The Respiratory Chain in Yeast Behaves as a Single Functional Unit*

Hans BoumansDagger , Leslie A. Grivell§, and Jan A. BerdenDagger

From the Dagger  E. C. Slater Institute and § Section for Molecular Biology, Department of Molecular Cell Biology, BioCentrum, University of Amsterdam, 1018 TV Amsterdam, The Netherlands

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Inhibitor titrations using antimycin have been used to study the pool behavior of ubiquinone and cytochrome c in the respiratory chain of the yeast Saccharomyces cerevisiae. If present in a homogeneous pool, these carriers should be able to diffuse freely through or along the membrane respectively and accept and subsequently donate electrons to an infinite number of the respective respiratory complex. However, we show that under physiological conditions neither ubiquinone nor cytochrome c exhibits pool behavior, implying that the respiratory chain in yeast is one functional unit. Pool behavior can be introduced for both small carriers by adding chaotropic agents to the reaction medium. We conclude that these agents disrupt the interaction between the respiratory complexes, thereby causing them to become randomly arranged in the membrane. In such a situation, ubiquinone and cytochrome c become mobile carriers, shuttling between the large respiratory complexes. Furthermore, we conclude from the respiratory activities found for different substrates that the respiratory units in yeast vary in composition with respect to the ubiquinone reducing enzyme. All units contain the cytochrome chain, supplemented with either succinate dehydrogenase or the internal or the external NADH dehydrogenase. This implies that when only one substrate is available, only a certain fraction of the cytochrome chain is used in respiration. The molecular organization of the respiratory chain in yeast is compared with that of higher eukaryotes and to the electron transfer systems of photosynthetic membranes. Differences between the organization of the respiratory chain of yeast and that of higher eukaryotes are discussed in terms of the ability of yeast to radically alter its metabolism in response to change of the available carbon source.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The mitochondrial respiratory chain consists of large multisubunit enzymes that are embedded in the inner membrane. These complexes are electronically connected by smaller components, ubiquinone and cytochrome c. Numerous studies have been performed to elucidate the molecular organization of the respiratory chain, and two extreme models have been considered (1). In the first, components of the respiratory chain are free to diffuse laterally and independently of one another in the membrane plane and electron transfer is coupled to diffusion-based collisions between these components (2-4). In the other model, respiratory components are arranged as an ordered macromolecular assembly or chain. Although the vast majority of studies performed lend weight to favor the first model, numerous observations have been reported indicating that multicomplex units can be isolated (see Ref. 1 and references therein).

The two models for the organization of the respiratory chain are kinetically distinguishable, the first implying that the components exhibit "pool behavior," whereas the latter implies the opposite. A homogeneous pool for ubiquinone was originally postulated by Kröger and Klingenberg (5) to account for the sigmoidal inhibition of mitochondrial respiration by antimycin A. The other small component of the respiratory chain, cytochrome c, was also shown to function in a common pool (3). These findings imply that the two small components of the respiratory chain are mobile carriers, mediating electron transfer between freely diffusible large respiratory complexes. Physical evidence for this organization was provided by the group of Hackenbrock, who showed that lateral diffusion of ubiquinone is the rate-limiting step in succinate-linked electron transport (6).

In the case of chloroplasts and intact cells of photosynthetic bacteria, a number of observations are incompatible with a model of freely diffusible electron transfer carriers (reviewed in Ref. 7). In chloroplasts, reduction of plastoquinone (PQ)1 displays only partial pool behavior, indicative of limitation of PQ diffusion in the membrane (8) and suggesting that small size domains exist which limit the rapid time-scale diffusion of PQ. In photosynthetic bacteria, ubiquinone is freely diffusible, but diffusion of the other mobile carrier of this system, cytochrome c2, is restricted to the domain of one supercomplex, which also includes two reaction centers and one cytochrome bc1 complex (9).

