(Received for publication, January 4, 1996)
From the
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.
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.
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. W303FLX1 and aW303
FLX1 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.
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 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
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).
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
W303FLX1; 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.
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 aW303FLX1 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
aW303FLX1 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
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
g for 20 min to remove remaining membrane fragments and was adjusted
to 80% saturation with solid ammonium sulfate. Following centrifugation
at 20,000
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
, 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
(
, W303-1A;
, aW303
FLX1), FMN (
,
W303-1A;
, aW303
FLX1), and riboflavin (
,
W303-1A;
, aW303
FLX1) 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.
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 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 (aW303
FLX1, 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 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 (
) and from the flx1 mutant aE536/LU1 (
). B, submitochondrial vesicles
were prepared from the transformant aE536/LU1/T1 in the presence
(
) and absence (
) of FAD and from the mutant
aW303
FLX1 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).
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.