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Originally published In Press as doi:10.1074/jbc.M403066200 on July 21, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54479-54486, December 24, 2004
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Mitochondrial Oxidative Phosphorylation Is Defective in the Long-lived Mutant clk-1*

Ernst-Bernhard Kayser{ddagger}§, Margaret M. Sedensky{ddagger}§, Phil G. Morgan{ddagger}§, and Charles L. Hoppel, Supported by the Veterans Affairs Hospitals Medical Services and by National Institutes of Health Grant PO1 AG15885¶||

From the {ddagger}Departments of Anesthesiology and Genetics, University Hospitals and Case Western Reserve University, Cleveland, Ohio 44106 and the Departments of Pharmacology and Medicine, Department of Veterans Affairs Medical Center, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, March 19, 2004 , and in revised form, July 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The long-lived mutant of Caenorhabditis elegans, clk-1, is unable to synthesize ubiquinone, CoQ9. Instead, the mutant accumulates demethoxyubiquinone9 and small amounts of rhodoquinone9 as well as dietary CoQ8. We found a profound defect in oxidative phosphorylation, a test of integrated mitochondrial function, in clk-1 mitochondria fueled by NADH-linked electron donors, i.e. complex I-dependent substrates. Electron transfer from complex I to complex III, which requires quinones, is severely depressed, whereas the individual complexes are fully active. In contrast, oxidative phosphorylation initiated through complex II, which also requires quinones, is completely normal. Here we show that complexes I and II differ in their ability to use the quinone pool in clk-1. This is the first direct demonstration of a differential interaction of complex I and complex II with the endogenous quinone pool. This study uses the combined power of molecular genetics and biochemistry to highlight the role of quinones in mitochondrial function and aging.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Caenorhabditis elegans gene clk-1 was one of the first reported gerontogenes, i.e. a gene that when mutated increases the life span of the worm. clk-1 mutants appear healthy, but many developmental processes and behavior patterns on a strict biological clock in the wild type are deregulated in the mutant. Hence, the name of the gene; clk stands for clock (1, 2). clk-1 codes for an enzyme required for the biosynthesis of ubiquinone (Fig. 1), the electron acceptor for both complex I- and complex II-dependent mitochondrial respiration (3). The best known function of ubiquinone is to serve as a redox carrier for two electrons and two protons between complex I or II and complex III of the respiratory chain in the inner mitochondrial membrane (see Fig. 2) (4). Regardless of the port of entry, all electrons are normally shuttled through ubiquinone. Ubiquinone, also known as coenzyme Q (CoQ), is composed of a redox-competent modified benzoquinone headgroup and a lipophilic polyisoprenyl tail (Fig. 1). The number of isoprenyl units forming the tail is species-specific. For C. elegans, this number is 9 (CoQ9, as is the case in mice and rats), whereas the principal species is CoQ8 in Escherichia coli (5-7).



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  		FIG. 1.
Quinones present in C. elegans mitochondria. CoQ9 is the ubiquinone produced by wild type C. elegans. CoQ8 is made by E. coli and incorporated by nematodes from their diet. DMQ9 is an intermediate in the biosynthesis of CoQ9 and the substrate for the wild type CLK-1 protein. Since this enzyme is defective in clk-1, DMQ9 accumulates in the mutant. RQ9 is another quinone produced by C. elegans and present in increased amounts in clk-1 mutants.

 



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  		FIG. 2.
The integration of steps necessary for oxidative phosphorylation. Electron donor substrates for the respiratory chain are transported across the inner mitochondrial membrane by different carriers. Pyruvate uses the pyruvate carrier; glutamate uses the amino acid carrier; malate and succinate each use the dicarboxylate carrier. In the matrix, electrons from pyruvate, glutamate, and malate are transferred to NAD+ by substrate-specific dehydrogenases and enter the respiratory chain via complex I. Succinate dehydrogenase is an integral activity of complex II. Electrons from complexes I and II are transferred to complex III by the common shuttle CoQ. The electrons reach oxygen, the terminal acceptor, via cytochrome c and complex IV. Electron transport down the respiratory chain (flat gray arrows) is absolutely linked to proton extrusion by complexes I, III, and IV (flat white arrows). The proton gradient across the inner membrane drives phosphorylation. ATP synthetase allows protons to re-enter the matrix and uses the energy released in this process to synthesize ATP. The substrates for phosphorylation, ADP and inorganic phosphate (Pi), enter the matrix via the ADP/ATP translocase (adenine nucleotide translocase) and the phosphate carrier. Respiration is high when the ATP synthetase is stimulated by the presence of ADP (state 3) and decreases when ADP is limiting (state 4). In cases in which the inhibition of phosphorylation is the cause for low respiration, respiration can be restored by adding a protonophore (such as DNP), allowing protons to re-enter the matrix without passing through ATP synthetase. The quinol analog DHQ can reduce complex III directly, whereas electrons from ascorbate are shuttled to cytochrome c by the redox carrier TMPD. Both artificial electron donors are useful to bypass substrate and electron transport upstream of coenzyme Q. Measuring respiration thus allows us not only to asses the integral function of mitochondria, but by varying electron donors, ADP conditions, and the use of DNP, it can also be used to locate the site of a mitochondrial defect.

