©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
FLX1 Codes for a Carrier Protein Involved in Maintaining a Proper Balance of Flavin Nucleotides in Yeast Mitochondria (*)

(Received for publication, January 4, 1996)

Alexander Tzagoloff (§) Jeanne Jang D. Moira Glerum (¶) Mian Wu

From the Department of Biological Sciences, Columbia University, New York, New York 10027

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Respiratory deficient mutants of Saccharomyces cerevisiae assigned to complementation group G178 were previously shown to have lowered concentrations of FAD and increased concentrations of FMN in mitochondria(1) . As a result of this nucleotide imbalance, the ratio of FAD to FMN is decreased 6-10-fold, and the mutants lack succinate dehydrogenase and lipoamide dehydrogenase activities, both of which depend on FAD. Overexpression of FAD1, coding for yeast FAD synthetase, partially corrects the respiratory defect and mitochondrial FAD deficiency by suppressing, rather than complementing, G178 mutants(1) . Based on the ability of FAD1 to partially restore the mitochondrial content of FAD, G178 mutants were rationalized to be defective in mitochondrial import of FAD. The ability of FAD1 to overcome the transport defect could be explained either by enhanced passive diffusion of FAD into mitochondria, due to more efficient synthesis of the coenzyme in the cytoplasm, or by mislocalization of FAD synthetase into mitochondria of strains overexpressing the gene from a multicopy plasmid. The presence of low but measurable FAD synthetase activity in mitochondria of yeast transformed with FAD1 on a high copy plasmid suggested mislocalization of a small fraction of the enzyme, because of its high cytoplasmic concentration, as the more likely explanation.

To gain a better understanding of the biochemical basis for the FAD deficiency, we have isolated a second gene that confers respiratory function to G178 mutants, in this case by complementation. This gene has been designated FLX1 (flavin exchange). In contrast to FAD1, mutants transformed with FLX1 grow as well as the wild-type strain on nonfermentable carbon sources. The deduced primary structure of the protein encoded by FLX1 indicates that it belongs to a family of mitochondrial transport proteins, with such members as the adenine nucleotide(2) , phosphate(3) , and several carboxylate carriers(4, 5) . The identification of the FLX1 gene product as an exchange carrier confirms the earlier proposal that the aberrant ratio of flavin nucleotides in the mutant is caused by a transport defect. This is also supported by studies on FAD permeability in submitochondrial particles prepared from wild-type and mutant yeast.


MATERIALS AND METHODS

Strains of S. cerevisiae and Growth Media

The genotypes and sources of the mutant and wild-type yeast strains used in this study are listed in Table 1. The compositions of the solid and liquid growth media have been described(1) .



Transformation of Yeast

aE536/LU1 (a leu2 ura3 flx1-1) was obtained from a cross of the original isolate E536 (9) to W303-1A. The mutant was grown in 100 ml of YPD to an approximate density of 10^7 cells/ml and transformed, by the method of Schiestl and Gietz(10) , with a yeast genomic library consisting of partial Sau3A fragments of yeast nuclear DNA (7-10 kb (^1)in length) ligated to the BamHI site of the shuttle vector YEp24(11) . This library was kindly provided by Dr. Marian Carlson (Department of Human Genetics, Columbia University).

Preparation of Mitochondria and Nucleic Acids from Yeast

Mitochondria were isolated from yeast grown in liquid YPGal by the method Faye et al.(12) , except that Zymolyase 20,000 instead of Glusulase was used to prepare spheroplasts. Yeast nuclear DNA was prepared by lysis of spheroplasts with 2% SDS, 0.1 M NaCl, 1 mM EDTA, followed by extraction with phenol:chloroform:isoamyl alcohol (1:1:0.2) and centrifugation in CsCl.

Disruption of FLX1

To construct a disrupted allele of FLX1, pG178/ST5 (see Fig. 3) was digested with BamHI, and the linearized plasmid was ligated to a 3.0-kb BglII fragment containing the yeast LEU2 gene. The disrupted flx1::LEU2 allele was recovered from this construct as a linear SmaI-SphI fragment and was substituted for the normal chromosomal copy of the gene in the respiratory competent haploid strains W303-1A and W303-1B by the one-step gene replacement procedure of Rothstein(13) .


