FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2044218758> ?p ?o ?g. }

• W2044218758 endingPage "102" @default.
• W2044218758 startingPage "95" @default.
• W2044218758 abstract "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 flx1Δ mutant strain was constructed. Coupled mitochondria isolated from flx1Δ 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 flx1Δ 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. 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 flx1Δ mutant strain was constructed. Coupled mitochondria isolated from flx1Δ 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 flx1Δ 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. The mechanism by which mitochondria obtain their own flavin cofactors is an interesting point of investigation because FMN and FAD are mainly located in mitochondria, where they act as redox cofactors of a number of dehydrogenases and oxidases that play a crucial role in both bioenergetics and cellular regulation (for reviews see Refs. 1.McCormick D.B. Physiol. Reviews. 1989; 69: 1170-1198Crossref PubMed Scopus (134) Google Scholar and 2.Lipton S.A Bossy-Wetzel E. Cell. 2002; 111: 147-150Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). As far as mammalian mitochondria are concerned, we have demonstrated that in rat liver the main source of intramitochondrial flavin cofactors is riboflavin (Rf) 1The abbreviations used are: Rf, riboflavin; SCM, Saccharomyces cerevisiae mitochondria; DASPMI, 2-(4-dimetylaminostyryl)-N-methylpyridinium iodide; HPLC, high pressure liquid chromatography; CS, citrate synthetase; PDC, pyruvate decarboxylase; LPDH, lipoamide dehydrogenase; Fp, flavoprotein; SDH, succinate dehydrogenase; SDH-Fp, flavoprotein subunit of succinate dehydrogenase; PDH, pyruvate dehydrogenase. taken up from the cytosol. FAD synthesis occurs inside the organelle from imported Rf and mitochondrial ATP, consistent with the presence of a mitochondrial riboflavin kinase (EC 2.7.1.26) and an FAD synthetase (EC 2.7.7.2) (3.Barile M. Passarella S. Bertoldi A. Quagliariello E. Arch. Biochem. Biophys. 1993; 305: 442-447Crossref PubMed Scopus (38) Google Scholar, 4.Barile M. Brizio C. Valenti D. De Virgilio C. Passarella S. Eur. J. Biochem. 2000; 267: 4888-4900Crossref PubMed Scopus (81) Google Scholar). Newly synthesized FAD can be either efficiently incorporated into newly imported apo-flavoproteins (5.Brizio C. Otto A. Brandsch R. Passarella S. Barile M. Eur. J. Biochem. 2000; 267: 4346-4354Crossref PubMed Scopus (19) Google Scholar, 6.Brizio C. Barile M. Brandsch R. FEBS Lett. 2002; 522: 141-146Crossref PubMed Scopus (13) Google Scholar) or can be exported into the outer mitochondrial compartments, where it is reconverted to Rf by FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2) in a recycling pathway, i.e. the Rf-FAD cycle (4.Barile M. Brizio C. Valenti D. De Virgilio C. Passarella S. Eur. J. Biochem. 2000; 267: 4888-4900Crossref PubMed Scopus (81) Google Scholar, 7.Barile M. Brizio C. De Virgilio C. Delfine S. Quagliariello E. Passarella S. Eur. J. Biochem. 1997; 249: 777-785Crossref PubMed Scopus (54) Google Scholar). This novel mitochondrial pathway is assumed to play a central role in cellular Rf homeostasis and in flavoprotein biogenesis (5.Brizio C. Otto A. Brandsch R. Passarella S. Barile M. Eur. J. Biochem. 2000; 267: 4346-4354Crossref PubMed Scopus (19) Google Scholar, 8.Vergani L. Barile M. Angelini C. Burlina A.B. Nijtmans L. Freda M.P. Brizio C. Zerbetto E. Dabbeni-Sala F. Brain. 