DENATURED PROTEINS INHIBIT TRANSLATION IN HEMIN-SUPPLEMENTED RABBIT RETICULOCYTE LYSATE BY INDUCING THE ACTIVATION OF THE HEME-REGULATED EIF-2-ALPHA KINASE

The heme-regulated inhibitor (HRI) of protein synthesis becomes activa ted in rabbit reticulocyte lysates in response to a variety of conditi ons including heme-deficiency, addition of oxidants, and heat shock. A ctivated HRI inhibits translation by catalyzing the phosphorylation of the alpha-subunit of eukaryotic initiation factor eIF-2. The molecula r nature of the ''signal'' that leads to the activation of HRI in resp onse to heat shock has not been characterized. We have recently report ed that HRI interacts with the 90- and 70-kDa heat shock proteins (hsp ) and a 56-kDa protein in hemin-supplemented lysates [Matts, R. L., Xu , Z., Pal, J.K., & Chen, J.-J. (1992) J. Biol. Chem. 267, 18160-18167] . In this report, we demonstrate that addition of denatured proteins, bovine serum albumin (BSA), beta-lactoglobulin, or alpha-lactalbumin, but not the addition of the native proteins, inhibits protein synthesi s in hemin-supplemented reticulocyte lysates. The inhibition was rever sed upon the addition of 10 mM cAMP or purified eIF-2B, classical crit eria for HRI-mediated translational inhibition. Denatured BSA, but not native BSA, stimulated the phosphorylation of the alpha-subunit of eI F-2. This stimulation of eIF-2alpha phosphorylation was inhibited by a monoclonal antibody to HRI, confirming that denatured BSA was causing the activation of HRI. The concentration of denatured BSA required to inhibit protein synthesis by 50% correlated with the levels of hsp70 present in each lysate preparation. Lysate hsp70 co-immunoadsorbed wit h denatured BSA, but not with native BSA. Hsp70 was co-adsorbed with H RI from lysate in the presence of native BSA, but not in the presence of denatured BSA. These observations indicate that the dissociation of hsp70 from HRI is strongly correlated with the activation of HRI caus ed by denatured proteins and suggest that denatured protein, through i ts capacity to sequester hsp70, is the ''signal' that leads to activat ion of HRI in response to heat shock.