It should be noted that most of the studies on the mitochondrial respiratory chain have been performed with mammalian mitochondria. In this study, we show that, in mitochondria from the yeast Saccharomyces cerevisiae under conditions of approximately physiological ionic strength, neither ubiquinone nor cytochrome c functions in a pool. These results indicate that, at least in yeast, the mitochondrial respiratory chain complexes form one functional respiratory unit.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Definition and Kinetics of a Pool Function of Ubiquinone and Cytochrome c-- When the respiratory components are arranged as an ordered macromolecular assembly, also referred to as the "solid state," the number of acceptors a carrier can reach (N) is 1. In the other model, where the carriers can freely diffuse through the membrane, the number of acceptors is determined by the lifetime of the reduced state of the carrier of interest and its diffusion constant, but will generally be large ("liquid state"). Other possible states have been described (1), which are intermediate between solid and liquid states. As a consequence, the number of potential acceptors (N) is larger than 1, yet smaller than in the liquid state.

In the situation that all ubiquinone or cytochrome c is present in a pool, respiratory activity (Vo) can be described as a function of the maximum rate of reduction (V1) of that component and the maximum rate of oxidation (V2) of that component (5).
V<SUB><UP>o</UP></SUB>=<FR><NU>V<SUB>1</SUB>×V<SUB>2</SUB></NU><DE>(V<SUB>1</SUB>+V<SUB>2</SUB>)</DE></FR> (Eq. 1)
We have studied pool behavior for both ubiquinone and cytochrome c mainly by titration of the respiratory activity with antimycin, a high affinity inhibitor of bc1 activity. When studying the Q pool, antimycin titrates the oxidation rate (V2). Data were fitted according to Equation 2, where x is the fraction of bc1 activity inhibited by antimycin.
V<SUB>x</SUB>=<FR><NU>V<SUB>1</SUB>×V<SUB>2</SUB>×(1−x)</NU><DE>(V<SUB>1</SUB>+V<SUB>2</SUB>×(1−x))</DE></FR> (Eq. 2)
In the case of the cytochrome c pool, antimycin titrates the reduction rate (V1). Data were fitted according to Equation 3.
V<SUB>x</SUB>=<FR><NU>V<SUB>1</SUB>×(1−x)×V<SUB>2</SUB></NU><DE>(V<SUB>1</SUB>×(1−x)+V<SUB>2</SUB>)</DE></FR> (Eq. 3)
The reaction that is being titrated, V2 in the case of succinate oxidation and V1 in the case of NADH oxidation, is also referred to as Vtitr.

Yeast Strain and Preparation of Mitochondria-- Saccharomyces cerevisiae strain DL1 (alpha , his3, ura3, leu2) was grown on lactate medium (0.5% yeast extract, 0.2% (w/v) magnesium sulfate, 0.6% (w/v) ammonium phosphate, 2% sodium lactate (70% w/v), 1.3% lactic acid (75% w/v), pH 4.5) until late logarithmic phase. Mitochondria were isolated as described previously (10). Protein concentrations were determined with the Lowry method (11). Mitochondria were diluted to 10 mg/ml in the mitochondrial respiratory buffer (see below) prior to the measurements.

Mitochondrial Oxygen Consumption Measurements-- Oxygen consumption rates were measured at 30 °C with a Clark oxygen electrode (Gilson) in a 1.8-ml thermostatically controlled chamber. Rates were determined from the slope of a plot of O2 concentration versus time. Mitochondria (0.5 mg of mitochondrial protein) were incubated in the mitochondrial respiratory buffer containing 0.6 M sorbitol, 25 mM potassium Pi, pH 7.0, 1 mM EDTA, 1 mM MgCl2 (also referred to as the standard buffer in this paper). Mitochondria were preincubated with specific inhibitors (antimycin A, KCN) for 2 min. Antimycin A was dissolved in methanol. Substrates (0.5 mM NADH, 5 mM succinate, 0.5% ethanol, 5 mM ascorbate + 0.1 mM N,N,N',N'-tetramethyl-p-phenylenediamine) were directly added to the reaction chamber. Titrations with different effectors were carried out by taking new mitochondria for each measurement. All measurements were carried out at least twice.

