Abstract
Yeast mitochondria (Saccharomyces cerevisiae) contain a permeability transition pore which is regulated differently than the pore in mammalian mitochondria. In a mannitol medium containing 10 mM Pi and ethanol (oxidizable substrate), yeast mitochondria accumulate large amounts of Ca2+ (>400 nmol/mg of protein) upon the addition of an electrophoretic Ca2+ ionophore (ETH129). Pore opening does not occur following Ca2+ uptake, even though ruthenium red-inhibited rat liver mitochondria undergo rapid pore opening under analogous conditions. However, a pore does arise in yeast mitochondria when Ca2+ and Pi are not present, as monitored by swelling, ultrastructure, and matrix solute release. Pore opening is slow unless a respiratory substrate is provided (ethanol or NADH) but also occurs rapidly in response to ATP (2 mM) when oligomycin is present. Pi and ADP inhibit pore opening (EC50 approximately 1 and 4 mM, respectively), however, cyclosporin A (7 microg/ml), oligomycin (20 microg/ml), or carboxyatractyloside (25 microM) have no effect. The pore arising during respiration is also inhibited by nigericin or uncoupler, indicating that an acidic matrix pH antagonizes the process. Pi also inhibits pore opening by lowering the matrix pH (Pi/OH- antiport). However, inhibition of the ATP-induced pore by Pi is seen in the presence of mersalyl, suggesting a second mechanism of action. Since pore induction by ATP is not sensitive to carboxyatractyloside, ATP appears to act at an external site and Pi may antagonize the interaction. Isoosmotic polyethylene glycol-induced contraction of yeast mitochondria swollen during respiration, or in the presence of ATP, is 50% effective at a solute size of 1.0-1.1 kDa. This suggests that the same pore is induced in both cases and is comparable in size with the permeability transition pore of heart and liver mitochondria.
Highlights
Combination with a second agent (e.g. Pi or an oxidant of matrix space components) is required for pore opening in most cases
Pi, which promotes opening of the mPTP, is an inhibitor of the yPTP through an action exerted via the matrix pH, and through a second action exerted at a site that is probably accessible from the extramitochondrial volume
Ca2ϩ Uptake by Isolated yeast mitochondria (yMt)—Intramitochondrial Ca2ϩ has a central role in regulating the mPTP [1, 3]; yMt lack a high activity Ca2ϩ uniporter [20, 33]
Summary
Combination with a second agent (e.g. Pi or an oxidant of matrix space components) is required for pore opening in most cases. The release of Ca2ϩ from rat liver mMt under either condition is accompanied by swelling and is inhibited by CsA (data not shown), indicating that the mPTP is responsible.
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