1999; 122: 2401-2411Crossref PubMed Scopus (81) Google Scholar). The origin of flavin cofactors in yeast mitochondria is still controversially discussed. It has been reported that yeast mitochondria do not contain their own FAD synthetase activity and that FAD is synthesized in the cytosol by Fad1p (9.Wu M. Repetto B. Glerum D.M. Tzagoloff A. Mol. Cell. Biol. 1995; 15: 264-271Crossref PubMed Scopus (77) Google Scholar). Thus, mitochondria have been supposed to get their FAD from the cytosol via an FAD uptake system, encoded by the FLX1 gene, a member of the mitochondrial carrier family (10.Tzagoloff A. Jang J. Glerum M. Wu M. J. Biol. Chem. 1996; 271: 7392-7397Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). On the other hand, we have proposed that in yeast as well as in mammals Rf imported from the cytosol is the main source of mitochondrial flavin cofactors. Our observations implied a mitochondrial localization for both riboflavin kinase and FAD synthetase (11.Pallotta M.L. Brizio C. Fratianni A. De Virgilio C. Barile M. Passarella S. FEBS Lett. 1998; 428: 245-249Crossref PubMed Scopus (46) Google Scholar). Consistently, the mitochondrial localization of riboflavin kinase, encoded by the FMN1 gene, has been recently confirmed by immunoblotting analysis (12.Santos M.A. Jimenez A. Revuelta J.L. J. Biol. Chem. 2000; 275: 28618-28624Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In order to elucidate further the mechanism by which Saccharomyces cerevisiae mitochondria (SCM) can provide their own flavin cofactors, Rf uptake and metabolism were studied in intact organelles. Moreover, the activity of FAD synthetase has been determined in solubilized mitochondria. To clarify the role of the Flx1p carrier in this pathway, mitochondria isolated from flx1Δ mutant cells were compared with wild-type mitochondria with respect to the ability to take up Rf, to synthesize FAD, and to export it out of the mitochondria. Moreover, the physiological role of Flx1p in maintaining the homeostasis of flavin cofactors has been studied as well as its influence on the amounts of FAD-binding enzymes. Materials—All reagents and enzymes were from Sigma, zymolyase from ICN, and Bacto yeast extract from Difco. Mitochondrial substrates were used as Tris salts at pH 7.0. Solvents and salts used for HPLC were from Baker. Yeast Strain, Media, and Growth Conditions—The wild-type S. cerevisiae strain (EBY157A, genotype MATα ura 3–52 MAL2–8c SUC2 p426MET25) used in this work is derived from the CEN.PK series of yeast strains and was obtained from P. Koetter (Institut fuer Mikrobiologie, Goethe-Universitaet Frankfurt, Frankfurt, Germany). Cells were grown aerobically at 28 °C with constant shaking in a semisynthetic liquid medium (3 g/l yeast extract, 1 g/l KH2PO4, 1 g/l NH4Cl, 0.5 g/l NaCl, 0.5 g/l CaCl2·2H2O, 0.6 g/l MgCl2·6H2O, 20 mg/l uracil, 0.05% glucose) supplemented with 2% ethanol or 2% glycerol as the carbon source. The pH of the medium was adjusted to 5.5 with HCl. Mitochondrial functionality in living cells was assessed by fluorescence microscopy, using the vital fluorescent dye 2-(4-dimetylaminostyryl)-N-methylpyridinium iodide (DASPMI) as described in Ref. 13.Rinaldi T. Ricci C. Porro D. Bolotin-Fukuhara M. Frontali L. Mol. Biol. Cell. 1998; 9: 2917-2931Crossref PubMed Scopus (66) Google Scholar. Construction of an flx1Δ Mutant Strain—An flx1Δ mutant strain was constructed by using a modification of the PCR targeting technique (14.Boles E. de Jong-Gubbels P. Pronk J.T. J. Bacteriol. 1998; 80: 2875-2882Crossref Google Scholar). A plasmid pUG6 (15.Güldener U. Heck S. Diedler T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1362) Google Scholar) has been used as a template to generate by PCR a DNA molecule consisting of a kanMX marker cassette (conferring G418 resistance) flanked by short homology regions to the FLX1 locus. For this purpose, appropriated oligonucleotides were constructed: S1-FLX1, 5′-ATGGTCGATCACCAGTGGACGCCACTGCAGAAGGAGGTTATCATCTCAGGTTTCGTACGCTGCAGGTCGAC-3′; and S22-FLX1, 5′-TTTATCGTTTGATATTTACAAGAAATTAAATCTATCATATAGCCTTTATGCATAGGCCACTAGTGGATCTG-3′. Short flanking homology regions allowed a specific recombination between wild-type chromosomal DNA containing the FLX1 gene and linear DNA containing kanMX. The transformation of EBY157A strain was done by electroporation. The mutant yeast cells (EBY167A, genotype MATα ura 3–52 MAL2–8c SUC2, flx1Δ:: kanMX) were selected for resistance to G418. Construction of Plasmid—The FLX1 gene was cloned by PCR with a pair of primers designed to amplify a DNA fragment enclosing the complete FLX1-open reading frame with its 5′ and 3′ regulatory regions. The amplified fragment, cleaved with BglII and PvuII, was cloned into the high-copy-number vector YEplac195 (16.Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2522) Google Scholar) with a URA3 marker, resulting in plasmid YEpFLX1. The transformation of the EBY167A S. cerevisiae strain was done by electroporation. Preparation of Spheroplasts and Mitochondria and Oxygen Uptake Measurements—Spheroplasts were prepared using zymolyase and mitochondria were isolated according to (17.Daum G. Bohn P.C. Schaltz G. J. Biol. Chem. 1982; 257: 13028-13033Abstract Full Text PDF PubMed Google Scholar). Mitochondrial protein was determined according to (18.Waddel W.J. Hill C. J. Lab. Clin. Med. 1956; 48: 311-314PubMed Google Scholar). The intactness of mitochondrial inner membrane was checked measuring the release of the matrix enzyme fumarase, as in Ref. 19.Neuburger M. Journet E.P. Bligny R. Carde J.P. Douce R. Arch. Biochem. Biophys. 1982; 217: 312-323Crossref PubMed Scopus (249) Google Scholar. Oxygen uptake measurements were carried out using a Gilson oxygraph as described previously (11.Pallotta M.L. Brizio C. Fratianni A. De Virgilio C. Barile M. Passarella S. FEBS Lett. 1998; 428: 245-249Crossref PubMed Scopus (46) Google Scholar). Flavin Uptake and Metabolism—Mitochondria (0.1–0.2 mg protein), isolated from cells grown on glycerol-containing medium, were incubated at 2 °C in 500 μl of a medium consisting of 0.6 m mannitol, 50 mm Tris-HCl, pH 7, 1 mm MgCl2. 1 min later, Rf, FAD, or FMN were added. At the appropriate time, the uptake reaction was stopped by rapid centrifugation. Rf, FMN, and FAD content of supernatants and pellets were measured in aliquots (5–80 μl) of neutralized perchloric extracts by means of HPLC (Gilson HPLC system including a model 306 pump and a model 307 pump equipped with a Kontron Instruments SFM 25 fluorimeter and Unipoint system software), and corrected for endogenous flavin content, essentially as described in Refs. 4.Barile M. Brizio C. Valenti D. De Virgilio C. Passarella S. Eur. J. Biochem. 2000; 267: 4888-4900Crossref PubMed Scopus (81) Google Scholar and 11.Pallotta M.L. Brizio C. Fratianni A. De Virgilio C. Barile M. Passarella S. FEBS Lett. 1998; 428: 245-249Crossref PubMed Scopus (46) Google Scholar. The amount of flavin actually taken up into the organelle was calculated after correction made for molecules present in the adherent space and/or nonspecifically bound to the membranes, as described elsewhere (4.Barile M. Brizio C. Valenti D. De Virgilio C. Passarella S. Eur. J. Biochem. 2000; 267: 4888-4900Crossref PubMed Scopus (81) Google Scholar, 20.Said H.M. McCloud E. Yanagawa N. Biochim. Biophys. Acta. 1995; 1236: 244-248Crossref PubMed Scopus (8) Google Scholar). FAD Synthetase Activity Assay—SCM (0.015–0.06 mg protein) were solubilized by treatment with Lubrol PX (0.3 mg/mg protein) for 15 min at 0 °C and pre-incubated for 1 min at 37 °C in 100 μl of a medium consisting of 50 mm Tris-HCl, pH 7.5, 5 mm MgCl2 in the presence of 5 mm sodium pyrophosphate. Then, FMN (10 μm) and ATP (5 mm) were added or not. After 2 min of incubation, the reaction was stopped by boiling at 100 °C for 3 min followed by centrifugation at 14,000 × g for 4 min at 4 °C. The amount of FAD present in the mitochondrial extracts was measured enzymatically, as described in Refs. 4.Barile M. Brizio C. Valenti D. De Virgilio C. Passarella S. Eur. J. Biochem. 