bc1 Electron Transfer Activity Measurements-- The Q2H2:cytochrome c oxidoreductase (bc1) assay was performed spectrophotometrically at 30 °C by measuring the reduction of 18 µM horse-heart ferricytochrome c at 550-540 nm by 25 µM Q2H2. The buffer used contained 2 mM EDTA, 0.5 mM potassium cyanide, and 20 mM potassium phosphate, pH 7.4, to obtain maximal activity with horse-heart cytochrome c as acceptor (12).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Antimycin Linearly Inhibits Electron Transfer Activity of the bc1 Complex-- Antimycin A is a specific inhibitor of the quinone reduction site and binds to the bc1 complex in a 1:1 ratio. Titration of the Q2H2:cytochrome c oxidoreductase activity of S. cerevisiae mitochondria with antimycin results in a linear relationship between inhibitor and electron transfer activity (Fig. 1). In other words, the relative saturation with inhibitor directly provides the relative inhibition of the bc1 activity.


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Fig. 1.   Titration of the rate of Q2H2:cytochrome c oxidoreductase activity with antimycin in isolated mitochondria.

Respiratory Activity through Different Pathways-- The respiratory chain of S. cerevisiae, unlike that of mammals, does not contain complex I. Instead, at least three NADH dehydrogenases are present, two of which transfer electrons to ubiquinone (13). When NADH is used as a substrate for respiration of isolated mitochondria, reduction of ubiquinone occurs via the external NADH dehydrogenase. This enzyme is bound to the mitochondrial inner membrane and faces the intermembrane space (IMS). In contrast, when ethanol is used as substrate, ubiquinone is reduced by the internal NADH dehydrogenase, which faces the matrix. Control experiments showed that no (significant) electron transfer occurs via the external NADH dehydrogenase during ethanol oxidation (data not shown), because the mitochondrial isolation procedure used depletes the IMS of NAD+. A third pathway, in which ubiquinone is reduced by succinate:ubiquinone oxidoreductase, is used with succinate as the substrate. As can be seen from Table I, this third pathway is the slowest, whereas use of NADH as a substrate results in the highest oxygen consumption rate. These differences must be due to different rates of ubiquinone reduction, while the total capacity of ubiquinol oxidation by the cytochrome chain remains constant.

                              
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Table I
Respiratory activities using various substrates

When studying a putative pool function for a carrier, the fastest reaction rate, either reduction (V1) or oxidation of that carrier (V2), should be titrated to get a clear distinction between pool or no pool. We have chosen to use antimycin, thus titrating the bc1 activity, in the study of a pool function for both ubiquinone and cytochrome c. In the case of ubiquinone, the bc1 complex is part of the oxidation reaction. Succinate was used as the substrate to provide a relatively slow Q reduction rate. When considering a possible cytochrome c pool, NADH was used as the substrate to provide a fast reduction rate, because the bc1 complex in this case is part of the reduction reaction. The validity of this set-up is confirmed by the respiratory activities after addition of Q or cytochrome c to the reaction medium; addition of Q increases the succinate oxidation activity, and addition of cytochrome c does not (Table I). In contrast, addition of Q does not increase the NADH oxidation rate, and addition of cytochrome c does.

We have also used the cytochrome c oxidase inhibitor KCN to analyze a putative pool function of cytochrome c, thus titrating the oxidation reaction, V2. However, the relatively low affinity of this inhibitor makes it unsuitable for distinguishing between pool or no pool. These measurements did show however that the maximum activity of cytochrome c reduction is approximately the same as the maximum cytochrome c oxidation activity, in agreement with previous findings (14).

