Purification of FLAG‐Tagged Eukaryotic Initiation Factor 2B Complexes, Subcomplexes, and Fragments from Saccharomyces cerevisiae

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation.

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“…GST-eIF5 was purified from Escherichia coli as described (23). eIF2B was purified from yeast strain GP5949, as described (39). …”

Section: Methodsmentioning

confidence: 99%

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control

Jennings¹,

Kershaw²,

White³

et al. 2016

Nucleic Acids Res

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“…Flag-tagged PKR was purified from a yeast strain resistant to the toxic effects of PKR expression (GP3299) and harboring plasmid pAV1412 as described previously (Krishnamoorthy et al 2001). eIF2B complexes, subcomplexes, and subunits were all purified from yeast as described (Mohammad-Qureshi et al 2007b) using protease-deficient strains bearing high-copy-number plasmids overexpressing the required factor genes from 2-mm plasmids BJ1995[GP3583] (MATa prb1-1122 pep4-3 leu2 trp1 ura3-52 gal2) or GP4597(MATa trp1D63 ura3-52 leu2-3 leu2-112 GAL2 gcn2D pep4TLEU2). Plasmids used are described in Supplemental Table S1.…”

Section: Yeast Geneticsmentioning

confidence: 99%

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

et al. 2013

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Protein synthesis factor eIF2 delivers initiator tRNA to the ribosome. Two proteins regulate its G-protein cycle: eIF5 has both GTPase-accelerating protein (GAP) and GDP dissociation inhibitor (GDI) functions, and eIF2B is the guanine nucleotide exchange factor (GEF). In this study, we used protein-protein interaction and nucleotide exchange assays to monitor the kinetics of eIF2 release from the eIF2•GDP/eIF5 GDI complex and determine the effect of eIF2B on this release. We demonstrate that eIF2B has a second activity as a GDI displacement factor (GDF) that can recruit eIF2 from the eIF2•GDP/eIF5 GDI complex prior to GEF action. We found that GDF function is dependent on the eIF2Be and eIF2Bg subunits and identified a novel eIF2-eIF2Bg interaction. Furthermore, GDF and GEF activities are shown to be independent. First, eIF2B GDF is insensitive to eIF2a phosphorylation, unlike GEF. Second, we found that eIF2Bg mutations known to disrupt GCN4 translational control significantly impair GDF activity but not GEF function. Our data therefore define an additional step in the protein synthesis initiation pathway that is important for its proper control. We propose a new model to place eIF2B GDF function in the context of efficient eIF2 recycling and its regulation by eIF2 phosphorylation.

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“…In support of the latter model, both the GEF domain of eIF2Bε and the eIF5-CTD share a common HEAT repeat structure 28,29 that bind to the eIF2β and γ subunits. 5,[30][31][32] In addition, it was shown that excess eIF5 antagonises GEF mutants (and vice versa). 10 Thus from our current knowledge it seems possible that stable binding between eIF5-GDI and eIF2βγ accompanies eIF2B binding to eIF2(αP) and that together both prevent eIF2Bε-eIF2γ interactions and inhibit GEF activity.…”

Section: How Do Gef and Gdi Regulation Combine To Control Eif2 Activity?mentioning

confidence: 99%

eIF5

Jennings

Pavitt

2010

Small GTPases

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Purification of FLAG‐Tagged Eukaryotic Initiation Factor 2B Complexes, Subcomplexes, and Fragments from Saccharomyces cerevisiae

Cited by 22 publications

References 43 publications

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control

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

eIF5

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