Figure 3.

Coimmune precipitation of the COX3 mRNA-specific activator proteins Pet54p, Pet122p, and Pet494p. Purified mitochondria containing the indicated epitope-tagged proteins were treated with 1 mM DSP, a membrane-permeable cross-linker at 25°C for 30 min followed by solubilization with 1% digitonin at 4°C for 30 min (see MATERIALS AND METHODS). After a clarifying spin, solubilized extracts (from 400 μg of mitochondria) were incubated with anti-HA affinity matrix (A) or with anti-Myc-agarose conjugate (B) for 4 h at 4°C under gentle shaking conditions and immunoprecipitates were analyzed by Western blotting with anti-HA-HRP and anti-Myc-HRP (see MATERIALS AND METHODS). Solubilized mitochondrial proteins (20 μg) were loaded in total extract lanes. Strains used were SN25, SN28, and CAB30 (Table 1).