The multisubunit translation initiation factor 3 ( eIF3) from fission yeast, Schizosaccharomyces pombe
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Eukaryotic translation initiation factor 3 (eIF3) is a complex multi-subunit translation initiation factor that plays a central role in translation initiation in eukaryotic cells. Mammalian eIF3 consists of 10 non-identical subunits. In contrast the unicellular budding yeast, Saccharomyces cerevisiae eIF3 consists of only five subunits. All five subunits of budding yeast eIF3 have corresponding homologues in mammalian eIF3. These five subunits are usually referred to as "core" eIF3 subunits. The additional subunits that are constituents of mammalian eIF3 but are not present in S. cerevisiae genome are commonly referred to as "non-core" eIF3 subunits. Interestingly, database searches revealed that fission yeast, Schizosaccharomyces pombe, contains structural homologues for four of these five "non-core" subunits.;In this thesis work we have used the molecular genetic system of the fission yeast S. pombe to characterize the function of two fission yeast proteins, Moe1 and spInt6, that are the homologues of two mammalian non-core eIF3 subunits, p66 (eIF3d) and p48/INT6 (eIF3e) respectively. We have cloned and characterized the fission yeast int6+ gene that encodes spInt6, while the gene moe1+ was identified by others as an interacting partner of Scd1, a Ras effector molecule. We show that in fission yeast, both spInt6 and Moe1 physically associate with 40S ribosomal particles as constituents of the eIF3 protein complex that also contains other eIF3 core subunits. However, strains lacking int6+(Deltaint6) or moe1+ (Deltamoe1) are viable and show only moderate inhibition in the rate of in vivo global protein synthesis. Polysome profile analysis shows no apparent gross defects in translation initiation. Mutant Deltamoe1 and Delta int6 cells are defective in certain physiological processes. In contrast to wild type cells mutant cells are hypersensitive to caffeine, which can be suppressed by the addition of an osmotic stabilizer. In meiosis both Delta moe1 and Deltaint6 cells produce incomplete tetrads frequently. Finally, both mutant cells are defective in nutrient uptake from the growth media. These phenotypes would imply that Moe1 and spInt6 are required for integrity of the cell membrane. Further analysis of eIF3 subunits in Delta int6 or Deltamoe1 cells shows that in these deletion strains, while all the eIF3 subunits are bound to 40S particles, dissociation of ribosome-bound eIF3 resulted in the loss of stable association between the eIF3 subunits. In contrast, eIF3 isolated from ribosomes of wild-type cells are associated with one another in a protein complex. These observations suggest that Moe1 and spInt6 are each required for stable association of eIF3 subunits in fission yeast. The implications of these findings in relation to function of eIF3 in translation initiation are discussed.
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