Abstract
The complex eukaryotic initiation factor 3 (eIF3) was shown to promote the formation of the 43 S preinitiation complex by dissociating 40 S and 60 S ribosomal subunits, stabilizing the ternary complex, and aiding mRNA binding to 40 S ribosomal subunits. Recently, we described the identification of RPG1 (TIF32), the p110 subunit of the eIF3 core complex in yeast. In a screen for Saccharomyces cerevisiae multicopy suppressors of the rpg1-1 temperature-sensitive mutant, an unknown gene corresponding to the open reading frame YLR192C was identified. When overexpressed, the 30-kDa gene product, named Hcr1p, was able to support, under restrictive conditions, growth of the rpg1-1 temperature-sensitive mutant, but not of a Rpg1p-depleted mutant. An hcr1 null mutant was viable, but showed slight reduction of growth when compared with the wild-type strain. Physical interaction between the Hcr1 and Rpg1 proteins was shown by co-immunoprecipitation analysis. The combination of Deltahcr1 and rpg1-1 mutations resulted in a synthetic enhancement of the slow growth phenotype at a semipermissive temperature. In a computer search, a significant homology to the human p35 subunit of the eIF3 complex was found. We assume that the yeast Hcr1 protein participates in translation initiation likely as a protein associated with the eIF3 complex.
Highlights
Eukaryotes initiate protein synthesis by dissociation of an 80 S ribosome into 40 S and 60 S subunits, formation of the Met-tRNAi1⁄7eIF211⁄7GTP ternary complex and binding of this complex to the 40 S subunit, joining the resulting 43 S preinitiation complex with mRNA, and formation of the 48 S initiation complex at the initiation codon, followed by addition of the 60 S ribosomal subunit
We have shown that the essential S. cerevisiae Rpg1 protein is the homologue of the mammalian eukaryotic initiation factor 3 (eIF3)-p170 protein [5] required for translation initiation in vivo and in vitro, interacts with Prt1p, and represents the largest component of the eIF3 core complex
Involvement of Rpg1p in the process of translation initiation was clearly demonstrated in a cell-free system dependent on exogenous eIF3 [25]: we showed that the sucrose gradient fraction containing Rpg1p possesses the biochemical activity ascribed to eIF3, e.g. the restoration of translation in an extract in which an endogenous eIF3 subunit had been inactivated
Summary
Eukaryotes initiate protein synthesis by dissociation of an 80 S ribosome into 40 S and 60 S subunits, formation of the Met-tRNAi1⁄7eIF211⁄7GTP ternary complex and binding of this complex to the 40 S subunit, joining the resulting 43 S preinitiation complex with mRNA, and formation of the 48 S initiation complex at the initiation codon, followed by addition of the 60 S ribosomal subunit. Another example is the STM1/TIF3 gene, the protein product of which was identified as a multicopy suppressor of a temperature-sensitive mutation in eIF4A and shown to represent the yeast homologue of mammalian eIF4B [24].
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