Figure 3: Disruption of FLX1. The construction of the flx1::LEU2 allele is illustrated in the lower part of the figure. pG178/ST3, linearized with BamHI, was ligated to a 3-kb BglII fragment containing the LEU2 gene. The locations of the BamHI (B), EcoRI (E), and BamHI-BglII (B/G) junctions are indicated on the maps. W303DeltaFLX1 and aW303DeltaFLX1 are two leucine-independent clones obtained by transformation of W303-1B and W303-1A, respectively, with a linear fragment of DNA containing the disrupted gene. Chromosomal DNA purified from the parental strain W303-1A (lane 1) and from each transformant (lanes 2 and 3) was digested with EcoRI, separated on a 1% agarose gel, and transferred to nitrocellulose. The blot was hybridized with a labeled 800-base pair fragment spanning the BamHI site used for the disruption. The probe detects a single 2-kb EcoRI fragment in the parental strain and two new fragments of approximately 1.8 and 3.2 kb in the mutants. The 1.8-kb fragment is consistent with the size of the region located between the EcoRI sites in the LEU2 and the FLX1 genes. The second fragment also has a size compatible with the presence of an EcoRI site 2 kb upstream of the EcoRI site in the gene. The migration of known DNA standards is indicated on the left.



Construction of a trpE/FLX1 Fusion Gene

The 1.45-kb BamHI-XbaI fragment containing the entire coding sequence of FLX1 starting from codon 21 was ligated in-frame to the amino-terminal coding sequence of trpE in pATH3(14) . The fusion protein was isolated in the insoluble fraction of Escherichia coli RR1 after lysis of cells in the presence of detergent and was size-fractionated on BiogelA 0.5 developed in the presence of 0.1% sodium dodecyl sulfate. Antibodies were raised in rabbits against the purified protein.

Manipulation of Nucleic Acids and Miscellaneous Methods

Standard methods were used for the transformation of E. coli, preparation of recombinant plasmids from E. coli, restriction enzyme mapping, and isolation and ligation of restriction fragments(15) . The sequences of FLX1 and flanking regions were determined by the method of Maxam and Gilbert, using 5` end-labeled single-stranded restriction fragments(16) . Samples of mitochondria and postmitochondrial supernatant proteins were separated on 12% polyacrylamide gels(17) , and after electrophoretic transfer to nitrocellulose, the Western blots were reacted with antiserum against a protein expressed from a trpE/FLX1 fusion gene.

Quantitation of Flavin Nucleotides

FAD and FMN, purchased from Sigma, were further purified on a preparative µBondapak C(18) (Waters) column developed isocratically with 25% methanol in 10 mM potassium phosphate buffer, pH 6. Riboflavin was used without any additional purification. The concentrations of the nucleotides were estimated spectrally using millimolar extinction coefficients of 11.3, 12.5, and 12.5 at 450 nm for oxidized FAD, FMN, and riboflavin, respectively(18) . For the enzymatic assays, samples were separated on a 3.9 times 300-mm 5 µBondapak column, with 25% methanol in 10 mM potassium phosphate, pH 6, as the mobile phase. The elution of flavin nucleotides was monitored both at 450 nm and by fluorescence emission at 530 nm, with an excitation wavelength of 450 nm. The concentration of each nucleotide was determined from the area under each peak. Standard curves obtained with the purified compounds were linear over a range of 0-1 nmol.

Enzyme Assays

Spectra of cytochromes were determined on detergent extracts of mitochondria(19) . Lipoamide dehydrogenase activity was measured by following lipoic acid-dependent oxidation of NADH at 340 nm(20) . Protein concentrations were measured according to Lowry et al.(21) .


RESULTS

Cloning and Sequence Analysis of FLX1

To better understand the biochemical basis for the respiratory deficiency of G178 mutants, attempts were made to clone the gene by transformation of aE536/LU1 with a yeast genomic library constructed with the yeast/E. coli shuttle vector YEp24(11) . This mutant was verified to have a phenotype similar to E536/AL1, the G178 mutant used previously to clone FAD1(1) . aE536/LU1 has virtually no lipoamide dehydrogenase activity (Fig. 1A) and displays an approximately 7-fold reduction in the mitochondrial FAD/FMN ratio (Fig. 1B).


