Channel Formation by Yeast F-ATP Synthase and the Role of Dimerization in the Mitochondrial Permeability Transition^,

Background: Whether channel formation is a general feature of F-ATP synthase dimers across species is unknown.

Results: Yeast F-ATP synthase dimers form Ca²⁺-dependent channels, and the e and g subunits facilitate pore formation in situ through dimerization.

Conclusion: F-ATP synthase dimers form the permeability transition pore of yeast.

Significance: Ca²⁺-dependent channel formation is a conserved feature of F-ATP synthases.

Abstract

Purified F-ATP synthase dimers of yeast mitochondria display Ca²⁺-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca²⁺ ionophore ETH129, which allows electrophoretic Ca²⁺ uptake, isolated yeast mitochondria undergo inner membrane permeabilization due to PTP opening. Yeast mutant strains ΔTIM11 and ΔATP20 (lacking the e and g F-ATP synthase subunits, respectively, which are necessary for dimer formation) display a striking resistance to PTP opening. These results show that the yeast PTP originates from F-ATP synthase and indicate that dimerization is required for pore formation in situ.

Introduction

Mitochondria from a variety of sources can undergo an inner membrane permeability increase, the permeability transition (PT),⁴ due to opening of a high conductance channel, the PT pore (PTP) (1). The PTP coincides with the mitochondrial megachannel (MMC) defined by patch clamp studies in mitoplasts (2–5). In mammals, PTP opening requires matrix Ca²⁺ and is favored by oxidative stress and P_i, inhibited by adenine nucleotides and Mg²⁺, and antagonized by cyclosporin A (CsA) through its interaction with matrix cyclophilin (CyP)D (6, 7). The mammalian PTP is today recognized to play a role in cell death in a variety of disease paradigms (8).

Inner membrane permeability pathways have been described in yeast (9) and in Drosophila melanogaster (10), but whether these coincide with the mammalian PTP remains an open question (11–14). The issue is particularly complex in the case of yeast, where multiple conductance pathways may exist including an uncoupling protein-independent permeability activated by ATP (15–17). Furthermore, the yeast PTP (yPTP) is inhibited rather than activated by P_i and insensitive to CsA (9), and due to the lack of a mitochondrial Ca²⁺ uniporter, its Ca²⁺ dependence has been more difficult to assess (18), although the Ca²⁺ content of Saccharomyces cerevisiae mitochondria is close to that of rat liver mitochondria (19). The problem of the Ca²⁺ dependence was solved by the Shinohara group (20), who showed that yeast mitochondria incubated with optimized substrate and P_i concentrations readily undergo a Ca²⁺-dependent PT upon treatment with ETH129, a Ca²⁺ ionophore that allows electrophoretic Ca²⁺ transport into the matrix of energized mitochondria. We recently demonstrated that dimers of mammalian F-ATP synthase reconstituted into planar bilayers give rise to Ca²⁺-activated currents with conductances ranging up to 1.3 nS in 150 mm KCl that closely match those displayed by the MMC-PTP (21). Here we have tested whether gel-purified F-ATP synthase dimers of S. cerevisiae form channels when reconstituted in lipid bilayers, and whether dimerization of the F-ATP synthase is necessary for PTP formation in intact mitochondria.

EXPERIMENTAL PROCEDURES

Yeast Strains and Materials

The S. cerevisiae strains BY4743 (4741/4742), as well as the mutants ΔCPR3 (MATa, his3Δ1, leu2Δ0, met5Δ0, ura3Δ0), ΔTIM11 (MATa, his3Δ1, leu2Δ0, met5Δ0, ura3Δ0), and ΔATP20 (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0), were purchased from Thermo Scientific. ΔTIM11ΔATP20 mutants were obtained by mating the ΔTIM11 and ΔATP20 strains and selecting the formed diploid by growth on synthetic defined (0.67% nitrogen base without amino acids, 2% dextrose) selective medium containing the required nutritional supplements except methionine and lysine. Diploids were then induced to sporulate in 1% potassium acetate, tetrads were dissected, and haploids were analyzed with semiquantitative PCR to detect null mutants for TIM11 and ATP20 genes. Digitonin was from Sigma, and ETH129 was from Sigma-Aldrich Japan and was dissolved in methanol. NADH, disodium salt was purchased from Roche Applied Science.

