Chapter 10 Purification of the cytochrome C reductase/cytochrome C oxidase super complex of yeast mitochondria.
The protein complexes of the respiratory chain interact by forming large protein particles called respiratory supercomplexes or "respirasomes". Biochemical characterization of these particles proved to be difficult because of their instability. Here we describe a strategy to isolate and characterize the cytochrome c reductase/cytochrome c oxidase supercomplex of yeast, also termed the III + IV supercomplex, which is based on lactate cultivation of yeast, gentle isolation of mitochondria, membrane solubilization by digitonin, sucrose gradient ultracentrifugation, and native gel electrophoresis. The procedure yields pure forms of two varieties of the III + IV supercomplex composed of dimeric complex III and one or two copies of monomeric complex IV. Supercomplex preparations can be used for physiological or structural investigations.
Braun
Hans-Peter
HP
Institute for Plant Genetics, Faculty of Natural Sciences, Universität Hannover, Hannover, Germany.
Sunderhaus
Stephanie
S
Boekema
Egbert J
EJ
Kouril
Roman
R
D003470:Culture Media; D003576:Electron Transport Complex IV / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D008928:Mitochondria / Q000201:enzymology*; D010088:Oxidoreductases / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014461:Ultracentrifugation
D003470:Culture Media; D010088:Oxidoreductases; D003576:Electron Transport Complex IV