2000; 267: 4888-4900Crossref PubMed Scopus (81) Google Scholar and 11.Pallotta M.L. Brizio C. Fratianni A. De Virgilio C. Barile M. Passarella S. FEBS Lett. 1998; 428: 245-249Crossref PubMed Scopus (46) Google Scholar. Briefly, the amount of FAD was determined by revealing the reconstituted holo-d-amino acid oxidase activity derived from the FAD binding to the apo-d-amino acid oxidase, using d-alanine (25 mm) as substrate. The oxidation of NADH in the l-lactate dehydrogenase coupled reaction was followed photometrically at 340 nm by means of a PerkinElmer λ19 spectrophotometer and its rate calculated as a tangent to the linear part of the progress curve. This rate was proven to be proportional to the FAD concentration. Calibration curves were obtained by using standard FAD solutions added to SCM. Corrections were also made to account for the inhibition due to FMN and ATP added to the assay. Other Enzymatic Assays—Citrate synthetase (CS) and pyruvate decarboxylase (PDC) activities were measured as in Refs. 21.Srere P.A. Methods Enzymol. 1969; 13: 3-11Crossref Scopus (2036) Google Scholar and 22.van Urk H. Schipper D. Breedveld G.J. Mak P.R. Scheffers W.A. van Dijken J.P. Biochim. Biophys. Acta. 1989; 992: 78-86Crossref PubMed Scopus (68) Google Scholar. Lipoamide dehydrogenase (LPDH) activity was assayed as reported in Ref. 23.Schnaitman C. Greenawalt J.W. J. Cell Biol. 1968; 38: 158-175Crossref PubMed Scopus (1013) Google Scholar, and succinate dehydrogenase (SDH) activity was measured as the reduction rate of 2,6-dichlorophenol-indophenol in the presence of phenazine methosulfate, as reported in Ref. 24.King T.E. Methods Enzymol. 1967; 10: 322-331Crossref Scopus (526) Google Scholar. Western Blotting—Proteins from SCM (0.04 mg) were separated by SDS-PAGE, according to Ref. 25.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar, and transferred electrophoretically onto a nitrocellulose membrane using a trans-blot semidry electrophoretic transfer cell (Sigma). The immobilized proteins were incubated overnight with a 1000-fold dilution of the specific polyclonal antibodies against FAD covalently bound to proteins (i.e. α-Fp antibodies containing antiserum raised in rabbits with the covalently flavinylated bacterial enzyme 6-hydroxy-d-nicotine oxidase, a kind gift from Prof. R. Brandsch, Freiburg, Germany) (26.Cicek G. Schiltz E. Hess D. Staiger G. Brandsch R. Clin. Exp. Immunol. 2002; 128: 83-87Crossref PubMed Scopus (9) Google Scholar). Immunoreactive materials were visualized with the aid of a secondary alkaline phosphatase conjugated anti-rabbit IgG. Quantitative evaluations were carried by densitometric analysis using the Chemi Doc system (Bio-Rad). UV-fluorescence Analysis—Proteins from SCM (0.04 mg) were separated by SDS-PAGE, then the unstained gel was incubated for 1 h in 10% acetic acid and inspected on an UV-transilluminator system. Upon illumination with UV light, the flavinylated flavoprotein subunit of SDH (SDH-Fp) was visible because of the fluorescence of the covalently bound flavin. Protein bands on the gel were then stained with Coomassie Blue. Quantitative evaluations were carried using the Chemi Doc system (Bio-Rad). Rf Uptake and FAD Synthesis in Isolated Intact SCM—To gain insight into the mechanism by which SCM can provide their own flavin cofactors, isolated mitochondria (from the wild-type strain EBY157A) were incubated with Rf in two different concentration ranges, i.e. 0–2.5 μm (Fig. 1A) and 0–30 μm (Fig. 1B). Then, the amounts of flavins in the acid-extractable fraction of both mitochondrial pellets and supernatants were measured by HPLC and compared with those of parallel samples in which no Rf was added. Experimental data were collected within the initial linear range of Rf uptake rates (i.e. 20 s incubation), and were corrected for adherent/bound vitamin as described under “Experimental Procedures.” Data were expressed as rates of flavin transport in dependence on Rf concentration. In the whole range of concentrations used (Fig. 1, A and B), Rf transport was accompanied by FAD formation (see also Refs. 4.Barile M. Brizio C. Valenti D. De Virgilio C. Passarella S. Eur. J. Biochem. 