Mitochondrial respiration in yeast whole cells is a valuable indicator of cell bioenergetics. Here, we present a protocol to quantify this phenotype applicable to different yeast species.

Metabolism is mainly coordinated by cellular energy availability and environmental conditions. Assays for knowing how cells adapt energetic metabolism to different nutritional and environmental conditions give valuable information to elucidate molecular mechanisms. Oxidative phosphorylation is the primary source of ATP in most cells, and mitochondrial respiration activity is a key component of oxidative phosphorylation, maintaining mitochondrial membrane potential for ATP synthesis. Mitochondrial respiration is often studied in isolated mitochondria that are missing the cellular context. Here, we present a method for quantifying mitochondrial respiration in yeast-intact cells. This method applies to any yeast species, although it has been generally used for Saccharomyces cerevisiae cells. First, the yeast growth in specific conditions is tested. Then, cells are washed and resuspended in deionized water with a 1:1 ratio (w/v). Cells are then placed in an oximeter chamber with constant stirring, and a Clark electrode is used to quantify oxygen consumption. Some molecules are sequentially placed into the chamber and selected according to this effect on the electron transport chain or ATP synthesis. ATPase inhibitor oligomycin is first added to measure respiration coupled to ATP synthesis. Afterward, an uncoupler is used to measure the maximal respiratory capacity. Finally, a mix of electron transport chain inhibitors is added to discard oxygen consumption unrelated to mitochondrial respiration. Data are analyzed using a linear regression to obtain the slope, representing the oxygen consumption rate. The advantage of this method is that it is specific for yeast mitochondrial respiration, maintaining the cellular context. It is essential to highlight that inhibitors used in mitochondrial respiration quantification could vary between yeast species.

Mitochondria plays a fundamental role in cellular bioenergetics since it is the main source of ATP for most cells, and several pathways converge and depend on the activity of mitochondrial pathways¹. Oxidative phosphorylation is needed for ATP synthesis that combines electron transport through the electron transport chain to reduce oxygen and F₁F₀- ATPase activity, synthesizing ATP using the mitochondrial membrane potential produced due to electron flux². Thus, mitochondrial respiration is part of the oxidative phosphorylation³.

The mitochondrial respiration chain comprises four complexes or, in some cases, yeast complex I-deficient (e.g., Saccharomyces cerevisiae), three complexes, and three dehydrogenase proteins⁴. Respiratory complexes are localized in the inner mitochondrial membrane; some yeast presents the canonical electron transport chain with complex I (NADH dehydrogenase) or three NADH dehydrogenase, ubiquinone (Co Q), complex II (succinate ubiquinone oxidoreductase), complex III (ubiquinone cytochrome oxidoreductase), cytochrome c (Cyt c), and complex IV (cytochrome c oxidase)^5,6. Electron transport through the complexes allows the pumping of protons from the mitochondria matrix to the intermembrane space, forming a mitochondrial membrane potential⁷. Protons located in the intermembrane space are pumped in reverse, towards the mitochondria matrix by F₁F₀ -ATPase to synthesize one ATP molecule per four protons pumped⁸.

Mitochondrial respiration measurements provide valuable insights into mitochondrial integrity, cell bioenergetic, and mitochondrial respiratory capacity⁹. Mitochondrial respiration function could be analyzed in vitro or in situ using diverse techniques. However, mitochondrial respiration analysis in intact cells could present a series of advantages since it comprises the entire cell metabolism, as cells' interactions with their environment and intracellular metabolism are considered when employing whole cells¹⁰. Moreover, using intact cells allows for easy evaluation of various experimental conditions and detects minimal changes that could not be observed in isolated mitochondria^10,11. Another advantage of mitochondrial respiration quantification in intact cells is that the effects of mitochondrial proliferation and localization are retained¹².

Mammalian mitochondria respiration in intact cells has been described in great detail¹², leaving aside the study of other eukaryotic organisms, such as yeasts. Here, we propose a technique for quantifying mitochondrial respiration adapted to yeast from mammalian protocols. Thus, concepts from mammalian quantification of mitochondrial respiration have been utilized in this technique. For example, this technique is divided into various stages as the mammalian protocol: 1) basal respiration (substrate dependent), 2) ATP-linked respiration (through the inhibition of ATP synthase), 3) maximal respiration capacity (employing an uncoupler that permits the permeability of protons through the inner mitochondrial membrane), 4) mitochondrial respiration inhibition (utilizing a mix of respiratory chain inhibitors to discard oxygen consumption for other sources different from mitochondrial respiration)^11,13. Therefore, this paper aims to present a protocol to quantify mitochondrial respiration in yeast.

