Fig 3.
Cytochrome c oxidase (COX) assembly in yeast strains is affected in heme biogenesis. (A) Alignment of a region in Cox15 homologues from different species (ClustalW2). Black boxes indicate identical residues in all species shown. The arrowhead indicates a conserved histidine at position 216 in B. subtilis, corresponding to H368 in S. cerevisiae. (B) Growth test on fermentable or nonfermentable medium. cox15Δ cells expressing either wild-type Cox15 (Cox15WT) or a catalytically inactive Cox15 (Cox15H368M) were spotted in serial 10-fold dilutions and incubated at the indicated temperatures (—, plasmid without insert). (C and D) For analysis of steady-state protein levels, mitochondria (Mito) isolated from the indicated strains were separated by SDS-PAGE or BN-PAGE and analyzed by Western blotting. (E) Coimmunoprecipitation of Coa3 from digitonin-solubilized mitochondria isolated from the indicated strains. Solubilized mitochondria were incubated with Coa3-specific or control antisera, and eluates were analyzed by SDS-PAGE and Western blotting. The amounts of protein loaded in the total samples correspond to 8% of the eluate.