Yeast dihydroxybutanone phosphate synthase, an enzyme of the riboflavin biosynthetic pathway, has a second unrelated function in expression of mitochondrial respiration.

aE280/U1 is a pet mutant of Saccharomyces cerevisiae partially deficient in cytochromes a, a3, and cytochrome b. The ability of this mutant to respire is restored by RIB3, a gene previously shown to code for 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBP synthase), an enzyme of the riboflavin biosynthetic pathway. The sequences of RIB3 from wild type and aE280/U1 indicated a single base change resulting in an A137T substitution. The alanine 137 is a conserved residue located in a cavity on the surface of the protein distant from the active site and from the subunit interaction domain involved in homodimer formation. The respiratory defect elicited by this mutation cannot be explained by a flavin insufficiency based on the following evidence: 1) growth of the aE280/U1 on respiratory substrates is not rescued by exogenous riboflavin; 2) the levels of flavin nucleotides are not significantly different in the mutant and wild type. We proposed that in addition to its known function in riboflavin synthesis, RIB3 also functions in expression of mitochondrial respiration. Restoration by riboflavin of growth of a rib3 deletion mutant on glucose but not glycerol/ethanol also supported this conclusion. An antibody against the N-terminal half of DHBP synthase was used to study its subcellular distribution. Most of the protein was localized in the cytosolic fraction, but a small fraction was detected in the mitochondrial intermembrane space.

aE280/U1 is a pet mutant of Saccharomyces cerevisiae partially deficient in cytochromes a, a3, and cytochrome b. The ability of this mutant to respire is restored by RIB3, a gene previously shown to code for 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBP synthase), an enzyme of the riboflavin biosynthetic pathway. The sequences of RIB3 from wild type and aE280/U1 indicated a single base change resulting in an A137T substitution. The alanine 137 is a conserved residue located in a cavity on the surface of the protein distant from the active site and from the subunit interaction domain involved in homodimer formation. The respiratory defect elicited by this mutation cannot be explained by a flavin insufficiency based on the following evidence: 1) growth of the aE280/U1 on respiratory substrates is not rescued by exogenous riboflavin; 2) the levels of flavin nucleotides are not significantly different in the mutant and wild type. We proposed that in addition to its known function in riboflavin synthesis, RIB3 also functions in expression of mitochondrial respiration. Restoration by riboflavin of growth of a rib3 deletion mutant on glucose but not glycerol/ethanol also supported this conclusion. An antibody against the N-terminal half of DHBP synthase was used to study its subcellular distribution. Most of the protein was localized in the cytosolic fraction, but a small fraction was detected in the mitochondrial intermembrane space.
Saccharomyces cerevisiae pet mutants have been used to study a broad range of problems related to mitochondrial function and biogenesis (1). Such mutants have been assigned to the following phenotypic classes based on their compositions of respiratory chain components: 1) mutants defective in individual carriers of the respiratory chain, 2) mutants having a normal set of respiratory enzymes (e.g. cytochrome spectra and ATPase), and 3) mutants with partial or severe pleiotropic deficiencies in respiratory chain components. Functional analysis of the latter is difficult because the phenotype fails to provide clues as to the nature of the biochemical lesion.
In the present study we have characterized a pet mutant (aE280/U1) with partial pleiotropic defects in respiratory chain components. The mutant has been determined to have a point mutation in RIB3, a conserved gene of which the product, 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBP 1 synthase), catalyzes the first step in the riboflavin biosynthetic pathway (2). Complementation of aE280/U1 by RIB3 was unexpected in view of previous studies indicating that rib3 mutants require riboflavin for growth (3). The lack of correspondence in the phenotype of the rib3 point mutant and a mutant with a complete deletion of RIB3 prompted us to examine the biochemical properties of the mutant further. We present evidence that the phenotype of aE280/U1 is not related to a flavin nucleotide deficiency and propose that in addition to its involvement in riboflavin synthesis, DHBP synthase has another function essential for expression of respiration in yeast.

MATERIALS AND METHODS
Strains and Media-The strains of yeast used in this study are listed in Table I. The respiratory-deficient mutant E280 (MAT␣ met6 rib3) was derived from S. cerevisiae D273-10B/A21 by mutagenesis with nitrosoguanidine (1). The media used for the growth of yeast have been described elsewhere (4). Escherichia coli RR1 was used for amplification of plasmid DNA and for expression of fusion proteins.
