The mitochondrial phosphate carrier interacts with cyclophilin D and may play a key role in the permeability transition - PubMed (original) (raw)
The mitochondrial phosphate carrier interacts with cyclophilin D and may play a key role in the permeability transition
Anna W C Leung et al. J Biol Chem. 2008.
Abstract
The mitochondrial permeability transition pore (MPTP) plays a key role in cell death, yet its molecular identity remains uncertain. Although knock-out studies have confirmed critical roles for both cyclophilin-D (CyP-D) and the adenine nucleotide translocase (ANT), given a strong enough stimulus MPTP opening can occur in the absence of either. Here we provide evidence that the mitochondrial phosphate carrier (PiC) may also be a critical component of the MPTP. Phenylarsine oxide (PAO) was found to activate MPTP opening in the presence of carboxyatractyloside (CAT) that prevents ANT binding to immobilized PAO. Only four proteins from solubilized CAT-treated beef heart inner mitochondrial membranes bound to immobilized PAO, one of which was the PiC. GST-CyP-D pull-down and co-immunoprecipitation studies revealed CsA-sensitive binding of PiC to CyP-D; this increased following diamide treatment. Co-immunoprecipitation of the ANT with the PiC was also observed but was insensitive to CsA treatment. N-ethylmaleimide and ubiquinone analogues (UQ(0) and Ro 68-3400) inhibited phosphate transport into rat liver mitochondria with the same concentration dependence as their inhibition of MPTP opening. UQ(0) and Ro 68-3400 also induced the "m" conformation of the ANT, as does NEM, and reduced the binding of both the PiC and ANT to the PAO column. We propose a model for the MPTP in which a calcium-triggered conformational change of the PiC, facilitated by CyP-D, induces pore opening. An interaction of the PiC with the ANT may enable agents that bind to either transporter to modulate pore opening.
Figures
FIGURE 1.
The ANT from beef heart mitochondria is not detected by a polyclonal antibody previously raised against purified rat ANT. Beef heart SMPs (5 mg/ml) were solubilized in _N_-dodecyl β-
d
-maltoside supplemented with 5 μ
m
atractyloside for 30 min at 4 °C. Extracts were passed through an anion exchange column (RESOURCE™ Q), the flow-through of which was then applied to a cation exchange column (RESOURCE™ S), as described under “Experimental Procedures.” Proteins were separated by SDS/PAGE and revealed by Coomassie Blue staining. ANT was detected by Western blotting using three different antibodies (see “Experimental Procedures”). Fractions are labeled across the_bottom_ of the figure: SMP; Sol SMP, solubilized SMPs;Q, Resource-Q flow-through fraction applied to the Resource-S column;S, Resource-S flow-through fraction.
FIGURE 2.
Pretreatment of mitochondria with CAT and BKA do not prevent activation of MPTP opening by PAO. MPTP opening was assayed under de-energized conditions in KSCN buffer (see “Experimental Procedures”) by the swelling of mitochondria (1 mg protein per ml). In A the mitochondrial suspension was pretreated for 1 min with either 4 μ
m
CAT or BKA prior to the start of recording and additions of PAO (20 μ
m
) or Ca2+ (total concentration of 1.4 m
m
to give 180 μ
m
free [Ca2+]) to the sample cuvette as indicated. In B, the mitochondrial suspension was pretreated with PAO (20 μ
m
) for 1 min prior to the start of recording and additions of BKA, CAT, and Ca2+ made at the concentrations used in A. In the inset, rat liver mitochondria (5 mg/ml) were pretreated with 0.1 m
m
PAO, 20 μ
m
CAT, or 20 μ
m
BKA for 10 min at room prior to solubilization with Triton X-100 and application to the PAO affinity matrix as described under “Experimental Procedures.” After extensive washing, the bound proteins were eluted with dithiothreitol, separated by SDS/PAGE and bound ANT revealed by Western blotting with the ANT antibodies shown.
FIGURE 3.
Pretreatment of mitochondria with CAT or BKA does not prevent PAO from overcoming the inhibition of MPTP opening by ADP. Mitochondria were preswollen in KSCN buffer as described under “Experimental Procedures” in the absence and presence of 5 μ
m
CAT, 20 μ
m
PAO or both as indicated. When present, CAT was added 1 min prior to addition of 20 μ
m
PAO. The PEG shrinkage technique was then used to determine the extent of MPTP opening in the presence of 70 μ
m
free [Ca2+] and ADP at the concentration indicated as described under “Experimental Procedures.” Rates of shrinkage have been expressed as a percentage of the rate in the absence of ADP derived by differentiation of the traces.
FIGURE 4.
The mitochondrial phosphate carrier binds to a PAO column. Inner mitochondrial membranes prepared from control and CAT-pretreated beef heart mitochondria were solubilized at 10 mg/ml in PCB buffer containing 3% (w/v) Triton X-100 and applied to and eluted from PAO affinity matrix as described in the legend to Fig. 2. Proteins were separated by SDS/PAGE and revealed by Sypro-Ruby stain. The proteins eluted from the CAT pretreated fraction were identified by mass spectrometry as (from order of highest molecular weight): Ubiquinol-cytochrome_c_ reductase complex core protein 2 mitochondrial precursor (QCR2_BOVIN); phosphate carrier protein (PiC, C53737); adenylate kinase-2 (AK-2, B29792); NIPSNAP-2 (Q3SWX4_BOVIN). The presence of PiC and AK-2 and the absence of ANT were confirmed by Western blotting. Further information from mass spectrometry analysis is given in supplemental Table S1.
FIGURE 5.
