Inactivation of eukaryotic initiation factor 2B in vitro by heat shock.
Protein synthesis in rat H35 Reuber hepatoma cells is rapidly inhibited on heat shock. At mild heat-shock temperatures the main cause for inhibition is the inactivation of the guanine nucleotide exchange factor eukaryotic initiation factor 2B (eIF2B); under more severe heat-shock conditions the activity of several initiation factors is compromised. eIF2B is required for GDP/GTP exchange on eIF2, which delivers methionyl-tRNA to the 40 S ribosomal subunit. We have tried to elucidate the mechanism underlying the inactivation of eIF2B by assays in vitro. Incubation of cell extracts at 41 degreesC or higher led to the inactivation of eIF2B. In agreement with observations in cells exposed to mild heat shocks, the thermal inactivation of eIF2B could be ascribed to neither eIF2alpha phosphorylation nor the induction of another inhibitor. With the use of glycerol gradients we show that eIF2B forms aggregates in heat-treated extracts. Furthermore eIF2B activity is protected against heat shock in thermotolerant cells. Taken together, these results suggest a role for chaperones in the control of eIF2B activity.
Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
G C
GC
Scheper
A A
AA
Thomas
R
R
van Wijk
D000818:Animals; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D020717:Eukaryotic Initiation Factor-2B; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D013997:Time Factors; D014407:Tumor Cells, Cultured
D015852:Eukaryotic Initiation Factor-2; D020717:Eukaryotic Initiation Factor-2B; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate