A High-Throughput Assay for Mitochondrial Membrane Potential in Permeabilized Yeast Cells

Analytical Biochemistry 293, 269 –276 (2001) doi:10.1006/abio.2001.5139, available online at http://www.idealibrary.com on

A High-Throughput Assay for Mitochondrial Membrane Potential in Permeabilized Yeast Cells Ellyn Farrelly, M. Catherine Amaral, Lisa Marshall, and Shu-Gui Huang 1 Tularik Inc., Two Corporate Drive, South San Francisco, California 94080

Received February 15, 2001; published online May 16, 2001

A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3ⴕdipropylthiadicarbocyanine iodide (DiSC 3(5); DiSC 3). In permeabilized cells, DiSC 3 fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K ⴙ and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H ⴙ transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential. © 2001 Academic Press Key Words: mitochondrial membrane potential; fluorescent probe; high-throughput screening; uncoupling protein 1.

In recent years, mitochondrial biology has become a subject of intense study in both the academic and the industry sectors. This is due to the mitochondrion’s pivotal role in cell metabolism and apoptotic cell death. A large number of diseases have been linked to mitochondrial dysfunction (1– 4), including the neurodegenerative Alzheimer’s disease and Parkinson’s disease and metabolic diseases such as Luft’s disease, obesity, and diabetes. 1

To whom correspondence should be addressed at Biology II, Tularik Inc., Two Corporate Drive, South San Francisco, CA 94080. Fax: (650) 825-7318. E-mail: [email protected]. 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

The mitochondrion’s essential role is to “combust” fuel or substrate molecules and to synthesize the energy molecule ATP for the cell. According to Mitchell’s chemiosmotic theory (5), H ⫹ ions are pumped during substrate oxidation to the cytosolic side of the mitochondrion, establishing a H ⫹ gradient that is solely coupled to ATP synthesis. The electrochemical potential across the mitochondrial inner membrane is an important index of the bioenergetic state of the cell and is precisely regulated to cope with cellular energy metabolism. For example, cancer cells typically grow fast and exhibit high mitochondrial membrane potential compared to normal cells (6). Nature has engineered an uncoupling protein (UCP1) 2 to increase energy expenditure in brown fat tissue. The mitochondria in brown fat cells display a low membrane potential with high oxidation capacity, allowing heat to be generated for the maintenance of body temperature in all hibernating animals and in small animals (7, 8). In recent years, the use of noninvasive fluorescent probes to measure mitochondrial membrane potential has contributed to our understanding of cellular bioenergetics and mitochondrial function. However, measurement of mitochondrial membrane potential still relies on slow, low-throughput procedures such as fluorescence microscopy and flow cytometry. Thus, it is desirable to have a robust, high-throughput assay to measure mitochondrial membrane potential. In this study, we establish such an assay in permeabilized yeast cells. We also show that this method can be employed in a high-throughput mode to screen random 2 Abbreviations used: DiSC 3, 3,3⬘-dipropylthiadicarbocyanine iodide (DiSC 3(5)); CCCP, carbonylcyanide m-chlorophenyl hydrazone; DNP, 2,4-dinitrophenol; UCP, uncoupling protein; WT cells, control yeast cells transformed with the vector alone; DMSO, dimethyl sulfoxide; CV, coefficient of variance; TMRE, tetramethylrhodamine ester; DiOC 6, 3,3⬘-dihexyloxacarbocyanine iodide; LiAc, lithium acetate; CSM-URA, uracil-free casamino acid mixture.

