A Defect in Coenzyme Q Biosynthesis Is Responsible for the Respiratory Deficiency in Saccharomyces cerevisiae abc1 Mutants*

Thai Q. Do, Adam Y. Hsu, Tanya Jonassen, Peter T. Lee, and Catherine F. ClarkeDagger

From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095

Received for publication, February 1, 2001, and in revised form, March 7, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ubiquinone (coenzyme Q or Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants designated coq1-coq8. Here we report that COQ8 is ABC1 (for Activity of bc1 complex), which was originally isolated as a multicopy suppressor of a cytochrome b mRNA translation defect (Bousquet, I., Dujardin, G., and Slonimski, P. P. (1991) EMBO J. 10, 2023-2031). Previous studies of abc1 mutants suggested that the mitochondrial respiratory complexes were thermosensitive and function inefficiently. Although initial characterization of the abc1 mutants revealed characteristics of Q-deficient mutants, levels of Q were reported to be similar to wild type. The suggested function of Abc1p was that it acts as a chaperone-like protein essential for the proper conformation and functioning of the bc1 and its neighboring complexes (Brasseur, G., Tron, P., Dujardin, G., Slonimski, P. P. (1997) Eur. J. Biochem. 246, 103-111). Studies presented here indicate that abc1/coq8 null mutants are defective in Q biosynthesis and accumulate 3-hexaprenyl-4-hydroxybenzoic acid as the predominant intermediate. As observed in other yeast coq mutants, supplementation of growth media with Q6 rescues the abc1/coq8 null mutants for growth on nonfermentable carbon sources. Such supplementation also partially restores succinate-cytochrome c reductase activity in the abc1/coq8 null mutants. Abc1/Coq8p localizes to the mitochondria, and is proteolytically processed upon import. The findings presented here indicate that the previously reported thermosensitivity of the respiratory complexes of abc1/coq8 mutants results from the lack of Q and a general deficiency in respiration, rather than a specific phenotype due to dysfunction of the Abc1 polypeptide. These results indicate that ABC1/COQ8 is essential for Q-biosynthesis and that the critical defect of abc1/coq8 mutants is a lack of Q.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ubiquinone (coenzyme Q or Q)1 is a prenylated benzoquinone found in both prokaryotic and eukaryotic membranes (1). In prokaryotes Q is found in the plasma membrane and functions in respiratory electron transport as well as in disulfide bond formation of periplasmic membrane proteins (2, 3). In eukaryotes Q is a an integral and essential component in mitochondrial electron and proton transport in the respiratory complexes I, II, and III (4). In addition to its function in respiratory electron transport, Q forms a critical link between a wide variety of other Q-linked dehydrogenases, including those acting on fatty acids, glycerol 3-phosphate, dihydroorotate, choline, sarcosine, and dimethylglycine (5). Recently, Q has been identified as an essential component of the H+ pumping function of UCP1, an uncoupling protein involved in thermogenesis (6). UCP1 depends on the availability of oxidized Q (QH2 is inactive), suggesting that the redox state of Q is important in regulating uncoupling activity. It is intriguing that another mechanism for regulating mitochondrial membrane potential, the permeability transition, is inhibited by Q and either inhibited or stimulated by various Q derivatives (7, 8). The permeability transition results in the opening of an unidentified, proteinaceous channel, releasing matrix contents with a molecular mass of up to 1500 Da. The opening of this channel reflects mitochondrial dysfunction, and is one of several pathways leading to programmed cell death (9).

Q is found in membranes throughout eukaryotic cells (10). In the plasma membrane, Q is involved in an enzyme-mediated trans-plasma membrane electron transport system that can reduce extracellular compounds such as the ascorbyl radical (11-13). By virtue of its ability to redox cycle, Q also has the capacity to function as a lipid soluble antioxidant, either directly as a chain-terminating antioxidant or indirectly by reducing the alpha -tocopheroxyl radical (14).

Studies of Q-biosynthesis in both prokaryotes and eukaryotes have enabled the elucidation of a putative Q-biosynthetic pathway (1, 15, 16). Tzagoloff and Dieckmann (17) described eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, coq1-coq8. The COQ1 gene product catalyzes the formation of the polyprenyldiphosphate precursor tail of Q (18). The COQ2 gene product catalyzes the attachment of the polyprenyldiphosphate tail to 4-hydroxybenzoate, leading to the formation of the early Q-biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid (HHB) (19). From HHB, a series of ring modifications produce Q and require the COQ3, COQ4, COQ5, COQ6, COQ7, and COQ8 genes (20). Yeast coq3-coq8 mutants lack Q and accumulate HHB as the predominant intermediate (21). Coq3p is located in the mitochondrial matrix, peripherally associated with the inner membrane and catalyzes both O-methylation steps in Q biosynthesis (22). Coq5p functions as a C-methyltransferase in the mitochondria (23, 24). Coq6p is likely to function as a flavin-dependent monooxygenase, adding one or more of the ring oxygens (20). Coq7p is tightly associated with the mitochondrial inner membrane, and is required for the last monooxygenase step in Q biosynthesis (25, 26). Coq4p is located in the mitochondrial matrix peripherally associated with the inner membrane (27). Although the precise functions of Coq4p and Coq7p in Q biosynthesis are not known, genetic evidence suggests that many of the Coq polypeptides may function in a multisubunit complex converting HHB to Q (27, 28).

Previous characterization of a coq8-1 mutant (strain C130) showed it to be respiratory defective, Q-deficient (29), and to accumulate HHB as the predominant intermediate (21). This study reports the isolation of the COQ8 gene and its identification as the previously described ABC1 gene. ABC1 was originally isolated as a multicopy suppressor of a cytochrome b mRNA translation defect (30). Studies of abc1 mutants have suggested that Abc1p acts as a chaperone-like protein essential for function of the bc1 complex (31). The data presented here show that Abc1/Coq8p is required for Q biosynthesis. The implications of our findings on the functional role of ABC1/COQ8 and its gene product are discussed.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains and Growth Media-- The strains used in this study are listed in Table I. Growth media for yeast were prepared as described (34) and included YPD (1% yeast extract, 2% peptone, 2% dextrose), YPE (1% yeast extract, 2% peptone, 2% ethanol), YPGal (1% yeast extract, 2% peptone, 2% galactose), YPG (1% yeast extract, 2% peptone, 3% glycerol), SDC (0.18% yeast nitrogen base without amino acids, 2% dextrose, 0.14% NaH2PO4, 0.5% (NH4)2SO4, complete amino acid supplement), SD-Leu (SDC minus leucine), and SD-Ura (SDC minus uracil). The complete supplement was modified so that the final concentration of each component was as described (23). Semisynthetic lactate media was prepared as described (35). Media for sporulation and tetrad analysis were prepared according to Adams et al. (34). All components of growth media were purchased from Difco, Fisher, or Sigma. 2% Agar was added for solid media.

                              
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Table I
Genotypes and sources of S. cerevisiae strains

In Vivo Labeling of Q Intermediates, Lipid Extraction, and Analysis by HPLC-- Yeast strains CH130-A1 (coq8-1) and FYDelta ABC1 (abc1Delta ::TRP1) were grown in 1 liter of SDC media supplemented with 0.65 µCi of [U-14C]p-hydroxybenzoic acid (365 Ci/mol), synthesized from L-[U-14C]tyrosine by alkali heat fusion (36). After 3 days of incubation at 30 °C with shaking (200 rpm), cultures were harvested (A600 nm = 10), and lipids were extracted as described (25). Approximately 10-12% of the 14C-radiolabel added to the cultures was recovered in the lipid extracts. Extracts were concentrated and aliquots containing 104 cpm were analyzed by normal phase HPLC employing a cyanopropyl column (Zorbax, 5 µm, 4.6 mm × 250 mm, MacMod Analytical, Chadds Ford, PA) as described by Poon et al. (37). The column was equilibrated with a mobile phase composed of 98% solvent A (hexane) and 2% solvent B (isopropyl alcohol/hexane/water/methylene chloride, 52:41:5:2) at a flow rate of 1 ml/min. Ten minutes after sample injection, the percentage of solvent B was increased linearly from 2 to 27% in 25 min (35 min from the start) and then from 27 to 100% in 20 min (55 min after the start). In the next 5 min, percentage of solvent B was decreased linearly from 100 to 2% and remained at 2% for 30 min to equilibrate the column before next sample injection. The radioactivity of 1-ml fractions were measured by scintillation counting in 10 ml of BIOsafe nonaqueous scintillation mixture (Research Products International) with a Beckman scintillation counter (model LS-3133P).

Cloning of the COQ8 Gene-- CH130-E1 (coq8-1) yeast were grown to early-log phase in YPD medium and transformed as described (38) with a YEp24-based plasmid library of S. cerevisiae genomic DNA (39) containing the URA3 gene as a selectable marker. A total of 44,000 transformants were obtained by selection on SD-Ura plates and, after a 3-day incubation at 30 °C, were replica plated onto YPG plates to test for respiratory growth. Thirty-eight colonies were complemented for respiratory deficiency as indicated by growth on YPG plate media. Eleven of the putative Q-prototrophic transformants were further purified and tested for cosegregation of uracil prototrophy and respiration competency following vegetative growth in rich media. All 11 showed co-segregation of the Ura- and YPG- phenotypes, indicating these traits were plasmid linked. Yeast plasmid DNA was recovered from each transformant and each of the 11 clones contained overlapping segments of genomic DNA inserts as confirmed by restriction enzyme mapping and Southern analysis (data not shown). One clone, pTM, was chosen for further study. A 4.0-kb HpaI/NheI fragment of pTM containing the ABC1 gene was isolated from pTM, processed for blunt end ligation and inserted into a SmaI-digested CEN-based vector pRS316 (40) generating p3HN4. Transformation of CH130-E1 with p3HN4 complemented the glycerol growth deficiency.

Disruption of the COQ8 Gene-- Two separate deletion alleles of ABC1 were prepared. In both cases, a 1.1-kb AflII/SunI fragment of p3HN4 was replaced (after preparation for blunt end ligation) with either a 1.1-kb HIS3 or 0.8-kb TRP1 fragment, derived from YDp-H or YDp-W, respectively (41). The resulting plasmids, pHQ8Delta and pWQ5Delta , were each used to obtain a linear BamHI/ClaI fragment with the disrupted gene. W303-1A was transformed with 1 µg of a linear fragment from pHQ8Delta by the procedure of Elble (42). Most of the histidine prototrophic clones from the transformations were respiration-defective and were complemented by the rho o strain, JM8, but not by the coq8-1 strain CH130-E1. Nuclear DNAs from 8 independent transformants were digested with BamHI and ClaI and separated on 1% agarose. The probe, a 4-kb HpaI/NheI fragment from pTM, hybridized to three fragments of 1.6-, 1.4-, and 1.2-kb in the genomic DNA of wild-type strain W303-1A. The genomic DNA of the mutant strain (W303Delta ABC1) contained two fragments of 1.4 and 2.8 kb as expected for the disrupted allele. Alternatively, linearized pWQ5Delta was used to transform FY251. Most of the tryptophan prototrophic clones issued from the transformations were respiration-defective and were complemented by the rho o strain JM8, but not by the coq8-1 strain CH130-E1. Nuclear DNA from one transformant was digested with BamHI and ClaI and separated on 1% agarose. The probe, a 4-kb HpaI/NheI fragment from pTM, hybridized to three fragments of 1.6, 1.4, and 1.2 kb in the genomic DNA of FY251. The genomic DNA of the mutant strain (FYDelta ABC1) contained two fragments of 1.4- and 2.5-kb, as expected for the disrupted allele.

