HEAT-SHOCK EFFECTS ON PHOSPHORYLATION OF PROTEIN-SYNTHESIS INITIATION-FACTOR PROTEINS EIF-4E AND EIF-2-ALPHA IN DROSOPHILA

Heat shock of mammalian cells causes changes in initiation factor phos phorylation that likely contribute to or cause the translational repro gramming characteristic of heat shock. In these investigations we have carried out a parallel analysis of Drosophila, focusing on eLF-4E and eLF-2 alpha. elF-4E plus associated proteins was purified from lysate s by m(7)GTP-Sepharose chromatography. A minor fraction (<10%) of eIF- 4E is phosphorylated under normal growth conditions, and phosphorylati on decreases during heat shock. Drosophila eIF-2 alpha has been identi fied by in vitro translation of T7 RNA polymerase-transcribed mRNA, an d immunoblotting with anti-Drosophila eIF-2 alpha antiserum. P-32-labe ling analysis (unfractionated cell lysates and immunoprecipitates) det ects phosphorylated eIF-2 alpha, whose amount increases approximately 2-3-fold upon heat shock. Immunoblotting analysis of two-dimensional g el-resolved proteins to determine the mass fraction of eIF-2 alpha pho sphorylated detects a single eIF-2 alpha spot in both normal temperatu re and heat shocked cells, indicating less than 5% phosphorylation aft er and before heat shock, Staining quantification is consistent with t his low prevalence. A major phosphoprotein which copurifies with eIF-4 E on m(7)GTP-Sepharose shows decreased overall phosphorylation and dec reased association with eIF-4E following heat shock. Several distincti ve characteristics of this phosphoprotein suggest it is Drosophila eIF -4B.