Disruption of RIB3-The linear SacI-PstI fragment containing RIB3 ( Fig. 1) was transferred to pUC18 (5). Two divergent primers each containing a BamHI site were used to amplify the entire plasmid except for the RIB3 coding region. The amplified plasmid was ligated to a 1-kb BamHI fragment containing the yeast HIS3 gene. The null allele, recovered as a linear SacI-PstI fragment, was introduced into the wild type diploid strain W303 by the one-step gene substitution procedure (6). Dissection of tetrads obtained from several histidine prototrophic diploid transformants indicated that growth of one-half of the meiotic progeny on glucose depended on the presence of riboflavin. The riboflavin auxotrophy co-segregated with the histidines prototrophy.
Preparation of an Antibody against DHBP Synthase-The region between the BclI and StyI sites coding for the amino-terminal half of DHBP synthase was ligated in frame to the E. coli expression vector pATH2 (7) digested with BamHI. This ligation required the introduction of a BamHI linker at the StyI site. E. coli RR1 cells harboring the plasmid were used to produce the trpE/RIB3 fusion product as described elsewhere (6). Following cells lysis, the overexpressed protein was recovered in the insoluble protein fraction and was dissolved in buffer containing 10 mM Tris-Cl, pH 7.5, 2% SDS, 10 mM ␤-mercaptoethanol, and 5 g/ml phenylmethylsulfonyl fluoride. The solubilized fusion protein was further purified by sizing on a Bio-Gel A 0.5 (BioRad) with 10 mM Tris-Cl, pH 7.5, 0.1 mM EDTA, and 5 mM ␤-mercaptoethanol as the eluting buffer. Fractions enriched for the fusion protein were pooled, concentrated by precipitation with acetone, and dissolved in 0.1% SDS, 1 mM ␤-mercaptoethanol for immunization of rabbits.
Cloning of the Wild Type and Mutant RIB3-A recombinant plasmid (pG164/T3) containing RIB3 was obtained by transformation of aE280/U1 with a yeast genomic library by the method of Schiestl and Gietz (8). This library, consisting of partial Sau3A fragments of yeast nuclear DNA cloned in the URA3-bearing shuttle plasmid YEp24 (9), was generously provided by Dr. Marion Carlson, Department of Human Genetics, Columbia University. The chemical method of Maxam and Gilbert (10) was used to sequence the ends of the nuclear DNA insert in pG164/T3.
To clone the mutant rib3 gene, nuclear DNA prepared from aE280/U1 was digested with a combination of PstI and SacI. Fragments averaging 1.9 kb were cloned in YEp352 (11) and used to transform E. coli. The resultant library was screened by colony hybridization with a 32 P-labeled 1.9-kb PstI-SacI fragment containing RIB3 (12). The identical procedure was used to clone RIB3 from the wild type strains W303-1A and D273-10B/A1. The two wild type and the mutant RIB3 genes were sequenced by the Sanger method (13) with a commercial sequencing kit (United States Biochemical, Cleveland, OH).
Miscellaneous Procedures-Standard methods were used for plasmid manipulations and colony hybridization (14). Yeast mitochondria were prepared by the method of Faye et al. (15) except that Zymolyase 20T (ICN Laboratories, Aurora, OH) instead of glusulase was used to convert cells to spheroplasts. Spectral analyses of mitochondrial cytochromes were performed as described previously (16). Flavin nucleotides were extracted and separated by HPLC on a C18 reverse phase column as described previously (17). Protein concentrations were determined by the method of Lowry et al. (18).

Phenotype of E280 and Identification of the Mutant Gene-
E280 is a respiratory-deficient strain of S. cerevisiae previously assigned to complementation group G164 of a pet mutants collection (1). Like other pet mutants, E280 grows on glucose but not on nonfermentable substrates such as glycerol/ethanol. Some growth of the mutant, however, is observed on glycerol/ ethanol after incubation of plates for prolonged periods. The mutant is complemented by o testers indicating that the growth defect on respiratory substrates is due to a recessive mutation in a nuclear gene. E280 displays normal translation of the mitochondrial gene products (Fig. 1A). Spectra of mitochondrial extracts, however, indicate a partial deficit of cytochromes a, a 3 , and b relative to cytochrome c (Fig. 1B). A 2-fold reduction of cytochrome oxidase in the mutant was measured enzymatically (Table II). The NADH and succinate oxidase activities are reduced, respectively, to 25 and 16% of wild type mitochondria. The different oxidase activities were not increased in mitochondria of the mutant grown in the presence of riboflavin.