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chemical libraries for modulators of mitochondrial membrane potential. MATERIALS AND METHODS

Materials. Fluorescent probes (DiSC 3, DiOC 6, JC-1, rhodamine, and TMRE) were purchased from Molecular Probes (Eugene, OR). Zymolyase was from Seikagaku Corporate (Tokyo, Japan). The diploid yeast Saccharomyces cerevisiae W303 (MAT␣ Ade2-1 Can1100 His3-11,15 Leu2-3,112 Trp1-1 Ura3) was used throughout this study. The plasmid for expression of hamster UCP1 (pEMBL-UCP1) and anti-UCP1 serum were kindly provided by Dr. M. Klingenberg (Munich, Germany). Yeast transformation was performed using the LiAc method (9). All other reagents were of the highest grade from Sigma. Permeabilization of yeast cells. UCP1-expressing yeast cells and wild-type (WT) cells (control cells transformed with the vector alone) were precultured overnight at 29.5°C in uracil-free glucose medium (0.7% yeast nitrogen base, 2% glucose, 25 mg/L tryptophan, 40 mg/L adenine, 0.1% CSM-URA). The cells were then diluted 1:100 and grown aerobically under vigorous shaking in uracil-free lactate medium (0.7% yeast nitrogen base, 2% lactate, 50 mg/mL tryptophan, 40 mg/mL adenine, and 0.1% CSM-URA, pH 6.0). Cells were harvested at an OD 600 ⬇ 3. Permeabilization was performed according to the method of Achleitner et al. (10) with minor modifications. After yeast spheroplasts were resuspended in lysis buffer (0.4 M sorbitol, 20 mM Hepes, 0.15 M potassium acetate, 2 mM magnesium acetate, 0.5 mM EGTA, pH 6.8) at an OD 600 ⫽ 150, they were aliquoted (0.8 mL) into 1.5-mL Eppendorf tubes and directly frozen at ⫺80°C. No loss of activity was observed after storage for 1 year. The degree of permeabilization of the yeast spheroplasts was evaluated by measuring the intracellular glucose 6-phosphate dehydrogenase activity (11). Permeabilized yeast cells were resuspended in the assay buffer (290 mM mannitol, 20 mM K 2HPO 4, 0.5 mM EGTA, pH 6.8) to yield a final OD 600 of 1.0. Substrates (0.2 mM NADP and 5 mM glucose 6-phosphate) were then added to initiate the reaction. NADPH formation was measured fluorometrically (␭ exc ⫽ 360 nm, ␭ em ⫽ 450 nm) every 0.5 min in an ISS PC1 photon counting spectrophotometer (ISS, Champaign, IL) DiSC 3 fluorescence measurement in permeabilized yeast cells. Permeabilized cells were quickly thawed and used within 2 h. Cells were added at an OD 600 ⫽ 0.1 to a 5 ⫻ 5-mm cuvet containing 400 ␮L assay buffer (290 mM mannitol, 20 mM K 2HPO 4, 0.5 mM EGTA, 10 mM Hepes, pH 6.9, freshly supplied with 5 mM ␣-glycerol phosphate, 2 ␮g/mL oligomycin, 0.02% bovine serum albumin, 0.5 mM phenylmethylsulfonyl fluoride, and 0.1 ␮M DiSC 3). DiSC 3 fluorescence intensity was

FIG. 1. Degree of permeabilization assessed by glucose-6-phosphate dehydrogenase reaction. High potassium acetate/freeze method and enzymatic assay conditions were as described under Materials and Methods. The rate constants of NADPH fluorescence increase were 0.058 ⫾ 0.001, 0.054 ⫾ 0.002, and 0.0021 ⫾ 0.0001 for permeabilized UCP1-expressing, permeabilized WT, and untreated WT cells, respectively. Data are expressed as means ⫾ SD (n ⫽ 3).

