The c subunit is an inner mitochondrial membrane (IMM) protein encoded by three nuclear genes. Best known as an integral part of the F0 complex of the ATP synthase, the c subunit is also present in other cytoplasmic compartments in ceroid lipofuscinoses. Under physiological conditions, this 75 residue-long peptide folds into an α-helical hairpin and forms oligomers spanning the lipid bilayer. In addition to its physiological role, the c subunit has been proposed as a key participant in stress-induced IMM permeabilization by the mechanism of calcium-induced permeability transition. However, the molecular mechanism of the c subunit participation in IMM permeabilization is not completely understood. Here we used fluorescence spectroscopy, atomic force microscopy and black lipid membrane methods to gain insights into the structural and functional properties of unmodified c subunit protein that might make it relevant to mitochondrial toxicity. We discovered that c subunit is an amyloidogenic peptide that can spontaneously fold into β-sheets and self-assemble into fibrils and oligomers in a Ca2+-dependent manner. C subunit oligomers exhibited ion channel activity in lipid membranes. We propose that the toxic effects of c subunit might be linked to its amyloidogenic properties and are driven by mechanisms similar to those of neurodegenerative polypeptides such as Aβ and α-synuclein.

The c subunit of the ATP synthase is an inner mitochondrial membrane (IMM) protein. Besides its role as the main component of the rotor of the ATP synthase, c subunit from mammalian mitochondria exhibits ion channel activity. In particular, c subunit may be involved in one of the pathways leading to the formation of the permeability transition pore (PTP) during mitochondrial permeability transition (PT), a phenomenon consisting of the permeabilization of the IMM due to high levels of calcium. Our previous study on the synthetic c subunit showed that high concentrations of calcium induce misfolding into cross-β oligomers that form low-conductance channels in model lipid bilayers of about 400 pS. Here, we studied the effect of cyclophilin D (CypD), a mitochondrial chaperone and major regulator of PTP, on the electrophysiological activity of the c subunit to evaluate its role in the functional properties of c subunit. Our study shows that in presence of CypD, c subunit exhibits a larger conductance, up to 4 nS, that could be related to its potential role in mitochondrial toxicity. Further, our results suggest that CypD is necessary for the formation of c subunit induced PTP but may not be an integral part of the pore.

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37The c subunit is an inner mitochondrial membrane (IMM) protein and is an integral part of the 38 F0 complex of the ATP synthase. Under physiological conditions, this short 75 residue-long 39 peptide folds into an a-helical hairpin and forms oligomers spanning the lipid bilayer. In 40 addition to its physiological role, the c subunit has been proposed as a key participant in stress-41 induced IMM permeabilization by the mechanism of calcium-induced permeability transition. 42 However, the molecular mechanism of the c subunit participation in IMM permeabilization is 43 not completely understood. Here we used fluorescence spectroscopy, atomic force microscopy 44 and black lipid membrane methods to gain insights into the structural and functional properties 45 of c subunit protein that make it relevant to mitochondrial toxicity. We discovered that c 46 subunit is an amyloidogenic peptide that can spontaneously fold into b-sheets and self-47 assemble into fibrils and oligomers. C subunit oligomers exhibited ion channel activity in lipid 48 membranes. We propose that the toxic effects of c subunit might be linked to its amyloidogenic 49properties and are driven by mechanisms similar to those of neurodegenerative polypeptides 50 such as Aβ and a-synuclein. 51 52 53