Figure 5.

Ksp1-dependent phosphorylation of eIF4G regulates mRNA decay of glycolytic genes. (A) The phosphorylated residues of Tif4631 under glucose deprivation conditions in WT and ksp1Δ cells. Immunoprecipitated Tif4631-TAP was analyzed by LC-MS/MS after tryptic digestion. (B) The potent residues that are phosphorylated by Ksp1. Twenty-four residues that were detected only in WT cells, not in ksp1Δ cells, are depicted above domain information of Tif4631. (C) Phosphorylation of Tif4631–24A under glucose deprivation conditions. C-terminally c-Myc tagged TIF4631 (WT) or tif4631–24A alleles were integrated into tif4631Δ cells. Cells were subjected to glucose deprivation for 30 min and analyzed by standard SDS-PAGE and Phos-tag SDS-PAGE. (D) Decay curves of glycolytic mRNAs after thiolutin treatment in TIF4631 and tif4631–24A cells. Data were fitted to one-phase exponential decay curve to determine mRNA half-lives. X-axis, time after thiolutin treatment. Error bars indicate SD of experiments performed in triplicate.