CHARACTERIZATION OF THE IN-VIVO PHOSPHORYLATION SITES OF THE MESSENGER-RNA-CENTER-DOT-CAP-BINDING COMPLEX PROTEINS EUKARYOTIC INITIATION FACTOR-4E AND P20 IN SACCHAROMYCES-CEREVISIAE

Eukaryotic translation is believed to be regulated via the phosphorylation of specific eukaryotic initiation factors (eIFs), including one of the cap-binding complex proteins, eIF-4E. We show that in the yeast Saccharomyces cerevisiae, both eIF-4E and another cap-binding complex protein, p20, are phosphoproteins. The major sites of phosphorylation of yeast eIF-4E are found to be located in the N-terminal region of its sequence (Ser(2) and Ser(15)) and are thus in a different part of the protein from the main phosphorylation sites (Ser(53) and Ser(209)) proposed previously for mammalian eIF-4E. The most likely sites of p20 phosphorylation are at Ser(91) and/or Ser(154). All of the major sites in the two yeast proteins are phosphorylated by casein kinase II in vitro. Casein kinase II phosphorylation of cap complex proteins should therefore be considered as potentially involved in the control of yeast protein synthesis. Mutagenesis experiments revealed that yeast eIF-4E activity is not dependent on the presence of Ser(2) or Ser(15). On the other hand, we observed variations in the amount of (phosphorylated) p20 associated with the cap binding complex as a function of cell growth conditions. Our results suggest that interactions of yeast eIF-4E with other phosphorylatable proteins, such as p20, could play a pivotal role in translational control.