Mitochondrial localization of human FAD synthetase isoform 1

作者：Torchetti Enza Maria; Brizio Carmen; Colella Matilde; Galluccio Michele; Giancaspero Teresa Anna; Indiveri Cesare; Roberti Marina; Barile Maria^*

来源：Mitochondrion, 2010, 10(3): 263-273.

DOI：10.1016/j.mito.2009.12.149

• FAD synthetase
• Mitochondria
• Riboflavin
• Intracellular trafficking
• Human

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 coli 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.

• 出版日期2010-4