Regulation of protein synthesis in rabbit reticulocyte lysate. Glucose 6-phosphate is required to maintain the activity of eukaryotic initiation factor (eIF)-2B by a mechanism that is independent of the phosphorylation of eIF-2 alpha.
Previous studies from other laboratories, using rabbit reticulocyte lysate filtered through Sephadex G-25 or G-50, have demonstrated that glucose 6-phosphate is required to maintain active rates of translation, but its mechanism of action is currently unsettled. We have tested whether glucose 6-phosphate is required to prevent activation of the hemin-controlled translational repressor and the phosphorylation of the smallest or alpha subunit of eukaryotic initiation factor 2 (eIF-2). We have found that antibody to the hemin-controlled translational repressor can completely restore protein synthesis in reticulocyte lysate, filtered through Sephadex G-25, that is incubated in the absence of hemin and presence of glucose 6-phosphate, but cannot restore protein synthesis in such lysate incubated in the presence of hemin and absence of glucose 6-phosphate. We have also found, using a modification of the method of Matts and London [1984) J. Biol. Chem. 259, 6708-6711) to measure the ability of gel-filtered lysate to dissociate and exchange GDP from eIF-2.GDP, that this endogenous eIF-2B activity is reduced to the same low level in the presence of hemin and absence of glucose 6-phosphate as it is in the absence of hemin and presence of glucose 6-phosphate. Although there is a low level of phosphorylation of eIF-2 alpha in gel-filtered lysate given hemin but no glucose 6-phosphate, it cannot account for the loss of eIF-2B activity, since this phosphorylation is removed by antibody to the hemin-controlled translational repressor or isocitrate, which do not restore protein synthesis or eIF-2B activity, and not by fructose 1,6-diphosphate, which does partially restore protein synthesis and eIF-2B activity. These findings suggest that sugar phosphates may exert a direct effect on eIF-2B and may be required for its proper function. Additional support for this conclusion is our finding that protein synthesis and eIF-2B activity in partially hemin-deficient lysate can be restored by high levels of glucose 6-phosphate or fructose 1,6-diphosphate without a reduction in the level of phosphorylated eIF-2 alpha, suggesting that such levels of sugar phosphate may permit restoration of normal function with a limiting amount of eIF-2B.
Gross
M
M
Department of Pathology, University of Chicago, Illinois 60637.
Rubino
M S
MS
Starn
T K
TK
D000818:Animals; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2; D005635:Fructosediphosphates / Q000494:pharmacology; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000494:pharmacology*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006427:Hemin / Q000494:pharmacology; D007074:Immunoglobulin G / Q000502:physiology; D007523:Isocitrates / Q000494:pharmacology; D007700:Kinetics; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects; D011494:Protein Kinases / Q000276:immunology / Q000502:physiology; D011500:Protein Synthesis Inhibitors; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D005635:Fructosediphosphates; D005958:Glucosephosphates; D007074:Immunoglobulin G; D007523:Isocitrates; D010448:Peptide Initiation Factors; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D006153:Guanosine Diphosphate; D019298:Glucose-6-Phosphate; D006427:Hemin; D006160:Guanosine Triphosphate; D003513:Cycloheximide; C034219:isocitric acid; D000242:Cyclic AMP; D011494:Protein Kinases; D019892:eIF-2 Kinase; C029063:fructose-1,6-diphosphate