To identify the gene responsible for the respiratory defect of E280, the uracil-requiring derivative strain aE280/U1 was transformed with a yeast genomic plasmid library. Two independent respiratory competent clones were obtained from the transformation. The plasmids had overlapping nuclear DNA inserts, one of which (pG164/T3) was used to localize the gene (Fig. 2). The results of subcloning indicated that the gene conferring respiration on aE280/U1 is located between a SacI and a PstI site (Fig. 2). This region of the insert contains RIB3, a gene of chromosome IV, previously shown to code for DHBP synthase (3). This enzyme catalyzes the conversion of ribose-5phosphate to 3,4-dihydroxy-2-butanone-4-phosphate, an early precursors in riboflavin synthesis (2). The failure of pG164/ ST31 and pG164/ST32, each containing different halves of RIB3, to restore respiration confirmed the correct identification of the gene. Site directed integration of a single copy of RIB3 in the chromosomal DNA of aE280/U1 restored normal growth on nonfermentable carbon sources (Fig. 3A), suggesting that RIB3 acts by complementation rather than suppression the mutant.
aE280/U1 Is a rib3 Mutant-Mutations in RIB3 have been shown to elicit a riboflavin auxotrophy on glucose (3). This phenotype was confirmed in the present study by deleting the gene in the diploid strain W303 (see "Materials and Methods" for the construction of the ⌬rib3:HIS3 null allele). Growth of the haploid strain W303⌬RIB3 containing the deletion allele on glucose is strictly dependent on the addition of riboflavin. This phenotype is different from that of E280 or aE280/U1, both of which grow on glucose in the absence of riboflavin. An unexpected property of the null mutant is its failure to grow on a   1. Phenotype of E280. A, in vivo labeling of mitochondrial translation products. The wild type strain D273-10B/A1 and the rib3 mutant E280 were labeled in the presence of cycloheximide (18). Mitochondria were prepared and separated on a 7.5-15% polyacrylamide gel (20). The ribosomal protein Var1, COX subunits 1 (Cox1), 2 (Cox2), and 3 (Cox3), cytochrome b (Cyt. b), and ATPase subunits 6 (Atp6), and 9 (Atp9) are identified in the margin. B, spectra of mitochondrial cytochromes in the wild type and mutant yeast. Mitochondria were prepared from the respiratory competent parental strain D273-10B/A1 and from the respiratory defective mutant E280. The mitochondria were extracted at a protein concentration of 6 mg/ml in the presence of 1% potassium deoxycholate and 1 M KCl (19). The difference spectra of the extracts oxidized with potassium ferricyanide versus those reduced with sodium dithionite were recorded at room temperature. nonfermentable carbon source supplemented with riboflavin ( Fig 3A). Like the rib3 null mutant, growth of the point mutants on glycerol/ethanol also fails to be rescued by riboflavin (Fig. 3A) The discrepancy in the growth properties of the rib3 null mutant and aE280/U1 on glucose raised the possibility that RIB3 might be suppressing rather than complementing the respiratory defect. To test linkage of the ⌬rib3:HIS3 null and E280 point alleles, the 1.98-kb SacI-PstI fragment containing RIB3 was transferred to the integrative vector YIp352 (11) yielding pG164/ST22. This plasmid was linearized at the BstXI site internal to RIB3 and was used to transform aE280/U1. A respiratory competent and Ura ϩ clone obtained from the transformation (aE280/U1/ST22) were crossed to W303-1B. Integration of RIB3 at its homologous locus in chromosomal DNA was confirmed by dissection of 12 complete tetrads issued from the resultant diploid cells. In each case all four meiotic spore progeny were ascertained to be respiratory competent. For the allelism test aE280/U1/ST22 was crossed to the rib3 null mutant W303⌬RIB3. Ten complete tetrads were analyzed following sporulation of the diploid cells. In every case the requirement of riboflavin for growth on glucose segregated 2:2. The ability of a single copy of RIB3 to restore the respiratory deficiency of aE280/U1 and the tight genetic linkage between the mutant allele of aE280/U1 and the rib3 null allele constitute strong evidence that the respiratory deficient phenotype of E280/U1 stems from a point mutation in RIB3.
