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 activated in rabbit reticulocyte lysates in response to a variety of conditions including heme-deficiency, addition of oxidants, and heat shock. Activated HRI inhibits translation by catalyzing the phosphorylation of the alpha-subunit of eukaryotic initiation factor eIF-2. The molecular nature of the ''signal'' that leads to the activation of HRI in response to heat shock has not been characterized. We have recently reported 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 synthesis in hemin-supplemented reticulocyte lysates. The inhibition was reversed upon the addition of 10 mM cAMP or purified eIF-2B, classical criteria for HRI-mediated translational inhibition. Denatured BSA, but not native BSA, stimulated the phosphorylation of the alpha-subunit of eIF-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 with denatured BSA, but not with native BSA. Hsp70 was co-adsorbed with HRI 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 caused by denatured proteins and suggest that denatured protein, through its capacity to sequester hsp70, is the ''signal' that leads to activation of HRI in response to heat shock.