Rescue of coq Mutants with Exogenous Q6-- Yeast cells were grown overnight in 5 ml of YPD to saturation. Overnight cultures were then diluted into 30 ml of YPE, in 125-ml Erlenmeyer flasks, to a density of ~0.25 A600 nm, with or without 15 µM Q6 supplementation. Q6 supplementation increased the absorbance of the culture by ~0.01 A600 nm. These cultures were subsequently incubated at 30 °C with shaking (200 rpm). Growth was monitored by A600 nm measurements over a period of 8 days. Samples were taken after 8 days of growth and plated on YPD plates to confirm that cultures were not contaminated.

Determination of Succinate-cytochrome c Reductase Activity-- Yeast cells were grown overnight in 5 ml of YPGal containing 0.1% dextrose. Overnight cultures were diluted into 100 ml of YPGal (0.1% dextrose) to a density of ~0.004 A600 nm. Cell cultures were grown and harvested at ~4 A600 nm. Yeast crude mitochondrial fractions were prepared as described (35) and aliquots were stored frozen at -80 °C. Succinate-cytochrome c reductase activities were assayed at 30 °C in 40 mM sodium phosphate buffer, pH 7.4, containing 20 mM sodium succinate, 500 µM EDTA, 250 µM potassium cyanide, and 50 µM horse heart cytochrome c (43). Briefly, 12.5 µg of mitochondrial protein was incubated in buffer without cytochrome c for 15 min. The reaction was initiated by addition of cytochrome c and monitored at 550 - 540 nm for 3 min. Specific cytochrome c reductase activities were determined from the initial linear rate of reaction at 550 - 540 nm using an extinction coefficient of 18.5 mM-1 cm-1 (43). All assays were performed in triplicate. For assays in the presence of added Q6, mitochondria were incubated 15 min in buffer containing 10 µM Q6, 1% EtOH, prior to initiation by addition of cytochrome c. This concentration of Q6 was sufficient to saturate the succinate-cytochrome c reductase activity.

Thermosensitivity Assays-- Crude mitochondria from various yeast strains of the CEN.PK2-1C background were prepared and stored as described above. Cytochrome c oxidase activities were assayed at 30 °C in 40 mM sodium phosphate buffer, pH 7.4, containing 25 µM reduced cytochrome c (43, 44). Horse heart cytochrome c was reduced in 40 mM sodium phosphate buffer, pH 7.4, with L-ascorbate, and the ascorbate was subsequently removed by dialysis. The reaction was initiated by adding 25 µg of mitochondrial protein and the reaction rate was monitored by following the oxidation of cytochrome c at 550 - 540 nm for 30 s (44). Cytochrome c oxidase activities were determined from the initial linear rate of reaction using an extinction coefficient of 18.5 mM-1 cm-1 (43). To determine the thermosensitivity of cytochrome c oxidase activities, mitochondria were incubated at 37 °C and over time aliquots were assayed. All assays were performed in triplicate.

Mitochondrial Import Assay-- An in vitro transcription template plasmid was constructed by inserting the ABC1/COQ8 open reading frame into the pBluescript SK(+) vector downstream of the T7 promoter. The ABC1/COQ8 open reading frame was polymerase chain reaction-amplified from pTM with 5' primer pHAC8-1 (5'-ACGCACGCGTCGACTTATGGTTACAAATATGGTGAA-3') and 3' primer p2H8R (5'-CGGAATTCTTAAACTTTATAGGCAAAAATC-3') with Vent DNA polymerase. The polymerase chain reaction product was gel-purified and kinase-treated and inserted into the EcoRV site of pBluscript SK(+) vector. The resulting clone, pBCOQ8, was linearized by XbaI and served as template for in vitro transcription (Promega Ribomax Large Scale RNA Production System). The resulting mRNA was then translated using Promega Flexi Rabbit Reticulocyte Lysate System in the presence of [35S]methionine (1000 Ci/mmol, at final concentration of 0.75 µM). Both the mRNA and the synthesized polypeptides were stored at -80 °C. The isolation of mitochondria (from D273-10B) and import reaction conditions were performed based on Yaffe (45) and described in Hsu et al. (46). Each import reaction contained 14 µl of radiolabeled in vitro translated product and mitochondria containing 200 µg of protein. Valinomycin was added to a final concentration of 10 µg/ml where appropriate. Following 30 min of incubation at 30 °C, the mitochondria were reisolated and washed once. Triton X-100 and proteinase K treatment after the import was performed by adding 1% Triton X-100 and/or 50 µg/ml proteinase K, final concentration, to resuspended mitochondria. The proteolytic digest was allowed to proceed at 0 °C for 20 min and was terminated by the addition of phenylmethylsulfonyl fluoride at a final concentration of 1 mM. The samples were mixed with sample buffer and heated at 100 °C for 5 min prior to analysis by SDS-polyacrylamide electrophoresis (12% polyacrylamide) and autoradiography.

