Figure 10.

Coa1 interacts with Shy1 and deletion of COA1 relieves the repression of Cox1 translation in shy1Δ cells. (A) Mitochondria (0.3 mg protein) from COA1-3HA or COA1-3HA SHY1-13Myc cells were solubilized in Tris 20 mM pH 7.4, 100 mM NaCl, 1 mM PMSF, 1% digitonin and clarified extracts were immunoprecipitated with rabbit polyclonal anti c-Myc antiserum and protein A agarose beads. The load representing 4% of the extracts and the entire fraction of the last wash and bead eluate were analyzed by immunoblotting. (B) Mitochondria (0.3 mg protein) from SHY1-13Myc or COA1-3HA, SHY1-13Myc cells were solubilized as in panel A. Clarified extracts were immunoprecipitated with rabbit polyclonal anti HA antiserum and protein A agarose beads and analyzed as in panel A. (C) In vivo labeling of mitochondrial translation products. Cells were incubated with ³⁵S-Met for 8 or 20 min before stopping the reaction. The samples were run on 12% SDS–PAGE, the gel was dried and exposed to autoradiographic film. (D) The gel from panel C and two other gels from similar experiments were quantified using the software Quantity One (Biorad). The intensity of the bands corresponding to Cox1, Cox2 and Cox3 were normalized to the intensities of the bands of Var1 and Cyt b. ^** Indicates a high statistical significance (P-value<0.0001) of the decrease of Cox1 translation in shy1Δ compared with WT. ^* Indicates statistical significance (P-value=0.0177) of the increase of Cox1 translation in coa1Δ shy1Δ compared with shy1Δ.