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J. Biol. Chem., Vol. 277, Issue 13, 10973-10981, March 29, 2002

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Uptake of Exogenous Coenzyme Q and Transport to Mitochondria Is Required for bc₁ Complex Stability in Yeast coq Mutants^*

Carlos Santos-Ocaña, Thai Q. Do, Sergio Padilla^§, Placido Navas^§, and Catherine F. Clarke^¶

From the Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1569 and the ^§ Laboratorio Andaluz de Biología, Universidad Pablo de Olavide, 41013 Sevilla, Spain

Received for publication, December 20, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery in Saccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q₆ by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-less coq7 mutants derived from four distinct laboratory yeast strains. Q₆ supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the four coq7 mutant strains. Following culture in medium containing dextrose, the added Q₆ was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q₆ in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q₆. These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q₆ into the plasma membrane. The two coq7 mutant strains rescued by Q₆ supplementation for growth on ethanol contained mitochondrial Q₆ levels similar to wild type. However, the content of Q₆ in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q₆, succinate-cytochrome c reductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q₆ content, succinate-cytochrome c reductase activity, and steady state levels of the cytochrome c₁ polypeptide. These studies show that transport of extracellular Q₆ to the mitochondria operates in yeast but is strain-dependent. When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc₁ complex is unstable. These results indicate that delivery of exogenous Q₆ to mitochondria is required fore activity and stability of the bc₁ complex in yeast coq mutants.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Coenzyme Q (ubiquinone or Q) is a lipophilic molecule bearing a fully substituted benzoquinone ring and a hydrophobic poly-isoprenoid tail that partitions the molecule to the membrane lipids. Q cycles between two different states, the oxidized ubiquinone state (Q) and the reduced ubiquinol state (QH₂). In eukaryotes Q is required for mitochondrial electron transport where it acts as an electron shuttle from complex I (NADH dehydrogenase) or complex II (succinate dehydrogenase) to complex III (1). Other mitochondrial functions of Q have recently been elucidated, including its involvement as an essential cofactor in the proton pumping activity of uncoupling proteins (2) and its action as an inhibitor of the mitochondrial permeability transition pore, which is involved in the initiation of apoptosis (3). Q is involved in respiratory electron transport in prokaryotic cells where it also serves as an acceptor of electrons in the introduction of disulfide bonds in periplasmic proteins (4). Recently, the oxidation state of Q (Q/QH₂ ratio) has been shown to be the signal that sets metabolism toward either fermentation or respiration, as sensed by the ArcB/ArcA two-component signal transduction system in Escherichia coli (5). In eukaryotic cells Q is present in a wide variety of other endomembranes (6), including Golgi and endoplasmic reticulum, where it functions as a chain-terminating antioxidant and also as a secondary co-antioxidant through the reduction of the -tocopheryl radical to -tocopherol (7). The redox chemistry of Q also plays a role in the electron transport chain present in the lysosomal membrane (8) and in the plasma membrane electron transport system of Saccharomyces cerevisiae (9) and mammalian cells (10).

The subcellular location of Q biosynthesis is a topic currently under investigation. Studies carried out in mammalian cells (6) suggest that Q synthesis is located not only in the mitochondria but also in the endoplasmic reticulum and Golgi apparatus. It was proposed that the high level of Q synthesis in Golgi apparatus serves as a reservoir for its subsequent distribution among membranes. However, studies in yeast indicate that synthesis of Q occurs on the matrix side of the mitochondrial inner membrane, as shown by the submitochondrial localization of several Coq polypeptides required for Q biosynthesis, including Coq2p, Coq3p, Coq4p, Coq5p, Coq7p, and Abc1p (11-16). These data indicate that Q is produced on the matrix side of mitochondria because the Coq3 polypeptide carries out both the initial and the product-forming O-methylation steps of Q biosynthesis (12). Both rat and human homologs of the yeast COQ3 and COQ7 genes rescue yeast for growth in media with non-fermentable carbon sources (17-20), and such rescue depends on mitochondrial localization of the gene product (21). Recent studies have identified Coq7 and its homologs as members of a di-iron carboxylate family of enzymes, responsible for the final hydroxylation step of Q biosynthesis (22), and have localized the Caenorhabditis elegans and mouse homologs of COQ7 (CLK-1) to mitochondria (23-25). This localization supports early biochemical studies identifying a mitochondrial site for Q biosynthesis within rat liver (26-28).

The fact that Q is distributed differentially among intracellular membranes indicates the existence of a mechanism for delivery of Q from its site of mitochondrial biosynthesis to other cellular membranes. Evidence for Q uptake and transport to the mitochondria is provided by experiments with yeast coq mutants where growth on a non-fermentable carbon source is rescued by the addition of Q₆ to the medium (11, 16). This transport mechanism may participate in the normal distribution of Q among membranes, including Q uptake by cells that either do not produce or produce low levels of Q and the mobilization of Q in response to metabolic changes or stress situations that require the redox functions of Q. Several studies provide support for Q mobilization within the cell. HL-60 cells treated with ethidium bromide (resulting in a loss of mitochondrial DNA) and respiratory deficient yeast mutants contain increased amounts of CoQ at the plasma membrane (29, 30). Q₁₀ is used as a therapeutic agent in the treatment of atherosclerosis (31), other cardiovascular diseases (32, 33), and neurodegenerative diseases (34, 35) including Parkinson's disease (36). The use of Q₁₀ in these clinical settings would benefit from a better understanding of its mode of transport to organs and tissues and of the mechanisms responsible for Q uptake by cells and intracellular transport among cell membranes. Although phospholipid intracellular transport has been widely studied in S. cerevisiae (37-39), the transport of Q₆ has not been addressed.