measured at 670 nm (␭ exc ⫽ 620 nm). The kinetics of fluorescence changes elicited by 2-bromopalmitate, GDP, and CCCP in UCP1 cells were fast. Generally fluorescence reached steady levels within 5 min and were stable for 60 min. Therefore, the reaction mixtures were incubated for 5 min before reading the fluorescence intensity. Microtiter plate assay. Microtiter plate assays were carried out in black 96- or 384-well plates. Using a 12-channel pipettor, DMSO (minus drug) was added to control wells and 8 ␮M CCCP in DMSO was added as a positive control. For high-throughput screening, 0.8 mL of freshly thawed cells was mixed into 400 mL of assay medium. A Quadra 384 Tomtec robot (Hamden, CO) was programmed to deliver 2 ␮L of compound (0.3 mM stock concentration in DMSO) and mix it with 48 ␮L of the cell suspension in 384-well plates. After an incubation of 5 to 40 min, the plates were read on an LJL Analyst fluorescence reader (Sunnyvale, CA). The excitation and emission filters were 640 ⫾ 20 and 682 ⫾ 22 nm, respectively. A beam splitter was used to cut off excitation light below 650 nm. RESULTS

Permeabilization of yeast cells. Yeast are monocellular eukaryotic walled cells. In order to establish a mitochondrial membrane potential assay in which the test compound rapidly equilibrates with the mitochondria, we prepared spheroplasts from aerobically grown yeast cells and permeabilized them using various methods, including treatment with digitonin (12), toluene/ethanol (13), or nystatin (11), and by the high potassium acetate/freeze method (10). We found the

MITOCHONDRIAL MEMBRANE POTENTIAL ASSAY

FIG. 2. Emission spectra of DiSC 3 in the presence of permeabilized yeast cells. DiSC 3 in the assay buffer exhibited high fluorescence with an emission maximum at 670 nm. When permeabilized WT yeast cells (OD 600 ⫽ 0.1) were present, the fluorescence decreased with a red shift of the peak wavelength to 680 nm. Abolition of the mitochondrial membrane potential by the addition of 8 ␮M CCCP increased the fluorescence level with a small blue shift of the peak wavelength to about 675 nm. Fluorescence excitation was at 620 nm.

high potassium acetate/freeze method yielded the best results in terms of the degree of permeabilization and fluorescence response (Fig. 1). Yeast cells permeabilized in this manner have been reported to retain intact mitochondria (10). Such cells have also been utilized to study the transport of phospholipid (10) and protein (14) into mitochondria. Figure 1 shows the degree of permeabilization monitored by the glucose-6-phosphate dehydrogenase reaction (11). Under our experimental conditions, this reaction is rate limited by the availability of the substrates NADP ⫹ and glucose 6-phosphate. The reaction was about 27-fold faster in the permeabilized cells than in the untreated cells, suggesting that the substrates can quickly equilibrate into the cytoplasma. DiSC 3 fluorescence measures the mitochondrial membrane potential. We characterized a number of mitochondrial membrane potential-sensitive fluorescent dyes (JC-1, DiOC 6, TMRE, rhodamine, and DiSC 3) by testing their responsiveness in permeabilized cells. In these assays, mitochondria in the permeabilized yeast cells were energized with the substrate ␣-glycerol phosphate to raise the mitochondrial membrane potential. The H ⫹ channel in mitochondrial ATP synthase was inhibited with oligomycin. Using the chemical uncoupler CCCP to abolish the membrane potential, we observed that, among the five dyes tested, DiSC 3 gave the strongest fluorescence response (a three- to fivefold increase) upon addition of 8 ␮M CCCP. The emission spectra of DiSC 3 (Fig. 2) showed a fluorescence maximum at 670 nm (␭ exc ⫽ 620 nm) in the assay buffer. When permeabilized cells were added, DiSC 3 fluorescence decreased rapidly, due to accumu-

