Fig. 1
Characterization of bovine F-ATP synthase. a Clear-native gel electrophoresis indicates the presence of monomeric, dimeric, and oligomeric bovine F-ATP synthase, and the absence of subcomplexes or other smaller protein complexes. Lane M: molecular weight markers (kDa); Lane 1: F-ATP synthase (30 μg). b Negative stain EM documents the presence of bovine F-ATP synthase monomers (blue rectangle), dimers (red circle), and tetramers (yellow rectangle). Scale bar 50 nm. c Subunit composition was determined by denaturing SDS gel electrophoresis. All expected subunits were detected, including the very weakly associated 6.8PL and DAPIT, see also data from mass spectrometry (Supplementary Table 1). Lane M: molecular weight markers (kDa); Lane 1: F-ATP synthase (30 μg). d, e NADH oxidation-coupled enzymatic assay showing that soluble F-ATP synthase (bFOF1 Sol, 10 μg added where indicated) has ATPase activity (addition of ATP 2.5 mM where indicated) that can be fully inhibited by oligomycin A (2 μM where indicated). Traces are representative of three independent experiments. f Cryo-EM image of a bovine F-ATP synthase proteoliposome. The clearly visible tram-track features (red double arrow) confirm the formation of lipid bilayers, and F1 domains protruding out of the membrane are easy to identify at the edge of the proteoliposome (white arrowheads). Scale bar 50 nm. g Negative stain EM of liposomes before and h after reconstitution of bovine F-ATP synthase. After reconstitution, F1 domains (open triangles) can be clearly distinguished from naked liposomes (closed triangles). Scale bar, 50 nm. i, j Nine-hundred and ninety-nine liposomes from 19 electron microscopy images taken after incorporation of F-ATP synthase were scored for the presence of F1 and their size measured with the aid of ImageJ software. Source data for panels d, e, i, j are provided as a Source Data file