Yeast Culture and Mitochondria Isolation

Yeast cells were cultured aerobically in 50 ml of 1% yeast extract, 1% bacto-polypeptone medium containing 2% glucose at 30 °C. When it reached an optical density of 2 at 600 nm, the culture was added to 800 ml of bacto-polypeptone medium supplemented with 2% galactose and incubated for 20 h at 30 °C under rotation at 180 rpm, yielding about 4.0 g of yeast cells. Yeast mitochondria were isolated as described (20) with the following modifications. Briefly, cells were washed, incubated for 15 min at 37 °C in a 0.1 m Tris-SO₄ buffer (pH 9.4) supplemented with 10 mm dithiothreitol (DTT), and washed once with 1.2 m sorbitol, 20 mm P_i, pH 7.4. Yeast cells were then suspended in the same buffer and incubated for 45 min at 30 °C with 0.4 mg/g of cells of Zymolyase 100T to form spheroplasts. The latter were washed once with sorbitol buffer and homogenized in 0.6 m Mannitol, 10 mm Tris-HCl, pH 7.4, and 0.1 mm EDTA-Tris with a Potter homogenizer. The homogenate was centrifuged for 5 min at 2,000 × g, and the supernatant was collected and centrifuged for 10 min at 12,000 × g. The resulting mitochondrial pellet was suspended in mannitol buffer, and protein concentration was determined from the A₂₈₀ of SDS-solubilized mitochondria (14).

Mitochondrial Calcium Retention Capacity

Mitochondrial Ca²⁺ uptake was measured with Calcium Green-5N (Molecular Probes) fluorescence using a Fluoroskan Ascent FL (Thermo Electron) plate reader at a mitochondrial concentration of 0.5 mg × ml⁻¹. Mitochondria were incubated as specified in the figure legends.

Gel Electrophoresis and Western Blotting

Mitochondria were suspended at 10 mg/ml in 150 mm potassium acetate, 30 mm HEPES, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride and solubilized with 1.5% (w/v) digitonin. After centrifugation at 100,000 × g with a Beckman TL-100 rotor for 25 min at 4 °C, supernatants were collected, supplemented with 50 mg/ml Coomassie Blue and 5 m aminocaproic acid, and quickly loaded onto a blue native polyacrylamide 3–12% gradient gel (BN-PAGE, Invitrogen). Electrophoresis was carried out at 150 V for 20 min and at 250 V for 2 h followed by gel staining with 0.25 mg/ml Coomassie Blue, 10% acetic acid or used for in-gel activity staining to detect bands corresponding to ATP synthase. Activity was monitored in 270 mm glycine, 35 mm Tris, pH 7.4, 15 mm MgSO₄, 8 mm ATP, Tris-buffered to pH 7.4, and 2 mg/ml Pb(NO₃)₂. Bands corresponding to monomeric and dimeric forms of ATP synthase were cut from the gels, and protein complexes were eluted overnight by incubation at 4 °C in 25 mm Tricine, 15 mm MgSO₄, 8 mm ATP, 7.5 mm Bis-Tris, 1% (w/v) n-heptyl β-d-thioglucopyranoside, pH 7.0. Samples were then centrifuged at 20,000 × g for 10 min at 4 °C, and supernatants were used for bilayer experiments. For cross-linking experiments, mitochondria were incubated 20 min at room temperature at 1 mg/ml in 250 mm sucrose, 2 mm P_i, and 2 mm CuCl₂. Five millimolar N-ethylmaleimide and 5 mm EDTA were then added to block the cross-linking reaction, and the incubations were transferred on ice for 10 min followed by centrifugation and preparation for BN-PAGE as described above. Total yeast mitochondria lysates and bands corresponding to dimers of F-ATP synthase cut out of BN-PAGE gels were subjected to SDS-PAGE followed by silver staining or transfer to nitrocellulose for Western blot analysis. Antibodies were polyclonal rabbit anti-ATP synthase γ subunit (a gift from Marie-France Giraud, Bordeaux, France), anti-Tom20 and anti-Tim54 (a gift from Nikolaus Pfanner, Freiburg, Germany).

Electrophysiology

Planar lipid bilayer experiments were performed as described in Ref. 31. Briefly, bilayers of 150–200 picofarads of capacitance were prepared using purified soybean asolectin. The standard experimental medium was 150 mm KCl, 10 mm Hepes, pH 7.5. All reported voltages refer to the cis chamber, zero being assigned to the trans (grounded) side. Currents are considered as positive when carried by cations flowing from the cis to the trans compartment. Freshly prepared F-ATP synthase dimers were added to the cis side. No current was observed when PTP activators were added to the membrane in the absence of F-ATP synthase dimers (n = 2).

