Its physiological role is to catalyze the transport of inorganic phosphate into the mitochondrial matrix (1). Uptake of phosphate into mitochondria is essential for the oxidative phosphorylation of ADP to ATP. Only one human gene for the PiC, that give rises to two alternatively spliced isoforms (A and B), has been detected.. The recombinant, reconstituted isoforms A and B exhibit similar substrate specificity and inhibitor sensitivity, but differ in their kinetic parameters and tissue distribution (2). We have analyzed the 5’-flanking region of the human PiC gene and have identified a single transcriptional initiation site, an activation domain and an inhibition domain (3). MATERIALS AND METHODS: Materilas and methods employed are reported in ref. 3. RESULTS: Through deletion analysis of the 5’ flanking regulatory region (-1213/-25 bp) and transient transfection of HeLa cells we have identified two distinct domains in the promoter of the PiC gene: an activation domain (-223/-25) and a inhibition domain (-1017/-814). The activation domain binds Sp1 and CREB with high specificity, as demonstrated by EMSA competion experiments. The CREB element is important not only for basal expression, but also for induced expression through the cAMP/protein kinase pathway. In fact, upon stimulation with foskolin, an activator of adenylate cyclase, we observed a clear increase in the level of both PiC mRNA and PiC protein and a two-fold activation of CAT expression. The silencer region was also characterized.. By Southwestern experiments two polypeptides (about 100 kDa and 55 KDa, respectively) were found to bind to a region of 25 nucleotides (from -943 to -919 bp). These two proteins were purified and identified as PSF (protein associated splicing factor) (100KDa) and p54 nrb/NonO (55KDa) by mass spectrometry. These findings may provide insight into the control of PiC gene expression. 1. Palmieri, F. (2004) Pflugers Arch. 446, 689-709 2. Fiermonte, G., Dolce, V., and Palmieri, F. (1998) J. Biol. Chem. 273, 22782-22787 3. Iacobazzi V, Infantino V, Costanzo P, Izzo P, and Palmieri F. (2005) Biochem. J., in press.

FUNCTIONAL ANALYSIS OF THE PROMOTER OF THE MITOCHONDRIAL PHOSPHATE CARRIER HUMAN GENE

INFANTINO, VITTORIA;
2005-01-01

Abstract

INTRODUCTION: The phosphate carrier (PiC) is a nuclear encoded protein that belongs to the mitochondrial carrier protein family. Its physiological role is to catalyze the transport of inorganic phosphate into the mitochondrial matrix (1). Uptake of phosphate into mitochondria is essential for the oxidative phosphorylation of ADP to ATP. Only one human gene for the PiC, that give rises to two alternatively spliced isoforms (A and B), has been detected.. The recombinant, reconstituted isoforms A and B exhibit similar substrate specificity and inhibitor sensitivity, but differ in their kinetic parameters and tissue distribution (2). We have analyzed the 5’-flanking region of the human PiC gene and have identified a single transcriptional initiation site, an activation domain and an inhibition domain (3). MATERIALS AND METHODS: Materilas and methods employed are reported in ref. 3. RESULTS: Through deletion analysis of the 5’ flanking regulatory region (-1213/-25 bp) and transient transfection of HeLa cells we have identified two distinct domains in the promoter of the PiC gene: an activation domain (-223/-25) and a inhibition domain (-1017/-814). The activation domain binds Sp1 and CREB with high specificity, as demonstrated by EMSA competion experiments. The CREB element is important not only for basal expression, but also for induced expression through the cAMP/protein kinase pathway. In fact, upon stimulation with foskolin, an activator of adenylate cyclase, we observed a clear increase in the level of both PiC mRNA and PiC protein and a two-fold activation of CAT expression. The silencer region was also characterized.. By Southwestern experiments two polypeptides (about 100 kDa and 55 KDa, respectively) were found to bind to a region of 25 nucleotides (from -943 to -919 bp). These two proteins were purified and identified as PSF (protein associated splicing factor) (100KDa) and p54 nrb/NonO (55KDa) by mass spectrometry. These findings may provide insight into the control of PiC gene expression. 1. Palmieri, F. (2004) Pflugers Arch. 446, 689-709 2. Fiermonte, G., Dolce, V., and Palmieri, F. (1998) J. Biol. Chem. 273, 22782-22787 3. Iacobazzi V, Infantino V, Costanzo P, Izzo P, and Palmieri F. (2005) Biochem. J., in press.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11563/22303
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