1. Culture media and inoculum preparation

1. Prepare 100 mL of yeast extract-peptone-dextrose medium (YPD) by adding 1 g of yeast extract, 2 g of casein peptone, and 2 g of glucose in 95 mL of distilled water. Distribute 3 mL into 10 mL glass test tubes, sterilize at 121 °C, and 1.5 psi for 15 min.
   NOTE: The medium can be stored at 4 - 8 °C for up to 1 month.
2. Inoculate a 10 mL glass test tube containing 3 mL of YPD medium with 250 µL of -20 °C glycerol-preserved yeast cells. Incubate overnight with constant agitation at 200 rpm and 30 °C.
   NOTE: Temperature incubation could vary in some yeast species (e.g., Kluyveromyces marxianus has an optimal growth temperature of 37 °C).
3. Fill a Petri dish (60 x 15 mm²) with sterile 2% YPD agar (add 10 g of yeast extract, 20 g of casein peptone, 20 g of glucose, and 20 g of bacteriological agar to 950 mL of distilled water) and streak the Petri dish with the yeast cells grown in the 10 mL glass test tube using a sterile loop. Incubate the Petri dish at 30 °C until isolated colonies appear.
4. Prepare a pre-inoculum by inoculating a sterile 10 mL glass test tube containing 3 mL of YPD medium at 2% glucose with two or three yeast-isolated colonies from the Petri dish. Incubate overnight with constant agitation at 30 °C.

2. Growth conditions and experiment design

1. Inoculate a 500 mL Erlenmeyer flask containing 100 mL of sterile synthetic complete medium (SC medium; 0.18 g of yeast nitrogen base without amino acids, 0.5 g of ammonium sulfate, 0.2 g of dipotassium phosphate, 1 g of yeast synthetic drop-out supplements and glucose according to the experimental design, in 98.02 mL of distilled water) with yeast culture at initial OD₆₀₀ ~ 0.1, that is approximately 3 mL of the overnight pre-inoculum (OD₆₀₀ ~ 3). Set the environmental condition or supplement with the chemical challenge to be tested in mitochondrial respiration (e.g., adding 100 µM resveratrol). Prepare a vehicle control in a separate flask for the chemical challenge. Test at least three biological replicates.
   NOTE: It is recommended to use a minimal media to discard nutrimental noise in the assay, and the minimal media proposed at this point is used for S. cerevisiae and could differ for other yeast species. Finally, the glucose concentration supplemented in the culture media is critical to avoid glucose repression or the Crabtree effect that inhibits mitochondrial respiration; concentrations below 0.5% (w/v) gave good responses in mitochondrial respiration accompanied by high-growth rates in S. cerevisiae. In Crabtree-negative yeasts, high glucose concentrations did not affect mitochondrial respiration (e.g., K. marxianus).
2. Weigh an empty 50 mL conical tube and use it to harvest cells at the mid-log phase (OD₆₀₀ ~ 0.5) by centrifugation at 4000 x g for 5 min.
   NOTE: The mid-log phase is recommended for testing mitochondrial respiration since it is the growth phase in which the energetic metabolism is more active.
3. Discard the supernatant and wash the pellet 3x with 25 mL of deionized water. Weigh the conical tube with the pellet cells (wet weight) and subtract the conical tube weight to obtain the cellular wet weight.
4. Resuspend the pellet in 2 mL of deionized water.
5. Record the cellular concentration by dividing the wet weight by 2 to obtain mg of cells/mL. This value is essential to determine how much volume needs to be added to have 50 mg of cells for the assay.