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lation of the dye in the energized mitochondria. The fluorescence maximum also red shifted to 680 nm, in accordance with earlier observations (15). Upon abolishing mitochondrial membrane potential with CCCP, the fluorescence intensity increased as a result of the release of dye from the mitochondria. DiSC 3 has been reported to respond to both the plasma membrane potential and the mitochondrial membrane potential in intact yeast cells (15, 16). To determine whether permeabilization of the cells abolishes the plasma membrane potential, permeabilized yeast cells were incubated in increasing KCl concentrations in the absence and presence of the K ⫹ ionophore valinomycin (Fig. 3A). In intact cells, a small but significant increase (20%) in the fluorescence intensity was observed with increasing K ⫹ concentration, indicating a progressive depolarization of the plasma membrane by K ⫹. This is in agreement with the observations by Gaskova and co-workers (15). Addition of valinomycin partially repolarized the plasma membrane, as indicated by a decrease in the fluorescence intensity. However, in the permeabilized cells, the fluorescence intensity was low and unaltered by the addition of K ⫹. In contrast, upon addition of valinomycin, the fluorescence intensity increased dramatically (threefold) to levels similar to those observed in the presence of CCCP. These findings suggest that the K ⫹ diffusion potential completely uncoupled the respiring mitochondria. In intact yeast cells, the plasma membrane can be depolarized by millimolar concentrations of glucose without influencing the mitochondrial membrane potential (16). We attempted to utilize this feature to differentiate between potentials across the plasma membrane and the mitochondrial membrane. In intact cells (Fig. 3B), glucose significantly raised fluorescence intensity with a 50% increase at about 2 mM. However, 8 ␮M CCCP did not depolarize the mitochondrial membrane potential. In respiring permeabilized cells, the fluorescence level was very low, and the glucose-induced rise was absent. CCCP increased the fluorescence about threefold and, again, the glucose-induced rise was lacking. Under nonrespiring conditions (i.e., in the presence of respiratory chain inhibitors rotenone and azide), the same fluorescence increase elicited by glucose was seen in intact cells (Fig. 3C). However, in the permeabilized cells, the glucose-elicited increase was absent, and the fluorescence levels were high; CCCP no longer increased the fluorescence intensity as it did under respiring conditions (Fig. 3B). These data suggest that, in intact cells, DiSC 3 fluorescence measures both the plasma membrane and the mitochondrial membrane potentials, whereas in permeabilized cells, DiSC 3 fluorescence reflects the mitochondrial membrane potential. We chose hamster UCP1 expressed in yeast cells to

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FIG. 3. DiSC 3 fluorescence measures mitochondrial membrane potential in permeabilized yeast cells. (Left column) Intact cells. (Right column) Permeabilized cells. (A) Influence of K ⫹/valinomycin in intact and permeabilized WT cells in the standard assay buffer. Influence of glucose on DiSC 3 fluorescence under (B) respiring and (C) nonrespiring conditions (in the presence of 10 ␮M rotenone and 1 mM azide). Final concentrations of valinomycin (A) and CCCP (B and C) were 2 and 8 ␮M, respectively. The data are expressed as means ⫾ SD (n ⫽ 4).

validate further our fluorescence assay. UCP1 is a H ⫹ transporter protein expressed in the mitochondrial inner membrane. UCP1 activation by free fatty acids results in the dissipation of the mitochondrial H ⫹ gradient, thus lowering the mitochondrial membrane potential. In our assays, we used the fatty acid 2-bromopalmitate as an activator and GDP as an inhibitor of UCP1 (7, 17) to modulate specifically the mitochondrial membrane potential. UCP1 expression was confirmed by immunoblotting (data not shown). In permeabilized cells, addition of 2-bromopalmitate (5 ␮M) elicited a 1.6-fold increase in fluorescence intensity. GDP (100 ␮M) was able to reverse completely this specific fluorescence increase by inhibiting UCP1 in the mitochondria (Fig. 4). These fluorescence responses were not observed in the UCP-minus control WT cells. These data show that the DiSC 3 fluorescence signal faithfully measures the mitochondrial membrane potential in permeabilized yeast cells.