Pool Function of Ubiquinone-- Titration of succinate oxidation using antimycin and using the standard buffer conditions (50 mM ionic strength) generates a linear relationship between activity and the relative amount of inhibitor (Fig. 2), thus indicating that under these conditions ubiquinone does not exhibit pool behavior. However, because the physiological ionic strength in yeast lies around 200 mM (15), we repeated the titration with 125 mM potassium Pi in the buffer, thereby providing an ionic strength of 250 mM for the reaction medium. As can be seen in Fig. 2, this results in a hyperbolic relationship between succinate oxidation and the relative amount of inhibitor, indicative for a pool function of ubiquinone under these reaction conditions. These data points can be fitted with the equation that describes pool-function kinetics, Vo = V1 × V2/(V1 + V2), as is described under "Experimental Procedures." The value for V2 found in the fitting procedure is given in Table II and is referred to as Vtitr, the maximum rate of the reaction that is titrated with antimycin relative to the overall rate (Vo). When the same procedure is applied to the data found with a buffer containing 25 mM potassium Pi, Vtitr is found to be close to 1, whereas V1 is calculated to be very large, indicating that the pool function kinetics in this case do not apply. In other words, the value found for Vtitr, and in particular its deviation from 1, reflects the degree of pool function of the carrier of interest, in this case ubiquinone.


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Fig. 2.   Titration of succinate oxidation with antimycin. Succinate respiration of isolated mitochondria was measured in the standard buffer (open circle ) and in the standard buffer supplemented with 100 mM potassium Pi (square ). In all experiments, a residual respiratory activity was found (5-8% of maximum activity) at saturating antimycin concentration.

                              
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Table II
Reaction rates of succinate and NADH oxidation titrated using antimycin under different ionic strength conditions

However, increase of the phosphate concentration in the reaction buffer may not only increase the ionic strength, but may in addition have an effect on the integrity of the respiratory complexes, because phosphate is a weak chaotropic agent. We therefore investigated which of the two properties of the high phosphate concentration is responsible for creating a pool function for ubiquinone by using different buffers in the reaction medium of the respiratory activity measurements. The results are summarized in Table II in terms of the Vtitr values found after fitting the titration data. Increasing the ionic strength using KCl instead of phosphate does not result in pool behavior, indicating that the pool function of ubiquinone is not dependent on the ionic strength. Furthermore, using the strong chaotropic agent trichloroacetic acid, adjusted to neutral pH, pool function can be introduced even at an intermediate ionic strength.

We conclude from these results that, under physiological conditions, there is no ubiquinone pool, due to the fact that succinate dehydrogenase and the bc1 complex together form a functional unit by some form of physical interaction. Chaotropic agents such as trichloroacetic acid and, to a lesser extent, phosphate can disrupt this interaction, thereby introducing pool behavior of ubiquinone.

A hyperbolic titration curve can also be obtained at low ionic strength, namely by adding Q2 to the reaction medium. The difference between this Q analogue and ubiquinone present in vivo (Q6) is the length of the hydrophobic isoprenoid site chain. It has been shown before that short-chain ubiquinones exhibit greater lateral mobilities within the membrane (16). We conclude that, even under conditions where succinate dehydrogenase and the bc1 complex physically interact, Q2 exhibits pool behavior because, due to its greater lateral mobility, it can transfer electrons between different succinate dehydrogenase-bc1 complex units.

