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.
Highlights
Tel.: 44-117-3312118; Fax: 44-117-3312168; E-mail: a.halestrap@ bristol.ac.uk. 3 The abbreviations used are: MPTP, mitochondrial permeability transition pore; AK2, adenylate kinase 2; ANT, adenine nucleotide translocase; BKA, bongkrekic acid; BSA, bovine serum albumin; CAT, carboxyatractyloside; kDa, which opens in the inner mitochondrial membrane (IMM) under conditions of calcium overload
Cyclosporin A (CsA), first shown to inhibit pore opening by Crompton et al [10], was the key to identifying the role for CyP-D, and we subsequently showed that the potency of several CsA analogues as inhibitors of the pore correlated with their ability to inhibit the activity of a matrix peptidyl-prolyl cis-trans isomerase (PPIase) [11]
We show that phenylarsine oxide (PAO), N-ethylmaleimide (NEM), and ubiquinone analogues, all known modulators of MPTP opening, can bind to both proteins
Summary
Materials—All reagents were obtained from Sigma unless otherwise stated. Ro 68-3400 was a kind gift of Hoffmann-La Roche Ltd, Basel, Switzerland. Following addition of mitochondria (7 mg of protein) to 7 ml of assay buffer, aliquots (3.5 ml) were incubated at 25 °C (MPTP assay) or 6 °C in the sample and reference cuvettes of a split-beam spectrophotometer for 5 min. Preswollen mitochondria (1 mg) were incubated in the sample cuvette of the split-beam spectrophotometer at 25 °C in 3 ml of KSCN buffer supplemented with 2 mM NTA, 2 M A23187, and the required concentration of Ca2ϩ and ADP. 2 and 9) IMMs were solubilized at 0.5 mg/ml in PCB containing 0.5% (w/v) Triton X-100 for 15 min at 4 °C and following removal of insoluble material a sample (1 ml) was incubated with 100 l of PAO beads (50% slurry) for 30 min at 4 °C. Thereafter, the purification followed the procedure described by Smith et al [36] with anion and cation exchange chromatography performed on an AKTA prime system (GE Healthcare UK Ltd, Little Chalfont, HP7 9NA, UK) using 1 ml of RESOURCETM Q and RESOURCETM S columns, respectively
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