To optimize the mitochondrial membrane potential assay conditions, we first varied the dye concentration while keeping the cell density and substrate concentration constant (Fig. 4A). Specific fluorescence responses were observed below 400 nM DiSC 3. At 400 nM, the modulations by 2-bromopalmitate and GDP were absent, due to a very high fluorescence background. The best sensitivity in terms of fluorescence response to 2-bromopalmitate (2.2-fold), GDP (2.1-fold), and CCCP (3.8-fold) was achieved at 100 nM DiSC 3. Second, we varied the cell density (Fig. 4B). A low cell density (OD 600 ⫽ 0.1– 0.2) yielded the best sensitivity, while higher cell densities diminished the response to the effectors. Also, high density is not desirable in fluorescence assays because of the increase in light scattering. Third, we investigated the DiSC 3 fluorescence at varying substrate concentrations (Fig. 4C). In the absence of external ␣-glycerol phosphate, weak responses to the effectors were observed, due to residual endogenous

MITOCHONDRIAL MEMBRANE POTENTIAL ASSAY

FIG. 4. Optimization of mitochondrial membrane potential assay conditions. UCP1-expressing cells were utilized to optimize (A) the DiSC 3 dye concentration, (B) cell density, and (C) substrate concentration. The standard assay condition was 5 mM ␣-glycerol phosphate, 0.1 ␮M DiSC 3, and a cell density of OD 600 ⫽ 0.1. Concentrations of 2-bromopalmitate, GDP, and CCCP were 5, 100, and 8 ␮M in DMSO, respectively. Control values were obtained in the presence of the same volume of DMSO.

substrates present in the cells. The best sensitivity was achieved at 5 to 10 mM ␣-glycerol phosphate. Furthermore, when the mitochondria became progressively energized with substrate, the basal fluorescence intensity decreased accordingly. This finding reinforces our notion that (i) DiSC 3 accumulation is dependent upon mitochondrial energization and (ii) the fluorescence intensity is an accurate measure of the mitochondrial membrane potential. The effects of known effectors on mitochondrial membrane potential were studied using our DiSC 3 fluorescence assay. Figure 5 shows dose–response data for chemical uncouplers (CCCP and DNP) and UCP1 effectors (bromopalmitate and GDP). CCCP showed a potency (IC 50) at about 1 ␮M, while DNP was much weaker as a chemical uncoupler with an IC 50 of ⬃30 ␮M. The potency was similar in both WT and UCP1-

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expressing cells. CCCP and DNP reached a similar maximum fluorescence intensity, which defines the zero membrane potential level. In UCP1-expressing cells, 2-bromopalmitate exhibited an IC 50 of 3.3 ␮M; however, the fluorescence intensity was only about 50% of the maximum fluorescence elicited by CCCP. These data indicate that the UCP1-specific activator could not fully uncouple the mitochondria. The effect of GDP on mitochondrial membrane potential was studied in the presence of 10 ␮M 2-bromopalmitate. An IC 50 of 0.75 ␮M was obtained for GDP, similar to previous reports (17). Pilot high-throughput screening. We adapted our fluorescence assay to measure mitochondrial membrane potential in microtiter plates. In UCP1-expressing yeast cells, similar sensitivity to modulations by 2-bromopalmitate (1.5- to 2-fold), GDP (2-fold), and CCCP (3- to 7-fold) were reproduced in both 96- and 384-well plates. We also investigated the influence of the solvent DMSO in the assay. A dose–response study showed that up to 8% DMSO was tolerated under the assay conditions. Then we evaluated the kinetics of fluorescence responses in the presence of 320 random single compounds. Once the cells and compounds were mixed and plated, fluorescence intensity was read every 5 min for 2 h (data not shown). In this plate, seven compounds increased the fluorescence intensity, and all seven reached maximal levels of fluorescence within 5 min. The signals were stable for about 50 min. As the incubation time was increased, there was a slow increase in the fluorescence intensity, presumably due to a loss of mitochondrial activity during prolonged uncoupling. In a pilot experiment, we screened 320 single compounds at a concentration of 12 ␮M in 384-well plates. In UCP1-expressing cells, we found 5 compounds that elicited a 1.5-fold or greater increase in fluorescence intensity. But 4 of the 5 also produced a hit in the UCP-minus control WT cells. Only 1 compound of the 320 compounds was a UCP1-selective activator hit, as it did not increase fluorescence in WT cells. We screened a small chemical library of 3300 random compounds in both cells. The data are summarized in Table 1. The assay is robust and reproducible with a coefficient of variance (CV) at ⬃7% and a Z⬘ factor of ⬃0.64 (18). In the activator screening, the hit rate was 1.2 to 1.3% in WT and UCP1-expressing cells. Of the 3300 compounds, we found only 4 UCP1-selective activator hits. The same fluorescence assay can be applied to screen for UCP1 inhibitors. In this case, mitochondria should become coupled and DiSC 3 fluorescence should be lower than the control level in UCP1-expressing cells. In the same library, we identified 31 compounds (0.9% hit rate) that decreased the DiSC 3 fluorescence intensity by greater than 30% in