2000; 267: 4888-4900Crossref PubMed Scopus (81) Google Scholar and 11.Pallotta M.L. Brizio C. Fratianni A. De Virgilio C. Barile M. Passarella S. FEBS Lett. 1998; 428: 245-249Crossref PubMed Scopus (46) Google Scholar), which resulted in the export of FAD into the extramitochondrial phase. A residual FAD fraction was retained in the pellet. Conversely, no significant variation in FMN content was observed. Due to the rapid conversion of Rf into FAD (that could be prevented neither by adding ADP plus oligomycin, nor by changing the temperature and the time of incubation of mitochondria), a detailed study of the kinetics of Rf uptake was prevented. Hence, the Rf determined in the pellet under these conditions is a transient value, lower than the true amount of Rf taken up. Thus, the time dependence of Rf increase in the pellet does not actually reflect a true Rf uptake rate. Despite the limitation imposed by this condition, the dependence of the apparent Rf uptake rate on vitamin concentration showed saturation characteristics (Fig. 1A). The sigmoidal feature of the curve probably depends on the combinations between transport and synthesis processes, rather than indicating true cooperativity. Nevertheless, data fitting was performed by means of Grafit software according to allosteric kinetics, and the resulting kinetic parameters, expressed as “pseudo” S0.5 and Vmax, were 1.2 μm and 28 pmol × min–1 × (mg protein)–1, respectively. At the lower concentration of Rf used (0.2 μm, Fig. 1A), FAD was synthesized with a rate of about 75 pmol × min–1 × (mg protein)–1 and it was entirely retained in the pellet. At higher Rf concentrations (0.4–2 μm range, in Fig. 1A), virtually no FAD could be detected in the pellet and the rate of FAD formation was described by the rate of FAD export, which reached a maximum of about 250 pmol × min–1 × (mg protein)–1 at 2 μm. When Rf concentration was increased up to 30 μm (Fig. 1B), the dependence of the apparent Rf uptake rate on the extramitochondrial vitamin concentration showed again a sigmoidal shape. The pseudo S0.5 and Vmax values were 18 μm and 7750 pmol × min–1 × (mg protein)–1, respectively. The rate of FAD formation starting from imported Rf increased with increasing external concentration of the vitamin, with a significant fraction of FAD remaining in the pellet. Under these conditions the rate of FAD export reached a maximum of about 600 pmol × min–1 × (mg protein)–1 at 10 μm Rf and it was inhibited at higher Rf concentrations. Despite the limitations described, these results strongly favor the existence of (at least) two different transport systems involved in Rf uptake into/FAD export out of mitochondria. Moreover, they imply the existence of an FAD synthetase activity inside mitochondria. To prove the existence of FAD synthetase in SCM, solubilized mitochondria were incubated with or without the substrates of FAD synthetase, i.e. FMN and ATP, and the FAD synthesis rate was measured enzymatically as in Refs. 4.Barile M. Brizio C. Valenti D. De Virgilio C. Passarella S. Eur. J. Biochem. 2000; 267: 4888-4900Crossref PubMed Scopus (81) Google Scholar and 11.Pallotta M.L. Brizio C. Fratianni A. De Virgilio C. Barile M. Passarella S. FEBS Lett. 1998; 428: 245-249Crossref PubMed Scopus (46) Google Scholar. A typical experiment is shown in Fig. 2. When solubilized SCM were incubated in the absence of substrates, a decrease of NADH absorbance was observed. Its rate increased with time up to a constant value (0.004 ΔA × min–1 after 6 min). This value corresponded to ∼100 pmol × (mg protein)–1 mitochondrial endogenous FAD, which is loosely and/or not bound to protein. When solubilized SCM were added with FMN and ATP for 2 min, the rate of the decrease in absorbance significantly increased (0.010 ΔA × min–1). This value corresponded to about 400 pmol × (mg protein)–1 of FAD. By subtracting the former FAD value from the latter, 300 pmol × (mg protein)–1 of newly synthesized FAD were calculated as a result of 2 min incubation with the substrate pair of FAD synthetase. It should be noted that in the FAD synthetase assay, sodium pyrophosphate was present to prevent FMN dephosphorylation, 2M. Barile, unpublished results. which competes with FAD synthesis and which is possibly the result of an FMN phosphohydrolase previously observed in SCM and rat liver mitochondria (10.Tzagoloff A. Jang J. Glerum M. Wu M. J. Biol. Chem. 1996; 271: 7392-7397Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 7.Barile M. Brizio C. De Virgilio C. Delfine S. Quagliariello E. Passarella S. Eur. J. Biochem. 1997; 249: 777-785Crossref PubMed Scopus (54) Google Scholar). The amount of FAD synthesized in solubilized SCM starting from FMN and ATP increased with the incubation time, reaching a maximum at 5 min incubation. Incubation for a longer time resulted in a progressive decrease of the newly synthesized FAD, probably due to FAD hydrolysis via FAD pyrophosphatase (7.Barile M. Brizio C. De Virgilio C. Delfine S. Quagliariello E. Passarella S. Eur. J. Biochem. 1997; 249: 777-785Crossref PubMed Scopus (54) Google Scholar, 8.Vergani L. Barile M. Angelini C. Burlina A.B. Nijtmans L. Freda M.P. Brizio C. Zerbetto E. Dabbeni-Sala F. Brain. 1999; 122: 2401-2411Crossref PubMed Scopus (81) Google Scholar). The rate of FAD synthesis, as measured in the interval of time in which the amount of newly synthesized FAD increased linearly with time, was found to be 150 ± 63 pmol × min–1 × (mg protein)–1, in three experiments carried out with different mitochondrial preparations. To exclude any contamination from cytosolic proteins, FAD synthesis was assayed in solubilized mitochondria and spheroplasts. FAD synthetase distribution was compared with those of CS and PDC, mitochondrial and cytosolic marker enzymes, respectively. In a typical assay (see the histograms in Fig. 2), the solubilized SCM were proven to synthesize FAD with a specific activity of 85 pmol × min–1 × (mg protein)–1. This value corresponded to about 37% of that measured in the spheroplasts. In the same experiment, the specific activity of PDC measured in the mitochondrial fraction was only 5% of that measured in the spheroplasts. The purity of the mitochondrial fraction was tested by measuring the enrichment of the CS specific activity, which was 4-fold higher than that measured in the spheroplasts. These results demonstrate that besides the cytosolic FAD synthetase, encoded by FAD1 (9.Wu M. Repetto B. Glerum D.M. Tzagoloff A. Mol. Cell. Biol. 1995; 15: 264-271Crossref PubMed Scopus (77) Google Scholar), mitochondria also possess an enzyme that performs FAD synthesis from the Rf imported from the cytosol. Involvement of the Flx1p Carrier in FAD Export out of Mitochondria—Due to the proposed absence of FAD synthetase inside mitochondria it had been proposed previously that Flx1p be involved in the mitochondrial import of cytosolically synthesized FAD (10.Tzagoloff A. Jang J. Glerum M. Wu M. J. Biol. Chem. 1996; 271: 7392-7397Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). To re-evaluate the function of the Flx1p carrier in awareness of the presence of FAD synthetase in SCM, and to gain further insights into the mechanisms of flavin traffic across the mitochondrial membrane, Rf uptake experiments were repeated by using mitochondria isolated from an flx1Δ mutant strain (Fig. 3). As in the wild-type strain, Rf uptake was accompanied by intramitochondrial FAD synthesis, both in 0–2 μm (Fig. 3A) and in 0–30 μm Rf concentration ranges (Fig. 3B), with no significant change of FMN content. Differently from the wild-type strain, no FAD export could be detected in the supernatant of mitochondria from the flx1Δ mutant strain. The dependence of the apparent Rf uptake rate on extramitochondrial Rf concentration in mitochondria from flx1Δ mutant cells (Fig. 3A) showed again saturation characteristics, with pseudo Km and Vmax values equal to 0.9 μm and 27 pmol × min–1 × (mg protein)–1, respectively. A sigmoidal shape curve fitted the data of Rf uptake obtained in the higher concentration interval (Fig. 3B), with pseudo S0.5 and Vmax values of the same extent of that found in the wild-type strain (20 μm and 11900 pmol × min–1 × mg protein–1, respectively). Despite the kinetical limitations, the results obtained with the flx1Δ mutant strain fit the same hypothesis made for the wild-type strain; i.e. (at least) two distinct uptake systems are involved in Rf transport into mitochondria. Moreover, FAD export into the extramitochondrial phase was proven to depend on the activity of a distinct export system, which presumably is the Flx1p carrier. Consistently, it was ascertained that the ability of FAD export, abolished in the mitochondria of the flx1Δ mutant strain, was restored in the mitochondria of the flx1Δ mutant strain transformed with the FLX1 gene on a multicopy plasmid (Fig. 4). A control was made that the inability to export FAD is specifically due to the FLX1 deletion and that it is not an undesired consequence of the poor growth capacity of the cell on the glycerol-containing medium (see below). Therefore, the same experiments were performed with mitochondria isolated from the wild-type and the flx1Δ mutant cells grown on medium containing ethanol as the carbon source. No significant change in Rf uptake efficiency was found, whereas the FAD export ability was reduced up to 80% in the mitochondria from flx1Δ mutant cells (not shown). The involvement of an export system for FAD, which is different from the Rf uptake systems, was further confirmed by studying the inhibitor sensitivity of the transport process. To this aim, the effect of lumiflavin, a Rf analogue able to inhibit plasma membrane transport of the vitamin (25.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar, 27.Said H.M. Ortiz A. Moyer M.P. Yanagawa N. Am. J. Physiol. Cell. Physiol. 2000; 278: 270-276Crossref PubMed Google Scholar), was studied. Results from a typical experiment carried out with wild-type SCM are shown in Table I. Lumiflavin, which apparently permeates the mitochondrial membrane, was found to significantly reduce the rate of FAD export. At 0.8 μm Rf, the export of FAD was completely inhibited by 15 μm lumiflavin. At 1.5 μm riboflavin, the FAD export rate was inhibited up to 45%. Conversely, lumiflavin did not significantly change the apparent rate of Rf uptake. When the same experiment was repeated by using mitochondria isolated from flx1Δ mutant cells, no effect of lumiflavin was observed both on the apparent Rf uptake rate and on the FAD synthesis rate in the pellet. This result allows us to exclude that lumiflavin inhibits either the Rf uptake system(s) or the intramitochondrial FAD synthetic machinery.Table IInhibition by lumiflavin of FAD export from SCM isolated from wild-type cellsAdditionFlavin transport rateRiboflavin uptakeFAD exportpmol × min-1 × mg protein-1Riboflavin (0.8 μm)4 ± 4119 ± 21Riboflavin (0.8 μm) + Lumiflavin8 ± 30a0, not detectable.Riboflavin (1.5 μm)16 ± 6254 ± 12Riboflavin (1.5 μm) + Lumiflavin28 ± 4140 ± 30a 0, not detectable. Open table in a new tab To prove that Flx1p is neither involved in flavin cofactor import nor strictly required in ma" @default.
• W2044218758 created "2016-06-24" @default.
• W2044218758 creator A5005692205 @default.
• W2044218758 creator A5020805217 @default.
• W2044218758 creator A5021242191 @default.
• W2044218758 creator A5031030356 @default.
• W2044218758 creator A5064656627 @default.
• W2044218758 creator A5074911112 @default.
• W2044218758 creator A5080543093 @default.
• W2044218758 date "2004-01-01" @default.
• W2044218758 modified "2023-10-02" @default.
• W2044218758 title "Riboflavin Uptake and FAD Synthesis in Saccharomyces cerevisiae Mitochondria" @default.
• W2044218758 cites W1408977967 @default.
• W2044218758 cites W1481378379 @default.
• W2044218758 cites W1503211689 @default.
• W2044218758 cites W1507851645 @default.
• W2044218758 cites W1540537257 @default.
• W2044218758 cites W1584337446 @default.
• W2044218758 cites W1590025317 @default.
• W2044218758 cites W1850569493 @default.
• W2044218758 cites W1923503578 @default.
• W2044218758 cites W1964021557 @default.
• W2044218758 cites W1964431087 @default.
• W2044218758 cites W1967390330 @default.
• W2044218758 cites W1984931558 @default.
• W2044218758 cites W1985955125 @default.
• W2044218758 cites W1990413808 @default.
• W2044218758 cites W1992657834 @default.
• W2044218758 cites W2000047924 @default.
• W2044218758 cites W2002363388 @default.
• W2044218758 cites W2003881327 @default.
• W2044218758 cites W2007676400 @default.
• W2044218758 cites W2031512086 @default.
• W2044218758 cites W2033774856 @default.
• W2044218758 cites W2034875354 @default.
• W2044218758 cites W2049977122 @default.
• W2044218758 cites W2054445480 @default.
• W2044218758 cites W2068744822 @default.
• W2044218758 cites W2075920699 @default.
• W2044218758 cites W2086623997 @default.
• W2044218758 cites W2093301014 @default.
• W2044218758 cites W2100837269 @default.
• W2044218758 cites W2101033784 @default.
• W2044218758 cites W2106450047 @default.
• W2044218758 cites W2113875636 @default.
• W2044218758 cites W2116585979 @default.
• W2044218758 cites W2118889062 @default.
• W2044218758 cites W2124859343 @default.
• W2044218758 cites W2125460607 @default.
• W2044218758 cites W2125562918 @default.
• W2044218758 cites W2131303188 @default.
• W2044218758 cites W954944586 @default.
• W2044218758 doi "https://doi.org/10.1074/jbc.m308230200" @default.
• W2044218758 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14555654" @default.
• W2044218758 hasPublicationYear "2004" @default.
• W2044218758 type Work @default.
• W2044218758 sameAs 2044218758 @default.
• W2044218758 citedByCount "95" @default.
• W2044218758 countsByYear W20442187582012 @default.
• W2044218758 countsByYear W20442187582013 @default.
• W2044218758 countsByYear W20442187582014 @default.
• W2044218758 countsByYear W20442187582015 @default.
• W2044218758 countsByYear W20442187582016 @default.
• W2044218758 countsByYear W20442187582017 @default.
• W2044218758 countsByYear W20442187582018 @default.
• W2044218758 countsByYear W20442187582019 @default.
• W2044218758 countsByYear W20442187582020 @default.
• W2044218758 countsByYear W20442187582021 @default.
• W2044218758 countsByYear W20442187582022 @default.
• W2044218758 countsByYear W20442187582023 @default.
• W2044218758 crossrefType "journal-article" @default.
• W2044218758 hasAuthorship W2044218758A5005692205 @default.
• W2044218758 hasAuthorship W2044218758A5020805217 @default.
• W2044218758 hasAuthorship W2044218758A5021242191 @default.
• W2044218758 hasAuthorship W2044218758A5031030356 @default.
• W2044218758 hasAuthorship W2044218758A5064656627 @default.
• W2044218758 hasAuthorship W2044218758A5074911112 @default.
• W2044218758 hasAuthorship W2044218758A5080543093 @default.
• W2044218758 hasBestOaLocation W20442187581 @default.
• W2044218758 hasConcept C181199279 @default.
• W2044218758 hasConcept C185592680 @default.
• W2044218758 hasConcept C197957613 @default.
• W2044218758 hasConcept C2775859393 @default.
• W2044218758 hasConcept C2776155809 @default.
• W2044218758 hasConcept C2777576037 @default.
• W2044218758 hasConcept C2778447961 @default.
• W2044218758 hasConcept C2779222958 @default.
• W2044218758 hasConcept C28859421 @default.
• W2044218758 hasConcept C55493867 @default.
• W2044218758 hasConceptScore W2044218758C181199279 @default.
• W2044218758 hasConceptScore W2044218758C185592680 @default.
• W2044218758 hasConceptScore W2044218758C197957613 @default.
• W2044218758 hasConceptScore W2044218758C2775859393 @default.
• W2044218758 hasConceptScore W2044218758C2776155809 @default.
• W2044218758 hasConceptScore W2044218758C2777576037 @default.
• W2044218758 hasConceptScore W2044218758C2778447961 @default.
• W2044218758 hasConceptScore W2044218758C2779222958 @default.
• W2044218758 hasConceptScore W2044218758C28859421 @default.

• next