Mechanism of inhibition of polypeptide chain initiation in heat-shocked Ehrlich cells involves reduction of eukaryotic initiation factor 4F activity.
Almost all living organisms studied respond to elevated temperature with a marked inhibition of overall protein synthesis but increased synthesis of a specific set of proteins, the so-called heat-shock proteins. We have prepared a cell-free protein synthesizing system (lysate) from heat-shocked Ehrlich ascites tumor cells that reflects the inhibition of protein synthesis in intact cells at elevated temperatures. We have isolated and partially purified a stimulator of the heat-shocked cell lysate from Ehrlich cells. Through four purification steps, the stimulator is chromatographically identical to eukaryotic initiation factor 4F (eIF-4F), an initiation factor which specifically binds mRNA cap structure. Therefore, we have tested the effects of highly purified reticulocyte eIF-4F on the heat-shocked cell lysate. Protein synthesis is strongly stimulated by addition of highly purified eIF-4F. Synthesis in the heat-shocked lysate is more inhibited at high (70 mM) KCl concentrations, than at lower concentrations, and stimulation by eIF-4F is correspondingly greater at higher KCl concentrations, so that the rate of protein synthesis is returned to control (non-heat-shocked lysate) levels at all KCl concentrations. Furthermore, at 70 mM KCl, in heat-shocked lysates, synthesis of the 68-kDa heat-shock protein is much less inhibited than synthesis of the bulk of non-heat-shock proteins, and eIF-4F stimulates synthesis of 68-kDa protein to a much lesser extent than non-heat-shock proteins. Thus, addition of purified eIF-4F reverses the effects of elevated temperatures on Ehrlich cells that are reflected in lysates. Therefore, we propose that the inhibition of translation in heat-shocked Ehrlich cells is the result of inactivation of eIF-4F function.
D000085:Acetates / Q000494:pharmacology; D019342:Acetic Acid; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002474:Cell-Free System; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D039562:Eukaryotic Initiation Factor-4F; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects*; D010448:Peptide Initiation Factors / Q000302:isolation & purification* / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism
D000085:Acetates; D039562:Eukaryotic Initiation Factor-4F; D006360:Heat-Shock Proteins; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D011189:Potassium Chloride; D019342:Acetic Acid