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FIG. 5. Dose responses of effectors of mitochondrial membrane potential in permeabilized yeast cells. Fluorescence measurements were performed in 384-well plates. (A) Chemical uncouplers in WT and UCP1-expressing cells. The best fitting yielded IC 50 values of 0.76 ⫾ 0.02 and 0.96 ⫾ 0.02 ␮M for CCCP in WT and UCP1-expressing cells, respectively, and 38.3 ⫾ 1.4 and 31.2 ⫾ 1.2 ␮M for DNP in WT and UCP1-expressing cells, respectively. Open symbols are data obtained in WT cells and filled symbols are data in UCP1-expressing cells. (B) Specific modulators in UCP1-expressing cells. For the GDP dose–response assay, 10 ␮M 2-bromopalmitate (BrPalm) was present at all GDP doses. The best fitting yielded IC 50 values of 3.3 ⫾ 1.2 and 0.75 ⫾ 0.23 ␮M for BrPalm and GDP, respectively. Values are expressed as means ⫾ SD (n ⫽ 4).

UCP1-expressing cells. However, only two compounds actually did not affect the fluorescence intensity in WT cells. Most of the hits decreased the fluorescence intensity independent of the mitochondrial membrane potential by quenching DiSC 3 fluorescence. DISCUSSION

Yeast cells have several merits that make them an ideal vehicle for mitochondrial membrane potential assays. First, the yeast cell harbors many copies of active mitochondria when grown aerobically. This feature makes yeast cells more favorable than most mammalian cell lines, because cultured mammalian cells tend to adapt to glycolysis and, therefore, maintain low mitochondrial activities. Second, yeast are of eukaryotic origin and can be engineered to express mammalian genes (19 –27). Furthermore, a number of mammalian genes (e.g., the mitochondrial UCPs) are not present in the yeast genome. The untransformed yeast cells can therefore serve as a clean negative control. Third, yeast cells can grow anaerobically; therefore, they can be exploited to express proteins such as the UCPs, which

can be potentially harmful to aerobic metabolism. Finally, yeast cells grow fast (doubling time 2 h) and can be cultured at very low costs. DiSC 3 has been applied to measure membrane potential in intact Escherichia coli cells (28), yeast (15, 16), and various mammalian cells (29 –31). In eukaryotic cells, the fluorescence intensity is a composite measure of the potential across the plasma membrane and the mitochondrial membrane, although the mitochondrial inner membrane exhibits much higher potentials than the plasma membrane. In this study, we permeabilized the plasma membrane and abolished its potential, while maintaining intact mitochondria. Thus, the mitochondrial membrane potential can be conveniently assessed by DiSC 3 fluorescence. Several lines of evidence suggest that DiSC 3 fluorescence measures mitochondrial membrane potential in permeabilized yeast cells. First, modulations of the plasma membrane potential by K ⫹ and glucose are absent in the permeabilized cells (Fig. 3). Second, dye accumulation strictly depends on mitochondrial energization. This is shown by the requirement of an oxi-