Figure 1: Phenotype of aE536/LU1. Panel A, lipoamide dehydrogenase activity of mutant and wild-type yeast mitochondria. Mitochondria were prepared from the respiratory competent strain W303-1A, from the flx1 mutant aE536/LU1 and from the mutant transformed with pG178/T1 (aE536/LU1/T1). Mitochondria permeabilized with 1% deoxycholate were assayed for lipoamide dehydrogenase in a 1-ml reaction mixture containing 20 mM potassium phosphate, pH 7.5, 30 µM NADH, 15 µM NAD, 0.1 mM KCN, and 2 mM lipoamide. The oxidation of NADH was measured at 340 nm after the addition of 50 µg of mitochondrial protein. The vertical bar corresponds to an absorbance (A) of 0.01. Trace a, aE536/LU1; trace b, W303-1A; trace c, aE536/LU1/T1; trace d, aE536/LU1/T1 with no lipoamide present in the assay. Panel B, analysis of flavin nucleotides in mitochondria of wild type and mutant yeast. Mitochondria of W303-1A, aE536/LU1, and aE536/LU1/T1 were extracted by heat treatment at 80 °C, and FAD, FMN, and riboflavin (RiF) were separated by reverse-phase chromatography as described under ``Materials and Methods.'' The tracings represent fluorescence emission at 530 nm with the excitation wavelength set at 450 nm. The elution times were 2.7-2.8 min for FAD, 3.8-3.9 min for FMN, and 7.1-7.3 min for riboflavin. The concentrations of FAD and FMN and their ratios in the three strains are reported.



Transformants initially selected for uracil prototrophy were further checked for complementation of the respiratory defect based on their ability to utilize glycerol. Out of 10^9 cells used in the transformation, 12 uracil-independent clones were also respiratory competent. These transformants contained the plasmids pG178/T1 and pG178/T2, with two different but overlapping inserts of nuclear DNA (Fig. 2). The region of DNA needed for complementation of the G178 mutation was narrowed down to a segment of approximately 2.7 kb, including one of the two BamHI sites in the pG178/T1 insert (see pG178/ST6 in Fig. 2). Smaller plasmids with deletions on either side of the BamHI site in pG178/ST6 did not complement aE536/LU1, indicating that this site was likely to be in an essential region of the gene.


Figure 2: Restriction map of pG178/T1, pG178/T2, and derivative plasmids. The location of BamHI (B), XbaI (X), SmaI (Sm), SacI (Sa), and SphI (Sp) sites are shown in the nuclear insert of pG178/T2. The unique SmaI and SphI sites of YEp24 are shown to indicate the orientation of the insert. The orientation of the insert in pG178/T1 is opposite to that of pG178/T2. pG178/ST3 was created by religation of pG178/T1 after removal of the region extending from the SmaI site in the insert to the SmaI site of YEp24. The unique BamHI site in pG178/ST3 is located in the coding region of FLX1 and was used to disrupt the gene (Fig. 3). pG178/ST6 was constructed by transferring the insert from pG178/ST3 to YEp352. Complementation, or lack thereof, by the subclones is indicated by the plus and minus signs, respectively. The direction of transcription and location of FLX1 are denoted by the solid arrow in the pG178/T2 insert.



The sequence of the pG178/ST6 insert revealed the presence of an open reading containing the BamHI site, inferred from the subcloning results to be internal to the gene (Fig. 2). The sequence (GenBank accession number L41168) is identical to a region of chromosome IX, all of whose sequence has recently been reported (GenBank accession number Z47047). This gene, henceforth referred to as FLX1, codes for a protein of 311 amino acids, with a M(r) of 34,544. The deduced amino acid sequence of the FLX1 reading frame (Flx1p) indicates that it is a member of a family of mitochondrial carrier proteins involved in transport of various metabolites (3, 4, 5) and coenzymes(2) .

In addition to their amino acid sequence similarity, members of this carrier family share other structural properties(22, 23) . They are invariably composed of three homologous repeats, each approximately 100 amino acids in length. There are two hydrophobic stretches in each repeat, accounting for a total of six transmembrane sectors in the entire protein(2) . The results of dot matrix comparisons and hydrophobicity plots confirm both of these features to be present in Flx1p (data not shown).

Disruption of FLX1

Several lines of evidence indicate that the restoration of respiratory competence in G178 mutants by FLX1 is due to complementation. Transformation of W303-1A and W303-1B with a disrupted allele of FLX1 (flx1::LEU2) on a linear DNA fragment yielded leucine-independent and respiratory defective clones, which were verified by Southern analysis of their genomic DNA to have acquired the mutant allele (Fig. 3). The respiratory block of the Leu transformants is not complemented by the G178 mutant E536, suggesting that the mutations in the two strains are genetically linked. This was corroborated by allelism tests. The FLX1 gene was transferred from pG178/ST6 to the integrative vector YIp352, which lacks the 2 µ origin of replication(24) . This new construct (pG178/ST9) was linearized at the unique BamHI in the gene to direct integration into the homologous chromosomal locus. Transformation of aE536/LU1 with the linearized plasmid produced uracil-independent and respiratory competent clones with a chromosomally integrated copy of the wild-type gene as determined by crosses to the respiratory competent parental haploid strain W303-1B and the isogenic mutant W303DeltaFLX1. Diploid cells obtained from the crosses were sporulated and analyzed by tetrad dissections. A total of 16 complete tetrads, analyzed from crosses of two independent respiratory competent transformants of aE536/LU1 to W303-1B, all showed a 2:2 segregation of the ura3 marker and a 4:0 segregation of the respiratory competent phenotype, confirming that the FLX1 and URA3 markers had integrated into chromosomal DNA. A similar number of tetrads derived from the same transformants crossed to W303DeltaFLX1 exhibited a 2:2 segregation of both the uracil-independent and respiratory competent phenotypes. In this case, only the respiratory competent segregants were uracil-independent, indicating that the mutation in aE536/LU1 is allelic to FLX1.

Mitochondrial Localization of Flx1p

The FLX1 product was localized in mitochondria with an antibody against a protein expressed from a trpE/FLX1 fusion gene. Mitochondria were prepared from the respiratory competent yeast W303-1B, from W303DeltaFLX1, a strain with a disrupted copy of FLX1, and from the transformant aE536/LU1/T1 harboring the wild-type gene on a multicopy plasmid. The immunoblot indicated the presence of a weakly reacting band with a size expected of Flx1p in the mitochondrial fraction of wild type (did not reproduce in Fig. 4A), but not the mutant with the disrupted gene. The antibody detected a much more abundant protein with the same electrophoretic mobility in mitochondria but not the postmitochondrial supernatant fraction of the transformant aE536/LU1/T1 (Fig. 4A). The mitochondrial localization of the protein expressed from the multicopy plasmid was further confirmed by purification of mitochondria from the transformant on an isopycnic gradient. Assays of the gradient fractions indicate a coincidence in the distribution of the protein detected by the antibody and of cytochrome oxidase activity (Fig. 4B).


Figure 4: Western blot analysis of Flx1p. In panel A, 30 µg of mitochondrial or postmitochondrial supernatant protein was separated by PAGE on a 12% polyacrylamide gel. The proteins were transferred to nitrocellulose and probed with antibody against the trpE/FLX1 fusion product. Lane 1, mitochondria of the respiratory competent strain W303-1B; lane 2, mitochondria of the mutant W303DeltaFLX1; lane 3, mitochondria of aE536/LU1/T1, a flx1 mutant harboring pG178/T1; lane 4, postmitochondrial supernatant of aE536/LU1/T1. The protein marked as Flxp1p on the right migrates with an apparent molecular weight of 33,000. A weakly stained protein of the same size is also detected in the lane with the wild-type (too faint to reproduce in the photograph) but not mutant mitochondria. In panel B, mitochondria (6.4 mg of protein) of aE536/LU1/T1 were layered on a 5-ml discontinuous gradient containing the concentrations of sucrose indicated in the figure. The gradient was centrifuged for 1 h at 65,000 rpm in a Beckman SW65Ti rotor, and 14 equal size fractions were collected. Each fraction was assayed for cytochrome oxidase activity and for Flx1p by Western blot analysis. The distribution of cytochrome oxidase across the gradient is shown by the bar graph. The relative concentration of Flx1p in the fractions is shown above the cytochrome oxidase activity. The bottom of the gradient is indicated by B.