Plasmid Construction of HA-tagged ABC1/COQ8-- Two yeast expression plasmids, one single copy and one multiple copy, were constructed to express the Abc1/Coq8 polypeptide containing a carboxyl-terminal peptide (MYPYDVPDYASLDGPMST) corresponding to the carboxyl terminus of the influenza hemagglutinin (HA) viral protein, an epitope for the 12CA5 monoclonal antibody (47). Construction began by directional cloning using SalI and NotI sites in the plasmid pADCL (48). The ABC1/COQ8 open reading frame region without the stop codon was polymerase chain reaction-amplified using 5' primer pHAC8-1 (described above) and 3' primer pHAC8-2 (5'-ATAAGAATGCGGCCGCAGAACTTTATAGGCAAAAATCT-3'). The polymerase chain reaction product was directionally cloned into the multiple copy pADCL vector (SalI and NotI at 5' and 3', respectively), to generate pHA8-1, a multiple copy vector containing the ABC1-HA construct. pRSHA8-1, a single copy version of the ABC1-HA construct, was generated by removing the 3.6-kb transcriptional cassette from pHA8-1 by BamHI partial digestion and insertion into the BamHI site of the vector pRS316 (40). Yeast abc1/coq8 mutants were transformed with pHA8-1, pRSHA8-1, or pTM (42). Transformants were selected for the presence of either the LEU2 gene (pHA8-1) or the URA3 gene (pRSHA8-1) on SD-Leu or SD-Ura plate media. Colonies obtained on the respective selective plate media were subsequently replica plated to YPG plate media. The Abc1-HA epitope fusion protein retained activity as assayed by the ability of either the single or multicopy plasmid construct to rescue abc1/coq8 null mutant yeast strains for growth on media containing a nonfermentable carbon source (YPG plates, data not shown). Yeast strain CH130-A1 harboring pHA8-1, pRSHA8-1, or pTM was used for subcellular localization by Western analysis.

Cell Lysis and Fractionation-- Cell cultures (1 liter) were grown in semisynthetic lactate media to saturation density. Cells were lysed and fractionated as described (35). Briefly, spheroplasts were prepared and lysed by Dounce homogenization with a tight fitting pestle. Total cell lysates were separated from nuclei and unlysed cells at 1,500 × g for 5 min. Crude mitochondria were separated from the cytosolic fraction by centrifugation of cell lysates at 12,000 × g for 10 min. Purified mitochondria were prepared using a linear Nycodenz gradient as described (35).

Western Analysis-- Fractions were assayed for protein concentration by the bicinchoninic acid assay. Equal amounts of protein from the mitochondrial fractions of cells containing the plasmids pTM, pRSHA8-1, or pHA8-1 were analyzed by electrophoresis on 12% Tris glycine gels and subsequently transferred to Hybond ECL nitrocellulose. Western analysis and membrane stripping were performed as described by Amersham Pharmacia Biotech. An exception to the stated protocol was the use of Western washing buffer: 10 mM Tris, pH 8.0, 154 mM NaCl, 0.1% Triton X-100. The primary antibodies recognizing the HA epitope or F1beta -ATPase were used in a 1:10,000 dilution. Horseradish peroxidase-linked secondary antibodies to rabbit and mouse IgG were used in a 1:1000 dilution.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The coq8-1 Mutant Lacks Q and Accumulates a Polar Intermediate That Co-elutes with HHB-- The coq8-1 mutant strain C130 (G75 complementation group) fails to respire or grow on nonfermentable carbon sources (17). The Q-deficient phenotype in the coq mutants is defined by a profound stimulation of both NADH- and succinate-cytochrome c reductase activities by the addition of decyl-Q, Q2, or Q6 to in vitro assays (29, 49). To study the biochemical defect responsible for Q-deficiency, D27310-B/A1, a wild-type strain, or strain CH130-A1 (coq8-1) were grown in SDC media in the presence of [U-14C]4-hydroxybenzoic acid, the aromatic ring precursor of Q. Lipid extracts were prepared, fractionated by normal phase HPLC, and monitored for 14C radioactivity (Fig. 1). In lipid extracts of wild-type yeast, the two predominant peaks of radioactivity correspond to Q6 and to the polar Q-biosynthetic intermediate HHB (Fig. 1A; Ref. 37). Lipid extracts of CH130-A1 (coq8-1) lack Q6 and contain one large peak of radioactivity in fraction 29 that co-eluted with HHB. HHB accumulates in cultures of CH130-A1 grown in the presence of [carboxyl-14C]4-hydroxybenozic acid (data not shown). HHB is present at high levels in wild-type yeast strains grown in media containing 2% glucose (SDC) and harvested at log phase (37), and in each of the coq3, coq4, coq5, coq6, and coq7 mutant strains (Refs. 21 and 25, and data not shown).


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Fig. 1.   Yeast coq8 mutants lack Q6 and accumulate a polar intermediate that corresponds to HHB. Lipid extracts were prepared from D27310-B/A1 (wild-type parent, Panel A), CH130-A1 (coq8-1 allele, Panel B), or from FYDelta ABC1 (abc1/coq8 deletion, Panel C) and separated by normal phase HPLC as described under "Experimental Procedures." 1-ml fractions were collected and 14C radioactivity determined by scintillation counting. Q6 and HHB standards eluted in fractions 7-9 and 28-30, respectively.

The Yeast coq8-1 Mutant Is Complemented by the ABC1 Gene-- To characterize further the nature of the defect in the coq8-1 mutant, the COQ8 gene was cloned by transformation of CH130-E1 (coq8-1) with a yeast genomic DNA library as described under "Experimental Procedures." Yeast plasmid DNA was recovered from each of 11 independent clones and found to contain overlapping segments of genomic DNA. One clone, pTM, contained an 8-kb insert and transformation of CH130-E1 with pTM restored growth on YPG media. Partial sequence determination of the insert DNA in pTM showed the insert to contain the previously characterized ABC1 gene (for Activity of bc1 complex) (Ref. 30; GenBankTM accession number X59027). A subclone containing the ABC1 gene on a 4.0-kb NheI/HpaI fragment (Fig. 2) complemented the glycerol growth deficiency of CH130-E1. The ABC1 gene encodes a 501-amino acid polypeptide that is required for respiration and was originally isolated as a multicopy suppressor of a cytochrome b mRNA translation defect (30). Characterization of an abc1 null mutant showed the phenotype to be reminescent of Q-deficient mutants, yet a quinone deficiency was ruled out at that time (31).


