Mitochondrial localization of human FAD synthetase isoform 1

Mitochondrion. 2010 Apr;10(3):263-73. doi: 10.1016/j.mito.2009.12.149. Epub 2010 Jan 7.


FAD synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Line
  • Cricetinae
  • Humans
  • Isoenzymes / analysis
  • Kinetics
  • Liver / chemistry
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Mitochondria / chemistry*
  • Molecular Weight
  • Nucleotidyltransferases / analysis*
  • Protein Transport
  • Rats


  • Isoenzymes
  • Nucleotidyltransferases
  • FMN adenylyltransferase