Abstract
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.
Highlights
Cell-free protein synthesizing systems prepared eukaryotic initiation factor 2 and cap-binding complex are from heat-shocked Ehrlich cells retain the inhibition involved
Crude extracts prepared from heat-shocked cells representing total cytosolic fraction (HSPRS) have reduced restoring activity compared with control cells
This reduction in activity corresponds to reduced levels of p220 p28 complex that can be obtained from heat-shocked cells compared with control cells by m7GTP-Sepharosechromatography
Summary
Protein Synthesis Restoring Actiuity inCell Extracts-Protein synthesis in heat-shockecdell lysates is severely inhibited yotic initiation factor; HRI, heme regulated inhibitor; HSPRS, high salt post-ribosomal supernatant; m‘GTP, 7-methyl-guanosinetri-. Dominant inhibitor, cell-free protein synthesis systems from control and heat-shocked cells were mixed in varying ratios. Small quantitiesof control lysate stimulate heat-shocked cell lysate proteinsynthesis. ~ 2 8scribed under “Materials and Methods” for stimulation of heatcomplex in the control cell lysate replenishes a deficiency of complex in the heat shock cell lysate. To confirm the deficiency of cap complex, the levels of restoring activity (ability to stimulate cell-free proteinsynthesis in lysates prepared shocked Ehrlich cell-free protein synthesis (restoringactivity). After centrifugation to remove ribosomes, thesupernatants (high-salt post-ribosomal supernatants (HSPRSs)) were compared for their ability to restore protein synthesis inheat-shocked cell. Complex withm’GTP-Sepharose-The marked reduction in cap-binding protein complex activity in heat-shocked Ehrlich cells prompted us to determine how much complex can be. Positionsof molecular weight markers are indicated as well as theposition of p220 and p28
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