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Fig. 2.   Restriction map and strategy of ABC1 deletion. A map of the 4.0-kb HpaI-NheI fragment in pTM is presented. The long black arrow indicates the position of the ABC1 open reading frame and its direction of transcription. Also present on this fragment is the amino-terminal two thirds of the PRP43 gene (YGL120C), with transcription proceeding in a direction opposite to ABC1. Deletion constructs were prepared by replacing a 1.1-kb AflII/SunI fragment (hatched bar) with either the HIS3 or TRP1 gene as described under "Experimental Procedures." Restriction sites are abbreviated and correspond to the following DNA restriction enzymes: A, AflII; B, BamHI; H, HindIII; Hp, HpaI; N, NheI; P, PvuII; S, SunI.

Yeast abc1 Null Mutants Lack Q and Accumulate HHB-- To further investigate the relationship of ABC1 to the coq8-1 mutation, a one-step gene replacement procedure (50) was used to obtain the abc1 null mutant strains W303Delta ABC1 and FYDelta ABC1 (Fig. 2). These yeast strains are either histidine or tryptophan prototrophic, respectively, and are respiration deficient. Both were complemented by a rho o strain, but not by the CH130-E1 (coq8-1) strain. These results imply that the abc1 null and the coq8-1 mutant are allelic. To verify this, W303Delta ABC1 was mated to CH130-E1(coq8-1), and the diploid cells were transformed with pTM to maintain the respiratory competency necessary for sporulation. Tetrads from this cross (28 complete sets) were dissected and segregated 2:2 for leucine and histidine prototrophy. After selection on 5-fluoro-orotic acid plate media for loss of pTM, all 28 sets of tetrads failed to grow on YPG and were rho +, confirming the allelism between the ABC1 gene and the original coq8-1 mutation.

To investigate whether the abc1 null mutation impairs the biosynthesis of Q, the yeast abc1 deletion strain FYDelta ABC1 was grown in the presence of [U-14C]4-hydroxybenzoic acid and lipid extracts were separated by normal phase HPLC. As shown in Fig. 1C, no Q was detectable in the abc1 null mutant strain, and HHB accumulated as the sole predominant intermediate, as observed for the coq8-1 mutant strain (Fig. 1B). Together these data show that ABC1 is the same as COQ8 and is essential for the biosynthesis of Q. Henceforth the gene will be called ABC1.

Q-deficient yeast mutants are characteristically hypersensitive to treatment with polyunsaturated fatty acids. This phenotype is attributed to the absence of Q and its role in protecting cells from oxidative stress due to autoxidation of the polyunsaturated fatty acids (21, 51). When stressed with the polyunsaturated linolenic acid, the abc1/coq8 null mutants also displayed this hypersensitive phenotype (data not shown), consistent with a deficiency in Q and its role as an antioxidant.

Rescue of the abc1 Null Mutant by Q6 Supplementation-- The Q-deficient strains coq3Delta and coq7Delta can be rescued for growth on ethanol, a nonfermentable carbon source, by supplementation of liquid media with Q6 (26). Such rescue by exogenous Q6 provides strong evidence that the sole defect leading to lack of growth is the availability of Q itself. To test whether the abc1 null mutant can be rescued for growth by the addition of exogenous Q6, wild-type yeast and various respiratory deficient mutants were incubated in growth media containing ethanol in the presence or absence of 15 µM Q6. Fig. 3 shows that both coq3Delta and abc1Delta mutants failed to grow on media containing ethanol, yet growth can be rescued by exogenous Q6. Although both strains showed slower growth in Q-supplemented media relative to the wild-type strain, the same final cell density was eventually achieved. As expected, the addition of exogenous Q6 did not rescue the respiratory deficient mutant cor1Delta (an essential subunit of the bc1 complex) on a nonfermentable carbon source. Since the growth defect of the abc1Delta mutant strain can be completely suppressed by the addition of Q6, the defect is due to Q availability.


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Fig. 3.   Exogenous Q6 rescues the abc1Delta and coq3Delta yeast mutants. Yeast strains CEN.PK2-1C (wild type: open circle  and ), CENDelta ABC1 (abc1Delta : Delta  and black-triangle), CENDelta COQ3 (coq3Delta : diamond  and black-diamond ), and CENDelta COR1 (cor1Delta :  and black-square) were grown in YPE supplemented without (open circle , Delta , diamond , and ) or with (, black-triangle, black-diamond , and black-square) 15 µM Q6 as described under "Experimental Procedures." Samples were taken over a period of 8 days and growth monitored by A600 nm measurements.

The effect of the addition of Q6 on the restoration of succinate cytochrome c reductase activity (complex II + III) was also investigated. As shown in Fig. 4, addition of Q6 to the growth media partially restored succinate-cytochrome c reductase activity in both coq3Delta and abc1Delta mutant strains, but failed to restore this activity in the cor1Delta mutant. A similar trend was observed when the Q6 was added to isolated mitochondria prior to the assay of succinate-cytochrome c reductase activities. The greatest degree of rescue was observed when Q6 was present during growth and also included in the subsequent in vitro assay. The extent of rescue by exogenous Q6 is similar in magnitude to the observed decyl-Q-mediated restoration of succinate-cytochrome c reductase activity in isolated mitochondria by Brasseur et al. (31).


