Characterization of translation initiation factor 5 (eIF5) from Saccharomyces cerevisiae. Functional homology with mammalian eIF5 and the effect of depletion of eIF5 on protein synthesis in vivo and in vitro.
Eukaryotic translation initiation factor 5 (eIF5) interacts in vitro with the 40 S initiation complex (40 S.AUG.Met-tRNAf.eIF2.GTP) to mediate the hydrolysis of ribosome-bound GTP. In Saccharomyces cerevisiae, eIF5 is encoded by a single copy essential gene, TIF5, that encodes a protein of 45,346 daltons. To understand the function of eIF5 in vivo, we constructed a conditional mutant yeast strain in which a functional but a rapidly degradable form of eIF5 fusion protein was synthesized from the repressible GAL promoter. Depletion of eIF5 from this mutant yeast strain resulted in inhibition of both cell growth and the rate of in vivo protein synthesis. Analysis of the polysome profiles of eIF5-depleted cells showed greatly diminished polysomes with simultaneous increase in free ribosomes. Furthermore, lysates of cells depleted of eIF5 were dependent on exogenously added yeast eIF5 for efficient translation of mRNAs in vitro. This is the first demonstration that the TIF5 gene encodes a protein involved in initiation of translation in eukaryotic cells. Additionally, we show that rat eIF5 can functionally substitute yeast eIF5 in translation of mRNAs in vitro as well as in complementing in vivo a genetic disruption in the chromosomal copy of TIF5.
Maiti
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Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Jack and Pearl Resnick Campus, Bronx, New York 10461, USA.
Maitra
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D000818:Animals; D003001:Cloning, Molecular; D040061:Eukaryotic Initiation Factor-5; D005800:Genes, Fungal; D005838:Genotype; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008322:Mammals; D010448:Peptide Initiation Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism*; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*
D040061:Eukaryotic Initiation Factor-5; D010448:Peptide Initiation Factors; D012331:RNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate