Fig. 4. Cycloheximide treatment did not alleviate mitochondrial aggregation. (A) Wild-type (SSC1) and the temperature sensitive mtHsp70 mutant (ssc1-2 and ssc1-3) cells were grown at 23°C in YPD medium to an early log phase. The culture was split into two and one half received 0.1 mg/ml of cycloheximide (+ CHX). Aliquots were taken at the indicated times after temperature shift to 37°C and cell extracts were prepared, which were then analyzed by SDS-PAGE and immunoblotting using anti-F₁ß antibodies. m and p shows the mature and the precursor forms of F₁ß, respectively. (B) Wild-type (SSC1, panels a-c) and the temperature sensitive mtHsp70 mutant (ssc1-2, panels d-f, ssc1-3, panels g-i) cells were grown at 23°C in YPD medium to an early log phase. Cycloheximide was added to each culture to 0.1 mg/ml. Aliquots from the cells grown at 23°C were shifted to 37°C for the periods indicated at the left of the panels. Cells were fixed and prepared for immunofluorescence microscopy with anti-Tom40 antibodies. Bar, 2 µm. (C) Percentage of cells containing aggregated mitochondria was plotted against time of incubation at 37°C. At least 150 cells were examined for each time point. , wild type (SSC1); , ssc1-2 mutant (ssc1-2); , ssc1-3 mutant (ssc1-3).