Figure 6.
Single particle electron microscopic analysis of the prohibitin complex. (A) Purification of prohibitin complexes from Δphb1 mitochondria harboring overexpressed Phb1His and Phb2 by metal chelating chromatography and glycerol gradient centrifugation as described in Materials and Methods. Samples were analyzed by SDS-PAGE and silver staining (left) or immunodetection against mitochondrial proteins as indicated (right). Prohibitin-containing fractions are shown. Lane 1, mitochondrial membranes (2 μg [silver staining] or 10 μg [immunodetection]); lane 2, eluate of Ni-NTA chromatography (1/200 or 1/40 of total); lane 3, prohibitin-containing fraction after glycerol gradient centrifugation (corresponding to a glycerol concentration of 24-26%; 1/40 or 1/8 of total). (B) Representative section of a micrograph showing a glycerol gradient fraction enriched in prohibitin complexes stained with 1% phosphotungstate. Bar, 200 nm. (C) Class averages of typical end-on or side-on projection views from prohibitin complexes. The data set was split in two subsets according to either elliptical ring-like (32%) or more rectangular views (68%). Percentages of particles of corresponding average images 1-5 are given as follows: for subset I (top), set to 100% class 1 counts 9%, class 2 6%, class 3 16%, class 4 12%, and class 5 11% of particles; for subset II (bottom), set to 100%, class 6 counts 17%, class 7 17%, class 8 8%, class 9 13% and class 10 8% of particles. Bar, 200 Å.