Sequence of the Mutant RIB3 Gene-To sequence the mutation, the rib3 gene was cloned from aE280/U1. Because this strain was derived from a cross of two non-isogenic strains (E280 and W303-1A), the gene was also isolated from D273-10B/A21 (parent of E280) and W303-1A. The genes obtained from the three strains were sequenced and compared with the previously reported sequence of RIB3. 2 The sequences of the D273-10B/A21 and W303-1A genes were identical to the sequence number reported in GenBank™. The rib3 gene isolated TABLE II Respiratory activities of mitochondria Mitochondria were isolated from the parental wild type strain D273-10B/A21 and from the rib3 mutant E280 grown in YPGal and in YPGal supplemented with 25 g/ml riboflavin. The NADH and succinate oxidase activities were measured at 24°C as described previously (21). Cytochrome oxidase was also assayed polarographically in the presence of N, N, NЈ, NЈ-tetramethyl-p-phenylenediamine and ascorbic acid (21). The values reported are averages of two assays with the ranges indicated.  The following strains were used to compare growth on the different media: the respiratory competent strains W303-1A (WT), the rib3 null mutants aW303⌬RIB3 (⌬rib3) and D273⌬RIB3 (⌬rib3*), the rib3 point mutant aE280/U1 (rib3), the rib3 point mutant aE280/U1/ST22U with RIB3 inserted at URA3 (rib3/i-RIB3), the rib3 point mutant aE280/U1/E280U with the mutant rib3 gene of E280 inserted at URA3 (rib3/i-rib3), and the null mutant aW303⌬RIB3 with the mutant rib3 gene of E280 (⌬rib3/i-rib3) or of the wild type gene (⌬rib3/i-RIB3). The different strains were spotted on rich glucose (YPD) or rich glycerol/ethanol (YEPG) plates without and without 25 g/ml riboflavin (ϩribo). The plates were incubated at 30°C for 36 h. from the aE280/U1 mutant, however, had a single G to A transition at nucleotide 409 of the gene, resulting in the substitution of alanine 137 by threonine.
The mutant gene isolated from aE280/U1 was cloned into the integrative plasmid YIp352. Uracil-independent clones obtained by transformation of the rib3 null mutant a/␣-W303⌬RIB3 with the E280 gene on the integrative plasmid were sporulated and the growth phenotype of the meiotic products analyzed. The presence of the mutant gene in a single copy restored normal growth of the null strain on glucose and glycerol in the absence of added riboflavin (Fig. 3B). The ability of the mutant gene to rescue the growth of the rib3 null strain is consistent with the growth properties of E280 and suggested that the A137T mutation does not affect riboflavin synthesis. Surprisingly, growth of the point mutant on glycerol was also partially restored when a single copy of the mutant gene was integrated into chromosomal DNA of the null mutant (Fig. 3C).
Flavin Content of Subcellular Fractions from Wild Type and Mutant Cells-The flavin nucleotides and riboflavin compositions of mitochondria and cytosolic fractions (post-mitochondrial supernatant) were compared in the wild type, the mutant aE280/U1, and the mutant transformed with RIB3 on a high copy plasmid. Flavin nucleotides were extracted and separated by HPLC on a C18 column. Although some differences were noted in the amount of riboflavin and flavin nucleotides in the three strains, there was no indication of any significant deficit of FMN or FAD in either the mitochondrial or post-mitochondrial fractions of the mutant (Fig. 4). Some reduction in riboflavin concentration is seen in the post-mitochondrial supernatant fraction. The increased ratio of FMN relative to FAD in mitochondria of the mutant and transformant may be because of strain differences, as this is also true of mitochondria in the D273-10B/A21 wild type strain (not shown). These results argue strongly against any significant effect of the A137T mutation on riboflavin synthesis.