3. Oxygen consumption assay (Polarography)

1. Employ a Clark-type oxygen electrode connected to a YSI5300A monitor and a computer for data acquisition.
   1. Place 5 mL of morpho-ethanol sulfonic acid adjusted to pH 6 with triethanolamine (MES-TEA buffer; 10 mM) and 50 mg of cells (wet weight) in the polarograph chamber. Gently place the electrode, taking care that there are no bubbles.
   2. Turn on the YSI5300A monitor, the computer for data acquisition, and the chamber for stirring. Set the YSI5300A monitor by selecting the Channel 1 Setting Dial in AIR. Adjust channel 1 to 100% with the channel 1 calibration dial and stabilize the signal for 5 min at 100%.
   3. Start recording data in the computer and measuring time using a chronometer.
      NOTE: The YSI5300A monitor does not have a data acquisition computer or program; this can be externally implemented by the user.
   4. Use lateral channeling of the electrode in the oximeter chamber to add the oxidizable substrate, oligomycin, the uncoupler, and the mix of respiratory chain inhibitors sequentially.
2. Add the oxidizable substrate (glucose, glycerol, L-lactate, or another carbon source according to the experimental design) to a final concentration of 10 mM with a chromatography syringe. Keep the chamber closed all the time and with constant agitation. Record air percentage consumption for 2-3 min to determine basal respiration.
   NOTE: Glucose, glycerol, and L-lactate are used to analyze the electron transport chain at different sites of the respiratory chain: glucose metabolism reduces NAD⁺ to form NADH and is utilized for analyzing the entire respiratory chain; glycerol transfers electrons to ubiquinone and is used to evaluate the respiratory chain from ubiquinone; finally L-lactate cedes its electrons to cytochrome c and is employed to determine the effect from cytochrome c¹⁴. These substrates could vary depending on the enzymatic capability of the yeast species.
3. Add oligomycin to obtain a final concentration of 0.01 mM in the oximeter chamber using a chromatography syringe to evaluate ATP-linked respiration and record air percentage consumption for 2-3 min.
4. Supplement the uncoupler, 3-chlorophenylhydrazone carbonyl cyanide (CCCP) to obtain a final concentration of 0.015 mM in the oximeter chamber with a chromatography syringe to determine maximal respiration capacity and record air percentage consumption for 2-3 min.
5. Use a chromatography syringe to add the electron transport chain inhibitors: first, 2- thenoyltrifluoroacetone (TTFA) and then, antimycin A (AA) at final concentrations of 1 mM and 1 mg/mL, respectively. Measure each inhibitor for 2-3 min.
   NOTE: Inhibitors of the electron transport chain could differ between yeast species; this should be considered in this step.
6. Screen each carbon source independently (i.e., glucose, glycerol, or L-lactate). Repeat steps 3.3 to 3.5 for each carbon source. Wash the chamber 3x with 70% ethanol and 3x with deionized water after each use.

4. Data processing

1. Use oxygen consumption curves (plotting air percentage versus time) to calculate the slopes of the oxidizable substrate (basal respiration), oligomycin (ATP-linked respiration), CCCP (maximal respiration), AA, and TTFA (not mitochondrial respiration; Figure 1). Calculate individual slopes for each supplemented compound using linear regression as described below.
   1. Create a new project file in the statistical software. Choose XY in the Create section. Select the option Enter or Import Data into a New Table in the section Data table.
   2. Select Numbers in the X subsection in the section Options. Click on Enter 3 replicate values in side-by-side subcolumns in the Y subsection in the section Options.
      NOTE: The table format has one X column and several Y columns, each with 3 subcolumns named A: Y1 and A: Y2.
   3. Enter time data in X columns (e.g., 0, 2, 4, 6, 8 s or 0, 1, 2, 3 min). Write the air percentage in the Y columns.
   4. Make a linear regression fitness. Click on Analyze in the menu bar. In the option Analysis, select Simple Linear Regression on XY analyses and click on OK.
   5. Obtain the slope value from the Results in Data tables on the left part of the screen.
2. Write slope values in a datasheet and subtract the slope value obtained from the respiratory inhibitors mix (AA and TTFA) from slopes obtained from the oxidizable substrate, oligomycin, and CCCP for discarding oxygen consumption from sources different from mitochondrial respiration.
3. Multiply the obtained values by 237 µM of O₂. This value is considered the solubility of O₂ in a buffered mitochondrial medium equilibrated with air (20.9% O₂) at 25 °C; this value varies according to the temperature, altitude, and buffer employed.
4. Convert to minute; the obtained values are µM of O₂ per time, depending on how time is plotted, to obtain µM of O₂/ min.
5. Divide by 50, that is, the mg of cells used; cell mg can vary according to the experimental design. Finally, the oxygen consumption is expressed as µM of O₂/mg of cells x min