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Fig. 4.   Q6 rescues the succinate-cytochrome c reductase activity in abc1Delta and coq3Delta mutants. Wild-type and mutant cells (CEN.PK2-1C background) were grown in liquid media containing galactose as carbon source (YPGal + 0.10% dextrose) in the presence or absence of 15 µM Q6. Mitochondria were isolated and succinate-cytochrome c reductase activities were measured in the presence or absence of 10 µM Q6 as described under "Experimental Procedures." No addition of Q6, (); addition of Q6 to isolated mitochondria (); addition of Q6 to growth media (); and addition of Q6 to both growth media and isolated mitochondria (black-square). The level of activity in the CEN.PK2-1C wild type strain, 522 nmol cytochrome c reduced min-1 mg of protein-1, has been set at 100%.

Thermosensitivy of abc1 Mutants-- The findings presented above indicated that abc1 mutants are Q-deficient. Characterization of abc1 mutants by Brasseur et al. (31) indicated that the activities of the various mitochondrial respiratory complexes, including cytochrome c oxidase, were thermosensitive when compared with wild-type. Considering the defect is in Q availability, these findings are intriguing, as they imply that Q itself may play a role in the proper assembly and/or stability of these complexes. To investigate this possibility, thermosensitivity assays were performed with mitochondria prepared from late log phase (A600 nm = 4.0) cultures of wild-type (CEN.PK2-1C) and the abc1 mutant. For these studies, the thermosensitivity of complex IV (cytochrome c oxidase) activity was determined as described under "Experimental Procedures." Our results, obtained with mitochondria isolated from late log phase cultures, indicated that complex IV activities were similarly thermosensitive in both wild-type and the abc1 mutant strain (Fig. 5A). Similar thermosensitivity assays were then performed with mitochondria isolated from stationary phase cultures of CEN.PK2-1C wild-type, or from the respiratory-deficient mutants abc1, coq3, cor1, or atp2. Cytochrome c oxidase activity present in wild-type mitochondria was less thermosensitive when compared with mitochondria isolated from Q-deficient mutants (abc1 and coq3) or from a respiratory deficient control cor1 mutant as well as an atp2 null (ATP2 encodes the beta  subunit of the ATPase) mutant (Fig. 5B). The data indicate that the stationary phase thermosensitivity of cytochrome c oxidase in the abc1 mutant strain is a general characteristic of respiration deficiency rather than a specific function of Abc1p.


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Fig. 5.   Thermosensitivity of cytochrome c oxidase activities. Wild-type (), abc1 (black-triangle), coq3 (black-diamond ), atp2 (cross ), and cor1 (black-square) mutants (CEN.PK2-1C background) were grown in YPGal (0.10% dextrose) and cultures harvested at late log phase (4.0 A600 nm, Panel A) or from stationary phase cultures (greater than 10 A600 nm, Panel B). Crude mitochondria were prepared and thermosensensitivity of cytochrome c oxidase activities were determined as described under "Experimental Procedures." Briefly, mitochondria were incubated at 37 °C and samples were assayed for cytochrome c oxidase activities at various time points. Activities are expressed as a percentage of the initial cytochrome c oxidase activities before heat shock, defined as 100%. In Panel A initial activities were 2244 and 1628 nmol of cytochrome c oxidized min-1 mg of protein-1, for the CEN.PK2-1C wild type and abc1 mutant, respectively. In Panel B, initial activities were 2128 cytochrome c oxidized min-1 mg of protein-1, for the CEN.PK2-1C wild type, and 509, 113, 143, and 25 nmol of cytochrome c oxidized min-1 mg of protein-1, for the abc1, coq3, cor1, and atp2 mutants, respectively.

Localization of Abc1p to the Mitochondria-- Previous studies have suggested that the Abc1 polypeptide is located in the mitochondria (30, 31). In vitro mitochondrial import assays were performed to determine whether the putative mitochondrial leader sequence of Abc1p functions to direct mitochondrial import. The Abc1p 35S-labeled in vitro translation products (Fig. 6, lane 1) generated an ~56-kDa precursor and several smaller products. Upon incubation with mitochondria, a new polypeptide corresponding to the mature form of Abc1p (53 kDa) accumulated (lane 2). The mature form was protected from treatment with proteinase K (lane 3), and the addition of Triton X-100 rendered the mature product susceptible to proteinase K treatment (lane 4). The processing of Abc1p was dependent on the membrane potential, as treatment with valinomycin inhibited the generation of mature Abc1p and its import (lanes 5 and 6).


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Fig. 6.   In vitro mitochondrial import and proteolytic processing of the Abc1 polypeptide. In vitro mitochondrial import assays were performed as described under "Experimental Procedures." Lane assignment: Lane 1, 10% of the radiolabeled in vitro translation product used in each import reaction. Lane 2, in vitro translation product incubated with isolated mitochondria. Lane 3, same as lane 2 but including a post-import proteinase K treatment. Lane 4, same as lane 3 except Triton X-100 was added to disrupt the mitochondria before proteinase K treatment. Lanes 5 and 6, in vitro translation product was incubated with mitochondria in the presence of the uncoupler valinomycin (10 µg/ml final concentration). After the incubation, the reisolated mitochondria were either treated with proteinase K, as in lane 5, or were directly analyzed in lane 6. The samples were mixed with sample buffer and heated at 100 °C for 5 min prior to analysis by SDS-polyacrylamide electrophoresis (12% polyacrylamide) and autoradiography. P and M indicate the positions of the precursor and mature form of Abc1p, respectively. The mitochondrial leader sequence of Abc1p is typical of mitochondrial leader sequences (52) and contains a 3-amino acid consensus sequence motif (designated by -10, -8, and -5) common to leader sequences of mitochondrial matrix polypeptides (53). Based on this motif, the predicted mature amino terminus is designated as +1.

