Figure 2.

Effects of methionine incubation on eIF2 phosphorylation and translation in C. neoformans. (A) Western blot analysis of eIF2 phosphorylation. Wild-type cells were grown to mid-log phase before resuspension in fresh minimal defined media containing 10 mM of either ammonium sulfate or methionine. Cells were collected in 30-min increments over a 3-h time course. Protein extracted from cells was analyzed by western blot for phosphorylated eIF2. Images shown are representative of three biological replicates. Densitometry analysis was performed by normalizing p-eIF2 signal to total protein signal, and data shown are the averages of three biological replicates. (B) Polysome profiling analysis of wild-type and gcn2Δ cells. Mid-log cells were incubated in minimal defined media with ammonium or methionine for 60 min. Cells were then treated with cycloheximide to arrest ribosomes, pelleted, and flash frozen in liquid nitrogen. Cells were lysed, and lysates were loaded onto sucrose gradients. Gradients were ultracentrifuged for 2 h at 39,000 rpm and 4°C to separate the contents of the lysate by density. After spinning, samples were run through a flow cell, where the absorbance of the sample at 254 nm was measured, and polysome profiles were generated. Images shown are representative of two biological replicates.