Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. Here we eject intact assemblies from membranes, without chemical disruption, and use mass spectrometry to define their composition. From E. coli outer membranes, we identify a chaperone-porin association and lipid interactions in the beta-barrel assembly machinery. Bridging inner and outer membranes we observe efflux pumps, and from inner membranes a pentameric pore of TonB, and the protein-conducting channel Sec YEG, in association with F1FO ATP-synthase. Intact mitochondrial membranes from Bos taurus yield respiratory complexes and fatty acid-bound dimers of the ADP/ATP transporter (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions and associated chaperones of the membrane proteome.

The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can transform the energy-conserving F-ATP synthase into an energy-dissipating device.