A carboxyl-terminal fusion of the Abc1 coding region with the hemagglutinin viral protein epitope (HA) was constructed in order to determine the subcellular localization of Abc1p. Both single copy and multicopy constructs expressing the Abc1-HA polypeptide were able to rescue W303Delta ABC1 for growth on media containing a nonfermentable carbon source (data not shown). Western analysis of yeast subcellular fractions indicated that the Abc1-HA fusion protein cofractionates with the mitochondria (Fig. 7). Although the expressed Abc1-HA fusion protein was targeted to mitochondria and rescued the abc1 mutant when present at single copy, submitochondrial localization studies revealed the majority of Abc1-HA-tagged polypeptide was found as an insoluble form, resistant to extraction with Triton X-100 in the presence of high salt. This problem was also observed upon analysis of other single copy HA-tagged constructs, including Coq6, Coq7, and Coq4.2 A similar problem has been reported in studies of COX3 mRNA-specific translational activator proteins (54). Submitochondrial localization studies will require antisera that recognize the non-tagged Abc1p.


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Fig. 7.   Abc1p localizes to mitochondria. W303Delta ABC1 mutant yeast harboring the plasmids pTM (N), pRSHA8-1 (S), or pHA8-1 (M) were generated as described. pTM is a multiple copy plasmid containing the wild-type ABC1 gene. pRSHA8-1 and pHA8-1 are single and multiple copy plasmids, respectively, harboring the HA-tagged ABC1 gene to express Abc1p with a carboxyl-terminal epitope tag from the hemagglutinin viral protein. These yeast strains were grown, lysed, and fractionated as described under "Experimental Procedures." 50 µg of protein from the total cell lysates (Total Lysate), cytosolic fractions (Cyto), or mitochondrial fractions (Mito) were subjected to Western analysis and visualized by chemiluminescence detection using purified 12CA5 antibodies to the HA-epitope tag (A) and antibodies to the mitochondrial protein F1beta -ATPase (B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The findings presented in this paper demonstrate that COQ8 encodes a mitochondrial protein necessary for Q biosynthesis and is allelic to the previously described ABC1 gene. ABC1 was initially isolated as a multicopy suppressor of a cytochrome b mRNA translation defect due to the cbs2-223 mutation (30). Cbs2p is a translational activator of the long untranslated leader in the mRNA of COB, a mitochondrial gene encoding cytochrome b (55). Yeast strains with either cbs2 null or cbs2-223 mutations fail to respire due to a defect in translation of cytochrome b. The ABC1 gene in multicopy cannot function in place of CBS2 because suppression is specific for the cbs2-223 mutation. Initial characterization of abc1 mutants revealed characteristics similar to Q-deficient mutants (30). However, in the assays performed by Brasseur et al. (31) no Q deficiency was detected in abc1 mutant strains. Further analysis of abc1 mutants revealed that various complexes of the mitochondrial respiratory chain, including complexes II, III, and IV, were thermosensitive when compared with complexes from wild-type cells. Based on this information, Abc1p was hypothesized to function as a chaperone-like protein essential for the proper conformation and efficient functioning of cytochrome b and complex III (31).

The results presented here show that Q is not detectable in either coq8-1 or abc1Delta mutants. This was determined by labeling yeast cells with the Q-biosynthetic intermediate [U-14C]4-hydroxybenzoic acid, subsequent extraction of lipids, and separation of Q and Q-intermediates by HPLC. This finding contradicts the previous characterization of abc1 mutants (31), in which a method of detection and measurement of Q relied on difference spectra of reduced versus oxidized total lipid extracts (56). It is possible that this latter method may be prone to interference from other components within the lipid extracts. Growth of the abc1 null mutant and other coq null mutants on a nonfermentable carbon source was rescued by supplementation of media with Q6 (Fig. 3). Such Q6 supplementation also restored, at least in part, the activity of succinate cytochrome c reductase. These findings indicate that a defect in Q biosynthesis is responsible for the respiratory deficiency in abc1 mutants.

Previous work by Brasseur et al. (31) showed the various complexes of the respiratory chain were more thermosensitive in mitochondria isolated from stationary phase cultures of abc1 mutant strains compared with wild-type strains. In light of the Q biosynthetic defect, this result suggested that the proper assembly and/or stability of these complexes may depend on Q availability. However, the findings presented here indicate that the thermosensitivity of the respiratory complexes in the abc1 mutant strain is a general characteristic of respiration deficiency rather than a specific function of either the ABC1 gene or Q itself. When mitochondria were isolated from log phase cells, the cytochrome c oxidase was similarly thermosensitive in both the wild-type and the abc1 mutant strain. However, in stationary phase cultures, an enhanced thermosensitivity of cytochrome c oxidase was observed in each of the respiratory deficient mutants when compared with wild-type mitochondria. In general, respiratory deficient mutants have defects in activating genes repressed by glucose (26, 57). Glucose repression is a global regulatory system in S. cerevisiae that affects expression of genes involved in gluconeogenesis, alternative sugar metabolism, and respiration (58). Hence, the observed thermosensitivity of cytochrome c oxidase observed in mitochondria isolated from the respiratory deficient cells may result from the defects in the glucose derepression regulatory system.