 
clk-1 mutants cannot produce CoQ9; instead, they accumulate large quantities of the precursor demethoxyubiquinone (DMQ9)1 and small amounts of rhodoquinone (RQ9) as well as dietary CoQ8 from E. coli (6, 7) (Fig. 1). Genetic, dietary, and biochemical studies have attempted to clarify the relative functional importance of different quinone species in clk-1 (6-11). In particular, considerable effort has gone into defining the relative contributions of CoQ8 and DMQ9 to electron transport. Nonetheless, it is not yet known which of the quinone species is the mitochondrial electron carrier in clk-1. Initially, it was hypothesized that the clk-1 defect in CoQ9 synthesis prolonged life span by slowing the metabolic rate of the mutant (2, 3). However, Braeckman et al. (12, 13) reported conflicting results about oxygen consumption of intact clk-1 animals relative to wild type (N2). This discrepancy has not been resolved (14, 15).

The overwhelming majority of respiration in animals occurs in mitochondria, yet no measurements have been done of the integrated activity of mitochondria from clk-1 animals. The measurement of oxidative phosphorylation is a test of mitochondrial functional integrity and overall function as it relies on the ability of the organelle to effectively transport substrates, transfer electrons to the electron transport chain, reduce electron acceptors (such as CoQ), and shuttle electrons to their ultimate electron acceptor, oxygen. Measurements done under conditions that require the coupling of the process to the formation of ATP therefore also necessitate an intact mitochondrial membrane. We isolated mitochondria from N2 and clk-1 animals to assess their fully integrated function by measuring oxidative phosphorylation. All of the endogenous components of the electron transport chain (ETC) were present. Using this approach, we found that complex I-dependent oxidative phosphorylation in clk-1 mitochondria is profoundly decreased, whereas complex II-dependent oxidative phosphorylation is normal.

The presence of a defect in oxidative phosphorylation represents a change that may explain the aging phenotype of clk-1. To further localize the defect causing the decrease in oxidative phosphorylation, we measured the activity of specific steps of the electron transport chain. We identified a defect in electron transport between complexes I and III of the electron transport chain when the endogenous quinone species were present. By contrast, under the same conditions, electron transport between complexes II and III was normal. We found no difference between N2 and clk-1 in the activity of any of the individual complexes of the ETC. These results indicate a difference in the relationship of complex I and complex II with the pool of quinones present in the long-lived mutant clk-1. The results uncover the potential importance of complex I activity in affecting life span. The power of mutational analysis in a simple animal model opens new avenues for the study of integrated mitochondrial function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Nomenclature—The conventions for C. elegans nomenclature have been followed throughout (16). Gene names are italicized three-letter abbreviations followed by a hyphen and a number, e.g. clk-1, the gene. This designation can also specify worms homozygous for a mutation in this gene, e.g. clk-1, the mutant worm. Individual allele names are represented by a combination of one or two letters and a number, either alone or added in parentheses, e.g. as in qm30 or clk-1(qm30).

Nematode Culture and Mitochondria Preparation—The wild type strain N2, the missense mutant clk-1(e2519), and the deletion mutant clk-1(qm30) were obtained from the Caenorhabditis Genetics Center. Stocks were maintained on nematode growth media (NGM) plates with a lawn of E. coli OP50 as food source. The gram amounts of nematodes necessary for mitochondria preparations were grown in liquid culture and fed with the wild type E. coli, K12. OP50 is a K12-derived uracil auxotroph. Both bacteria strains produce ubiquinone CoQ8. Culture conditions, separation of worms from remaining E. coli, and preparation of mitochondria were performed as described previously (17, 18).

