Effect of prooxidants on yeast mitochondria.

Tightly coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts were used in this study. The two yeasts are aerobes containing the fully competent respiratory chain with three energy conservation sites. Interaction of the yeast mitochondria with prooxidants (diamide, menadione, oxaloacetate, phenylarsine oxide, hydrogen peroxide, t-butyl peroxide, and ascorbate plus Fe(2+)) was studied. The prooxidants, depending on their chemical nature, either caused uncoupling (e.g., activated state 4 respiration) or inhibited oxidation of respiratory substrates. All of the agents dissipated the membrane potential without megachannel formation (no large-scale swelling of mitochondria was observed). Except for combined application of ascorbate and Fe(2+), the prooxidant-induced decrease in the membrane potential was specifically prevented by ATP, even in the cases when classic antioxidants, e.g., N-acetylcysteine, were ineffective. No permeabilization of yeast mitochondria was observed under concerted action of prooxidants and Ca(2+), suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with Ca(2+) uptake.

Mitochondria from Dipodascus (Endomyces) magnusii and Yarrowia lipolytica yeasts did not undergo a Ca²âº-dependent permeability transition even under anaerobic conditions.

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts. The two yeast strains are good alternatives to Saccharomyces cerevisiae, being aerobes containing well structured mitochondria (thus ensuring less structural limitation to observe their appreciable swelling) and fully competent respiratory chain with three invariantly functioning energy conservation points, including Complex I, that can be involved in induction of the canonical Ca²âº/P(i)-dependent mitochondrial permeability transition (mPTP pore) with an increased open probability when electron flux increases(Fontaine et al. J Biol Chem 273: 25734­25740, 1998; Bernardi et al. FEBS J 273:2077­2099, 2006). High amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating pore opening. Previously (Kovaleva et al. J Bioenerg Biomembr 41:239­249, 2009; Kovaleva et al. Biochemistry (Moscow) 75: 297­303, 2010) we have shown that mitochondria from Y.lipolytica and D. magnusii were very resistant to the Ca²âºoverload combined with varying concentrations of P(i),palmitic acid, SH-reagents, carboxyatractyloside (an inhibitor of ADP/ATP translocator), as well as depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high [P(i)]. Here we subjected yeast mitochondria to other conditions known to induce an mPTP in animal and plant mitochondria, namely to Ca²âº overload under hypoxic conditions (anaerobiosis). We were unable to observe Ca²âº-induced high permeability of the inner membrane of D. magnusii and Y. lipolytica yeast mitochondria under anaerobic conditions, thus suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with the Ca²âº uptake. The results provide the first demonstration of ATP-dependent energization of yeast mitochondria under conditions of anaerobiosis.