Minimum Requirements for the Function of Eukaryotic Translation Initiation Factor 2

Abstract

Eukaryotic translation initiation factor 2 (eIF2) is a G protein heterotrimer required for GTP-dependent delivery of initiator tRNA to the ribosome. eIF2B, the nucleotide exchange factor for eIF2, is a heteropentamer that, in yeast, is encoded by four essential genes and one nonessential gene. We found that increased levels of wild-type eIF2, in the presence of sufficient levels of initiator tRNA, overcome the requirement for eIF2B in vivo. Consistent with bypassing eIF2B, these conditions also suppress the lethal effect of overexpressing the mammalian tumor suppressor PKR, an eIF2α kinase. The effects described are further enhanced in the presence of a mutation in the G protein (γ) subunit of eIF2, gcd11-K250R, which mimics the function of eIF2B in vitro. Interestingly, the same conditions that bypass eIF2B also overcome the requirement for the normally essential eIF2α structural gene (SUI2). Our results suggest that the eIF2βγ complex is capable of carrying out the essential function(s) of eIF2 in the absence of eIF2α and eIF2B and are consistent with the idea that the latter function primarily to regulate the level of eIF2·GTP·Met-tRNAiMet ternary complexes in vivo.

eIF2B Mechanisms of Action and Regulation: A Thermodynamic View

“…However, catalysis and inhibition differ in their sensitivity to the strength of the interaction, with inhibition being affected first, while catalysis may become impaired only if eIF2α binding in the eIF2B reg pocket is severely disrupted. A role for the eIF2α–eIF2B reg interaction in catalysis is also supported by the observation that overexpression of eIF2βγ and tRNA i in S. cerevisiae suppresses the lethality of both eIF2B deletion and eIF2α deletion …”

“…The resulting cells have a slow-growth phenotype, which is partly ameliorated by a mutation in eIF2γ that increases the rate of spontaneous GDP dissociation. 15 The lethal phenotype of eIF2Bγ and eIF2Bε depletion can be suppressed by overexpression of only eIF2, without overexpressing tRNA i , while overexpressing only tRNA i is sufficient to suppress the lethality of eIF2Bδ depletion. 16 Therefore, the essential functions of eIF2Bγ and eIF2Bε appear to be related to nucleotide exchange, while that of eIF2Bδ appears to be related to binding of Met-tRNA i to eIF2-GTP.…”

“…A role for the eIF2α− eIF2B reg interaction in catalysis is also supported by the observation that overexpression of eIF2βγ and tRNA i in S. cerevisiae suppresses the lethality of both eIF2B deletion and eIF2α deletion. 15 ■ EIF2B FUNCTIONS AND MECHANISM OF ACTION Slow Rate of Dissociation of GDP from eIF2. The rate of dissociation of GDP from eIF2 is ∼1 × 10 −1 min −1 for S. cerevisiae eIF2 18,19 and even slower, ∼5 × 10 −3 min −1 , for mammalian eIF2.…”

“…Their essential function must be related to eIF2 nucleotide exchange, because the lethality of an eIF2B deletion is suppressed by co-overexpression of eIF2 and tRNA i . The resulting cells have a slow-growth phenotype, which is partly ameliorated by a mutation in eIF2γ that increases the rate of spontaneous GDP dissociation . The lethal phenotype of eIF2Bγ and eIF2Bε depletion can be suppressed by overexpression of only eIF2, without overexpressing tRNA i , while overexpressing only tRNA i is sufficient to suppress the lethality of eIF2Bδ depletion .…”

Phosphorylation of Human Eukaryotic Initiation Factor 2γ: Novel Site Identification and Targeted PKC Involvement

“…As the largest of the three subunits, the 472 amino acid human eIF2γ protein contains several binding domains, has a molecular weight of roughly 51.8 kDa, and shares 72% sequence homology to that of its counterpart in Saccharomyces cerevisiae. 9,13,23 The γ-subunit binds directly to GDP/GTP and Met-tRNA i and serves as the heterotrimer's core, which binds to the other subunits. 24À28 The N-terminus of eIF2γ binds glycoprotein p67 which has a direct role in protecting eIF2α ser-51 phosphorylation .…”