Oxidative Phosphorylation Assays—Oxygen uptake of intact mitochondria was measured polarigraphically with a Clark electrode as described previously (19). The external electron donor substrates malate, glutamate, or pyruvate with malate were used to drive complex I-dependent electron transport. C. elegans mitochondria differ from mammalian mitochondria in that malate is capable of fueling complex I by itself. We have previously reported this in our studies of oxidative phosphorylation in the C. elegans mutant, gas-1 (17). The presence of the gloxylate pathway in C. elegans mitochondria suggests a pathway for respiration of malate (20, 21).

Each of the three complex I-dependent substrates employs a different transporter to cross the mitochondrial membrane. Succinate was used as the electron donor for complex II. Succinate uses the same dicarboxylate transporter as malate to cross the mitochondrial membrane (22, 23). Thus, by comparing the effects of these substrates on oxidative phosphorylation, one can also determine whether the absence of a specific transporter plays a role in causing defects in respiration. Duroquinol (DHQ), an electron donor that supplies electrons directly to complex III, was used to measure the distal steps of electron transport that are common to both complex I- and complex II-dependent electron flow (24, 25). At the end of each oxidative phosphorylation experiment, uncoupled respiration through complex IV was measured using N,N,N',N'-tetramethyl-p-phenylenediamine) (TMPD)/ascorbate. This serves as a quality control for mitochondrial function during every assay performed.

ETC Assays—Freshly prepared mitochondria were solubilized with cholate, and zero order rates were determined spectrophotometrically for the following enzyme activities: citrate synthase, rotenone-sensitive NADH cytochrome c reductase, antimycin A-sensitive succinate cytochrome c reductase, antimycin A-sensitive decylubiquinol cytochrome c reductase, NADH ferricyanide reductase, rotenone-sensitive NADH decylubiquinone reductase, malonate-sensitive succinate-DCIP reductase, and duroquinone-stimulated malonate-sensitive succinate DCIP reductase. The first order rate constant was determined for cytochrome c oxidase.

Assays and calculations were performed as described by Hoppel and co-workers (24, 25). Of note, for complex II, complexes II-III, and succinate dehydrogenase activities, the complexes were exposed to phosphate buffer and additional succinate prior to measuring activity. Progress curves were followed for 10 min to ensure that maximum rates were attained.

Statistics—Data are presented in the form of averages with S.D.s of at least three independent mitochondria preparations. Averages are compared using analysis of variance (26). Significance is taken as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Oxidative Phosphorylation
The measurement of oxidative phosphorylation provides a unique physiologic tool to examine the integrated function of the intact mitochondrion (Fig. 2). This process requires the function of membrane transporters and substrate dehydrogenases, as well as membrane integrity, a functional ETC, and ATP synthetase. The ratio of state 3 respiration (ADP present) to state 4 respiration (ADP-limiting), termed the respiratory control ratio (RCR), reflects the dependence of oxidation of the mitochondrial substrates upon the availability of the phosphate acceptor, ADP. By varying those substrates that are fuels for NADH formation (malate, glutamate, pyruvate), the different transporters within the inner mitochondrial membrane are assessed, as well as different dehydrogenases that generate NADH. Electrons from NADH enter the ETC at complex I and utilize complexes III and IV (1, 27). In a similar manner, succinate oxidation utilizes the mitochondrial dicarboxylate transporter (the same one as malate), but the dehydrogenase is an integral part of complex II. Electrons are then passed through complexes II-IV. Complex V (ATP synthetase) uses the proton gradient formed to drive ATP synthesis. CoQ is the crucial molecule serving as the electron acceptor for either complex I or II, passing electrons from either to complex III. CoQ is considered to be unrestricted in its availability to both complex I and complex II (4).

