Abstract
The protein encoded by the fission yeast gene, moe1(+) is the homologue of the p66/eIF3d subunit of mammalian translation initiation factor eIF3. In this study, we show that in fission yeast, Moe1 physically associates with eIF3 core subunits as well as with 40 S ribosomal particles as a constituent of the eIF3 protein complex that is similar in size to multisubunit mammalian eIF3. However, strains lacking moe1(+) (Deltamoe1) are viable and show no gross defects in translation initiation, although the rate of translation in the Deltamoe1 cells is about 30-40% slower than wild-type cells. Mutant Deltamoe1 cells are hypersensitive to caffeine and defective in spore formation. These phenotypes of Deltamoe1 cells are similar to those reported previously for deletion of the fission yeast int6(+) gene that encodes the fission yeast homologue of the p48/Int6/eIF3e subunit of mammalian eIF3. Further analysis of eIF3 subunits in Deltamoe1 or Deltaint6 cells shows that in these deletion strains, while all the eIF3 subunits are bound to 40 S particles, dissociation of ribosome-bound eIF3 results 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.
Highlights
Eukaryotic translation initiation factor 31 is the most complex initiation factor known both with respect to its function and subunit composition [1,2,3,4,5]
We show that in fission yeast, Moe1 physically associates with Eukaryotic translation initiation factor 3 (eIF3) core subunits as well as with 40 S ribosomal particles as a constituent of the eIF3 protein complex that is similar in size to multisubunit mammalian eIF3
We show that Moe1/ eIF3d as well as spInt6/eIF3e physically associate with the eIF3 core subunit spPrt1 in fission yeast in a protein complex that is similar in size to multisubunit mammalian eIF3
Summary
Strains and Media—The strains used in this study (Table I) were derived from wild-type strain 972 hϪ. Preparation of Yeast Postribosomal Supernatant and Ribosomal Salt-wash Protein Fractions—Exponentially growing cultures of yeast strains (70 ml each) were harvested, washed with buffer B (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 15 mM MgCl2, 0.1 mM EDTA, 2.5 mM 2-mercaptoethanol), and lysed by vortexing with glass beads in buffer B containing a mixture of protease inhibitors as described above. After incubation at 0 °C for 30 min, the suspension was centrifuged again at 100,000 rpm for 30 min, and the supernatant containing ribosomal salt-wash proteins was dialyzed against 600 ml of NET buffer containing 70 mM NaCl and 0.5 mM phenylmethylsulfonyl fluoride for 3 h Both the postribosomal supernatant and dialyzed ribosomal 0.5 M KCl-wash proteins were analyzed by SDS-PAGE followed by Western blotting. Other Methods—Methods used for (a) measuring the rate of in vivo protein synthesis, (b) polysome profile analysis, and (c) detecting the association of Moe and other fission yeast eIF3 subunits to 40 S particles were as described [18]
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