The aim of this study is to characterize the distribution of Q in the plasma membrane and mitochondria in yeast coq mutant strains with defects in Q₆ biosynthesis. The culture of coq mutant strains in YPD (dextrose-containing) medium supplemented with Q₆ provides an assay for the ability of exogenous Q₆ to restore Q-dependent functions within distinct cell compartments, such as Q-dependent redox activities in the plasma membrane and succinate-cytochrome c reductase activity in the mitochondria. The data show that intracellular transport of Q₆ is dependent on the genetic background of the yeast strain and that in the Q-less yeast the delivery of exogenous Q₆ to mitochondria appears to be required to stabilize the cytochrome c₁ polypeptide and to maintain a productive complex III.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains and Materials-- The yeast strains used in this study are described in Table I. Yeast were grown in rich YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) and in YPE (1% yeast extract, 2% peptone, and 2% ethanol) when growth rescue experiments were performed. Defined media contained 0.18% yeast nitrogen base without amino acids, 2% dextrose, 0.14% NaH₂PO₄, 0.5% (NH₄)₂SO₄, and either a complete amino acid supplement (SDC) or the amino acid supplement minus one or more components (SD selective) (40). Cultures used in all experiments were tested to ascertain rho status. Aliquots of cultures were diluted and plated on YPD. After 4 days at 30 °C, 20 colonies were randomly selected and mated with a ⁰ tester strain (JM6 or JM8). Diploids were selected by transferring the cells to SD selective medium with glucose and respiratory competence assessed by transfer to YPG plates (glycerol). Subcellular fractionation experiments were completed and analyzed only after verifying that 100% of the colonies tested showed growth on the glycerol plate medium. Enhanced chemiluminescence reagents and bicinchoninic acid assay for proteins were from Pierce, reagents for SDS-PAGE were from Bio-Rad, and nitrocellulose was from Amersham Biosciences, Inc. Zymolyase 20T was purchased from ICN Pharmaceuticals. Coenzyme Q₆, coenzyme Q₉, and Nycodenz were from Sigma. Anti-mouse and anti-rabbit IgG peroxidase conjugated were from Calbiochem. Methanol was from EM Science, and ethanol was from Pharmco. All other chemicals were from Fisher. Antisera to Sec62p and cytochrome c₁ polypeptides were kindly donated by Gay Bush and Alexander Tzagoloff. Antisera to plasma membrane ATPase (Pma1p)¹ were a gift from Greg Payne, OM45 was from Michael Yaffe, and porin and -KDH were from Gottfried Schatz.

Plasma Membrane and Mitochondria Purification-- Yeast plasma membranes were purified as described (41). This procedure requires two separate sucrose gradient separations and an alkaline protein extraction to remove extrinsic proteins and to open plasma membrane vesicles to release vesicles derived from other endomembranes. This method produces a low yield of plasma membrane but with a concomitant high purity. In brief, yeast cells were lysed by vortexing with glass beads and centrifuged (10 min at 700 × g) to remove debris. The supernatant was centrifuged (30 min at 20,000 × g) to obtain a crude membrane pellet. Crude membranes were resuspended in sucrose buffer (20% w/w sucrose, 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, and 1 mM dithiothreitol) and applied to a sucrose step gradient of 4 ml (43%) (w/w) sucrose and 2 ml (53%) (w/w) sucrose in the same buffer. After centrifugation (4 h at 100,000 × g), plasma membranes were recovered at the 43/53 interphase and reapplied to a second sucrose step gradient as before. To remove non-intrinsic plasma membrane proteins, the samples were treated with 100 mM Na₂CO₃, pH 11.5 (42), and suspended in the same buffer with 0.33 M sucrose.

Yeast spheroplasts and subcellular fractions enriched in mitochondria were isolated by published procedures (43). Mitochondria were purified using a Nycodenz gradient. Crude mitochondria were suspended in buffer A (0.6 M sorbitol, 5 mM K⁺MES, pH 6.0), layered on top of a discontinuous step Nycodenz gradient (5 ml (20%) and 5 ml (14.5%) in buffer A), and subjected to 1 h of centrifugation at 100,000 × g. Purified mitochondria were harvested from the interface, diluted 3-fold with buffer A, and centrifuged for 10 min at 12,000 × g. The pellet of purified mitochondria was washed once with buffer B (0.6 M mannitol, 10 mM Tris-HCl, pH 7.4) and suspended in a small volume of buffer B.

Western Analysis-- The protein concentrations of plasma membrane and mitochondrial fractions were assayed by the bicinchoninic acid assay. Equal amounts of protein (25 µg) from both fractions were analyzed by electrophoresis on 12.5% Tris-glycine gels and subsequently transferred to a nitrocellulose membrane. The incubation with the antibodies and the subsequent washes were performed with 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5% skimmed milk, and 0.05% Tween 20. The primary antibodies were used at the following dilutions: 1:2,000 Pma1; 1:500-5,000 Sec62p; 1:10,000 porin; 1:5,000 OM45; 1:10,000 -KDH, and 1:10,000 cytochrome c₁. Horseradish peroxidase-linked secondary antibodies to rabbit and mouse IgG were used in a 1:10,000 dilution. Blots were washed free of all antibodies by incubating the nitrocellulose membranes 30 min at 55 °C in 60 mM Tris-HCl (pH 6.8), 2% SDS, and 0.7% -mercaptoethanol. Densitometry analysis was performed with the Alphaimager 3.3 software from Alpha Innotech (San Leandro, CA).