Pool Function of Cytochrome c-- A putative pool function of cytochrome c was studied by titrating NADH oxidation using antimycin. Using the standard buffer conditions a linear relationship between respiratory activity and amount of inhibitor is obtained (Fig. 3), reflected by the value for Vtitr found after fitting these data (Table II). Increasing the phosphate concentration gives similar results as for ubiquinone; titration with antimycin results in a hyperbolic inhibition curve and a value for Vtitr (in this case corresponding to V1) clearly higher than 1 is found. When the standard buffer containing 25 mM phosphate is supplemented with 200 mM KCl, thereby raising the ionic strength to 250 mM, cytochrome c exhibits pool behavior as well. The phosphate concentration was subsequently lowered to 5 mM, which is close to the physiological concentration of 3.6 mM reported for yeast cells in stationary phase (17). As can be seen in Fig. 3 and Table II, titration of NADH oxidation with antimycin produces an inhibition curve intermediate between a straight line and the curve obtained in case of pool behavior of cytochrome c. The results obtained with a buffer containing trichloroacetic acid indicate that, as for ubiquinone, pool behavior of cytochrome c can be introduced at an ionic strength lower than the physiological value. Replacing trichloroacetic acid by the same concentration of KCl, thus giving the same ionic strength, confirms that the chaotropic nature of trichloroacetic acid is responsible for the observed pool behavior of cytochrome c (Table II). Addition of cytochrome c to the reaction medium did not induce pool behavior (Table II), presumably because at low ionic strength all cytochrome c is bound to the inner membrane, independent of concentration.


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Fig. 3.   Titration of NADH oxidation with antimycin. NADH respiration of isolated mitochondria was measured in the standard buffer (open circle ), in the standard buffer supplemented with 100 mM potassium Pi (square ), and in buffer containing 5 mM potassium Pi + 200 mM KCl (triangle ).

The conclusions drawn from these data are depicted in a model in Fig. 4. The standard conditions are reflected in Fig. 4A. At this low ionic strength cytochrome c diffuses in two dimensions, parallel to the inner membrane (2). Because the bc1 complex and cytochrome c oxidase physically interact, a cytochrome c molecule that is reduced by a given bc1 complex can only donate its electron to the adjacent cytochrome c oxidase (N = 1). At higher ionic strength, cytochrome c diffuses in three dimensions, part of it being free in the intermembrane space (2) (Fig. 4B). Although the bc1 complex and cytochrome c oxidase still physically interact, an intermediate pool function of cytochrome c is present because the IMS-soluble cytochrome c is able to reach other respiratory units. This, however, results only in a "partial pool function," because the diffusion in the IMS is relatively slow and, as a consequence, the number of possible acceptors (N) limited. Chaotropic agents present in the reaction buffer cause the dissociation of the respiratory units into separate complexes (Fig. 4C). A cytochrome c molecule can now theoretically donate its electron to any cytochrome c oxidase molecule (N is large), thus showing maximal pool behavior.


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Fig. 4.   Model for the pool function of cytochrome c under different reaction conditions. A, low ionic strength and in the absence of chaotropic agents; B, high ionic strength and in the absence of chaotropic agents (conditions most closely mimicking the physiological situation); C, in the presence of chaotropic agents, irrespective of the ionic strength (see "Results" for details).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The mitochondrial inner membrane is generally viewed as a fluid structure in which the two-dimensional movement of the respiratory chain components is a central feature of the process of electron transfer. This view has been confirmed by a large number of studies on the mammalian respiratory chain, where the components behave as independent lateral diffusants. It has been well established that there is a Q pool in mammals as well as a cytochrome c pool, irrespective of the ionic strength (3). Although there are reports that do not fit with the Q-pool model, Ragan and Cottingham (18) have shown that, using minor adjustments in the calculation methods, even these data, which at first sight are in conflict with the Q-pool model, can be fitted accordingly. The organization of the respiratory chain in mammals is described by the "random-collision model," which was proposed by the group of Hackenbrock, based on a large number of studies on diffusion coefficients of the respiratory components (2-4, 6, 19). These studies on mitochondrial membranes show the free mobility of the respiratory complexes in vivo. Although structures as complex I/III and complex II/III units could be isolated from mammalian cells (see Ref. 1, and references therein), indicating physical interaction between these complexes under certain in vitro conditions, this is not a reliable criterion for association in vivo.

