Publications

• Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism.

  Peng M.Z., Shao Y.X., Li X.Z., Zhang K.D., Cai Y.N., Lin Y.T., Jiang M.Y., Liu Z.C., Su X.Y., Zhang W., Jiang X.L., Liu L.

  The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32^Y174C/Y174C and Slc25a32^K235R/K235R) and compo ... >> More

  The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32^Y174C/Y174C and Slc25a32^K235R/K235R) and compound heterozygous (Slc25a32^Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32^-/-) mouse was also generated. The Slc25a32^K235R/K235R and Slc25a32^Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32^-/- mice die in utero with NTDs. None of the Slc25a32 mutations hindered the mitochondrial uptake of folate. Instead, the mitochondrial uptake of flavin adenine dinucleotide (FAD) was specifically blocked by Slc25a32^Y174C/K235R, Slc25a32^K235R/K235R, and Slc25a32^-/- mutations. A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid β-oxidation and amino acid metabolism being impaired, Slc25a32^-/- embryos also had a subunit of glycine cleavage system-dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulation. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32^-/- embryos. The Slc25a32^K235R/K235R and Slc25a32^Y174C/K235R mice had no glycine accumulation, but had another formate donor-dimethylglycine accumulated and formate deficiency. Meanwhile, they suffered from the absence of all folate intermediates in mitochondria. Formate supplementation increased the folate amounts, but this effect was not restricted to the Slc25a32 mutant mice only. In summary, we established novel animal models, which enabled us to understand the function of SLC25A32 better and to elucidate the role of SLC25A32 dysfunction in human disease development and progression. << Less

  Cell. Mol. Life Sci. 79:375-375(2022) [PubMed] [EuropePMC]

• FLX1 codes for a carrier protein involved in maintaining a proper balance of flavin nucleotides in yeast mitochondria.

  Tzagoloff A., Jang J., Glerum D.M., Wu M.

  Respiratory defective mutants of Saccharomyces cerevisiae previously assigned to complementation group G178 are characterized by an abnormally low ratio of FAD/FMN in mitochondria. A nuclear gene, designated FLX1, was selected from a yeast genomic library, based on its ability to confer wild-type ... >> More

  Respiratory defective mutants of Saccharomyces cerevisiae previously assigned to complementation group G178 are characterized by an abnormally low ratio of FAD/FMN in mitochondria. A nuclear gene, designated FLX1, was selected from a yeast genomic library, based on its ability to confer wild-type growth properties to a representative G178 mutant. Genetic evidence has confirmed that the flavin nucleotide imbalance of G178 mutants is caused by mutations in FLX1. The sequence of FLX1 is identical to a reading frame recently reported to be present on yeast chromosome IX (GenBank Z47047). The sequence and tripartite repeat structure of the FLX1 product (Flx1p) indicate it is a member of a protein family consisting of mitochondrial substrate and nucleotide carriers. In yeast, FAD synthetase is present in the soluble cytoplasmic protein fraction but not in mitochondria. Riboflavin kinase, the preceding enzyme in flavin biosynthesis, is present in both subcellular fractions. The absence of FAD synthetase in mitochondria implies that FAD is imported from the cytoplasm. The lower concentration of mitochondrial FAD in flx1 mutants suggests that Flx1p is involved in flavin transport, a role that is also supported by biochemical evidence indicating more efficient flux of FAD across mitochondrial membrane vesicles prepared from wild-type strains than membrane vesicles from flx1 mutants. << Less

  J. Biol. Chem. 271:7392-7397(1996) [PubMed] [EuropePMC]

• Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria: involvement of the Flx1p carrier in FAD export.

  Bafunno V., Giancaspero T.A., Brizio C., Bufano D., Passarella S., Boles E., Barile M.

  We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside ... >> More

  We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1Delta mutant strain was constructed. Coupled mitochondria isolated from flx1Delta mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1Delta mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation. << Less

  J Biol Chem 279:95-102(2004) [PubMed] [EuropePMC]