Reduced Formation of Initiation Complexes between Met-tRNAf and 40-S Ribosomal Subunits in Rabbit Reticulocyte Lysates Incubated at Elevated Temperatures. Activity of the Met-tRNAf Binding Factor

Abstract

Rabbit reticulocyte lysates preincubated at 42 -45 "C and subsequently assayed for protein synthesis at 37 "C show (a) a decrease in their rate of polypeptide chain initiation and (b) decreased Met-tRNAf binding to native 40-S subunits and 80-S ribosomes. Similarly, the amount of [3sS]Met-tRNAf . 40-S-subunit initiation complexes formed in vitro with native 40-S subunits isolated from supraoptimally heated lysates is only 10 -30 of that formed with native 40-S subunits prepared from control lysates preincubatedat 0 "C or 37 "C. This decrease is not due to reduced formation of Met-tRNAr ternary complex or to increased destruction of this complex, since we find that 0.5 M KC1 ribosomal washes extracted from lysates preincubated at 0 "C, 37 "C, or 43-45 C have almost identical activities in Met-tRNAf . GTP . eIF-2 ternary complex formation and Met-tRNAf hydrolysis, whether assayed at 37 "C or 45 "C. Similarly, the results of binding assays of [35S]Met-tRNAf to 40-S subunits performed at both temperatures show that the control and heated washes, added at either limiting or optimal amounts, stimulate almost equally the binding of Met-tRNAf to (a) salt-washed derived 40-S subunits and (b) native 40-S subunits, prepared from control or supraoptimally heated lysates. Moreover the factor-directed binding of Met-tRNAf by the derived 40-S subunits is not affected by the presence of either the control or heated postribosomal supernatant. We conclude that at elevated temperatures there is no irreversible inactivation of the Met-tRNAf binding factor for ternary complex formation and Met-tRNAf binding to 40-S subunits in vitro and discuss some possible explanations for the lowered Met-tRNAf binding observed in vivo.Incubation of rabbit reticulocyte lysates at 42 -45 "C leads to a rapid decrease in their rate of protein synthesis with a concomittant breakdown of polysomes and a reduction in the amount of [35S]Met-tRNAf . 40-S-ribosomal-subunit initiation complex formed [1,2]. This and other evidence implicates a lesion in polypeptide chain initiation which superficially resembles that seen in lysates incubated without haemin [3 -51. We have already presented evidence that the haemin-independent high-temperature-sensitive lesion in polypeptide chain initiation, although mediated by a soluble inhibitor which resembles the haemincontrolled repressor, does not lead to any apparent unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm when measured in a l-cm pathlength cell.Enzyme. Creatine kinase (EC 2.7.3.2).l?efinirion.inactivation of initiation factors when assayed at 30-37 "C (see preceding paper [6]). Moreover, addition of optimal amounts of control initiation factors, contained in the 0.5 M KCl ribosomal wash of unincubated lysates, to the 45 "C preheated lysates [2,6] or of a mixture of partially purified factors, including the Met-tRNAf binding factor, to preheated KCIwashed ribosomes and reticulocyte supernatant [7], fail to restore their rate of protein synthesis to the cont...

Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate

“…activity under stress conditions that activate HRI in heminsupplemented RRL. Besides heme-deficiency, HRI becomes activated in hemin-supplemented lysate in response to heatshock [12,36,37], the presence of denatured protein [15], or oxidative stress induced by sulfhydryl reactive agents, such as Hg 2ϩ [19,20] or GSSG [38]. However, earlier experiments suggested that supernatant factor was ineffective in preventing protein synthesis inhibition due to GSSG-induced activation of HRI [17].…”

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

“…HRI is present in an inactive form in hemin-supplemented reticulocyte lysates (33,34) and becomes active under conditions of heme deficiency (33,34) or heat shock (4,15) or upon treatment with sulfhydryl reagents such as N-ethylmaleimide or o-iodosobenzoate (20), oxidized glutathione (16,19,25), and heavy metal ions (22). It has been shown that hemin binds directly to HRI (17) and that this binding promotes intersubunit disulfide bond formation (8).…”

Phosphorylation and the control of protein synthesis

“…A variety of mild physical stresses, of which high hydrostatic pressure (Henderson & Hardesty 1978) and somewhat elevated temperature (Bonanou-Tzedaki 1978) are canonical examples, cause an inhibition of protein synthesis that shares characteristics with the other conditions listed above.…”