RESULTS AND DISCUSSION

Properties of the Ca²⁺-dependent Permeability Transition of Yeast Mitochondria

We used ETH129 to allow Ca²⁺ uptake by energized yeast mitochondria (20) and monitored the propensity of the yPTP to open based on the Ca²⁺ retention capacity (CRC), i.e. the maximal Ca²⁺ load retained by mitochondria before onset of the PT (22). In keeping with previous observations (20), (i) energized yeast mitochondria were able to accumulate Ca²⁺ provided as a train of pulses (Fig. 1A) until onset of the PT, which causes depolarization followed by Ca²⁺ release; and (ii) increasing concentrations of P_i increased the matrix Ca²⁺ load necessary to open the yPTP (Fig. 1, A and B), possibly following formation of matrix P_i-Ca²⁺ complexes. As in mammalian mitochondria, Mg²⁺-ADP increased the CRC, an effect consistent with yPTP inhibition (Fig. 1C). The CRC was not affected by decavanadate (results not shown), which inhibits the ATP-induced, voltage-dependent anion channel (VDAC)-dependent yeast permeability pathway (23, 24).

FIGURE 1.

Properties of the permeability transition of yeast mitochondria. The incubation medium contained 250 mm sucrose, 10 mm Tris-MOPS, 1 mm NADH, 10 μm EGTA-Tris, 5 μm ETH129, 1 μm Calcium Green-5N, final pH 7.4, 0.5 mg/ml bovine serum albumin, and 0.1 mg of mitochondria in a final volume of 0.2 ml. A, the medium was supplemented with 1 mm (trace a), 2 mm (trace b), 5 mm (trace c), or 10 mm P_i (trace d), and where indicated, Ca²⁺ was added. Traces shown are representative of 13 independent experiments. a.u., arbitrary units. B, experimental conditions as in panel A with the indicated P_i concentrations. Values on the ordinate refer to the amount of Ca²⁺ accumulated prior to the precipitous release that follows the PT (n = 13 ± S.E.). C, the experimental conditions were as in panel A with 2 mm P_i, and the medium was supplemented with 2 mm MgCl₂, 1 μm oligomycin, and the stated concentrations of ADP (n = 8 ± S.E.). D–F, the experimental conditions were as in panel A with 2 mm P_i, and the medium was supplemented with the stated concentrations of PhAsO (D), diamide (E), or Cu(OP)₂ (F). For panels D–F, n (± S.E.) was 6, 4, and 7, respectively.

The mammalian PTP is modulated by two classes of redox-sensitive thiols whose oxidation increases the pore sensitivity to Ca²⁺, i.e. (i) matrix thiols that react with phenylarsine oxide (PhAsO) and can be oxidized by diamide (25); and (ii) external thiols that can be oxidized by copper-o-phenanthroline (Cu(OP)₂) (26). The threshold Ca²⁺ load required for yPTP opening was moderately affected by PhAsO (Fig. 1D), whereas it was very sensitive to diamide (Fig. 1E) and to Cu(OP)₂ (Fig. 1F). These experiments indicate that the yeast PTP is affected by the redox state of thiol groups as also suggested by a previous study (18).

CsA desensitizes the mammalian pore to Ca²⁺ through matrix CyPD, a peptidyl-prolyl cis-trans isomerase that behaves as a PTP inducer (27, 28). Through studies of CyPD-null mitochondria, it became clear that CyPD is a modulator but not an obligatory constituent of the PTP and that a PT can occur in the absence of CyPD, or in the presence of CsA, albeit at higher matrix Ca²⁺ loads (8). Yeast mitochondria possess a matrix CyP (CPR3), which facilitates folding of imported proteins in the matrix and is sensitive to CsA (29); however, the yPTP is not affected by CsA (9), as also confirmed in the CRC assay (Fig. 2A, compare traces a and b). These findings suggest either that CPR3 does not interact with the pore or that CsA does not interfere with CPR3 binding. To resolve this issue, we tested the CRC of ΔCPR3 mutants, which displayed a lower rate and slightly lower extent of Ca²⁺ accumulation (Fig. 2A, trace c), indicating that CPR3 does not sensitize the yPTP to Ca²⁺, at variance from the effects of CyPD in mammalian mitochondria (30). The small decrease of CRC in the mutants (Fig. 2B) may be due to slower protein import and defective respiratory chain assembly and/or function (31). It was recently established that rotenone is a good inhibitor of the PTP in mammalian mitochondria lacking CyPD, possibly because of decreased production of reactive oxygen species through inhibition of reverse electron flow (32). Rotenone did not affect the yPTP (Fig. 2A, trace d), in keeping with the lack of a rotenone-sensitive, energy-conserving complex I and with the lack of “off-site” effects. Taken together, the above results suggest that, despite the lack of a fast Ca²⁺ uptake system (19), S. cerevisiae mitochondria can undergo a Ca²⁺-induced PT, which displays some similarities with the mammalian PT (sensitization by matrix Ca²⁺ and oxidative stress, inhibition by Mg²⁺-ADP), but also some differences (inhibition by phosphate and lack of sensitivity to CPR3 and rotenone).