Malate—Using malate as a substrate, state 3 rates (ADP present in nonlimiting amounts) in mitochondria from two clk-1 alleles, e2519 (missense) and qm30 (deletion), are 22 and 31%, respectively, of those in mitochondria from the wild type (N2) (Table I). State 4 rates are not significantly decreased in clk-1 as compared with N2. In clk-1, "high ADP" rates, determined in the presence of 10 times as much ADP as that used to measure state 3 respiration, are decreased to the same extent as state 3 rates (Table I, Fig. 3A). With malate as the substrate, the RCR of clk-1 is 1.5 as compared with 3.0 for N2 (Table I). The decrease of the RCR results purely from the decrease of state 3 respiration. For complex I-dependent substrates, the functional maximum for ADP/O is about 2.5 (29). The fact that we measured 2.8 ± 0.3 as the actual ADP/O for wild type mitochondria indicates that our preparation protocol leaves the inner membrane intact. The lack of a clear transition between state 3 and state 4 respiration in clk-1 animals makes determination of the ADP/O ratio less accurate in the mutants. Our previous work with N2 and gas-1 (a complex I mutant) also showed high ADP/O and RCR values, indicating that the isolation procedures do not damage the mitochondrial membrane (9).


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  		TABLE I
Oxidative phosphorylation assay Intact C. elegans mitochondria were supplied with electron donor substrates, and respiration (oxygen uptake) was followed with a Clark electrode. Respiration rates were measured under conditions in which the ATP synthetase substrate ADP was either not limiting (state 3) or limiting (state 4). (See also Fig. 3 for "high ADP" state 3 rates as well as DNP-uncoupled conditions.) Rates are given in nanoAtom/min/mg of protein as mean and S.D. N is the number of independent determinations. The RCR is the quotient of state 3 and state 4 respiration rates. ADP/O denotes the number of ADP molecules phosphorylated per oxygen atom reduced assuming that all the ADP added to elicit state 3 has completely been phosphorylated at the point of transition to state 4. Reported values have been corrected as suggested by Hinkle et al. (29). * indicates different from N2, p < 0.05.

 



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  		FIG. 3.
Oxidative phosphorylation assays. Isolated mitochondria were supplied with saturating amounts of ADP (high ADP) and varying electron donor substrates: malate (MAL), glutamate (GLU), pyruvate with malate (PYR), succinate (SUC), or DHQ. Respiration rates were measured as the rate of disappearance of oxygen. Bars and error bars represent means and standard deviations, respectively, in nanoAtom O/min/mg of protein of at least three independent determinations. * indicates a value different from that of N2, p < 0.05. After the determination of the above respiration rate, DNP was added to each experiment to uncouple respiration from phosphorylation. Note: the uncoupler does not release the depression of oxygen uptake in mutant mitochondria. Thus, clk-1 affects mitochondrial function upstream of phosphorylation. * indicates value different from that of N2, p < 0.05.

 
Since respiration and phosphorylation are coupled, low respiratory rates in clk-1 during state 3 or high ADP could result from the inhibition of ATP synthetase or failure of transport of ADP or phosphate into the mitochondrion. However, the addition of the uncoupler 2,4-dinitrophenol (DNP) to mitochondria did not increase oxygen consumption, neither in the mutants nor in the control (Table I, Fig. 3B). Failure of DNP to release the depressed high ADP respiration in clk-1 rules out a defect in the adenine nucleotide translocase or the ATP synthetase as the underlying mechanism.

In order for malate to generate NADH, the mitochondrial membrane dicarboxylate transporter must transport malate into the mitochondrial matrix in which malate dehydrogenase transfers electrons to NAD+ to generate NADH (Fig. 2). The depression seen when using malate as a substrate could occur at either of these steps or downstream in the electron transport chain. To determine whether the malate transporter or dehydrogenase was defective, we substituted glutamate or pyruvate plus malate as substrates. Both of these substrates also generate NADH, but each is dependent on a different transporter and dehydrogenase (Fig. 2).

Glutamate—When glutamate was used as a substrate, state 3 rates for both clk-1 alleles, e2519 and qm30, were depressed (37 and 41% of normal, respectively), whereas state 4 respiration remained roughly the same (Table I). High ADP rates with glutamate were also depressed as seen for malate (Fig. 3A). DNP-uncoupled rates, as well as the coupling parameters RCR and ADP/O, were also similar between the two substrates (Table I, Fig. 3B).

Pyruvate—A third complex I-specific donor, pyruvate, uses a third transporter and dehydrogenase. When pyruvate was used as a substrate, state 3 rates for both clk-1 alleles, e2519 and qm30, were depressed (to 25 and 29% of normal, respectively). The data for pyruvate are similar to those for glutamate and for malate (Table I, Fig. 3). Since oxidative phosphorylation is decreased for all three substrates, specific membrane transporters or dehydrogenases are eliminated as causative for the changes seen in clk-1 mitochondria. Again, uncoupling studies indicate that the defect is not in the adenine nucleotide translocase or in the ATP synthetase. Rather, the inhibition must lie downstream of the production of NADH, i.e. in the electron transport chain.