Lipid Extraction and Determination of CoQ₆-- Q₉ (10 µl of a 2 mM stock) was added as an internal standard to aliquots of plasma membrane and mitochondria (500 µl, 0.5-1 mg of protein), and samples were incubated 20 min at room temperature. The samples were mixed with an equal volume of 2% SDS and vortexed 1 min. Two milliliters of 5% isopropyl alcohol in ethanol was added, and samples were vortexed again for 1 min. To recover CoQ_, 5 ml of hexane were added, and the mixture was vortexed at top speed for 1 min and centrifuged at 1000 × g for 5 min. The upper phases recovered from three extractions were pooled and dried in a rotatory evaporator. Lipid extracts were suspended in 1 ml of 9:1 methanol/ethanol, dried in a speed-vac and kept at 20 °C. Samples were suspended in a suitable volume of 9:1 methanol/ethanol prior to HPLC injection.

Q₆ and Q₉ were separated by reversed-phase high pressure liquid chromatography with a C18 column (Alltech Econosphere 5-µm, 4.6 × 250-mm column) and quantified with an ESA Coulochem II electrochemical detector and a 5010 analytical cell (E1, 450 mV; E2, 500 mV). A separate precolumn cell (ESA 5020) was set to an oxidizing mode (E, +450 mV) to convert all hydroquinones to quinones. The mobile phase was adjusted to a flow rate of 1 ml/min and was composed of methanol/ethanol/2-propanol (88/24/10) and 13.4 mM lithium perchlorate. Q₆ and Q₉ were quantified from the electrochemical detector results with Q₆ and Q₉ as external standards. The use of Q₉ as an internal standard indicated a recovery of 90-100% of total Q₉ added to samples.

Ascorbate Stabilization Assay-- The ascorbate stabilization assay was described previously (30). Yeast cultures were harvested at mid-log phase (OD_600 nm = 4) and washed twice in cold water. Cells were resuspended at 10⁷ cells/ml in 0.1 M Tris-HCl buffer (pH 7.4) with 0.06 mM CuSO₄. Ascorbate oxidation was followed by the direct reading of absorbance at 265 nm, with an extinction coefficient E is 14.5 mM¹ cm¹ at pH 7.4 (44). The addition of ascorbate (final concentration, 0.15 mM) to the cell suspension initiated the ascorbate oxidation caused by the presence of Cu²⁺. Cells were removed by centrifugation, and the supernatants were used to measure the ascorbate oxidation rates. Ascorbate stabilization is defined as the difference between the oxidation rate of ascorbate in the presence of cells and the oxidation rate without cells.

Assays of Mitochondrial Complexes-- Yeast cells were grown at 30 °C overnight in 10 ml of YPD with shaking at 200 rpm and were used to inoculate 200 ml of YPD to a density of 0.25 OD_660 nm/ml. The cultures were incubated at 30 °C as before until cell density was 9-10 OD_660 nm/ml, and then the cultures were divided in two flasks, one receiving 100 µl of 2 mM CoQ₆ in ethanol and the other 100 µl of ethanol alone. Both cultures were incubated at 30 °C, 200 rpm for 48 h. Crude mitochondrial fractions were prepared as indicated above and were assayed directly without freezing. Succinate-cytochrome c reductase activity was measured in 40 mM sodium phosphate buffer (pH 7.4) containing 20 µM sodium succinate and 250 µM potassium cyanide. Samples of mitochondria (50 µg of protein) were incubated in the assay buffer without cytochrome c for 5 min. The reaction was initiated by the cytochrome c addition and was monitored at 550 nm minus 540 nm. The specific activity was determined with an E of 18,500 M¹ cm¹ (45). For assays in the presence of exogenous Q₆ the assay buffer contained 10 µM Q₆ or 1% ethanol.

Cytochrome c oxidase activity was measured in 40 mM sodium phosphate buffer (pH 7.4) containing 25 µM reduced cytochrome c (45). Horse heart cytochrome c was reduced with L-ascorbate until ratio A₅₅₀/A₅₆₅ was between 6 and 9. The reaction was started with the addition of 15-25 µg of mitochondrial samples, and the cytochrome c oxidation was monitored measuring the decrease of A₅₅₀ using an Aminco DB-3500 spectrophotometer. The specific activity was determined using an E of 18,500 M¹ cm¹.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Supplementation of Ethanol Growth Medium with Q₆ Rescues Only a Subset of the coq7 Mutant Strains-- Previous studies have shown that yeast coq3 and coq7 mutants with defects in Q biosynthesis are rescued for growth on non-fermentable carbon sources (such as ethanol) by the addition of exogenous Q₆ (11, 16). To test the generality of this rescue in coq7 mutants, four null coq7 mutants from distinct wild-type genetic backgrounds (W303.1B, CEN.PK2-1C, EG103, and FY250) were used (Table I). The strains were incubated in growth medium containing ethanol (YPE) in the presence or absence of 15 µM exogenous Q₆ for 7 days. Growth was monitored by measuring the OD at 600 nm, and aliquots of cultures were transferred to YPD plate medium to verify the absence of contamination. In the absence of Q₆ supplementation the coq7 mutants were unable to grow on YPE medium (Fig. 1A). The addition of Q₆ to YPE medium rescued the growth of CEN.MP3-1A and W303COQ7 strains (Fig. 1B). Growth was slower than in wild-type strains, although after 4 days the cultures reached a density similar to the parental strains. However, FY250 coq7 and EG103 coq7 strains showed no growth in the Q₆-supplemented YPE medium after 7 days of culture. This lack of rescue by exogenous Q₆ indicates a defect either in Q₆ uptake or in a hypothetical Q₆ transport system.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Supplementation of YPE medium with Q6 rescues the growth of only two of the four Q-less yeast coq7 mutant strains tested. Yeast cells were cultured in YPD, washed with water, diluted in YPE medium to 0.25 OD/ml, and grown for 7 days (A). Q6 diluted in ethanol was added to 15 µM final concentration to the cultures (B). Cells were cultured at 30 °C with shaking, and samples were taken at the indicated times. The wild-type strain CEN.PK2-1C was used as control, and this growth is representative of the other wild-type parental strains (data not shown).

