Control of protein synthesis by hemin. Purification of a rabbit reticulocyte hsp 70 and characterization of its regulation of the activation of the hemin-controlled eIF-2(alpha) kinase.
We have purified a soluble rabbit reticulocyte protein, previously termed the supernatant factor, that reverses the inhibition of protein synthesis in hemin-deficient lysate by promoting the inactivation of the hemin-controlled eIF-2 alpha kinase (HCR) mediating the effect of hemin deficiency. We have identified the supernatant factor as a member of the heat shock protein 70 family, denoted hsp 70(R), based upon its size (72 kDa), specific reaction to a monoclonal antibody against eukaryotic hsp 70, strong binding affinity for ATP, and endogenous ATPase activity. We have investigated the role of hsp 70(R) and hemin in the regulation of the activation of HCR from its latent precursor (ProHCR) and the translational control of protein synthesis in rabbit reticulocyte lysate. We find that autophosphorylation of Pro-HCR is reduced by about 75% by adding saturating hsp 70(R) and almost completely reduced by adding either saturating hemin or limiting hemin plus limiting hsp 70(R). In contrast, autophosphorylation of HCR, which is similar in magnitude to that of ProHCR, is unaffected by adding either saturating hsp 70(R), saturating hemin, or limiting amounts of both. The activation of HCR (measured by inhibition of protein synthesis) from isolated ProHCR is completely prevented by hsp 70(R) in the presence, but not absence, of dithiothreitol. This suppression appears to be due to the association of hsp 70(R) with ProHCR, since hsp 70(R) action is prevented by ATP/Mg2+ and because activation of HCR from less purified ProHCR, that has associated hsp 70(R), is suppressed by dithiothreitol alone. This association is confirmed by sucrose gradient centrifugation, which shows co-sedimentation of some hsp 70(R) with ProHCR following preincubation together that is prevented by ATP/Mg2+ and does not occur after conversion of ProHCR to HCR. Limiting hsp 70(R) reduces the concentration of hemin required to prevent activation of HCR from isolated ProHCR from 0.75 to 0.15 microM and the optimal hemin concentration needed to maintain protein synthesis in reticulocyte lysate from 25 to 10 microM. Limiting hsp 70(R) also allows the delayed addition of hemin to suppress activation of HCR from ProHCR and to reverse inhibition of protein synthesis in hemin deficient lysate. The association of hsp 70(R) with ProHCR also underlies the observation that much more protein is synthesized in reticulocyte lysate in the absence of hemin at 25 degrees C than at temperatures of 30 degrees C or greater. These observed effects may be specific to hsp 70(R), since they are not observed with rabbit reticulocyte eIF-2 or eIF-2B, and since the comparable hsp 70 from bovine brain is incapable of maintaining or restoring protein synthesis in hemin-deficient lysate.
Gross
M
M
Department of Pathology, University of Chicago, Illinois 60637.
Olin
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A
Hessefort
S
S
Bender
S
S
D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2 / Q000097:blood / Q000302:isolation & purification; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000097:blood* / Q000302:isolation & purification; D006427:Hemin / Q000494:pharmacology* / Q000502:physiology; D006706:Homeostasis; D015151:Immunoblotting; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000097:blood*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D006360:Heat-Shock Proteins; D011506:Proteins; D008623:Mercaptoethanol; D006427:Hemin; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D004229:Dithiothreitol