Succinate—Succinate is the specific substrate for complex II and uses the same dicarboxylate transporter as malate (22, 23). Results for succinate (Table I, Fig. 1) show that rates for state 3, high ADP, and state 4 respiration are not significantly decreased in clk-1 as compared with rates for N2. These results also confirm that the dicarboxylate transporter is not the cause of the decrease seen with malate as a substrate. All other parameters of respiration, including measurements of coupling (RCR and ADP/O), are similar for clk-1 and N2. Thus, clk-1 mitochondria are capable of normal complex II-dependent respiration. These results indicate that the integrated function of complex II, quinones, and complexes III, IV, and V is normal. The coupling results also indicate that the inner mitochondrial membrane of the mutant is intact and that the isolation procedure did not selectively damage the clk-1 mitochondria in any functional manner.

Duroquinol—The reduced form of the CoQ analog duroquinone (DHQ or duroquinol) donates electrons directly into the respiratory chain at complex III, thus bypassing all upstream elements of the respiratory chain, including complex I, complex II, and endogenous quinone species. Using DHQ as an electron donor, none of the measured parameters shows a marked difference between mitochondria from clk-1 and N2 (Fig. 1, Table I). Thus, the inhibition of electron transport seen with the complex I-dependent electron donors must occur upstream of complex III. Data obtained with DHQ are consistent with the results obtained with the complex II-dependent substrate, succinate.

TMPD/Ascorbate—TMPD donates electrons to the respiratory chain via cytochrome c. TMPD/ascorbate was added at the end of each oxidative phosphorylation assay (after uncoupling with DNP). The resulting respiration rates were similar in clk-1 mutants to those observed in N2 (Table I), indicating that, for this point of entry, electron transport capacities are equal for the mutants and wild type. The defect seen with malate, glutamate, and pyruvate is therefore located upstream of cytochrome c, congruent with the results for duroquinol and succinate.

Cytochrome c is easily lost from mitochondria with damaged outer membranes. High respiration rates with TMPD/ascorbate without added external cytochrome c are therefore another indicator that our preparation leaves the mitochondria intact. Furthermore, assuming that the respiration rate with TMPD/ascorbate is limited by the amount of cytochrome c in the assay, this rate can be used as a measure for the actual amount of mitochondria in each assay.

Electron Transport Chain
The above results unequivocally demonstrate a profound functional defect in integrated oxidative phosphorylation in clk-1 when NADH is the electron donor. The clk-1 gene is required for the biosynthesis of CoQ9, which is expected to transport electrons from both complex I and complex II to complex III. However, the identified defect is only associated with complex I, whereas the integrated function of complexes II-V is normal. To dissect the molecular basis of this defect, we measured the activities of individual components of the ETC. It is important to note that the following studies of the ETC complement the studies of oxidative phosphorylation. Oxidative phosphorylation represents the intact, fully integrated function of the mitochondria with endogenous quinone species. The ETC studies localize the defects seen in oxidative phosphorylation by assaying specific mitochondrial components.

Classically, characterization of the electron transport chain is done in several steps. First is the use of primary assays. Citrate synthase and complex IV are used as primary mitochondrial marker enzymes. Secondly, the two arms of the initial part of the electron transport chain, complexes I-III and complexes II and III, are studied.

Citrate Synthase—Citrate synthase is a soluble enzyme of the mitochondrial matrix and serves as a mitochondrial marker for an intact inner mitochondrial membrane. Mitochondria isolated from mutant animals were comparable with those from wild type animals in citrate synthase activity (Table II) and did not suffer damage to the inner mitochondrial membrane with resulting leakage of the matrix.


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  		TABLE II
Mitochondrial marker enzymes Succinate dehydrogenase was measured as succinate-phenazine ethosulfate/DCIP reductase. The units given are 1/min/mg of protein for cytochrome c oxidase activity and nmol/min/mg of protein for the other activities. * indicates different from N2, p < 0.05.

 
Complex IV—Complex IV activity was measured as the first order rate constant. As compared with the activity in N2, it was normal for qm30 and e2519 (Table II). Thus, the downstream portions of the ETC are normal in clk-1.

