FAD synthetase or ATP:FMN alenylyltransferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS-PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coil showed apparent K(m) and V(max) values for FMN equal to 1.3 +/- 0.7 mu M and 4.4 +/- 1.3 nmol x min(-1) x mg protein(-1), respectively, and was inhibited by FMN at concentration higher than 1.5 mu M. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Mitochondrial localization of human FAD synthetase isoform 1

GALLUCCIO, Michele;INDIVERI, Cesare;
2010

Abstract

FAD synthetase or ATP:FMN alenylyltransferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS-PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coil showed apparent K(m) and V(max) values for FMN equal to 1.3 +/- 0.7 mu M and 4.4 +/- 1.3 nmol x min(-1) x mg protein(-1), respectively, and was inhibited by FMN at concentration higher than 1.5 mu M. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.11770/156857
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