Mitochondrial localization of human FAD synthetase isoform 1.
FAD synthetase or ATP:FMN adenylyl transferase (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 microM 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 microM. 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.
Torchetti
Enza Maria
EM
Dipartimento di Biochimica e Biologia Molecolare E. Quagliariello, Università degli Studi di Bari, Via Orabona 4, I-70126 Bari, Italy. em.torchetti@biologia.uniba.it
Brizio
Carmen
C
Colella
Matilde
M
Galluccio
Michele
M
Giancaspero
Teresa Anna
TA
Indiveri
Cesare
C
Roberti
Marina
M
Barile
Maria
M
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D007527:Isoenzymes / Q000032:analysis; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry*; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000032:analysis*; D021381:Protein Transport; D051381:Rats
D007527:Isoenzymes; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase