FIGURE 3.

eEF3 overexpression leads to reduced eIF2α phosphorylation. Yeast strains from Fig. 1 were grown to exponential phase and subjected to amino acid starvation by adding 3AT to a final concentration of 10 mm 20 min before harvesting (A and C) or by adding SM to a final concentration of 1.25 μm 15 min or 1 h before harvesting (E). As control, no 3AT or SM was added (unstarved). Whole cell extract was generated and subjected to SDS-PAGE and immunoblotting using antibodies against eIF2α and the phosphorylated form of eIF2α (eIF2α-P), respectively. The amount of eIF2α and eIF2α-P was quantified using the program ImageJ and plotted for 3AT starvation (B and D) and SM starvation (F), respectively, relative to the total amount of eIF2α and relative to the eIF2α-P/eIF2α ratio of the strain expressing GST under replete conditions.