Purification of Yeast Plasma Membrane and Mitochondria Fractions-- The ability of CEN.MP3-1A and W303COQ7 yeast strains to grow with a non-fermentable carbon source in the presence of exogenous Q₆ indicated that exogenous Q₆ is transported to the mitochondria. The first barrier to a possible Q₆ transport pathway is likely to be the plasma membrane; thus, insertion of Q₆ into the cell plasma membrane may be a critical first step of Q₆ uptake. To analyze this process we quantified the amounts of Q₆ present in the plasma membrane and mitochondria of the eight strains (four wild-type and four coq7 mutants) cultured with or without exogenous Q₆. YPD culture medium was used to allow for growth of the FY250 coq7 and EG103 coq7 mutant strains. Yeast were harvested at a cell density of OD_660 nm = 10-14 representing post-diauxic shift in wild-type cells and increased reliance on the use of Q₆ in respiratory energy metabolism.

Yeast plasma membrane fractions were purified as described under "Experimental Procedures." The quality of the plasma membrane samples was tested with several antibodies against endomembrane marker proteins that can co-purify with plasma membrane (Fig. 2A). Purified plasma membrane was compared with the starting material of crude membranes. Western analysis shows that there is a 15-fold enrichment of the Pma1p marker in plasma membrane as compared with crude membranes. The abundance of this marker is consistent with the high expression level of Pma1p in yeast grown in glucose medium (46). The presence of contaminating membranes was assayed with antibodies to marker proteins of the endoplasmic reticulum (Sec62), mitochondrial outer membrane (porin and OM45), and mitochondrial inner membrane (cytochrome c₁). None of these marker proteins were detected in plasma membrane fractions. The determination of Q₆ content in plasma membrane requires fractions with a high purity to minimize cross-contamination with membranes from organelles enriched in Q₆ such as mitochondria or endoplasmic reticulum. The extent of purification of the plasma membrane shown in Fig. 2A discounts both mitochondrial and endoplasmic reticulum as contributors of Q₆. Two markers from the outer mitochondrial membrane were used because although porin is considered to be a specific marker of mitochondria, several authors have described its presence in other intracellular membranes of mammalian cells (47). Thus, the OM45 protein marker was used to detect the presence of outer mitochondrial membrane specifically and showed the same results as porin.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Yeast plasma membrane and mitochondria fractions are highly purified. Aliquots of plasma membrane and mitochondria (25 µg of protein) were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The Western blots were probed with several antibodies to marker proteins including cytochrome c1 (mitochondrial inner membrane), OM45 protein and porin (mitochondrial outer membrane), Sec62 (endoplasmic reticulum), and Pma1 (plasma membrane H+-ATPase). Blots were stained with a secondary antibody obtained in goat against rabbit IgG, except for Sec62, which was obtained against mouse IgG. Secondary antibodies were labeled with horseradish peroxidase and were developed with the enhanced chemiluminescence system. A, plasma membrane fraction from the CEN.PK2-1C strain. CM, crude membranes after cell disruption; PM, plasma membrane. B, mitochondrial fractions from the CEN.PK2-1C strain. Crude, mitochondrial fraction after the osmotic cell lysis; Pure, purified mitochondria after the Nycodenz gradient. Mitochondrial results correspond to those of the same blot stripped twice. Plasma membrane results (except Pma1) correspond to those of the same blot stripped three times.

Mitochondria were purified according to a widely used method (43) that involves a final Nycodenz gradient. The validity of this procedure is well established but was also tested during this work with antibodies that recognized specific proteins from several mitochondrial compartments and endoplasmic reticulum (Fig. 2B). The results indicate that the purification step using the Nycodenz gradient results in an enrichment of 150% in the cytochrome c₁ marker and 191% of porin, whereas the signal produced by Sec62 antibody decreased dramatically to only 3% of that present in mitochondrial crude preparations.

