Characterization of mammalian translation initiation factor 5 (eIF-5). Demonstration that eIF-5 is a phosphoprotein and is present in cells as a single molecular form of apparent M(r) 58,000.
Eukaryotic translation initiation factor 5 (eIF-5) promotes the hydrolysis of GTP bound to the 40 S initiation complex (40 S.mRNA.Met-tRNA(f).eIF-2.GTP). Using assays that measure (a) the release of 32Pi from [gamma-32P]GTP bound to the 40 S initiation complex and (b) the eIF-5-dependent formation of an 80 S initiation complex from a preformed 40 S initiation complex containing bound [35S]Met-tRNA(f), we devised a novel and rapid procedure for purifying eIF-5 from rabbit reticulocyte lysates in high yield. Highly purified eIF-5 is a monomeric protein with a M(r) of about 58,000. However, in partially purified preparations, 58-kDa eIF-5 is associated with other cellular protein(s) and sediments in glycerol gradient centrifugation as a protein of M(r) approximately 160,000. Chicken antibodies to native eIF-5 were isolated from egg yolks of laying hens immunized with rabbit reticulocyte eIF-5. 35S-Labeled eIF-5, isolated from rat anterior pituitary GH3 cells using affinity-purified anti-eIF-5 antibodies, also has an apparent M(r) of 58,000. eIF-5 immunoprecipitated from extracts of 32P-labeled GH3 cells was phosphorylated on serine residues. Phosphopeptide mapping revealed two major sites of phosphorylation, which are distinct from rabbit reticulocyte eIF-5 sites phosphorylated in vitro by casein kinase II. These results demonstrate that eIF-5 is a phosphoprotein that is present in cells as a single molecular form of apparent molecular weight 58,000.
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461-1602.
J
J
Chevesich
J
J
Chaudhuri
U
U
Maitra
D000818:Animals; D001156:Artemia; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D040061:Eukaryotic Initiation Factor-5; D006160:Guanosine Triphosphate / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D040061:Eukaryotic Initiation Factor-5; D010448:Peptide Initiation Factors; D010710:Phosphates; D010748:Phosphopeptides; D010750:Phosphoproteins; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D013462:Sulfur Radioisotopes; C024988:tRNA, formylmethionine-; D006160:Guanosine Triphosphate