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. 2011 Jan 21;286(12):10137–10146. doi: 10.1074/jbc.M110.205625

FIGURE 3.

FIGURE 3.

The absence of Mne1 affects early steps of Cox1 biogenesis in the strains containing mitochondrial introns. A, in vivo labeling of mitochondrial translation products in WT and mne1Δ cells with or without ([Δi]) mitochondrial introns. Cells were pulsed for 15 min with [35S]methionine at 30 °C. The reaction was stopped by addition of cold methionine; following a 60-min chase at 30 °C samples were subjected to SDS-PAGE and analyzed by autoradiography. B, MNE1 was deleted in the arg8Δ strain with ectopic ARG8 replacing the COX1 codons (cox1Δ::ARG8m). The cells were grown in complete synthetic medium containing 2% glucose and 0.2× arginine (4 mg/liter), serially diluted, and spotted on glucose-containing SC with (+Arg) or without (−Arg) 1× arginine (20 mg/liter) and SC containing 2% glycerol/lactate. The plates were incubated at 30 °C for 2 (glucose plates) or 4 days (glycerol plates). The arg4 mutant strain was used as a control. C, BN-PAGE analysis of Cox1 early assembly intermediates. Mitochondria (30 to 50 μg) purified from the WT and mne1Δ strains containing a 13xMyc epitope-tagged version of MSS51 or COA1 were analyzed by native electrophoresis and immunoblot with anti-Myc antibodies. The monomeric form of complex V that served as a loading control was visualized with anti-F1 serum. D, steady-state levels of the indicated proteins in WT and mne1Δ mitochondria assessed by SDS-PAGE. The outer membrane protein porin, visualized by the respective antibody was used as a loading control. E, respiratory growth of mne1Δ strains with and without mitochondrial introns. Cells were handled as described in the legend to Fig. 1A, except that plates were incubated at 30 °C for 2 (glucose plates) or 4 days (glycerol plates). F, oxygen consumption by mne1Δ cells with or without ([Δi]) mitochondrial introns. Cells were grown overnight in liquid complete medium with 1% glucose and oxygen consumption was measured. The results are shown as a percentage of WT oxygen consumption and represent the averages of three independent experiments. Error bars indicate S.D.