Content of Q₆ in Plasma Membrane and Mitochondria-- The Q₆ present in plasma membrane and purified mitochondria was quantified by HPLC separation and electrochemical detection. The addition of Q₆ to YPD cultures led to general but variable increases of Q₆ content at the plasma membrane in wild-type strains (Fig. 3). In CEN.MP3-1A and FY250 coq7 the amount of exogenous Q₆ incorporated into plasma membrane was slightly higher than in the corresponding wild-type strains. EG103 coq7 and W303COQ7 displayed slightly lower amounts than wild-type strains. Yet in all coq7 mutant strains tested the level of plasma membrane Q₆ either approximated or exceeded the level of Q₆ in plasma membrane of the unsupplemented wild-type strain. Hence, these results suggest that the defect in the rescue of growth of EG103 coq7 and FY250 coq7 strains with exogenous Q₆ in ethanol as carbon source cannot be due to a defect of Q₆ uptake or insertion in the plasma membrane.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   The addition of exogenous Q6 to cultures of coq7 mutant strains increases the plasma membrane Q6 content, but two strains have defects in delivering exogenous Q6 to the mitochondria. Plasma membrane and Nycodenz-purified mitochondria were isolated from each of the strains indicated following culture in YPD in the absence or presence of 2 µM exogenous Q6. Lipid extractions and Q6 quantification were performed as described under "Experimental Procedures." Results are expressed as the average ± S.D from two independent extractions. For each graph cross-hatched bars correspond to yeast strains cultured in the presence of 2 µM Q6. The open and shaded bars correspond to mitochondrial and plasma membrane Q6 content, respectively. Q6 was not detectable in coq7 strains cultured without exogenous Q6 (data not shown).

Supplementation of media with exogenous Q₆ had little effect on the level of Q₆ in Nycodenz-purified mitochondria from the EG103 or W303.1B wild-type strains (Fig. 3). Q₆ supplementation produced an increase in mitochondrial Q content in the CEN.PK2-1C wild-type strain, whereas a decrease was observed in the FY250 wild-type strain. Mitochondria from the coq7 mutant strains (CEN.MP3-1A and W303COQ7), following culture in medium supplemented with Q₆, showed a high Q₆ content comparable with the level of Q₆ present in the unsupplemented wild-type parent (Fig. 3). Although it is possible to detect Q₆ in mitochondria from EG103 coq7 and FY250 coq7 the Q₆ content is only 35 and 8% of the level present in the corresponding wild-type parental strains. These data indicate that delivery of exogenous Q₆ to mitochondria is strain-dependent and is higher in strains that can be rescued by exogenous Q₆.

Exogenous Q₆ Functions at the Plasma Membrane as Measured by Ascorbate Stabilization Activity-- In S. cerevisiae ascorbate stabilization has been characterized as an activity of the plasma membrane redox system that maintains extracellular ascorbate in its reduced form (48, 9). In this system Q₆ acts as an electron shuttle that transfers electrons from a cytosolic donor such as NADH to an external acceptor, reducing ascorbate free radical to ascorbate. This activity is partially dependent on plasma membrane Q₆ (30) and can serve as an assay to test whether the exogenous Q₆ incorporated into plasma membrane is functional. Ascorbate stabilization activity was measured in EG103 and W303.1B yeast strains and in the corresponding coq7 mutants (Fig. 4). In wild-type strains supplementation of growth medium with Q₆ led to only a slight increase of the ascorbate stabilization activity, probably because the amount of Q₆ present naturally in these strains is sufficient to support this activity. In coq7 mutant strains Q₆ supplementation of media increased the activity by 75 and 50% in the W303COQ7 and EG103 coq7 strains, respectively, and restored ascorbate stabilization activity to wild-type levels. These data indicate that each of the four coq7 mutant strains tested retains the ability to take up exogenous Q₆ and incorporate it into the plasma membrane.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   The increase of Q6 at the plasma membrane correlates with the augmentation of ascorbate stabilization activity. Cells were harvested at mid-log phase to measure the ascorbate stabilization rate as described under "Experimental Procedures." Specific activity is shown as the average ± S.D. of two separate experiments. Closed bars correspond to the ascorbate stabilization activity in cells cultured in the absence of exogenous Q6, and open bars correspond to the activity of cells cultured in presence of 2 µM exogenous Q6.

Function of Exogenous Q₆ in the Mitochondria as Measured by Succinate-Cytochrome c Reductase Activity-- Q₆ participates in mitochondrial complexes I, II, and III of S. cerevisiae (1, 49). To test whether exogenous Q₆ restored activities in the mitochondrial respiratory chain, the succinate-cytochrome c reductase activity was measured in crude mitochondrial fractions obtained from both wild-type and coq7 deletion strains cultured in the presence or absence of exogenous Q₆. The effect of adding Q₆ to the in vitro assays of succinate-cytochrome c reductase was also determined (Fig. 5A). The succinate-cytochrome c reductase activity in wild-type strains was high but quite variable among the different yeast strains analyzed. In some wild-type strains the addition of Q₆ to the in vitro assay led to a moderate increase in activity, whereas in the EG103 wild-type strain a 70% increase was observed. Succinate-cytochrome c reductase activity was barely detectable in the coq7 mutant strains even after addition of Q₆ to the in vitro assay. However, succinate-cytochrome c reductase activity is readily detected in mitochondria isolated from the coq7 mutant strains CEN MP3-1A, W303COQ7, and FY250 coq7 cultured in YPD plus Q₆. This activity was further increased by addition of Q₆ to the in vitro assay (300% for CEN MP3-1A, 250% for W303COQ7, and 200% for FY250 coq7).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   The effect of Q6 supplementation on succinate-cytochrome c reductase activity and steady state levels of cytochrome c1 and other mitochondrial proteins. Succinate-cytochrome c reductase activity, steady state levels of cytochrome c1, porin, and -ketoglutarate dehydrogenase proteins, and Q6 content were measured in crude mitochondria from yeast as described under "Experimental Procedures." For both panels, wt corresponds to the wild-type strain of the designated genetic background,  corresponds to the coq7 null mutant strain, and Q6 corresponds to the coq7 mutant cultured in medium supplemented with 2 µM Q6. A, succinate-cytochrome c reductase activity is expressed as the average ± S.D. of three assays. The (+) or () sign under each bar indicates the presence or absence of 10 µM CoQ6 in the in vitro assay. B, aliquots of crude mitochondrial protein (25 µg) assayed in A were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The Western blots were probed for mitochondrial cytochrome c1, porin, and -ketoglutarate dehydrogenase. The relative intensity of the cytochrome c1 signal was measured by densitometry and is shown over each lane. The Q6 content (pmol/mg protein; average of three determinations) of each mitochondrial preparation is given under each lane. The S.D. was less than 5% of Q6 content.