FIGURE 2.

CPR3 deletion does not affect the yeast permeability transition. A, the experimental conditions were as in Fig. 1 with 2 mm P_i; 0.8 μm CsA was added in trace b only, and 2 μm rotenone was added in trace d only. Where indicated, Ca²⁺ was added to wild-type (traces a, b, and d) or ΔCPR3 (trace c) mitochondria (traces are representative of three independent experiments). B, the experimental conditions were as in Fig. 1 with P_i as indicated (n = 4 ± S.E.). Closed symbols, wild-type mitochondria; open symbols, ΔCPR3 mitochondria. Two-way analysis of variance test was performed, *, p < 0.05.

Purified F-ATP Synthase Dimers Possess Channel Activity

To test whether yeast F-ATP synthase dimers can form channels similar to those found in mammals (21), we separated mitochondrial protein extracts by BN-PAGE, identified dimers by in-gel activity staining, and eluted them for incorporation into a planar asolectin membrane (see Fig. 4A for an example of the dimer used). The addition of 1–10 pmol of the dimers to the bilayers in symmetrical 150 mm KCl did not elicit current activity unless Ca²⁺, PhAsO, and Cu(OP)₂ were also added (Fig. 3A). We observed a clear activity in 12 out of 14 reconstitutions, with channel unit conductance usually ranging between 250 and 300 pS (multiples of this unit conductance were often observed; in one case 1000 pS was reached). This conductance is compatible with the values exhibited by a channel observed in mitoplasts from a porin-less yeast strain, which was insensitive to CsA, ADP, or protons and in which the combination of ADP and Mg²⁺ was not tested (33). The activity studied here was characterized by rapid oscillations between closed and open states (flickering), which is typical of the mammalian MMC-PTP, and by variable kinetics. A typical flickering behavior is illustrated in the bottom part of Fig. 3A. As is the case for the mammalian F-ATP synthase (21) and for the MMC-PTP measured in mitoplasts (4), the addition of Mg²⁺-ADP induced a clear-cut inhibition of the channel in five out of six experiments (total inhibition was observed in two cases, and partial inhibition was observed in three cases). The representative experiment of Fig. 3B shows activity recorded before and immediately after the addition of Mg²⁺-ADP in one case of full inhibition, which is illustrated in the corresponding amplitude histograms (Fig. 3B). Taken together, these data provide evidence that under conditions of oxidative stress, yeast F-ATP synthase can form Ca²⁺-activated channels with features resembling the MMC-PTP (although with lower conductance). It should be noted that the dimer preparation did not contain Tom20 or Tim54 (Fig. 4A) and therefore that channel activity cannot be due to the twin pore translocase (34).

FIGURE 4.

ΔTIM11, ΔATP20, and ΔTIM11ΔATP20 mutants lacking subunits involved in dimerization of F-ATP synthase are resistant to PTP opening. A, mitochondrial protein extracts were separated with BN-PAGE and stained with Coomassie blue (lanes labeled Coomassie) or subjected to in-gel activity staining (lanes labeled Activity) to identify bands of F-ATP synthase dimers (D) and monomers (M) (note also a faint band corresponding to F₁ (F¹)). The gel region corresponding to the dimers of the BY4743 strain was cut out and subjected to SDS-PAGE together with a mitochondrial extract from the same strain followed by silver staining (lanes labeled Silver staining) or blotting and probing with the indicated antibodies (lanes labeled Western). OSCP, oligomycin sensitivity-conferring protein. B, the experimental conditions were as in Fig. 1 with 1 mm P_i. Where indicated, Ca²⁺ was added to wild type, ΔTIM11ΔATP20, ΔTIM11, or ΔATP20 mutants (traces are representative of 13, 6, 7, and 6 independent experiments for the corresponding genotypes). C, experimental conditions as in A with 1 mm P_i. One-way analysis of variance test was performed to analyze CRC differences between BY4743 and mutants, *, p < 0.01, **, p < 0.001. D, BN-PAGE (left lanes) and activity staining (right lanes) of mitochondria with the indicated genotypes after treatment with 2 mm CuCl₂.

