FIGURE 6.

Mne1 is a component of RNase-sensitive high molecular weight complex containing aI5β intron. A, mitochondria (50 μg) purified from the WT MNE1::Myc strain were lysed in 1% digitonin and clarified lysates were treated with either 4 units of RNase A or Turbo-DNase, or left untreated. Following a 30-min incubation at room temperature, samples were subjected to BN-PAGE and analyzed by Western blot with antibodies against Myc and F₁. B, distribution of Mne1-Myc containing complexes in MNE1::Myc strain with (rho⁺) and without (rho^O) mitochondrial DNA analyzed by native electrophoresis as described above. Anti-porin antibody was used to detect the respective complex that served as a loading control. C, steady-state levels of Mne1-Myc in the strains with and without mitochondrial DNA were assessed by immunoblotting as described in the legend to Fig. 2D. D, clarified lysates obtained from 450 μg of untagged or MNE1::Myc mitochondria with and without introns were immunoprecipitated with goat polyclonal anti-Myc beads under RNA-protecting conditions. Half of the entire fraction of each respective bead eluate was analyzed by immunoblotting with anti-Myc antibodies; the second half served as a template for an RT-PCR analysis with the primers to aI5β intron or ligated COX1 E5β-E6γ exon. The PCR products were amplified with 30 and 24 reaction cycles, respectively. E, BN-PAGE analysis of the Mne1 complexes in mitochondria with and without introns was performed as described above.