FIG. 1.

Kinetics of disappearance of 60S ribosomal subunits and inhibition of protein synthesis in eIF6-depleted cells. (A) Exponentially growing cultures of KSY603[GAL10::Ub-HA-TIF6] cells in SGal−Met medium were transferred to SD−Met medium and allowed to grow. At the indicated times, cell lysates were prepared from 1 A₆₀₀ unit of cells, and approximately 100 μg of protein from each cell lysate was analyzed by Western blotting using monoclonal anti-HA antibodies as probes. (B) Total ribosomes were isolated from KSY603 (●) and control wild-type (■) cells at indicated times following a shift of each culture from SGal-Met to SD-Met medium. Ribosomes were dissociated into subunits and sedimented through 15 to 40% sucrose gradients as described in Materials and Methods and in reference 29. (C) Rates of protein synthesis. At the indicated times following shift of cells from SGal-Met to SD-Met medium, 1 A₆₀₀ unit of cells from each culture was harvested and suspended in 300 μl of SD-Met medium containing 0.5 μCi of [³⁵S]methionine (1,175 Ci/mmol), and cells were shaken for 5 min at 30°C. The pulse was then terminated by the addition of a stop buffer containing unlabeled methionine (1.8 mg/ml) and cycloheximide (50 μg/ml). The rate of protein synthesis at each time point was calculated as described previously (29). The data presented in panels B and C are averages of two independent experiments.