Subcellular localization of fad1p in saccharomyces cerevisiae: A choice at post-transcriptional level?

FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In Saccharomyces cerevisiae, the gene encoding for FAD synthase is FAD1, from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that FAD1 generates two echoforms— that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3′ region of FAD1 mRNA was analyzed by 3′RACE experiments, which revealed the existence of (at least) two FAD1 transcripts with different 3′UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3′UTRs allowed us to predict the existence of a cis-acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3′UTR context. Here, we propose that the long FAD1 transcript might be responsible for the generation of mitochondrial Fad1p echoform

Silencing of FAD synthase gene in Caenorhabditis elegans upsets protein homeostasis and impacts on complex behavioral patterns

Background: FAD synthase is a ubiquitous enzyme that catalyses the last step of FAD biosynthesis, allowing for the biogenesis of several flavoproteins. In humans different isoforms are generated by alternative splicing, isoform 1 being localized in mitochondria. Homology searching in Caenorabditis elegans leads to the identification of two human FAD synthase homologues, coded by the single copy gene R53.1. Methods: The C. elegans R53.1 gene was silenced by feeding. The expression level of transcripts was established by semi-quantitative RT-PCR. Overall protein composition was evaluated by two-dimensional electrophoresis. Enzymatic activities were measured by spectrophotometry and oxygen consumption by polarography on isolated mitochondria. Results: From R53.1 two transcripts are generated by trans-splicing. Reducing by 50% the transcription efficiency of R53.1 by RNAi results in a 50% reduction in total flavin with decrease in ATP content and increase in ROS level. Significant phenotypical changes are noticed in knock-down nematodes. Among them, a significant impairment in locomotion behaviour possibly due to altered cholinergic transmission. At biochemical level, impairment of flavoenzyme activities and of some KCN-insensitive oxygen-consuming enzymes is detected. At proteomic level, at least 15 abundant proteins are affected by R53.1 gene silencing, among which superoxide dismutases. Conclusion and General Significance: For the first time we addressed the existence of different isoforms of FAD-metabolizing enzymes in nematodes. A correlation between FAD synthase silencing and flavoenzyme derangement, energy shortage and redox balance impairment is apparent. In this aspect R53.1-interfered nematodes could provide an animal model system for studying human pathologies with alteration in flavin homeostasis/flavoenzyme biogenesis

FAD Synthase (FADS) localization and function in neuronal cell models: a possible involvement of FADS in the pathogenesis of neurodegenerative diseases.

BACKGROUND AND AIMS: FAD synthase (FADS, EC 2.7.7.2), coded in humans by FLAD1 gene, is the last enzyme in the pathway converting riboflavin into the redox cofactor FAD, essential for the activity of hundreds of flavoenzymes. In non-neuronal cells FADS performs a citosolic, mitochondrial and nuclear localization (Giancaspero TA et al., 2013). Interestingly, a case report (Lin J et al., 2009) showed that in a ALS patient with an IgA gammopathy, the neuronal surface antigen was represented by FADS. Subcellular localization of FADS in human neuronal cells is still unknown. Also, a significant reduction of FADS mRNA levels was observed in the blood of ALS patients. Thus, here we aimed at studying the subcellular localization and the effect of altered expression levels of FADS on cellular bioenergetics in experimental models, i.e. neuronal cell models. METHODS: Confocal and sub-fractionation studies were performed on human neuroblastoma (SK-N-SH) and mouse motor neuron (NSC-34) cell lines. After overexpression and silencing, FADS levels were assessed by RT-PCR and WB, while Riboflavin, FMN and FAD content was measured by HPLC. The effects of altered FADS levels on neuronal bioenergetics were evaluated by measuring oxygen consumption rate and levels of ATP, ROS and glutathione reductase. RESULTS: Confocal data collected in neuronal cells showed a partial co-localization of FADS with a lipid raft marker and a high degree of co-localization with a vesicle marker in both the neuronal cell lines. Also, a clear toxic effect on bioenergetics was shown in FADS overexpressing SK-N-SH cells, as indicated by the specific reduction in complex I functionality. CONCLUSIONS: These data straightforwardly demonstrate the localization of FADS in membrane domains of two neuronal cell models, suggesting a novel role for FADS in neuronal physiology and, possibly, in neurotransmission. The relationships with results collected in other models will be discussed

The role of cysteine residues in human FAD synthase (isoform 2)

FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into FAD. At the moment two isoforms of the human FADS have been characterized, being involved in FAD synthesis in mitochondria and cytosol (1). hFADS is organized in two domains: the 3\u2032-phosphoadenosine-5\u2032-phosphosulfate (PAPS) reductase domain, which is the catalytic domain, and the resembling molybdopterin-binding domain (MPTbd), which performs a kinetic regulatory role (2). Using C. glabrata FMNAT as a template, we built the homology model of the PAPS reductase domain. Mass spectrometry and DTNB titration experiments allowed us to reveal the presence of four disulfide bridges in hFADS2, three of them located in close proximity to the PAPS reductase domain, and to validate the proposed model. In the frame of the hypothesis that hFADS is a component of a machinery that delivers FAD to cognate apo-flavoproteins in a tightly controlled process, the redox residues of cysteines require particular attention (2, 3). Redox changes of hFADS are ascertained here by the shift of the bands observed in non-reducing vs reducing conditions on SDS-PAGE. The involvement of cysteines in hFADS catalytic cycle is demonstrated by the strong inhibitor effects of thiol reagents on FAD synthesis rate. To verify the structure/function relationships of these redox residues we performed single substitutions of cysteines with alanine by site directed mutagenesis using overlap extension procedure starting from the most conserved residues, and from those closest to the active site in the homology model. All the purified mutants are able to synthesize FAD, C303A being the most significantly impaired. (1)Torchetti, E.M. et al. Mitochondrion-2010, 10, 263\u2013273 (2)Miccolis A. et al. Int. J. Mol. Sci.-2012, 13, 16880-16898 (3)Torchetti, E.M et al. FEBS J.-2011, 278, 4434\u20134449 Acknowledgments: This work was supported by grants from PON-ricerca e competitivita 2007-2013 (PON project 01_00937) to M.B