Abstract
Purified mitochondrial ATP synthase has been shown to form Ca2+-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore (mPTP) but the oligomeric state required for channel formation is being debated. We reconstitute purified monomeric ATP synthase from porcine heart mitochondria into small unilamellar vesicles (SUVs) with the lipid composition of mitochondrial inner membrane and analyze its oligomeric state by electron cryomicroscopy. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86o angle relative to the first. We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca2+-activated channels with the key features of mPTP. Based on our findings we conclude that the ATP synthase monomer is sufficient, and dimer formation is not required, for mPTP activity.
Highlights
Purified mitochondrial ATP synthase has been shown to form Ca2+-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore but the oligomeric state required for channel formation is being debated
The mitochondrial permeability transition was first found to be correlated with mitochondrial inner membrane channel activity several decades ago: patch-clamp recordings of mitoplasts showed that the inner membrane contains a nonselective, high-conductance megachannel, which is sensitive to calcium, adenine nucleotides, and suspected to be the mPT pore[9,10,11,12,13,14]
Despite extensive research in the field, the exact molecular composition of the mitochondrial permeability transition pore (mPTP) is still mysterious. mPTP was suggested previously to form from different mitochondrial inner membrane proteins including the adenine nucleotide translocator (ANT), the voltage-dependent anion channel (VDAC) and the phosphate carrier (PiC)[16,17,18,19]
Summary
Purified mitochondrial ATP synthase has been shown to form Ca2+-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore (mPTP) but the oligomeric state required for channel formation is being debated. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86o angle relative to the first We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca2+-activated channels with the key features of mPTP. Despite its role as the main energy-producing enzyme, ATP synthase has been reported recently to play a role in the mitochondrial inner membrane depolarization caused by the channel activity of the mitochondrial permeability transition pore (mPTP)[5,6,7,8]. Recent reports have shown that purified and detergent-solubilized dimers of F1FO ATP synthase, when incorporated into lipid bilayers, form Ca2+-activated channels, with the key features of the megachannel associated with the mPT5,8. The question still remains open whether the ATP synthase dimer or the monomer alone can form a channel
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