Mitochondrial localization of human FAD synthetase isoform 1
Introduction
Mammals must obtain riboflavin (Rf, vitamin B2) from diet, whereas plants, as well as fungi and bacteria, have the ability to synthesize Rf de novo. The primary role of Rf in cell metabolism derives from its conversion into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), the redox cofactors of a large number of dehydrogenases, reductases and oxidases (Joosten and van Berkel, 2007, Powers, 2003). Most of these flavoenzymes are compartmented in the cellular organelles, where they ensure the functionality of mitochondrial electron transport, photosynthesis, metabolism of fatty acids, some amino acids, choline and betaine, synthesis of protoporphyrin and of vitamins B6, B12 and folate. FAD is also the coenzyme of glutathione reductase, which mediates regeneration of reduced glutathione, a scavenger of free radicals and reactive oxygen species and a modulator of protein function by S-glutathionylation (Werner et al., 2005, and Refs. therein). Ero1 and sulfhydryl oxidases, which mediate the oxidative folding of secretory proteins (Thorpe et al., 2002, Tu et al., 2000), apoptosis-inducing factor (AIF), which acts as a caspase-independent mitochondrial effector of apoptotic cell death (Modjtahedi et al., 2006), lysine-specific demethylase 1 and 2 (LSD1 and LSD2), which are involved in chromatin remodeling, are also flavoenzymes that play a crucial roles in cellular regulation (Forneris et al., 2008, Joosten and van Berkel, 2007, Karytinos et al., 2009). Consistently, deficiency of Rf in humans and experimental animals has been linked to several diseases, such as cancer, cardiovascular diseases, anemia, abnormal fetal development, different neuromuscular and neurological disorders (Depeint et al., 2006, Powers, 2003, Valenti et al., 2006).
Recent studies indicate that, beside acting as classical enzyme cofactor, Rf and/or Rf derived cofactors (or related metabolites) could play additional regulatory roles, as observed for other vitamin B derived cofactors (Depeint et al., 2006, Valenti et al., 2006). A post-transcriptional control on the expression of the succinate dehydrogenase flavoprotein subunit (SDH-Fp) by flavin cofactors has been suggested in Saccharomyces cerevisiae (Giancaspero et al., 2008). In plants, Rf treatment is able to activate signal transduction pathways, thus conferring resistance to fungal infections (Roje, 2007, and Refs. therein). Transcriptional or post-transcriptional control has also been evoked to explain the beneficial biochemical effect of Rf therapy in patient suffering from RR-MADD, Rf-Responsive Multiple Acyl-CoA Dehydrogenase Deficiency (Antozzi et al., 1994, Bugiani et al., 2006, Gianazza et al., 2006, Gregersen, 1985, Vergani et al., 1999). Rf deficiency seems to alter the affinity of transcriptional factors for DNA or to modulate translational efficiency in HepG2 and Jurkat lymphoid cells (Manthey et al., 2006). The relevance of such processes merits further research aimed to better describe Rf metabolism and flavoenzymes biogenesis.
Rf to FAD conversion occurs via the sequential actions of riboflavin kinase or ATP:riboflavin 5′-phosphotransferase (RK, EC 2.7.1.26) which converts the vitamin into FMN and FAD synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 2.7.7.2). In mammals RK and FADS are two distinct mono-functional enzymes, which have been purified from rat tissues, and biochemically characterized (Bowers-Komro et al., 1989, McCormick et al., 1997, Oka and McCormick, 1987, Yamada et al., 1990).
The activities of RK and FADS has been found to change during respiratory infection in mice hepatocytes (Brijlal et al., 1999) and Phenobarbital administration (Hamajima et al., 1979) and to depend on changes in thyroid hormones (Lee and McCormick, 1985) and Rf status (Lee and McCormick, 1983). The expression of both RK and FADS decreased in response to Rf depletion in HepG2 cells (Werner et al., 2005). It is noteworthy that RK and FADS expression level is significantly reduced in patients with amyotrophic lateral sclerosis (Lin et al., 2009).
The first eukaryotic genes encoding mono-functional RK and FADS have been identified in S. cerevisiae and named FMN1 (Santos et al., 2000) and FAD1 (Wu et al., 1995), respectively. Fad1p is a 35 kDa soluble enzyme which is essential for yeast life. The structure of human recombinant RK has been solved (Karthikeyan et al., 2003), whereas the molecular structure of FADS in higher eukaryotes is still missing. While this manuscript was in preparation, the structural characterization of FADS from the pathogenic yeast Candida glabrata has been reported (Huerta et al., 2009). The eukaryotic FADS belongs to the PAPS-reductase family and has little or no sequence similarity to the bacterial enzymes such as ribF in Escherichia coli, which is a bi-functional enzyme with both RK and FADS activities (Efimov et al., 1998, Kearney et al., 1979, Manstein and Pai, 1986) as in all prokaryotes. Together with bi-functional one, in prokaryotes, mono-functional enzymes with the only RK activity have also been described (Solovieva et al., 2003). No mono-functional prokaryotic FADS has yet been found. Since the bi-functional FAD-forming enzyme is required for bacterial viability and it is unrelated to mammalian FADS, this enzyme is a potential target for the development of novel antimicrobial drugs (Gerdes et al., 2002).