This mitochondrial respiration technique can be used for yeast species other than S. cerevisiae, such as Scheffersomyces stipitis¹⁵ and K. marxianus¹⁶. However, for representative purposes, we only present results from S. cerevisiae. It is well-known that S. cerevisiae presents a predominant respiratory metabolism in low glucose concentrations (below 0.8 mM)^17,18. Thus, to corroborate the respiratory phenotype of S. cerevisiae, it was grown in two glucose concentrations for stimulating (0.5% glucose) and limiting (5% glucose) mitochondrial respiration^18,19. As expected, the results obtained in basal respiration (oxidizable substrate), ATP-linked respiration (oligomycin), and maximal respiratory capacity (CCCP) validate the characteristic energetic metabolism of S. cerevisiae (Figure 2). We observed higher values of oxygen consumption (basal respiration, ATP-linked respiration, and maximal respiratory capacity) in cells grown with 0.5% glucose compared to values obtained in cells grown with 5% glucose (Figure 2).

Additionally, the assays used three substrates: glucose, glycerol, and L-lactate. The objective of employing diverse substrates is to evaluate different parts of the mitochondrial respiratory chain. In these conditions, mitochondrial respiration did not show significant substrate-dependent changes since no treatment to disturb the respiratory chain was added (Figure 2).

We used quercetin to evaluate the technique's capacity, a compound that negatively influences mitochondrial respiration²⁰. The data obtained showed that quercetin significantly decreased mitochondrial respiration in cells grown in media supplemented with 0.5% glucose (Figure 3). On the other hand, cells grown in media supplemented with 5% glucose did not present a significant negative influence from quercetin (Figure 4).

Figure 1: Representative oxygen consumption curve. Oxygen consumption curve obtained in S. cerevisiae cells grown in SC media (0.5% glucose). Colors represent different slopes obtained at substrate, uncouplers, and inhibitor supplementation. Green: The substrate utilized glucose and slope-corresponding basal respiration. Blue: oligomycin, ATP synthase inhibitor, slope employed for determination ATP-linked respiration. Yellow: CCCP, uncoupler, slope used to evaluate maximal respiration. Grey and red: inhibitor complex II and III, respectively; slope employed for determining oxygen consumption not dependent on mitochondria. Abbreviations: CCCP = 3-chlorophenylhydrazone carbonyl cyanide; TTFA = 2- thenoyltrifluoroacetone; AA = antimycin A. Please click here to view a larger version of this figure.

Figure 2: Oxygen consumption assays in S. cerevisiae cells grown at 0.5% and 5% glucose. Mitochondrial respiration is evaluated through oxygen consumption measured at basal respiration, maximal respiratory capacity, and ATP-linked respiration in S. cerevisiae cells grown in SC supplemented with 0.5% and 5% glucose. Three substrates were used to evaluate their specific influence on the electron transport chain: glucose (NADH dehydrogenases), glycerol (ubiquinone pool), and L-lactate (cytochrome c). (A) Basal respiration at 5% and 0.5% glucose with glucose, glycerol, and L-lactate as substrates; (B) maximal respiration at 5% and 0.5% glucose with glucose, glycerol, and L-lactate as substrates; (C) ATP-linked respiration at 5% and 0.5% glucose with glucose, glycerol and L-lactate as substrate. Results are presented as mean ± S.D. from three independent experiments, with each experiment having two technical repetitions. One-way ANOVA followed by the Tukey test (p ≤ 0.05; different letters: significant difference, same letters: no significant difference) for statistical analysis. Please click here to view a larger version of this figure.

Figure 3: Quercetin limited mitochondrial respiration in low glucose concentration in S. cerevisiae cells grown. Oxygen consumption measurement was performed at basal respiration, maximal respiratory capacity, and ATP-linked respiration in S. cerevisiae cells grown in SC media (0.5% glucose) quercetin supplemented (100 μM). Ethanol was used to dissolve the quercetin solution. Results are presented as mean ± S.D. from three independent experiments, with each experiment having two technical repetitions. Statistical analyses were done using a student t-test paired (*, ** p ≤ 0.05). Please click here to view a larger version of this figure.

