Heat shock impairs the interaction of cap-binding protein complex with 5' mRNA cap.
Cell-free protein synthesizing systems prepared from heat-shocked Ehrlich cells retain the inhibition of translation that is seen at the cellular level. Recently, we showed that a highly purified cap-binding protein complex composed of the p220 and p28 subunits of eukaryotic initiation factor 4F, in a 1:1 molar ratio, restores protein synthesis in these cell-free translation systems (Lamphear, B.J., and Panniers, R. (1990) J. Biol. Chem. 265, 5333-5336). Here we have estimated the amount of cap-binding complex in cell extracts that can restore protein synthesis in heat-shocked cells. We find reduced restoring activity in heat-shocked cell extracts. Further, less cap-binding complex can be purified by 7-methyl-guanosine triphosphate Sepharose affinity chromatography from heat-shocked cell extracts, and we conclude that heat shock impairs the binding of complex to 5' mRNA cap. We have ruled out proteolysis and competitive inhibitors as mediators of this impairment. However we cannot distinguish between two possible explanations: (i) reduced association of p220 with p28 or (ii) a non-competitive inhibitor blocks complex binding to cap. We have also examined the affect of heat shock on the phosphorylation state of two forms of p28, p220.p28 complex and p28 free of p220. Both forms have reduced levels of phosphorylation during heat shock. The significance of these changes is discussed.
Lamphear
B J
BJ
Cancer Center, University of Rochester, New York 14642.
Panniers
R
R
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002474:Cell-Free System; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000378:metabolism*; D006358:Hot Temperature; D010766:Phosphorylation; D039381:RNA Cap-Binding Proteins; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits
D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D039381:RNA Cap-Binding Proteins; D012333:RNA, Messenger