Protein synthesis in brine shrimp embryos. Regulation of the formation of the ternary complex (Met-tRNAf X eIF-2 X GTP) by two purified protein factors and phosphorylation of Artemia eIF-2.
We have purified from the ribosomal wash of dormant and developing embryos of Artemia two proteins, Co-eIF-2(A) and Co-eIF-2(B). These factors are essential for ternary complex formation and binding of [35S]-Met-tRNAf to 40-S ribosomal subunits with 15-30 microgram eIF-2/ml of reaction mixture. On polyacrylamide gel electrophoresis in dodecylsulfate, Co-eIF-2(A) is composed of a single polypeptide of Mr 65 000, whereas Co-eIF-2(B) contains polypeptides of Mr 105000 and 112000. Co-eIF-2(A) is sensitive to 4.5 microM aurintricarboxylic acid but Co-eIF-2(B) requires approximately 15 microM aurintricarboxylic acid to give 50% inhibition of ternary complex formation. The stimulatory activity of both factors is abolished by pretreatment of the proteins with N-ethylmaleimide. Artemia eIF-2 rapidly bonds [3H]GDP or [3H]GTP and at 15 degrees C the initiation factor rapidly equilibrates bound nucleotides with free GDP or GTP. Both Co-eIF-2(A) and Co-eIF-2(B) have no effect on the exchange or the amount of nucleotide bound. The small subunit (Mr 43 000) of Artemia eIF-2 is phosphorylated in the presence of the rabbit reticulocyte heme-repressible kinase. Tryptic digestion of [32P]phosphorylated eIF-2 produces a single major phosphopeptide and several minor ones. Acid hydrolysis of these phosphopeptides, as well as of [32P]phosphorylated eIF-2, demonstrates that the radioactivity is predominantly associated with phosphoserine. Phosphorylated Artemia eIF-2 is active in ternary complex formation, in AUG-dependent binding of [35S]Met-tRNAf to 40-S ribosomal subunits and in cell-free protein synthesis. Both Co-eIF-2(A) and Co-eIF-2(B) stimulate ternary complex formation with phosphorylated eIF-2. A kinase that phosphorylates the small subunit of eIF-2 is present in the post-ribosomal supernatant as well as in the ribosomal wash of developing Artemia embryos.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001156:Artemia / Q000378:metabolism*; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D004625:Embryo, Nonmammalian / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D010448:Peptide Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met
D000596:Amino Acids; D015852:Eukaryotic Initiation Factor-2; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D001312:Aurintricarboxylic Acid; D006160:Guanosine Triphosphate; D008274:Magnesium