The results presented here argue against the model that the Abc1 polypeptide acts as a chaperone essential for the assembly or stability of the respiratory complexes (30, 31). However, the multicopy ABC1 suppression of the cytochrome b mRNA translation defect in cbs2-223 mutants remains an intriguing observation. Could higher levels of Q, hypothetically resulting from the ABC1 gene present at multicopy, mediate the suppression of the cbs2-223 mutation? In this model the function of the mutant Cbs2p as a translational activator of COB mRNA would be rescued, albeit inefficiently, by an increased amount of Q acting as a cofactor. Another scenario is that increased levels of Q might be expected to stabilize a very low amount of cytochrome b polypeptide produced in the cbs2-223 mutant. Although cytochrome b in the cbs2-223 mutant is undetectable in whole cell absorption spectra (59), it has been hypothesized that low levels might be translated (30). One prediction of these models of Q as cofactor (either with Cbs2 in translational activation or in stabilizing cytochrome b) is that supplementation of media with Q6 would rescue the respiratory defect in mutants harboring the cbs2-223 allele. Alternatively, the suppression of the cbs2-223 allele could be mediated by a function of Abc1p distinct from its role in Q biosynthesis. However, since exogenous Q6 restores growth of the abc1 null mutant on media containing a nonfermentable carbon source, such a secondary function of Abc1p would not be essential for translation of COB mRNA or assembly of the bc1 complex.

Unlike Cbs2, a protein that appears to be specific to S. cerevisiae (60), proteins with amino acid sequence similarity to ABC1 have been identified in many species of eukaryotes, including human, mouse, and Caenorhabditis elegans (61). The ABC1 homologs from Schizosaccharomyces pombe (62) and Arabidopsis thaliana (61) show functional conservation to ABC1 in S. cerevisiae, since both were isolated by complementation of the S. cerevisiae abc1 mutant. These studies strongly suggest Q biosynthesis in these eukaryotes is highly conserved, while the suppression of the cbs2-223 mutation is not. The respiratory chain complexes in the S. pombe abc1Sp::LEU2 mutant were severely affected since cytochrome aa3 was undetectable, cytochrome c oxidase activity was decreased 7-fold, and the mutant cells showed very slow growth on glucose containing media (62). Thus Q deficiency in S. pombe produced drastic effects on respiratory complexes and growth. These respiratory defects were rescued by transformation with either the S. pombe or S. cerevisiae ABC1 gene. In S. cerevisiae abc1 mutants, the ABC1 homolog of A. thaliana only partially restored the activity of succinate-cytochrome c reductase and NADH-oxygen oxidoreductase (complexes I-IV), because levels of activity were only 24 and 13% of wild type (61). It seems likely that this observed inefficient rescue is due to incomplete restoration of Q biosynthesis. A similar phenomenon of partial restoration of Q biosynthesis was observed in the coq3 S. cerevisiae mutant rescued with a human COQ3 homolog (63). In this case, Q levels in the coq3 yeast mutant harboring the human COQ3 cDNA on a multicopy plasmid were only 20% of wild type, and Q was undetectable when the human COQ3 cDNA was present on a single copy plasmid.

The specific function of Abc1p in the biosynthesis of Q has yet to be defined. Like many mutants in the Q-biosynthetic pathway, abc1 mutants accumulate HHB as the predominant intermediate (21). It has been suggested that the accumulation of this single intermediate may result from the role of these polypeptides in a multisubunit protein complex involved in the biosynthesis of Q (28). Yeast abc1 mutants have greatly reduced levels of Coq3p and its corresponding O-methyltransferase activity (28). These findings suggest that Abc1p may be an essential component of this complex, required for one or more of the various monooxygenase and/or methylation steps in the modification of the quinone ring. However, it is unlikely that Abc1p is itself catalyzing this step as analysis of the encoded amino acid sequence does not reveal any similarities to any known hydroxylases or methyltransferases. Interestingly, both prokaryotic and eukaryotic ABC1 homologs possess conserved kinase motifs belonging to a class of eukaryotic serine/threonine protein kinases (64). One prokaryotic homolog of ABC1 is E. coli ubiB, a gene required for the first monooxygenase step in Q-biosynthesis; the conversion of 2-octaprenylphenol to 2-octaprenyl-6-hydroxyphenol. E. coli ubiB mutants lack Q and accumulate 2-octaprenylphenol as the predominant intermediate (65-67). Based on the conserved kinase motifs in UbiB, it has been suggested that UbiB plays an indirect role in the first monooxygenase step of Q biosynthesis, perhaps activating proteins necessary for monooxygenase activity via phosphorylation (67). The analogous hydroxylation step in Q biosynthesis in yeast catalyzes the hydroxylation of HHB to 3,4-hydroxy-5-hexaprenylbenzoic acid. Based on the homology between Abc1p and UbiB, and their involvement in Q biosynthesis, it is tempting to speculate that Abc1p may share a similar function in yeast Q biosynthesis. Work is ongoing to investigate this possibility.

    ACKNOWLEDGEMENTS

We thank Wayne Poon for help with the analysis of 14C-labeled yeast lipid extracts and the members of the Clarke laboratory, and Drs. Pamela L. Larsen, Patrice Hamel, and Carlos Santos-Ocaña for helpful comments and suggestions. The yeast coq mutant strains were generously provided by Dr. Alexander Tzagoloff, Columbia University.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM45952 (to C. F. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive E., Los Angeles, CA 90095-1569. Tel.: 310-825-0771; Fax: 310-206-5213; E-mail: cathy@mbi.ucla.edu.

Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M100952200

2 T. Jonassen, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: Q, ubiquinone or coenzyme Q; decyl-Q, coenzyme Q with a n-decane tail; HHB, 3-hexaprenyl-4-hydroxybenzoic acid; Q2 and Q6, coenzyme Q with either 2 or 6 isoprene groups in the tail; kb, kilobase(s); HA, hemagglutinin; HPLC, high performance liquid chromatography.

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DISCUSSION
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