Complexes I-III—Electron transport from complex I to complex III via endogenous quinones is measured as rotenone-sensitive NADH:cytochrome c reductase activity (Fig. 4). This activity in the clk-1 mutants is only 20% that of N2 (Fig. 5). The percentage of decrease of electron transport for complexes I-III is similar to the decrease seen in the complex I-dependent oxidative phosphorylation assays in clk-1 mitochondria. Thus, by two independent methods, complex I-dependent function, using endogenous quinones, is defective.



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  		FIG. 4.
Mitochondrial respiratory chain. Bold arrows depict the flow of electrons from either NADH or succinate to the terminal acceptor oxygen (ox) through the electron transport chain consisting of the complexes I-IV (indicated by I, II, III, and IV), CoQ, and cytochrome c (cyt.c). The sites in which electrons are delivered/diverted by external electron donors/acceptors respectively are depicted by thin arrows. Blunted lines indicate the sites of action for inhibitors relevant to our electron transport chain assays. UQ, ubiquinone; PES, phenazine ethosulfate; duroQ, duroquinol; DCPIP, dichlorophenol indophenol.

 



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  		FIG. 5.
Electron transport chain assay. Mitochondria from clk-1(qm30), clk-1(e2519), and wild type N2 were solubilized with cholate, and electron transport activities were measured as follows: I-III, rotenone-sensitive NADH-cytochrome c reductase; II-III, antimycin A-sensitive succinate-cytochrome c reductase; I, rotenone-sensitive NADH-decylubiquinone reductase; II, malonate-sensitive succinate-DCIP reductase using either the endogenous quinone species or duroquinone (DQ); III, antimycin A-sensitive decylubiquinol-cytochrome c reductase. Bars represent means and standard deviation in nmol of substrate/min/mg of protein. * indicates value different from that of N2, p < 0.05.

 
Complexes II and III—Electron transport from complex II to complex III via endogenous quinones was assayed as succinate: cytochrome c reductase, either as the malonate-sensitive or as the antimycin A-sensitive moiety of the total electron flow (Fig. 4). (Malonate, rather than the classic complex II inhibitor 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione, was used since we found that malonate is a better inhibitor of C. elegans complex II.) Regardless of the inhibitor used, the activities for both mutants and wild type are equal (Fig. 5). This finding is consistent with the results for succinate-driven oxidative phosphorylation.

The above data indicate that the components of the mitochondria are not damaged and that complex I-dependent activity is defective. Since the clk-1 gene is responsible for the synthesis of CoQ9, we isolated the steps in the ETC proximal to the expected role of the quinones. To localize the defect in clk-1 mitochondria, we measured the individual activities of complexes I, II, and III. These studies do not reflect the interaction of these complexes with endogenous quinones but rather determine whether the individual complexes are similar between N2 and clk-1.

Complex I—The activity of complex I, i.e. the flow of electrons from NADH to quinones, was measured as the rotenone-sensitive NADH-dependent reduction of the CoQ analog, decylubiquinone (Fig. 4). The assay is independent of endogenous quinones as the external CoQ analog is present in excess. Measured complex I activity was equal in clk-1(e2519) and N2 and elevated in clk-1(qm30) (Fig. 5). Therefore, complex I activity is not defective in clk-1. We also evaluated the activity of proximal subunits of complex I and found them to be the same in clk-1 as in N2 (data not shown).

Complex III—Complex III activity was assayed as the antimycin A-sensitive electron transport from the reduced CoQ analog, decylubiquinol, to cytochrome c (Fig. 4). As in the case of the complex I assay, the excess of external CoQ obviates the dependence of electron flow upon endogenous quinones. The measured complex III activities for both clk-1 alleles are the same as those for N2 (Fig. 5).

Complex II—Complex II activity was measured as malonate-sensitive electron transport from succinate to the artificial chromogenic electron acceptor DCIP (also known as succinate: quinone reductase) (Fig. 4). Using the endogenous quinone species, activities are equal for wild type and both clk-1 mutants (Fig. 5). When the ubiquinone analog duroquinone was used as the quinone electron acceptor from complex II (Fig. 4), rates were increased but remained equal between N2 and both clk-1 mutants (Fig. 5). Succinate dehydrogenase (also known as phenazine ethosulfate-mediated succinate:DCIP oxidoreductase) activity measures electron transport through the proximal two subunits of complex II from succinate to phenazine ethosulfate and ultimately to DCIP. The succinate dehydrogenase activity in both clk-1 alleles is more than double that of N2 (Table II).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the presence of a fully integrated mitochondrial electron transport chain, complex I-dependent activity is severely defective in clk-1 mitochondria. All other components of mitochondrial function are normal. The defect in oxidative phosphorylation through complex I is not relieved by uncoupling respiration from ATP synthesis. ETC studies indicate that the activity of complexes I-III is low despite the normal activity of isolated complex I as well as complex III. Since quinones bridge the gap between complexes I and III, these data indicate that the defect in the combined complex I-III activity is likely the result of alterations in the endogenous quinone species in clk-1. In contrast, succinate-driven oxidative phosphorylation is normal in clk-1, as is complex II and III activity. Furthermore, the isolated complex II activities of N2 and clk-1 are equal, regardless of the quinone species used as the electron acceptor, indicating normal complex II function. The ability of complex I and complex II to distinguish between endogenous quinone species, as seen in clk-1, has never been described.

CoQ is the pivotal molecule that shuttles electrons from either complex I or complex II to complex III. clk-1 animals have a known mutation that results in a lack of CoQ9 and the accumulation of DMQ9, RQ9, and bacterial CoQ8 (3, 6-8). Jonassen et al. (7, 8) showed that, when grown on its usual E. coli diet, the predominant quinone species in clk-1 is DMQ9, an intermediate in the synthesis of CoQ9. It is clear that DMQ9 cannot completely substitute for CoQ9, for clk-1 animals raised on a CoQ-less diet die as larvae. However, eliminating CoQ8 from the diet of clk-1 in adulthood lengthens life span. Thus, DMQ9 or RQ9 may adequately support respiration in later development (5, 10, 11, 30). Regardless of the functional quinone species used by clk-1 animals, the metabolic defect unmasks a surprising differential effect of the quinone species that accumulate in the mitochondria of this long-lived mutant. If all quinone species functioned equally well with either electron donor (i.e. reduced complex I or II), then coupling through the quinone species should be the same for electrons coming from either complex I or complex II. However, complex I cannot effectively use these molecules to transport electrons to complex III in clk-1, whereas complex II uses this pool effectively to transport electrons to complex III.

Previous attempts to directly demonstrate a metabolic defect in clk-1 have studied components of the electron transport chain rather than fully integrated mitochondrial function. In agreement with our results, Felkai et al. (30) measured electron transport from succinate to cytochrome c from submitochondrial particles and showed no significant difference in clk-1(qm30) and clk-1(qm51) as compared with N2 controls. Although their assay is similar to our II-III assay, the absolute activity for N2 is ~30% of what we measure. Of note, they did not confirm that the activity measured was specific to complexes II and III by inhibition with 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione, malonate, or antimycin A. Jonassen et al. (10) measured complex II and III activity in mitochondria from clk-1 animals fed different quinones and obtained values similar to ours. However, they did not compare these rates to those of N2.

Miyadera et al. (6) assayed electron transport in clk-1 mitochondria in a manner conceptually similar to our I-III and II-III assays. They treated mitochondria with cholate and then used NADH and succinate as electron donors for complexes I and II, respectively. The authors found no difference between mutants (e2519, qm30) and control (N2) when the endogenous quinones were used to transport electrons to complex III. These data qualitatively agree with ours except for the decrease we see in complex I-dependent activity. The complex I-III activity we measure is consistent with our oxidative phosphorylation results. It is unclear why our results differ from the earlier report. However, it is of note that those reported complex I-III rates were quite low (~30% of our measured activity) as compared with those in our studies for N2. In addition, they did not report the activity measured as sensitive to rotenone. As a result, it is impossible to know whether the rates reported by them reflect complex I activity or nonspecific oxidation of NADH. Since we report only the rotenone-inhibited rates, we are confident that our rates reflect complex I-specific activity. Miyadera et al. (6) also measured rates of electron transport using the exogenous quinones CoQ2 and DMQ2 as the final electron acceptors. They report that NADH-quinone reductase activity is similar whether CoQ2 or DMQ2 is the acceptor, whereas the activity with DMQ2 as acceptor for succinate-quinone reductase is much less than the rate with CoQ2. However, with decylubiquinone as the acceptor, the rotenone-sensitive complex I activities we report (Fig. 5) are 7-10 times those rates reported by Miyadera et al. (6). Thus, the complex I activities are similar between N2 and clk-1 regardless of the electron acceptor used. With either duroquinone as the acceptor (Fig. 5) or CoQ2 as the acceptor (6), there is also no difference between N2 and clk-1 in complex II activity. The activity is markedly decreased for N2 when DMQ2 is the acceptor, which is consistent with the well known specificity of electron transport on the particular quinone species available (Refs. 31 and 32, and see above for complex I). Additionally, when DMQ2 is used as the acceptor, they found no difference between N2 and clk-1.