Localization of DHBP Synthase-The subcellular localization of DHBP synthase was studied with an antibody raised against the protein expressed from a trpE/RIB3 fusion gene. The antibody detected a protein of ϳ28 kDa in the post-mito-chondrial supernatant fraction of wild type yeast (Fig. 5A). The identity of this protein as DHBP synthase is confirmed by its absence in the rib3 null mutant and its presence at much higher concentrations in the mutant transformed with the wild type gene on a multicopy plasmid. The mass of DHBP synthase derived from the sequence of RIB3 is 22.5 kDa. The reason for the apparent larger mass obtained by SDS-PAGE is not clear.
Although most of the DHBP synthase was detected in the post-mitochondrial supernatant fraction corresponding to the soluble cytosolic proteins of yeast, a small fraction was also present in mitochondria. This was true both for the transformant overexpressing the protein (Fig. 5A) and for wild type (see longer exposure in Fig. 5B). Several lines of evidence indicated that this was not an artifactual association. The mitochondrial fraction isolated from an over-expresser was further purified on a Nycodenz gradient (23). The gradient fractions were probed with antibody against DHBP synthase and cytochrome b 2 , used here as a mitochondrial marker. The antibody detected two peaks, one of which co-sedimented with the mitochondrial cytochrome b 2 marker (Fig. 5C). The second peak was well separated in the upper part of the gradient. No attempts were made to identify the nature of the material in this fraction.
Mitochondria purified on a Nycodenz gradient were diluted under isotonic and hypotonic conditions. Even though the hypotonic treatment causes the outer membrane to burst, it remains attached to and fractionates with the mitoplasts. Both mitochondria and mitoplasts were treated with proteinase K (24). The results of this experiment indicate that disruption of the outer membrane released most of the DHBP synthase and of the intermembrane marker cytochrome b 2 (Fig. 5D). The small fraction of the two proteins remaining in the mitoplasts was completely susceptible to the protease. In contrast, DHBP synthase and cytochrome b 2 were largely resistant to the protease in unlysed mitochondria (Fig. 5D). The loss of DHBP synthase from the mitoplasts suggests that the protein is located in the intermembrane space. The possibility that DHBP synthase may be associated with the outer membrane and is dissociated from it under hypotonic conditions, however, cannot be excluded. The mutation in aE280/U1 does not affect the steady-state concentration of DHBP synthase in the cytoplasm (data not shown). This result excludes the possibility that the respiratory deficiency is due to a lower cytosolic concentration of the enzyme. It is interesting to note that the mitochondrial and cytoplasmic concentrations of DHBP synthase are higher in transformants harboring an integrated copy of the gene than in wild type (Fig. 6). This suggests that the chromosomal context (URA3 site in the case of the integrated genes) influences the expression of the gene. The estimated 4 -5-fold increase of DHBP synthase in the strain with the integrated mutant allele (aW303⌬RIB3/i-E280) may explain the ability of this transformant to grow on nonfermentable substrates (Fig. 3B). DISCUSSION In the present study, the RIB3 gene encoding DHBP synthase, an enzyme of the riboflavin biosynthetic pathway, was cloned based on its ability to restore respiration in the pet mutant, aE280/U1. The rescue by RIB3 is the result of genetic complementation. This is supported by tight linkage of the mutation in aE280/U1 to a rib3 null allele and sequence analysis of the rib3 gene in aE280/U1, which confirmed the presence of a mutation in the gene. The single base mutation causes a relatively innocuous substitution of a threonine for an alanine at residue 137 of the protein.
The rib3 null mutant used in this study shows an auxotrophic requirement of riboflavin for growth on glucose as reported previously (3). In contrast, the rib3 point mutant, like other pet mutants, grows on glucose in the absence of added riboflavin. Moreover, riboflavin does not restore the growth of the mutant on nonfermentable substrates. This suggests that the phenotype of the rib3 point mutant is unrelated to the activity of DHBP synthase in riboflavin synthesis. Instead it must have another function required for expression of mitochondrial respiration. This is also supported by the following evidence. 1) Analysis of FAD and FMN failed to show any substantive decrease in the mitochondrial and cytosolic concentrations of these nucleotides in the mutant aE280/U1. Some decrease in the riboflavin concentration was seen in the cytosolic fraction of the mutant. This, however, could be because of the different genetic background of the two strains used for the analysis.
2) The rates of oxidation of NADH or succinate in isolated mitochondria were not increased when the point mutant was grown in the presence of riboflavin in the growth medium. 3) Riboflavin rescued growth of the rib3 null mutant on glucose but not on nonfermentable substrates.