Figure 4: Quercetin does not affect mitochondrial respiration in high glucose concentration in S. cerevisiae. Oxygen consumption was measured at basal respiration, maximal respiratory capacity, and ATP-linked respiration in S. cerevisiae cells grown in SC media (5% glucose) quercetin supplemented (100 µM). Ethanol is used to dissolve the quercetin solution. Results show mean ± S.D. from three independent experiments, each with two technical repetitions. Statistical analyses were performed using student t-test paired (p ≤ 0.05; ns: non-significant). Please click here to view a larger version of this figure.

Mitochondrial respiration phosphorylation plays a fundamental role in several pathways that depend on mitochondrial membrane potential and maintain ATP levels through oxidative phosphorylation. Understanding how environmental and nutritional conditions impact yeasts' mitochondrial respiration serves as a tool to elucidate molecular mechanisms.

It is essential to consider the following critical steps to obtain reliable results from this method. Agitation >200 rpm is critical to obtaining sufficient biomass under respiratory growth conditions to test mitochondrial respiration; agitation conditions lower than 200 rpm cause poor growth and biomass formation, taking higher times to reach the mid-log growth phase and could impact oxygen consumption rates since cells survive in a less oxygenated environment. Glucose concentration in the media is set according to the yeast phenotype (Crabtree-positive or negative); this is critical in the case of Crabtree-positive yeasts since high-glucose concentrations inhibit mitochondrial respiration and could exert a bias in the data interpretation. For example, in S. cerevisiae, glucose causes catabolic repression, inhibiting mitochondrial respiration at high glucose concentrations^21,22. Based on this evidence, lower values in basal respiration, ATP-linked respiration, and maximal respiration of cells grown at high glucose concentration (5%) are obtained compared with yeast cells grown at low glucose concentration (0.5%). Respiratory chain inhibitors should be carefully selected according to yeast susceptibility; this is critical to discard sources of cellular oxygen consumption other than mitochondrial respiration.

Substrates, mitochondrial uncouplers, and inhibitors are used to determine different parameters of mitochondrial respiration in intact cells. Nevertheless, their use has some limitations. For instance, the unspecific targets outside mitochondria of mitochondrial inhibitors and uncouplers in intact cells must be considered to avoid bias in the oxygen consumption interpretation. Furthermore, the use of alternative carbon sources for screening the site of action in the respiratory chain should be tested with more detail since by-products of glycerol or L-lactate metabolism could be oxidized to reduce NAD⁺ or FAD⁺, which cedes electrons and activate the respiratory chain since NADH dehydrogenases and Complex II, respectively. Finally, S. cerevisiae and other yeast harbor PDR-transporters such as Yor1p (yeast oligomycin resistance protein), which suggests the efflux of oligomycin from the cells²³. Further, assays using PDR-deficient cells, e.g., the AD1-8 strain, which has major PDR transporters and PDR1/PDR3 transcription factors deleted, are needed to validate the use of oligomycin in S. cerevisiae for mitochondrial respiration quantification.

The use of intact cells for mitochondrial respiration quantification maintains the cellular context, giving a more robust technique for knowing the nutrimental or environmental effects on yeast bioenergetics. Moreover, using yeast-intact cells for mitochondrial respiration quantification is more economical and, at the technical level, easier than isolated mitochondria. Additionally, as in isolated mitochondria assays, diverse carbon sources indicate the site of action in the respiratory chain of some molecules. As proof of concept, this technique corroborates that resveratrol supplementation affects the respiratory chain at NADH dehydrogenase level since resveratrol inhibition on basal respiration is only observed with glucose and not with glycerol or L-lactate²⁴. In vitro studies with solubilized complex I demonstrated that resveratrol competes with NAD^+, affecting complex I activity²⁵. This corroborates the phenotype observed in intact cells with the technique presented here. Besides, this technique is also reliable for exploring the negative effects of chemicals on yeast mitochondrial respiration. For example, quercetin is a polyphenol widely studied in recent decades. Among the primary evidence found, it has been shown to limit mitochondrial respiration in intact cells, which has been validated by complex respiratory chain activity^26,27. Importantly, diverse research has reported quercetin inhibitory effects on mitochondrial respiration in different model studies²⁸.

It is important to have a detailed description of yeast metabolism, including bioenergetics, since these microorganisms could be animal pathogens, including humans, and are also utilized as cell factories or in biotechnological applications.

The authors have nothing to disclose.

This work was supported by the Tecnológico Nacional de México (Grant 20026.24-PD) awarded to LAMP.