The restoration of succinate-cytochrome c reductase activity in coq7 mutants by Q₆ supplementation of growth medium was strain-dependent and was higher in strains that can be rescued by exogenous Q₆. The measure of Q₆ in pure mitochondrial fractions (Fig. 3) showed the presence of Q₆ even in EG103 coq7 and FY250 coq7 strains. These data were confirmed by quantifying Q₆ in the crude mitochondrial fractions used to measure succinate-cytochrome c reductase activity (Fig. 5B). Interestingly, there is a correlation between high levels of Q₆ (CEN.MP3-1A and W303COQ7), high levels of succinate-cytochrome c activity, and the ability to be rescued by Q₆ supplementation of YPE medium. However, coq7 strains that show wild-type contents of mitochondrial Q₆ did not show wild-type levels of succinate-cytochrome c reductase activity. It is possible that a lack of Q₆ during respiratory complex assembly renders the complex unstable and retards the metabolic switch from fermentation to respiration in CEN MP3-1A and W303COQ7. To investigate this possibility, the same samples used to measure the Q₆ content were subjected to Western analysis with antibodies against cytochrome c₁ (a polypeptide component of complex III), porin, and -ketoglutarate dehydrogenase (Fig. 5B). The cytochrome c₁ level in the different wild-type strains was variable, but it correlated well with both Q₆ content and succinate-cytochrome c reductase activity. A similar correlation was observed in coq7 mutants cultured with Q₆. The cytochrome c₁ polypeptide was virtually absent in the coq7 mutant strains. The defect shown by coq7 mutant strains in the assembly or stability of complex III is unlikely to be from the lack of respiration per se, because other respiratory defective mutants (such as those in cytochrome c oxidase) retain significant levels of succinate-cytochrome c reductase activity (50).

The labeling of the same Western blots with anti-porin antibodies shows that porin levels are similar for all strains (Fig. 5B). These results suggest that the lack of cytochrome c₁ is unlikely to result from a lack of glucose derepression in coq7 mutants because, like porin, expression of cytochrome c₁ is enhanced by low glucose (51) or a switch to non-fermentable carbon source (52). During the diauxic switch, several genes involved with respiratory metabolism are expressed under the action of the Hap1 and Hap2/3/4 complexes. These complexes are activated in low glucose conditions. CYT1 (encoding the cytochrome c₁ polypeptide) and KDG1 (encoding -ketoglutarate dehydrogenase) are among the genes controlled by Hap complexes. The steady state expression of Kdg1p is similar for all strains and culture conditions (Fig. 5B) and is similar to the porin results. This is in marked contrast to the Cyt1p results, although these genes share the same transcriptional regulation pathway (52, 53).

Although the effect of Q₆ in the succinate-cytochrome c reductase activity was not related directly to the lack of respiration, it is possible that the lack of Q₆ also affects other mitochondrial complexes. To investigate this possibility we measured a respiratory enzyme activity that does not depend directly on Q₆, cytochrome c oxidase. The results indicate that in coq7 null strains, cytochrome c oxidase activity was present at levels 20-30% that of wild-type activity. Similar decreases in cytochrome c oxidase activity have been noted for other coq mutants (16, 54), and this has been attributed to a general defect in respiration rather than the lack of Q per se. Supplementation of growth medium with Q₆ restored cytochrome c oxidase activity in the CEN.MP3-1A, W303COQ7, and FY250 coq7 mutant strains (Fig. 6). However, the restoration of cytochrome c oxidase activity in response to Q₆ supplementation was not uniform. In W303COQ7 and CEN.MP3-1A strains activities were completely restored, but activities were only partially restored in FY250 coq7, and very low activities were found in EG103 coq7.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Cytochrome c oxidase activity is not strictly dependent on the presence of mitochondrial Q6. Cytochrome c oxidase activity was measured in crude mitochondria as described under "Experimental Procedures." For each genetic background, wt corresponds to the wild-type strain,  corresponds to the coq7 null mutant, and Q6 corresponds to the coq7 mutant cultured in medium supplemented with 2 µM Q6. Cytochrome c oxidase activity is expressed as the average ± S.D. of three assays.