We show in this study that the two small carriers of the respiratory chain of S. cerevisiae, ubiquinone and cytochrome c, do not exhibit pool behavior under physiological conditions. This behavior differs clearly from that of the corresponding components of the mammalian respiratory chain, and it implies that these carriers in yeast can only transfer electrons to one specific acceptor molecule. We conclude from this that the respiratory chain complexes in this organism physically interact, thereby forming a functional unit. Because different respiratory pathways exist, differing in the ubiquinone reduction enzyme, different units must exist. A unit consists of the cytochrome chain, supplemented with either succinate dehydrogenase or one of the NADH dehydrogenases.

The existence of such units, as based on the kinetic data presented here, does not exclude the possibility that association/dissociation events take place at rates significantly lower than the rate of electron transfer. If occurring, the relative low rates of these events will make them irrelevant with respect to the kinetic behavior of the system and will therefore not introduce pool behavior. This is in contrast to the situation proposed for the respiratory chain in bovine heart by Heron et al. (20). In their model, electron transfer from NADH to cytochrome b requires interaction between complexes I and III (21), but to explain the Q-pool behavior in this organism, it is assumed that these supercomplexes are formed and re-formed at rates higher than that of electron transfer.

The interpretation of an organization of the yeast respiratory chain in units is additionally supported by the respiratory activities found using different substrates (see Table I). The activity of succinate oxidation plus that of ethanol oxidation equals the respiratory activity when both substrates are used together, inconsistent with the formula for pool function kinetics. This implies that both ubiquinone reduction pathways operate independently, only a fraction of the ubiquinone available being involved and only a fraction of the quinol oxidase system being used in each pathway. When a combination of substrates including NADH is used, the outcome is not so clear. When succinate or ethanol are added together with NADH, the respiratory activity is higher than that of oxidation of NADH alone, although only partial additivity is obtained. We propose that this is because of the fact that the interaction of the external NADH dehydrogenase with the unit is weaker, thereby resulting in a higher rate of associations/dissociations. Based on the fact that partial additivity is observed, despite the fact that the reaction capacity of external NADH:Q6 oxidoreductase is about 11 times higher than that of the total flux of NADH to oxygen (22), it has to be concluded that this rate is within the same order as the rate of electron transfer. Thus, using NADH as a substrate partial pool behavior is introduced (which, however, is not observed using antimycin) because of the fact that the Q reduction rate is so much higher than the Q oxidation rate.

In the case of succinate and ethanol oxidation, pool behavior can be introduced for both ubiquinone and cytochrome c by addition of chaotropic agents, which are known to weaken hydrophobic interactions between proteins inside the membrane. In relation to the view that associations and dissociations take place at a low rate under normal conditions, it can be imagined that addition of chaotropic agents increases this rate to a rate higher than that of electron transfer.

Alternatively, the interaction between the respiratory complexes can be disrupted by solubilization of the mitochondrial membranes. When the mild detergent lauryl maltoside was added to the reaction medium, respiratory activity dropped to less than 10% and was only restored by addition of both Q and cytochrome c to the reaction chamber (data not shown). This behavior clearly differs from that of bovine heart, where solubilization of submitochondrial particles did not decrease the succinate:cytochrome c oxidoreductase activity (23). These results provide strong support for the idea that the respiratory complexes physically interact and that after solubilization both ubiquinone and cytochrome c become carriers that shuttle between the different respiratory complexes. It has been shown for mammalian mitochondria that an increase of the lipid volume results in a corresponding decrease in the electron transfer reaction rates (16, 19), that could be restored by addition of ubiquinone. We propose that, in solubilized yeast mitochondria, the respiratory complexes diffuse freely and a similar organization exists as in "diluted" mammalian mitochondrial membranes.

