Change in Nutritional Status Modulates the Abundance of Critical Pre-initiation Intermediate Complexes During Translation Initiation in Vivo

Abstract

In eukaryotic translation initiation, eIF2GTP-Met-tRNA(i)(Met) ternary complex (TC) interacts with eIF3-eIF1-eIF5 complex to form the multifactor complex (MFC), while eIF2GDP associates with eIF2B for guanine nucleotide exchange. Gcn2p phosphorylates eIF2 to inhibit eIF2B. Here we evaluate the abundance of eIFs and their pre-initiation intermediate complexes in gcn2 deletion mutant grown under different conditions. We show that ribosomes are three times as abundant as eIF1, eIF2 and eIF5, while eIF3 is half as abundant as the latter three and hence, the limiting component in MFC formation. By quantitative immunoprecipitation, we estimate that approximately 15% of the cellular eIF2 is found in TC during rapid growth in a complex rich medium. Most of the TC is found in MFC, and important, approximately 40% of the total eIF2 is associated with eIF5 but lacks tRNA(i)(Met). When the gcn2Delta mutant grows less rapidly in a defined complete medium, TC abundance increases threefold without altering the abundance of each individual factor. Interestingly, the TC increase is suppressed by eIF5 overexpression and Gcn2p expression. Thus, eIF2B-catalyzed TC formation appears to be fine-tuned by eIF2 phosphorylation and the novel eIF2/eIF5 complex lacking tRNA(i)(Met).

Computational analysis of miRNA-mediated repression of translation: Implications for models of translation initiation inhibition

“…In contrast, the level of eIF4F, as the canonical limiting factor, was set significantly lower so translation would be dependent on its concentration as observed experimentally (e.g., Mathonnet et al 2007). Finally, the amount of subunit joining factors for the 60S large ribosomal subunit were estimated to be more abundant than eIF4F but still substoichiometric when compared to 40S levels, consistent with in vivo levels (Singh et al 2007).…”

“…eIF4F was conversely set very low, so that the protein output would depend on it, as it is the rate-limiting factor in initiation. Finally, the subunit joining factors are substoichiometric to the small ribosomal subunit, consistent with their in vivo abundance (Singh et al 2007). Small ribosomal subunit: 100 Subunit joining factors for the 60S large ribosomal subunit: 25 eIF4F: 6 FIGURE 4.…”

eIF2B promotes eIF5 dissociation from eIF2•GDP to facilitate guanine nucleotide exchange for translation initiation

“…This is consistent with a GDF function for eIF2B, meaning that eIF2B is able to readily access eIF2 from preformed eIF2/eIF5 complexes and promote nucleotide exchange. In vivo eIF2 and eIF5 are equimolar, while eIF2B levels are ;10-fold lower (Singh et al 2007). In our assay, 42 nM eIF2B mimics this ratio.…”

“…Thus, in summary, we provide here evidence for the importance of GDF in vivo because two mutants in eIF2Bg that were isolated as regulators of GCN4 translational control have a major defect in eIF2B GDF activity but only a minor or no defect in GEF function. Discussion eIF2B is a dual-function protein with GDF and GEF activities eIF2 and eIF5 interact with high affinity (Algire et al 2005) and have been shown to exist as an abundant cellular fraction (Singh et al 2006(Singh et al , 2007. The nature and high affinity of such a G-protein•GDP/GAP complex is atypical of most G-protein systems studied.…”

“…This region is highly conserved with the CTD of eIF5 (Bieniossek et al 2006;Wei et al 2006), and both eIF2Be and eIF5 interact with eIF2 in a similar manner, predominantly interacting with the same site on eIF2, a lysine-rich eIF2b region termed the ''K boxes'' (Asano et al 1999; Alone and Dever 2006;Luna et al 2012). eIF2 and eIF5 are equally abundant, and in yeast, there is a large cellular fraction of the eIF2/eIF5 complex (Singh et al 2006(Singh et al , 2007. eIF2B is considerably less abundant (;10-fold).…”