FIGURE 3.

F-ATP synthase dimers reconstituted in planar lipid bilayers display Ca²⁺-induced currents. Dimers were excised (see Fig. 4A, wild-type) and eluted for planar bilayer experiments. A, upper part, representative current traces recorded at +80 and −100 mV (cis) (upper and lower traces, conductance (g) = 125 and 250 pS) upon incorporation of purified dimeric F-ATP synthase following the addition of 3 mm Ca²⁺ (added to the trans side) plus 0.1 mm PhAsO and 20 μm Cu(OP)₂ (added to both sides). Lower part, typical, most often observed channel kinetics (see also expanded portion of the recording obtained at −60 mV (cis); g = 250 pS). a.u., arbitrary units. B, top, effect of 2 mm ADP plus 1.6 mm Mg²⁺ added to the trans side on channel activity (−60 mV, g = 250 pS); current trace before and immediately after the addition of the modulators is shown. Bottom, amplitude histograms obtained from the same experiment before (left panel) and after (right panel) the addition of ADP/Mg²⁺. Gaussian fitting (green lines) was obtained using the Origin 6.1 Program Set.

Dimerization of F-ATP Synthase Is Required for PTP Formation

Dimers of F-ATP synthase are the “building blocks” of long rows of oligomers located deep into the cristae, which contribute to formation of membrane curvature and to maintenance of proper cristae shape and mitochondrial morphology (35–42). Mammalian F-ATP synthase dimers also appear to be the units from which the PTP forms in a process that is highly favored by Ca²⁺ and oxidative stress (21), events that are required for channel formation (8, 21). To test the hypothesis that yPTP formation requires the presence of F-ATP synthase dimers, we studied mutants lacking subunits involved in dimerization/oligomerization of the enzyme, i.e. subunit e (TIM11) and subunit g (ATP20) (35, 43–45). Strains lacking these subunits display balloon-shaped cristae with ATP synthase monomers distributed randomly in the membrane (39). The ΔTIM11, ΔATP20, and ΔTIM11ΔATP20 mutants lacked dimers when analyzed by BN-PAGE, whereas the monomeric F-ATP synthase was assembled and active (Fig. 4A), consistent with their ability to grow on non-fermentable carbon sources, and developed a normal membrane potential upon energization with NADH (results not shown). CRC assays with ETH129 demonstrated that mitochondria from ΔTIM11, ΔATP20, and ΔTIM11ΔATP20 strains take up a larger Ca²⁺ load than wild-type strains (Fig. 4B), with a doubling of the CRC (Fig. 4C).

Dimers may transiently form also in ΔTIM11 and ΔATP20 strains (46), a finding that could explain why Ca²⁺ release is eventually observed also in the “dimerization-less” mutants. Consistent with this possibility, we did detect dimers in BN-PAGE after treatment with CuCl₂ (Fig. 4D), which promotes formation of disulfide bridges between adjacent cysteine residues of the monomers (45, 47, 48). Not all of the monomers dimerized after CuCl₂ treatment (Fig. 4D), suggesting that cysteine oxidation stabilizes pre-existing dimers that are otherwise dissociated by detergent treatment, but does not induce cross-linking of monomers.

In summary, our data provide the first demonstration that yeast F-ATP synthase dimers form high conductance channels analogous to the mammalian MMC-PTP, and thus that channel formation is a conserved feature of F-ATP synthases; show that yeast mitochondria can undergo a bona fide PT activated by oxidative stress; and indicate that dimers of F-ATP synthase are required for PTP formation in situ (21). Our findings do not exclude the existence of other permeability pathways that may involve the voltage-dependent anion channel (23, 24), nor the possible regulation of yPTP by outer mitochondrial membrane proteins (8). We think that it will now be possible to unravel the many open questions about the structure and function of the PTP (8) with the powerful methods of yeast genetics.