The yeast mitochondrial permeability transition is regulated by reactive oxygen species, endogenous Ca2+ and Cpr3, mediating cell death

Abstract

We investigated the properties of the permeability transition pore (PTP) in Saccharomyces cerevisiae in agar-embedded mitochondria (AEM) and agar-embedded cells (AEC) and its role in yeast death. In AEM, ethanol-induced pore opening, as indicated by the release of calcein and mitochondrial membrane depolarization, can be inhibited by CsA, by Cpr3 deficiency, and by the antioxidant glutathione. Notably, the pore opening is inhibited, when mitochondria are preloaded by EGTA or Fluo3 to chelate matrix Ca, or are pretreated with 4-Br A23187 to extract matrix Ca, prior to agar-embedding, or when pore opening is induced in the presence of EGTA; opened pores are re-closed by sequential treatment with CsA, 4-Br A23187 plus EGTA and NADH, indicating endogenous matrix Ca involvement. CsA also inhibits the pore opening with low conductance triggered by exogenous Ca transport with ETH129. In AEC, the treatment of tert-butylhydroperoxide, a pro-oxidant that triggers transient pore opening in high conductance in AEM, induces yeast death, which is also dependent on CsA and Cpr3. Furthermore, AEMs from mutants lacking three ADP/ATP carrier (AAC) isoforms and with defective ATP synthase dimerization exhibit high and low conductance pore openings with CsA sensitivity, respectively. Collectively, these data show that the yeast PTP is regulated by Cpr3, endogenous matrix Ca, and reactive oxygen species, and that it is involved in yeast death; furthermore, ATP synthase dimers play a key role in CsA-sensitive pore formation, while AACs are dispensable.

Bisindolylpyrrole triggers transient mitochondrial permeability transitions to cause apoptosis in a VDAC1/2 and cyclophilin D-dependent manner via the ANT-associated pore

“…Time course experiments were then performed over 20 min with mitochondria that were immobilized in agar 45 As shown in Fig. 4 Bi and iii, upon BP treatment, the TMRM signals started to drop within 2–5 min, and calcein release occurred over 15 min; both effects were completely blocked by 3 μM CsA (Fig.…”

“…Agar-embedded mitochondria were prepared 45 : aliquots (5 μl) of mitochondrial suspensions, which were prepared by diluting calcein-loaded mitochondria 4 times in assay buffer containing DMSO, 3 μM CsA, 50 μM bongkrekate (Calbiochem) or 50 nM tubulin (Cytoskeleton), were mixed with 5 μl of 2% (w/v) type VII agarose in assay buffer (37 °C), with half of these mixtures placed in circular areas with a diameter of 15 mm circumscribed with a PAP pen on a 24 mm × 32 mm cover glass. After solidification, agar was covered with 0.3 ml of assay buffer.…”

Properties of the Permeability Transition of Pea Stem Mitochondria

“…Nevertheless, Drosophila appears not to contain a mitochondrial cyclophilin and the mitochondrial CyP isoform in S. cerevisiae appears not to regulate the PTP, consistently with the insensitivity of the PTP of both organisms to CsA (Bernardi et al, 2015). It should be mentioned that agar-embedded yeast mitochondria display a CsA-sensitive PT, adding a further layer of complexity to the known factors of PTP modulation (Kamei et al, 2018). We suggest that in pea mitochondria increasing Pi concentrations lead to decreased matrix free Ca 2+ (Zoccarato and Nicholls, 1982), which prevails on any PTP-promoting effect of Pi.…”

“…The physical components of PTP that were tentatively proposed in the past are the voltage-dependent anion channel (VDAC), the benzodiazepine receptor, the adenine nucleotide translocase (ANT) and the phosphate carrier. However, isolated mitochondria from different organisms, in which the expression of each of these proteins was suppressed, still showed PT (Kokoszka et al, 2004; Krauskopf et al, 2006; Baines et al, 2007; Gutiérrez-Aguilar et al, 2014; Šileikytė et al, 2014; Kamei et al, 2018), indicating that they are not essential for the PT to occur. Recent evidence shows that, in addition to their enzymatic and structural roles, F-ATP synthase may be involved in PTP formation in mammalian (Giorgio et al, 2013; Alavian et al, 2014), Saccharomyces cerevisiae (Carraro et al, 2014; Kamei et al, 2018) and Drosophila melanogaster (von Stockum et al, 2015) mitochondria.…”

Role ofCNC1gene in TDP-43 aggregation-induced oxidative stress-mediated cell death inS. cerevisiaemodel of ALS

“…For this, the yeast cell suspensions expressing URA3 marked plasmid encoded TDP-43-YFP or vector were prepared as described earlier [67]. First, yeast cultures were grown overnight in SRaf-Ura and inoculated into fresh SGal-Ura for the overexpression of the TDP-43-YFP for 24 h. Then, the cells were pelleted and re-suspended in 100 μL of water and incubated for 1 h with 1.5 μM CellROX deep red in dark.…”