Abstract
Although the mitochondrial permeability transition pore (PTP) is presumably formed by either ATP synthase or the ATP/ADP carrier (AAC), little is known about their differential roles in PTP activation. We explored the role of AAC and ATP synthase in PTP formation in Saccharomyces cerevisiae using bisindolylpyrrole (BP), an activator of the mammalian PTP. The yeast mitochondrial membrane potential, as indicated by tetramethylrhodamine methyl ester signals, dissipated over 2–4 h after treatment of cells with 5 μM BP, which was sensitive to cyclosporin A (CsA) and Cpr3 deficiency and blocked by porin1/2 deficiency. The BP-induced depolarization was inhibited by a specific AAC inhibitor, bongkrekate, and consistently blocked in a yeast strain lacking all three AACs, while it was not affected in the strain with defective ATP synthase dimerization, suggesting the involvement of an AAC-associated pore. Upon BP treatment, isolated yeast mitochondria underwent CsA- and bongkrekate-sensitive depolarization without affecting the mitochondrial calcein signals, indicating the induction of a low conductance channel. These data suggest that, upon BP treatment, yeast can form a porin1/2- and Cpr3-regulated PTP, which is mediated by AACs but not by ATP synthase dimers. This implies that yeast may be an excellent tool for the screening of PTP modulators.
Highlights
The permeability transition pore (PTP) has long been hypothesized to be formed by the voltage-dependent anion channel (VDAC) or the adenine nucleotide translocase (ANT); this hypothesis has been questioned based on gene knockout experiments with VDAC1/2/3 genes [9] and ANT1/2 genes [10]
It has been recognized that yeast PTP is not sensitive to cyclosporin A (CsA) [36,37] or the CypD homolog Cpr3 [38], which can function as a chaperone in protein folding in the mitochondrial matrix through catalyzing peptidyl-prolyl cis-trans isomerization [39], we showed that the swelling of agar-embedded yeast mitochondria is sensitive to CsA/Cpr3 and mediated by endogenous matrix Ca2+ [40]
In the preliminary experiments to elucidate the cytoprotective mechanism of these compounds, we found that the cytoprotective effect against Ca2+ -mediated oxidative cell death in mammalian cells could be counteracted, paradoxically, by CsA or CypD ablation; we hypothesized that the mechanism was associated with the PTP
Summary
The mitochondrial permeability transition pore (PTP) is a Ca2+ -dependent non-specific hydrophilic channel with an exclusion size of 1.5 kDa within the inner mitochondrial membrane, which leads to mitochondrial membrane depolarization and swelling [1,2,3] and is associated with cell death under a variety of pathologies [4]. The PTP has long been hypothesized to be formed by the voltage-dependent anion channel (VDAC) or the adenine nucleotide translocase (ANT); this hypothesis has been questioned based on gene knockout experiments with VDAC1/2/3 genes [9] and ANT1/2 genes [10].
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