A still controversial matter in FADS biochemistry is its sub-cellular localization in eukaryotes. De Luca and Kaplan first demonstrated that the enzyme responsible for FAD formation is located in the cytosol of rat liver (De Luca, 1958). Since then, FADS has been purified from rat liver cytosolic fractions only as a protein with molecular mass (m) of about 53 kDa by SDS–PAGE (McCormick et al., 1997, Oka and McCormick, 1987). A cytosolic localization has also been demonstrated for the yeast Fad1p (Wu et al., 1995). Thus, for many years it has been assumed that in eukaryotes FAD biosynthesis occurred only in the cytosol. However, using cell fractionation and activity measurements, we have demonstrated the presence of FADS activity in mitochondria from rat liver (Barile et al., 2000, Barile et al., 1993), S. cerevisiae (Bafunno et al., 2004, Pallotta et al., 1998) and Nicotiana tabacum Yellow Bright-2 (Giancaspero et al., 2009). Nevertheless, the protein responsible for FAD synthesis in mitochondria remains to be identified and characterized. This is probably why, up to now, the sub-cellular location (cytosolic or mitochondrial) of lower and higher eukaryotic FADS is still controversial and remains to be fully investigated (Bafunno et al., 2004, Barile et al., 2000, Spaan et al., 2005, Tzagoloff et al., 1996).
The hypothesis that different isoforms with compartment-specific functions may exist in eukaryotes has been recently supported by the cloning and functional characterization of two products of the human FLAD1 gene, the putative human ortholog of yeast FAD1. FLAD1 encodes different transcript variants among which the transcript variant 1 and transcript variant 2. The two corresponding protein products differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1), which contains a putative mitochondrial targeting peptide predicted by bioinformatics (Brizio et al., 2006).
In this paper we report experiments aimed to: (i) definitively prove the existence of a FADS isoform in rat liver mitochondria (RLM); (ii) directly demonstrate that hFADS1 is the human mitochondrial FADS.
Section snippets
Materials
pCMVTNT™ vector and TNT® SP6 Coupled Reticulocyte Lysate System were purchased from Promega. The pSPT19-pMe2GlyDH plasmid was a kind gift from R. Brandsch, Freiburg, Germany. Restriction endonucleases and other cloning reagents were purchased from Fermentas. Redivue™ L-[35S] Methionine was from Amersham-GE Healthcare. Nitrocellulose membrane Optitran BA-S 85 was from Schleicher & Schuell. The dye reagent for protein assay (Bio-Rad protein assay) was from Bio-Rad. Anti-OxPhos Complex II 70 kDa
The existence of FADS in rat liver mitochondria
Our previous results, based on functional approaches, showed that, besides in the cytosol, FADS activity can be specifically revealed in mitochondria from rat liver (Barile et al., 1993, Barile et al., 2000).
Experiments described in this paper benefit of the use of a novel policlonal antibody raised against the recombinant hFADS2 which was produced according to (Galluccio et al., 2007).
To test the specificity of this antibody in a first series of experiments, the cell lysates of BHK-21 cell
Discussion
For many years it was assumed that the biosynthesis of FAD from Rf from yeast to mammals occurred only in the cytosol (McCormick, 1989). In agreement with this hypothesis, the databases MITOP (http://www.mitop.de:8080/mitop2/) and BRENDA (http://www.brenda-enzymes.info/) still show that mitochondria do not contain FADS.
On the other hand, using cell fractionation and activity measurements, we have demonstrated the presence of FADS activity in mitochondria from rat liver (Barile et al., 2000,
Acknowledgments
This work was supported by grants from MIUR (FIRB 2003 project RBNE03B8KK: “Molecular recognition in protein–ligand, protein–protein and protein–surface interaction: development of integrated experimental and computational approaches to the study of systems of pharmaceutical interest” to M.B) and from Università degli Studi di Bari (Fondi di Ateneo per la ricerca to M.B.). C.B. was supported by a post-doctoral research fellowship (Giovani Ricercatori) financed by FIRB 2003 project RBNE03B8KK.
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These authors contributed equally to this work.