How do the quinone species present in clk-1 transport electrons between complexes II and III but not between complexes I and III? It is possible that complex I is unable to use the quinone species available in clk-1 mitochondria to effectively transport electrons to complex III. Studies in mammalian mitochondria have reported that complex I is sensitive to the length of the isoprenyl side chain of CoQ but only when the number of isoprenyl units is six or less (31, 32). Alternatively, the quinones present in clk-1 may not be equally available to both complexes I and II. The possibility that different pools of quinones function preferentially for one or the other electron donor has not been considered previously. It may be that supercomplexes of the ETC (33, 34) sequester their own quinones; in this case, the functional quinone(s) are not effectively packaged with the I-III supercomplex. As a third possibility, a component of the clk-1 quinone pool may act as a specific antagonist of electron transfer from complex I to complex III. For example, an excess of DMQ9 may inhibit the interaction of CoQ8 with complex I.

The results presented here necessitate a reassessment of the cause of the life span extension seen in clk-1 mutants and emphasize the importance of integrated mitochondrial function in determining longevity (28, 35). The simplest explanation of the aging phenotype of clk-1 is that its profound decrease in complex I-dependent respiration slows metabolism, and thus, slows development and extends life span. However, the postembryonic development times reported by Hekimi and co-workers (30) (and confirmed in our laboratory under our growth conditions) for N2 (44 h), qm30 (75 h), and e2519 (60 h) do not follow the same order as the NADH-dependent oxidative phosphorylation rates. We do see a difference between N2 and the clk-1 mutants, but no difference in respiration is noted between the clk-1 alleles that correlates to their difference in developmental times.

The C. elegans mutant gas-1, which contains a mutant complex I subunit, has a similar decrease in complex I-dependent rates but has a much shortened life span. Elsewhere, we have shown that oxidative damage to mitochondrial proteins is increased in gas-1 but decreased in clk-1 as compared with N2 (18). The mechanism by which oxidative phosphorylation is slowed is therefore crucial to the life span of the animal. In this regard, the role of complex I and its interaction with different endogenous quinone species in reactive oxygen species production is very important. The power of mutational analysis in C. elegans, combined with the ability to rigorously assess integrated mitochondrial function in the nematode, holds great promise for better understanding the manner in which mitochondrial function determines life span. For example, recent data show that the double mutant, clk-1;gas-1, lives twice as long as N2 (18). The mechanism of this phenomenon is under active investigation.

Oxidative phosphorylation, a measure of integrated mitochondrial function, is affected by a profound metabolic defect in clk-1. This defect is specific to complex I-dependent substrates. ETC studies localize this defect to the transfer of electrons from complex I to complex III. The site of the dysfunction is consistent with the predicted genetic defect of clk-1. However, the startling finding is that only one avenue of electron transfer to quinones is selectively disrupted in this mutant. The relationship of the phenotype of clk-1 to the selective disruption of complex I-mediated respiration requires further study.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported in part by National Institutes of Health Grant GM58881. Back

|| To whom correspondence should be addressed: Louis Stokes Veterans Affairs Medical Center, Medical Research Service (151W), 10701 East Blvd., Cleveland, OH 44106. Tel.: 216-791-3800 (ext. 5657); Fax: 216-707-5973; E-mail: charles.hoppel{at}case.edu.

1 The abbreviations used are: DMQ, demethoxyubiquinone; DHQ, duroquinol; RQ, rhodoquinone; ETC, electron transport chain; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; DCIP, dichlorophenol-indophenol; RCR, respiratory control ratio. Back


   ACKNOWLEDGMENTS
 
We are deeply appreciative for excellent technical assistance of Judy Preston, Julie Rosenjack, Hiral Patel, and Hassan Golchini.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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