Subcellular Compartmentation of Flavin Nucleotide Biosynthesis in Yeast

FAD synthetase, the enzyme responsible for the conversion of FMN to FAD was previously found to be localized in the cytoplasm (1) . In view of the limited information available on compartmentation of flavin biosynthesis in S. cerevisiae, a more complete analysis was done of the subcellular distribution of both FAD synthetase and the preceding enzyme of the pathway, riboflavin kinase (25, 26) .

Mitochondria were isolated from the respiratory competent haploid strain W303-1A, grown under derepressed conditions on galactose. Proteins of the postmitochondrial supernatant fraction were cleared of small membrane fragments and polyribosomes and were concentrated by precipitation with ammonium sulfate. This fraction catalyzes a time-dependent conversion of FMN to FAD in the presence of ATP with a specific activity of 20 pmol/min/mg of protein (data not shown). The identical fraction prepared from aW303DeltaFLX1 had a similar specific activity, indicating that the flx1 mutation does not affect expression of FAD synthetase.

In agreement with earlier findings, mitochondria of respiratory competent yeast, permeabilized by sonic disruption, failed to promote any significant conversion of FMN to FAD (data not shown). This was also true of the separated soluble protein and membrane fractions obtained from sonically disrupted mitochondria. In these assays, mitochondria or submitochondrial fractions were noted to cause a progressive decrease of FMN. The loss of FMN is probably due to a phosphatase-catalyzed dephosphorylation of FMN to riboflavin. The presence of phosphatase(s) in mitochondria and the postmitochondrial supernatant fractions, capable of converting FMN to riboflavin, is evident from the results of assays done identically to those used to measure FAD synthetase, except for the omission of ATP. Under these conditions, disappearance of FMN is accompanied by a nearly stoichiometric appearance of riboflavin (data not shown). The postmitochondrial fraction is only slightly more active than mitochondria. Submitochondrial membrane vesicles and the soluble protein fraction of mitochondria both promote dephosphorylation of FMN.

The synthesis of FMN from riboflavin, catalyzed by riboflavin kinase, is difficult to quantitate because of the competing phosphatase reaction. Incubation of riboflavin and ATP in the presence of postmitochondrial supernatant proteins leads to the appearance of both FMN and FAD (Fig. 5). Since the decrease in riboflavin is equivalent to the FAD and FMN synthesized, the riboflavin kinase rate in the presence of excess substrate (50 µM riboflavin) must be at least equal to the sum of the rates of FMN accumulation and dephosphorylation. The true rate is probably even higher since some of the FMN synthesized is also converted to FAD. The net increase in FMN indicates that FAD synthetase catalyzes the rate-limiting step during the conversion of riboflavin to FAD.