	DISCUSSION

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This study shows that uptake of exogenous Q₆ and transport to the mitochondria operate in yeast. The rescue of growth of the Q-less coq7 mutants on non-fermentable carbon sources by exogenous Q₆ is dependent on the genetic background of the yeast strains under study, because supplementation of growth medium with Q₆ rescued only two (CEN.MP3-1A and W303COQ7) of the four coq7 mutant strains tested. The defect in the two non-rescued strains (EG103 coq7 and FY250 coq7) does not lie in uptake because Q₆ supplementation of growth medium restored the content of plasma membrane Q₆ to wild-type levels. The exogenous Q₆ present in the plasma membrane is functional (as indicated by the restoration of ascorbate stabilization), a plasma membrane redox activity dependent on Q₆. Instead, a defect in the transport of Q₆ from the plasma membrane to mitochondria appears to be responsible for the inability of the EG103 coq7 and FY250 coq7 strains to grow in YPE supplemented with Q₆. These two non-rescued strains contained much lower levels of mitochondrial Q₆ than did the two strains rescued by Q₆ supplementation. In the rescued strains the presence of mitochondrial Q₆ was functional, as indicated by the restoration of succinate-cytochrome c reductase activity, a coupled assay for complexes II and III that depends on Q. These data indicate that uptake of exogenous Q₆ and its delivery to the mitochondria of coq mutant yeast strains restores respiratory electron transport function.

Our results suggest that mitochondrial Q₆ influences the content of the cytochrome c₁ polypeptide in complex III. There was a strong correlation between the level of exogenous Q₆ delivered to the mitochondria, the level of succinate-cytochrome c reductase activity, and the steady state level of the cytochrome c₁ polypeptide (Cyt1p). The effects on Cyt1p could be caused by changes in synthesis and/or stability. The data presented here argue against the regulation being exerted at the level of Cyt1p synthesis. In the absence of exogenous Q₆ Cyt1p is still detected in yeast coq7 mutants (Fig. 5B). Although Q₆ supplementation produces a dramatic increase in Cyt1p levels (especially in those strains rescued by Q₆), this increase is probably not because of increased synthesis per se. The transcription of the CYT1 gene is regulated similarly to other mitochondrial genes in response to low glucose levels by the Hap1 and Hap2/3/4 complexes (52). The Hap complexes also regulate levels of porin and -ketoglutarate dehydrogenase. However, unlike Cyt1p, the steady state levels of porin and -ketoglutarate dehydrogenase are high in each of the coq7 mutant strains. These data agree with studies of porin import into mitochondria isolated from yeast coq5 mutant strains (14) demonstrating that the porin import is independent of Q₆ content or state of respiration. Some studies have indicated that porin is hyperexpressed in stationary phase, upon diauxic shift, and upon glucose deficiency (55, 56). Other authors have indicated that the in vitro import of porin to mitochondria does not require ATP (57). Therefore, unlike Cyt1p, the levels of porin and -ketoglutarate dehydrogenase polypeptides are not correlated with the Q₆ content and provide an indirect indication that synthesis of the Cyt1p is not repressed.

Previous studies of other Q-less yeast mutants also suggest that synthesis and assembly of the bc₁ complex is operational in the absence of Q₆. For example, yeast abc1 mutants completely lack Q₆ (16), but cytochrome absorption spectra indicate that these mutants retain synthesis and assembly of cytochromes b, c₁, c, and aa₃, although each is present at lower levels (58, 59). The presence of assembled respiratory complexes as determined by cytochrome absorption spectra was also noted during the original characterization of the coq mutants (54, 50). Indeed, complex III can be purified from Q-less yeast mutant strains, but this purification requires large amounts of starting material (80 liters of culture) and produces a complex III with altered catalytic properties as compared with wild-type strains (60).

We favor the hypothesis that the decreased level of Cyt1p in the coq7 mutant strains reported here is caused by decreased stability. The observed decrease in Cyt1p levels in the absence of Q₆ shows a striking parallel with the decreased stability of cytochrome b₆ in the FUD2 mutant of Chlamydomonas reinhardtii (61). Cytochrome b₆ is one of the polypeptide components of the chloroplast cytochrome b₆f complex that mediates quinol oxidation, electron transport, and proton pumping in a manner analogous to the mitochondrial bc₁ complex. In the C. reinhardtii FUD2 mutant, a 12-amino acid insertion mutation in the cytochrome b₆ polypeptide produces a decreased affinity for plastoquinol in the Q_o site. The cytochrome b₆f complex was found to be much less stable in the FUD2 mutant than in its wild-type counterpart, and this decreased stability was especially pronounced when cultures were harvested at a high cell density (e.g. stationary phase). It is intriguing that the decreased affinity for plastoquinol in the FUD2 mutant and the increased turnover noted for the b₆f complex may be analogous to the decreased availability of Q₆ in the coq yeast mutants and the decreased level of Cyt1p. Because the yeast cultures examined in Fig. 5B were harvested at a high cell density (9-10 OD_660 nm per ml), the decreased level of Cyt1p may be even more pronounced than it would be under log phase growth.

A large body of evidence indicates that increased proteolysis is responsible for removing photosynthetic and respiratory subunit polypeptides synthesized in the absence of heme cofactors or partner polypeptides (50, 62, 63). Similarly, a lack of Q may render the bc₁ complex less stable and more prone to proteolytic degradation. Q₆H₂ functions as both substrate for the quinol oxidation step of the bc₁ complex and as an essential cofactor for proton pumping. Recent evidence indicates that each bc₁ complex monomer contains three molecules of tightly bound Q (64). Based on the current models of Q-dependent proton pumping by the bc₁ complex (49), it is reasonable to suppose that the stability of this complex in vivo would depend at least in part on the availability of Q.