In chloroplasts, rapid diffusion of PQ is restricted to isolated domains (8, 24), which may vary in size. The structural interpretation given to account for these domains is the crowding of the membrane by integral proteins. The surface fraction of the membrane occupied by proteins in chloroplasts is close to the two-dimensional percolation threshold, resulting in a network of barriers to diffusion (8, 24). The absence of pool behavior for ubiquinone in the respiratory chain in yeast, however, cannot be explained by such a model. First, an organization in isolated domains will result in partial pool behavior (the fast pool fraction in chloroplasts represents 50-70% of the total; Ref. 8). In the case of the yeast respiratory chain, no Q pool is observed. Second, if such an organization were to exist in yeast, addition of chaotropic agents would not result in introduction of pool behavior. Furthermore, crowding cannot explain the differences in pool behavior of the small carriers in yeast compared with those in higher eukaryotes, inasmuch as the fraction of the mitochondrial inner membrane in mammals occupied by protein is at least as high as in yeast (and in heart tissue even higher).

In the photosynthetic bacterium Rhodobacter sphaeroides, reaction centers and cytochrome c2 and bc1 complexes form supercomplexes, thereby limiting diffusion of cytochrome c2. About 70% of these supercomplexes include two reaction centers, one cytochrome c2, and one bc1 complex, whereas the remaining ones only include two reaction centers and one cytochrome c2 (9). In contrast to the organization in chloroplasts, these structures have a precise stoichiometry and correspond to reproducible oligomeric entities. Such an organization thus corresponds to that of the respiratory chain in yeast. It is proposed that the cytochrome c2 molecules trapped in a supercomplex lacking a bc1 complex, can slowly escape and react with bc1 complexes located in another area of the membrane (9), similar to the model given in Fig. 4B to account for the partial pool behavior of cytochrome c at high ionic strength. It is interesting to note that the binding affinity of cytochrome c2 to reaction centers is dependent upon the ionic strength of the medium (25).

Our findings with the yeast S. cerevisiae, showing that both ubiquinone and cytochrome c are not in a pool under physiological conditions and the subsequent interpretation of these data resulting in a unit-model, permit a rational explanation of intercomplex effects observed by others. Bruel et al. (26) found that certain mutants of subunit VIII of the bc1 complex result in a decrease of the succinate:ubiquinone oxidoreductase activity by 40-60%, while leaving the bc1 complex activity unaltered (26). Interaction between the bc1 complexes and cytochrome c oxidase in yeast is suggested by the studies carried out by Pearce and Sherman (27), who showed that certain labile forms of cytochrome c are protected from degradation by the interaction with its physiological partners. Because both cytochromes a·a3 and c1 are required for protection, it can be concluded that cytochrome c is in close proximity to both of these cytochromes (27). Recently, it was found that disruption of the ABC1 gene in yeast results in a decrease of activity of complexes II, III, and IV (28). It was concluded that the ABC1 gene product acts as a chaperone-like protein essential for correct folding of the bc1 complex, and that the effects on complexes II and IV might result from interactions with the modified bc1 complex. Beattie and co-workers (29) have shown that the reduction of exogenous quinones by the internal NADH dehydrogenase could be almost completely inhibited by myxothiazol, indicating that the exchange of endogenous ubiquinone with the added quinone does not occur at the level of the Q reduction site of NADH dehydrogenase, but at the level of the bc1 complex, implying that these two enzymes physically interact during electron transfer. Reduction of quinones by the external NADH dehydrogenase could only be inhibited by 18% using myxothiazol, which made the authors conclude that the external NADH dehydrogenase does not interact with the bc1 complex (29). However, we propose that these data do not argue against the unit-model. As discussed above, we propose that the interaction of the external NADH dehydrogenase in the respiratory unit is rather weak, thus resulting in partial pool behavior. This interpretation is in agreement with the partial sensitivity to myxothiazol of ubiquinone reduction by the external NADH dehydrogenase (29).

Furthermore, the unit-model clarifies our recent findings with certain yeast mutants, which exhibit a reduced assembly efficiency of the bc1 complex, resulting in a lower steady-state level of this complex. Parallel to this, a lower steady-state level of cytochrome c oxidase is found in these mutants, implying a regulatory mechanism that prevents an overcapacity of this complex with respect to the bc1 complex activity.2 The ability to "sense" the steady-state level of the bc1 complex in our view supports the idea that physical interaction exists between the two complexes.