Figure 5: Riboflavin kinase activity in mitochondria and postmitochondrial supernatant fractions. The respiratory competent strain of yeast W303-1A, and the flx1 mutant aW303DeltaFLX1 were grown to stationary phase in YPGal, and mitochondria were prepared. Mitochondrial suspensions at a protein concentration of 20 mg/ml in 0.4 M sorbitol, 20 mM Tris-HCl, pH 7.5 (ST) were sonically irradiated for 5 s and centrifuged at 105,000 times g for 20 min. The supernatant consisting of matrix proteins was decanted, and the submitochondrial membranes recovered in the pellet were washed with an excess of ST and resuspended in the starting volume of ST. The postmitochondrial supernatant was centrifuged at 105,000 times g for 20 min to remove remaining membrane fragments and was adjusted to 80% saturation with solid ammonium sulfate. Following centrifugation at 20,000 times g for 15 min the protein precipitate was dissolved in one-fifth the starting volume of 20 mM Tris-HCl, pH 7.5 and dialyzed overnight against the same buffer. The mitochondria (MIT), the ammonium sulfate-precipitated proteins of the postmitochondrial supernatant (PMS), the matrix fraction (MATRIX), and the submitochondrial membranes (SMP) were tested for riboflavin kinase activity. Conversion of riboflavin to FMN was assayed in 20 µl of a reaction mixture containing 50 mM Tris-HCl, pH 7.5, 5 mM MgCl(2), 5 mM ATP, 50 µM riboflavin, and 200 µg of the postribosomal supernatant or mitochondrial proteins. The volumes of the matrix and submitochondrial fractions added to the assay were the same as mitochondria. After the indicated times (0, 15, 30 min) at 37 °C, the reactions were quenched by the addition of 180 µl of 10 mM potassium phosphate, pH 6, preheated to 80 °C. The mixtures were incubated for 5 min at 80 °C, adjusted to 25% methanol, and centrifuged to remove denatured proteins. The extracted flavins were separated by reverse-phase chromatography on a 5 µBondapak column as detailed under ``Materials and Methods.'' The values reported correspond to differences in FAD (circle, W303-1A; box, aW303DeltaFLX1), FMN (bullet, W303-1A; , aW303DeltaFLX1), and riboflavin (up triangle, W303-1A; , aW303DeltaFLX1) in the total sample.



Phosphorylation of riboflavin, as measured by the appearance of FMN, occurs with a 3-4 times lower specific activity when permeabilized mitochondria are used instead of the postmitochondrial supernatant fraction. The specific activity of this reaction measured with mitochondria is approximately 5 pmol/min/mg protein. This rate, however, does not take into account dephosphorylation of FMN under the assay conditions used. A more realistic estimate of the mitochondrial riboflavin kinase activity can be obtained from the difference in FMN breakdown in the presence and absence of ATP. This calculation yields a value of 20 pmol/min/mg of protein. Most of the mitochondrial riboflavin kinase activity is recovered in the soluble protein fraction obtained after sonic disruption of mitochondria, suggesting that it is a matrix enzyme.

Efflux of FAD from Submitochondrial Particles

The presence in mitochondria of riboflavin kinase but not FAD synthetase is consistent with a role of the Flx1p in import of cytoplasmically synthesized FAD into mitochondria. A mutation restricting the flow of FAD into mitochondria would be expected to shift the mitochondrial ratio of flavin nucleotides in favor of FMN, which is in agreement with the phenotype of flx1 mutants. To test the possible involvement of Flx1p in transport of FAD into mitochondria, efflux of FAD was measured in submitochondrial vesicles preloaded with this nucleotide. The rate of efflux of entrapped FAD was compared in vesicles prepared from wild-type yeast, a flx1 mutant, and the same mutant transformed with FLX1. In these experiments, mitochondria were sonically treated with or without FAD in the suspending buffer, and the resultant membrane vesicles were washed several times to remove residual external nucleotide. The concentrations of endogenous and trapped FAD, determined for the native and preloaded membranes are reported in Table 2. In agreement with previous analyses of unfractionated mitochondria, submitochondrial membrane vesicles prepared from two different mutant strains have 5 times less FAD than the comparable fractions derived from the respiratory competent strain and from the mutant transformed with FLX1. The increase in the flavin content of the membrane ranges from 0.12 to 0.18 nmol/mg protein when mitochondria are disrupted in the presence of 0.06 mM FAD in the suspending medium.



After incubation at 37 °C over a period of 30 min, the submitochondrial particles were sedimented by high speed centrifugation, and the concentration of FAD was measured in the supernatant. The rate of release of FAD depends on the source of the membranes and whether or not they were preloaded with FAD. The highest rate of FAD escape was measured in preloaded particles from the respiratory competent strain D273-10B and the transformant aE536/LU1/T1 (Fig. 6). Efflux of FAD occurred at about 10 pmol/min/mg of protein. Similar rates were observed in the respiratory competent haploid strain W303-1A and in a ^o derivative of W303-1A (data not shown). This rate was approximately 2 times higher than that measured in the same particles that had not been preloaded. Submitochondrial particles of two different mutants (aW303DeltaFLX1, and aE536/LU1) preloaded with FAD, showed 3 times lower rates of efflux.


Figure 6: Efflux of FAD from submitochondrial vesicles. Submitochondrial vesicles prepared in the presence or absence of external FAD as described in the legend to Table 2, were suspended at a protein concentration of 8-10 mg/ml in 0.4 M sorbitol and were incubated for the indicated times at 37 °C. The membrane vesicles (2 mg of protein) were sedimented by centrifugation at 105,000 times g for 20 min. The supernatants were collected, and their FAD concentrations were determined as described in the legend to Fig. 5. A, submitochondrial vesicles were prepared in the presence of FAD from the wild-type strain D273-10B/A1 (bullet) and from the flx1 mutant aE536/LU1 (circle). B, submitochondrial vesicles were prepared from the transformant aE536/LU1/T1 in the presence (bullet) and absence (box) of FAD and from the mutant aW303DeltaFLX1 in the presence () and absence of FAD ([circo).



The addition of FMN did not increase the rate of FAD efflux (data not shown). It was not possible to measure FMN efflux because of the rapid rate of dephosphorylation by the particles of the nucleotide to riboflavin (see above).


DISCUSSION

The respiratory defect of mutants in complementation group G178 was previously ascribed to an imbalance in the mitochondrial FAD/FMN ratio(1) . The decreased concentration of FAD severely affects the enzymatic activity of succinate dehydrogenase and lipoyl dehydrogenase, both of which employ FAD as coenzyme. The yeast FLX1 gene, cloned in the present study, confers respiratory competence to G178 mutants and restores the FAD/FMN ratio to that seen in wild-type yeast. Allelism tests indicate that FLX1 exerts its effect by complementing mutations in the endogenous gene. This is in contrast to previous findings that partial restoration of respiration in flx1 mutants by FAD1 (in multicopy), the structural gene for yeast FAD synthetase, is due to suppression (1) .

The primary structure of Flx1p indicates it is a new member of a protein family consisting of mitochondrial substrate and nucleotide carriers. In addition to being homologous to previously reported carrier proteins, Flx1p also exhibits the tripartite repeat structure characteristic of this class of carriers(22, 23) . The identification of a mitochondrial carrier protein as the target of the mutations defining complementation group G178 indicates that the lower mitochondrial concentration of FAD in this group of mutants stems from a transport defect. Although mitochondrial levels of FAD could be influenced by transport of a component other than FAD, several lines of evidence suggest that Flx1p is directly involved in transport of FAD. Assays of FAD synthetase and riboflavin kinase in respiratory competent yeast indicate that the kinase is present in both mitochondria and the soluble protein fraction of yeast. FAD synthetase activity, however, is present in the soluble fraction but is absent in mitochondria, implying that mitochondrial FAD is imported from the cytoplasm. The dual distribution of enzymes catalyzing a biosynthetic pathway whose end product functions in both mitochondria and the cytoplasmic compartment has precedents in amino acid biosynthesis (e.g. arginine, leucine) and heme biosynthesis. In the case of heme, which in most cells is associated predominantly with the respiratory chain, the mitochondrial localization of ferrochelatase may favor fine tuning the regulation of this pathway. The presence of FAD synthetase in the cytoplasm of yeast may similarly be of advantage in regulating the production of this coenzyme, the bulk of which is used by cytoplasmic enzymes.

The lower ratio of FAD to FMN in flx1 mutants is consistent with a role of Flx1 in transport of FAD into the organelle. Further support for a role of Flx1p in FAD transport comes from studies of FAD efflux from submitochondrial particles. Vesicles prepared from wild-type mitochondria or from mitochondria of mutants transformed with FLX1 display a 2-3 times higher rate of FAD transfer than membranes obtained from flx1 mutants. Attempts to measure efflux of FMN were not successful because of the rapid rate of conversion of entrapped FMN to riboflavin. The rates of FAD transfer from either wild-type or mutant vesicles were not increased by the presence of FMN in the assay buffer. It is not clear at present, therefore, whether FMN is also a ligand for Flx1p.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant HL22174. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

Recipient of a post-doctoral fellowship from the Medical Research Council of Canada.

(^1)
The abbreviation used is: kb, kilobase(s).


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