TABLE 1

Summary of Pilot High-Throughput Screening Data Cell type

F DMSO

F CCCP

F CCCP/F DMSO

CV (%)

Z⬘ factor

Activator hits

Inhibitor hits

UCP1 cells WT cells

15.7 ⫾ 1.4 13.2 ⫾ 1.1

100 ⫾ 5.1 100 ⫾ 6.6

6.4 ⫾ 0.3 7.5 ⫾ 0.5

7.1 ⫾ 2.9 7.4 ⫾ 1.1

0.64 ⫾ 0.13 0.65 ⫾ 0.07

43 39

31 29

Note. High-throughput screening was performed on 3300 random single chemical compounds in 384-well plates. F DMSO and F CCCP are relative fluorescence intensities for the basal DMSO control and positive control with 8 ␮M CCCP. Fluorescence values are normalized against the CCCP controls, which were 1,431,660 and 1,539,781 for UCP1-expressing cells and WT cells, respectively. The Z⬘ factor was calculated according to Zhang et al. (18). An activator hit is defined as a compound that elicits DiSC3 fluorescence by 1.5-fold or greater and an inhibitor hit as a compound that decreases the fluorescence intensity by 30% or greater. Data are given as means ⫾ SD (n ⫽ 14).

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dation substrate (␣-glycerol phosphate, Fig. 4C) and by the lack of dye accumulation in nonrespiring permeabilized cells or respiring cells treated with CCCP (Fig. 3C). Third, DiSC 3 fluorescence responds vigorously to specific modulation of the mitochondrial membrane potential in permeabilized UCP1-expressing cells. This novel assay can be readily adapted to highthroughput screening of compounds that modulate the mitochondrial membrane potential. As an example, we screened a small random chemical library. In this pilot high-throughput screening experiment, we found a high hit rate (1.3%) in UCP1 activator screening. Most of these primary hits were chemical uncouplers, inhibitors of the mitochondrial respiratory chain, or compounds that cause fluorescence artifacts. Therefore, they increase fluorescence intensity nonspecifically in UCP1-expressing and WT cells. Both chemical uncouplers and inhibitors of the respiratory chain decrease the mitochondrial membrane potential, but they can be readily distinguished in mitochondrial oxygen consumption assay in which the chemical uncoupler stimulates, while the respiratory chain inhibitor inhibits, this reaction. Fluorescence artifacts most likely arise from fluorescent compounds and can be readily assessed by reading the fluorescence intensity in the absence of the cells. In the inhibitor screening, a lower hit rate (0.9%) was observed. Fluorescence artifacts are more pronounced in this case due to quenching of DiSC 3 fluorescence by the compounds. Therefore, it is more desirable to employ the “differential display” strategy by performing the counter screen in control WT cells in parallel with UCP1-expressing cells. This assay is simple, robust, and cost-effective. The permeabilization procedure yielded reproducible results in our hands. Cells permeabilized in this way can be preserved at ⫺80°C for at least 1 year without any loss of activity. An obvious advantage is the uniformity of a single-batch preparation that can allow screening and follow-up assays for a large chemical library. From the quantities of cells used in this study, we estimate that a single culture of 4 L of yeast cells would allow screening of ⬃500,000 compounds in 384-well plates. Finally, this assay is versatile and can be applied to study the effects of known modulators on mitochondrial membrane potential. This includes any effectors that are directly or indirectly involved in regulating the mitochondrial membrane potential, such as chemical uncouplers, inhibitors of mitochondrial respiratory chain, or apoptotic agents. As shown in this study, our assay can be used to screen a random chemical library for modulators of known target proteins (e.g., the UCPs and homologs). Alternatively, by expressing a cDNA library in yeast cells, it is possible to identify target genes involved in regulating mitochondrial membrane potential or cellular energy expenditure.

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ACKNOWLEDGMENTS We thank Drs. Jin-Long Chen, Marc Learned, and Larry McGee for helpful discussions during the course of this work.

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