Figure 5.

EM visualization of rat heart mitochondria FAO–ETC protein complexes. A, FAO–digitonin-permeabilized rat heart mitochondria were separated by a sucrose gradient centrifugation, and the gradient fractions were analyzed by BN-PAGE. The left panel shows a protein stain of the gel, and the right panel shows a complex I activity stain. The complex I and supercomplex fractions of the sucrose gradient were combined for EM analysis. B, EM grid with negative staining shows that there are variable forms of SC particles. C, panel a, particles are the main supercomplex form identified, representing one molecule each of complexes I, III, and IV. Panel b, additional particle binds on top of the short arm of complex I in some particles (as indicated by arrowheads). Panel c, extra particle also is seen binding to the top of complex III. In the final image in this row, both extra masses are seen simultaneously with the short (matrix) arm of complex I and the top of complex III (as indicated by arrowheads). Schematics to the right of each image stylize the three types of association with blue representing complex I; pink representing complex III; and orange representing complex IV. D, gold-labeled antisera as indicated to the left of the figure were incubated with supercomplexes on EM grids and then imaged. The black dots are the gold particles, and a summary of each set of binding experiments is cartooned to the right of the images.