Figure 2. Decreased NAC and ribosomes association with OM14Δ mitochondria.

(a) Cells were subjected to cellular fractionation and samples from the cytosolic (Cyt.) or mitochondrial (Mito.) fractions were subjected to western analysis with the indicated antibodies. The histogram presents the average of the ratio between NAC signals in the two fractions from several independent biological repeats (n), each entailing the entire procedure, from cells’ growth to western analysis. Error bars represent s.e.m. P value was calculated by independent-samples one-sided t-test. Normal distribution was verified by standard tests (either Shapiro–Wilk or Kolmogrov–Smirnov). (b) Purification of mitochondria depleted of ER or ribosomes. Following translational arrest, cell lysate (T) was prepared in the presence of EDTA and separated into cytosolic (C) and membrane (M) fractions by differential centrifugation. The M fraction was further separated through a sucrose gradient into six fractions. Aliquot from each fraction was subjected to western analysis with the indicated antibodies. (c) ER and ribosome-depleted mitochondria were isolated either from OM14⁺ or OM14Δ cells, incubated with ribosomes and subjected to centrifugation. Sup (S) and pellet (P) samples were analysed by western blot with the indicated antibodies. The arrow indicates the horizontal line in which the membrane was cut, to allow simultaneous incubation with the indicated primary antibodies. Also, unrelated lane was cropped between lanes 2 and 3. (d) Ribosomes with a nascent chain of mitochondria protein (MDH1) were incubated with mitochondria and separated to S and P, as above. Samples were analysed by western blot with the indicated antibodies. The presented panels are from the same image, that was cropped to remove unrelated lanes. Histograms present the results of three independent biological repeats and statistics were calculated as above. Y axis is the (P/P+S) signal for Rpl3, normalized to the signal of Tom20.