Transformation of the rib3 null mutant with the rib3 allele of E280 either in single (integrated into chromosomal DNA) or in multiple copies (episomal plasmid) conferred growth on glucose. Surprisingly, the transformants also grew on the nonfermentable substrates glycerol/ethanol. We attribute this to the  (22), transferred to nitrocellulose, and probed with an antiserum against the trpE-DHBP synthase fusion protein. Antibody-antigen complexes were detected by a secondary reaction with 125 I-protein A. The arrow on the right points to DHBP synthase. The migration of molecular size markers is shown on the left. B, wild type mitochondria and post-mitochondrial supernatant proteins were separated and probed as described in A, but the Western blot was exposed to x-ray film for a longer time. C, purification of mitochondria on a Nycodenz gradient. The gradient was prepared by layering 0.9 ml of 5, 10, 15, 20, and 25% Nycodenz containing 0.6 M sorbitol, 20 mM K-MES, pH 6, in a 5-ml Beckman Ultracentrifuge tube 4 h before usage. Mitochondria (5 mg protein in 0.5 ml) from the transformant aW303⌬RIB3/E280, prepared by the method of Glick and Pon (23), were layered on the gradient and centrifuged at 75,000 ϫ g for 90 min in a Beckman SW 65 rotor. A total of 15 fractions were collected, separated on a 12% polyacrylamide gel, and assayed for DHBP synthase and the mitochondrial intermembrane space marker, cytochrome b 2 . DHBP synthase (DHBP Synth.) and cytochrome b 2 (b2) are identified by the arrows on the right. D, localization of DHBP synthase in the intermembrane space of mitochondria. Mitochondria of aW303⌬RIB3/E280 were purified on a Nycodenz gradient as described in B. The mitochondria (Mt) at a protein concentration of 13.5 mg/ml were diluted to 1 mg/ml in either 0.6 M sorbitol or 20 mM K-Hepes pH 7.4 or under hypotonic conditions in 20 mM K-Hepes pH 7.4 to prepare mitoplasts (Mp). One-half of the mitochondria and mitoplasts were exposed to proteinase K (0.1 mg/ml) for 45 min on ice. The proteinase-treated and nontreated controls were centrifuged at 12,000 ϫ g for 20 min, and the pelleted particles were resuspended and analyzed for DHBP synthase and cytochrome b 2 as described in A. DHBP synthase and cytochrome b 2 are identified by the arrows on the right. elevated level of the mutant protein in the integrant as a result of increased expression of the gene in the new chromosomal context (URA3 locus). This explanation is consistent with the partial restoration of growth of aE280/U1 on glycerol/ethanol when transformed with an extra copy of the mutant gene. These observations together with the slow growth phenotype of aE280/U1 on glycerol/ethanol suggest that the E280 allele reduces but does not abolish the activity of DHBP synthase needed for respiration. Overexpression of the mutant protein, therefore, is able to compensate for the partial loss of function.
The role of DHBP synthase in expression of mitochondrial respiration is not clear. The presence, even at somewhat elevated levels, of riboflavin in the mitochondria of the point mutant excludes DHBP synthase from involvement in riboflavin transport. The presence of riboflavin in the mutant also excludes 3,4-dihydroxy-2-butanone 4-phosphate, the precursor of riboflavin formed by the synthase, from being needed in some other pathway essential for respiration. The more likely alternative is that mitochondrial respiration depends on a function of DHBP unrelated to riboflavin synthesis. The small fraction of DHBP synthase detected in the mitochondrial intermembrane space suggests that this function is likely to occur in mitochondria.
On the basis of the reported structure of the Magnaporthe grisea DHBP synthase (25), the A137T mutation in aE280/U1 occurs in a region distant from the binding sites for the substrate and metal cofactor. The mutation is also removed from the interface of the homodimer (25). The alanine is located in a depression on the surface of the protein with the methyl group pointing outward. The threonine, with an extra carbon and hydroxyl, can be accommodated without interfering sterically with the neighboring residues in this surface structure. Although the alanine is conserved in the M. grisea enzyme, in some organisms the corresponding position is occupied by serine (26). Conceivably, this domain may be involved in the binding of a molecule that is delivered to mitochondria by DHBP synthase and is required for biogenesis/maintenance of the respiratory chain.