Why should yeast organize its respiratory chain differently from higher eukaryotes? The single-unit organization we describe is reminiscent of that found in many bacteria. The organization of the respiratory chain in many prokaryotes is very complex, in that it contains alternative terminal oxidases in addition to the pathway similar to that in eukaryotes. The possession of respiratory branches enables the bacterium to adapt its efficiency of oxidative phosphorylation to changes in the environmental conditions (30). One of the respiratory branches exists of a ubiquinol oxidase, which can be considered to be a supercomplex constituted of both a bc1 complex and a cytochrome c oxidase and which can be found in, e.g., Paracoccus denitrificans (31), Sulfolobus acidocaldarius (32), and the thermophilic bacterium PS3 (33), and can be considered to be a primitive characteristic. The physical interaction between the bc1 complex and cytochrome c oxidase in ubiquinol oxidase implies that a pool function for cytochrome c is absent.

In respect of the supracomplex organization in S. cerevisiae, it can be imagined that this offers advantages in terms of response to rapidly fluctuating availability of different substrates. S. cerevisiae is known for its capacity to radically alter metabolism by availability of a fermentable carbon source. When glucose is available, synthesis of respiratory chain components is strongly repressed. If the respiratory chain in yeast would be organized in separate complexes, a decrease in concentration of these complexes would result in a dramatic decrease in respiratory capacity. It has been shown for mammalian mitochondria that when the phospholipid to protein ratio increases, the respiratory activity proportionally decreases, due to an increase in average distance between respiratory complexes (19). When the concentration of respiratory complexes decreases by, e.g., a factor of 5, the respiratory capacity of a chain organized in freely diffusing respiratory components will decrease by approximately a factor 25, inasmuch as the activity per protein also decreases. However, in the case of a unit-organized chain, the respiratory capacity will only decrease by a factor of 5. In other words, the organization in units might serve to "buffer" respiratory capacity at low concentrations of respiratory complexes under glucose-repressive conditions.

For multicellular organisms, there is much less need for a compact organization of the respiratory chain in a unit (or even a supercomplex), because of cellular homeostasis. An organization of separate, independently diffusing complexes, however, provides the advantage that all enzyme complexes of the cytochrome chain can participate in electron transfer, irrespective of the substrate available.

How might interaction between the complexes in yeast be achieved? Two possibilities can be envisaged; either differences exist in function of subunits of the individual complexes, or the accessory factors required for assembly of the respiratory chain differ between yeast and mammals. The latter is made less likely in view of the strong evolutionary conservation of many of these proteins (from prokaryotes to higher eukaryotes in the case of, e.g., Abc1p (34), Sco1p (35), and Oxa1p (36)) and examples that show functionality of human homologues in yeast (36, 37). We therefore consider it more likely that some of the supernumerary subunits (subunits that are not directly involved in electron transfer), especially those which show a high level of sequence variation and have been shown to be involved in intracomplex assembly, are involved. As an example, subunit VIII of the yeast bc1 complex can be mentioned (only 18% sequence identity with the bovine heart homologue), which was recently shown to be involved in intercomplex interaction with succinate:ubiquinone oxidoreductase (26).

    ACKNOWLEDGEMENT

We thank A. F. Hartog for the synthesis of ubiquinone-2.

    FOOTNOTES

* This work was supported in part by grants from the Netherlands Organization for the Advancement of Pure Research under auspices of the Netherlands Foundation for Chemical Research.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.

To whom correspondence should be addressed: E. C. Slater Institute, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands. Tel.: 20-5255114; Fax: 20-5255124; E-mail: jan.berden{at}chem.uva.nl.

1 The abbreviations used are: PQ, plastoquinone; Q, ubiquinone; Q2H2, 2,3-dimethoxy-5-methyl-6-geranyl-1,4-benzoquinol; IMS, intermembrane space.

2 H. Boumans, J. A. Berden, L. A. Grivell, and K. van Dam, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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