Addition of Q₆ to the culture medium failed to restore Cyt1p levels in the coq7 mutants that have inefficient transport of Q₆ from the plasma membrane to mitochondria. It is likely that the defects of Q₆ transport are different in these two strains. Mitochondria from EG103 coq7 cultured with exogenous Q₆ contain significant levels of Q₆ (35% of wild-type levels), yet growth in ethanol is not rescued, Cyt1p is not detected, and there is no appreciable succinate-cytochrome c reductase activity. It is possible that in this strain only a small fraction of the mitochondrial Q₆ is delivered to the inner membrane. In contrast, mitochondria from FY250 coq7 contain low levels of Q₆ (8% of wild-type), but although its growth in ethanol medium cannot be sustained by exogenous Q₆ there are low but significant levels of Cyt1p and succinate-cytochrome c reductase activity. In this case it is possible that Q₆ is delivered to the mitochondrial inner membrane in a functional way, but the amount delivered is extremely low. Hence, although the nature of the defects in Q₆ transport from plasma membrane to mitochondria appear to differ in the two non-rescued stains, the outcome is similar in that neither strain is able to utilize exogenously provided Q_6. Genetic analysis of these strains may help elucidate two complementary aspects of Q₆ intracellular transport.

There is considerable controversy in the literature regarding the ability of cells and animals to assimilate and transport exogenous Q to mitochondria. In general, much work with animal models indicates that dietary supplementation with Q₁₀ increases levels of Q₁₀ in lipoproteins and in the liver but has little or no effect on increasing Q₁₀ levels in peripheral tissues such as muscle, heart, or brain (65-68). Such feeding studies have sometimes been interpreted as indicating that there is little or no intracellular transport of dietary Q to mitochondria (69). However, when dietary Q₁₀ supplements have been given to old rats (70, 71) or to human patients with deficiencies in Q₁₀ (72), significant uptake of Q₁₀ into mitochondria has been documented. In general, such uptake restores Q-dependent assays of respiratory function. These apparently disparate results imply that the uptake of dietary Q and its transport to the mitochondria may be dictated by the need for Q or may even depend on a state of mitochondrial Q deficiency. The situation may be similar to that of sterol uptake in yeast ergosterol mutants (73) in which the amount of sterol taken up reflects the need for sterol. Cells with adequate levels of sterol do not take up added sterols, whereas ergosterol biosynthetic mutants take up sterols readily. However, once adequate sterol levels are achieved any further accumulation depends on growth; hence sterol depletion. An analogous phenomenon is observed in the yeast model presented here. For example, Q₆ supplementation of growth medium has little or no effect on the mitochondrial Q₆ content of wild-type yeast strains, whereas a dramatic increase in the Q₆ content is observed in the mitochondria of the rescued coq7 mutant strains (Fig. 3). A similar uptake and transport of Q₈ to mitochondria has been postulated to account for the rescue of growth and fertility of the C. elegans Q-deficient clk-1 mutants by dietary sources of Q₈ (74).

Although this study documents the uptake of exogenous Q₆ and delivery to the mitochondria, in yeast and in most eukaryotic cells capable of Q biosynthesis it seems more likely that this Q transport pathway usually functions in the opposite direction, namely transport of Q₆ from mitochondria (the site of synthesis) to the plasma membrane. Hence, we speculate that the delivery of exogenous Q₆ to the mitochondrial inner membrane may be inefficient, and the assimilation of exogenous Q₆ within respiratory complexes may produce respiratory complexes with decreased stability as compared with complexes containing endogenously synthesized Q₆. Even in strains that are rescued by Q₆ and that have wild-type levels of Q₆ in mitochondria, both succinate-cytochrome c reductase activity and Cyt1p levels are lower than in wild-type strains having the same Q₆ concentration. Whereas in rescued strains the amount of operational complex III is sufficient to permit growth in non-fermentable carbon sources, there is nonetheless a dramatic (200-500%) increase in succinate-cytochrome c reductase activity when Q₆ is added directly to isolated mitochondria in the in vitro assay.

In summary, we have shown that the inability of certain yeast strains to be rescued by Q₆ complementation of culture medium containing a non-fermentable carbon source is caused by a defect in Q₆ transport from plasma membrane to mitochondria. Our hypothesis is that this defect can be attributed to a general problem in the Q₆ transport between organelles. Future work will focus on identifying the genes and polypeptides involved in the uptake and intracellular transport of Q.

	ACKNOWLEDGEMENTS

We thank Drs. Patrice Hamel and Tanya Jonassen for comments on the manuscript and discussions of Q transport. This work was supported by the National Institutes of Health Grant GM45952 (C. F. Clarke) and by a Fulbright Scholarship Award (C. Santos-Ocaña).

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM45952 and Fulbright Project 99104.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.

Recipient of a Fulbright postdoctoral fellowship from the Spanish Ministry of Education and Science.

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

Published, JBC Papers in Press, January 11, 2002, DOI 10.1074/jbc.M112222200

	ABBREVIATIONS

The abbreviations used are: Pma1p, plasma membrane ATPase; HPLC, high pressure liquid chromatography; Q, ubiquinone or coenzyme Q; Q₆, Q with a tail with 6 isoprene units; Q₁₀, Q with a tail of 10 isoprene units.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES