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. Author manuscript; available in PMC: 2013 Mar 7.
Published in final edited form as: Cell Metab. 2012 Mar 7;15(3):348–360. doi: 10.1016/j.cmet.2012.02.006

Figure 2. Rcf1 physically interacts with respiratory Complex III and IV.

Figure 2

(A) Mitochondria from an EV-containing wild-type strain or an rcf1Δ mutant strain expressing Rcf1-His6/HA3 were subjected to two-step purification. The final eluate was analyzed by silver staining. Discrete bands were excised and the protein identity was determined by mass spectrometry as indicated in the text. (B) Digitonin-solubilized mitochondria extracted from yeast strains either expressing Rcf1-His6/HA3 (+) or not (−) were immunoprecipitated using anti-HA antibody. One percent of the mitochondrial lysate and the final eluate were immunoblotted using anti-Cor1&Qcr2, Cyt1, Cox1, Cox2, Cox3, Cox4 and HA antibodies. * indicates IgG light chain. (C) Rcf1-His6/HA3 was purified from digitonin and DDM-solubilized mitochondria, respectively. The final eluate was visualized by silver stain and protein identities were determined by mass spectrometry. Cox3 is indicated based on SDS-PAGE/Western blot as it was not detected by mass spectrometry. (D) Mitochondria extracted from a strain harboring a plasmid expressing Rcf1 or Rcf1-His6/HA3 were solubilized by digitonin or DDM and subjected to BN-PAGE/Western blot. Rcf1, Complex III and Complex IV were immunoblotted by anti-HA, Cor1&Qcr 2 and Cox2 antibodies, respectively.