The mitochondrial phosphate carrier binds to GST-CyP-D in a CsA-sensitive manner. Rat liver mitochondria (5 mg/ml) were pretreated with 5 μ
m
CsA, 10 μ
m
CAT, or 1 m
m
diamide, alone or in combination, for 10 min at room temperature prior to preparation of IMMs, solubilization with Triton X-100 and passage through a GST-CyP-D affinity column as described under “Experimental Procedures.” After elution of bound proteins with glutathione, samples were separated by SDS/PAGE and analyzed by Western blotting with PiC and ANT antibodies as shown. Where indicated, the GST-CyP-D column was preincubated with 25 μ
m
CsA before addition of the solubilized IMMs containing 2 μ
m
CsA.
FIGURE 6.
CsA-sensitive co-immunoprecipitation of CyP-D with the PiC and ANT. Mitochondria (2 mg/ml) were treated at room temperature with 2 μ
m
CsA or 2 μ
m
CAT for 10 min, before their solubilization and incubation with immunoprecipitating antibodies raised against the C terminus of the PiC (A) or ANT (B), as described under “Experimental Procedures.” Where CsA and CAT were added in combination, mitochondria were incubated with CsA for 10 min at 22 °C before the addition of CAT and further incubation for 10 min. Immunocomplexes were separated by SDS/PAGE and analyzed by Western blotting with the antibodies indicated.
FIGURE 7.
Concentration dependence of the inhibition of MPTP opening and mitochondrial phosphate transport by _N_-ethylmaleimide. In_B_, phosphate transport was assayed according to the scheme shown in_A_ by the swelling of rat liver mitochondria (1 mg/ml) in de-energized buffer as described under “Experimental Procedures.” When present, CsA (0.5 μ
m
) was added 1 min before 40 m
m
NaPi addition whereas NEM (20 nmol/mg protein) was added to mitochondria (40 mg/ml) for 2 min at 0 °C before their dilution to 1 mg/ml in assay buffer and the initiation of swelling by addition of 40 m
m
NaPi. In C, MPTP opening in response to 80 μ
m
free [Ca2+] was assayed in parallel under similar de-energized conditions (see “Experimental Procedures”). D shows data for phosphate transport following treatment with increasing concentrations of NEM while_E_ shows the same data corrected by subtraction of the maximally inhibited trace as described in the text. To calculated initial rates of transport, first order regression analysis was performed using GraphPad Prism to the equation A_520 = initial rate/k × exp(–_kt) + plateau, where k is the rate constant,t is time in seconds, and plateau the final _A_520 once swelling is completed. The initial rates were used to calculate the percentage inhibition of transport at each NEM concentration and mean data (±S.E.) of three separate experiments are given in F (squares) where data for MPTP inhibition are also given (circles).
FIGURE 8.
Concentration dependence of the inhibition of MPTP opening and mitochondrial phosphate transport by UQo and Ro 68-3400. Experiments were performed as described in the legend to Fig. 7. A shows light scattering traces for phosphate transport following treatment with increasing concentrations of Ro 68-3400 for 5 min prior to addition of 40 m
m
Pi to initiate transport. These data and similar data for UQo were corrected by subtraction of the maximally inhibited trace and initial rates determined in order to calculate the percentage inhibition of transport as described in the legend to Fig. 7. Mean data (±S.E.) of three separate experiments are given (closed squares) for Ro 68-3400 in B and for UQo in_C_. Parallel data for MPTP inhibition are also presented (closed circles).
FIGURE 9.
Ubiquinone analogues inhibit PiC and ANT binding to the PAO column and PAO activation of MPTP opening. In A, rat liver mitochondria (5 mg/ml) were pretreated with 0.1 m
m
PAO, 20 μ
m
CAT, 20 μ
m
BKA, 50 μ
m
UQo, or 20 μ
m
Ro 68-3400 for 10 min at room temperature prior to solubilization with Triton X-100 and application to a PAO column. Subsequent washing and elution of bound proteins was performed as described under “Experimental Procedures.” In B, MPTP opening was assayed under de-energized conditions in KSCN buffer as described in the legend to Fig. 2. For the top 6 traces, the mitochondrial suspension was pretreated for 1 min with 50 μ
m
UQo, 20 μ
m
Ro 68-3400, or without further addition prior to the start of recording and subsequent additions of PAO (20 μ
m
) or Ca2+ (180 μ
m
free [Ca2+]) to the sample cuvette. In the bottom 3 traces, the mitochondrial suspension was pretreated with PAO (20 μ
m
) for 1 min prior to the start of recording and additions of 20 μ
m
UQo, 20 μ
m
Ro 68-3400, and 180 μ
m
Ca2+.
FIGURE 10.
Ubiquinone analogues induce the m conformation of the ANT at similar concentrations to those inhibiting MPTP opening. In A and_B_, conformational changes of energized beef heart mitochondria were detected in a split-beam spectrophotometer at 30 °C as described under “Experimental Procedures.” Where indicated, additions were made to the sample cuvette as follows: Ca2+ (0.47 m
m
total to give 1 μ
m
free [Ca2+]), ADP (0.2 m
m
), CAT (10 μ
m
), and BKA, Ro 68-3400, and UQ0 at the concentrations indicated. In C, the change in_A_520 induced by increasing concentrations of Ro 68-3400 were determined from the traces shown in B.
FIGURE 11.
Ro 68-3400 inhibits transport of ADP by the ANT. Respiration was measured in rat liver mitochondria suspended at 0.5 mg/ml in mildly hypotonic KCl buffer (see “Experimental Procedures”) supplemented with substrates, either 5 m
m l
-glutamate + 2 m
m l
-malate (A) or 5 m
m
succinate + 0.5 μ
m
rotenone (B). Where indicated, additions were made as follows: 1 m
m
ADP, 5 μ
m
BKA, 50 μ
m
Ro 68-3400, and 40 n
m
FCCP.
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