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eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation.

Jennings MD, Pavitt GD - Nature (2010)

Bottom Line: We find that this activity is independent of the GAP function and identify conserved residues within eIF5 that are necessary for this role.Second we show that eIF5 is a critical component of the eIF2(alphaP) regulatory complex that inhibits the activity of the guanine-nucleotide exchange factor (GEF) eIF2B.Together our studies define a new step in the translation initiation pathway, one that is critical for normal translational controls.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.

ABSTRACT
In protein synthesis initiation, the eukaryotic translation initiation factor (eIF) 2 (a G protein) functions in its GTP-bound state to deliver initiator methionyl-tRNA (tRNA(i)(Met)) to the small ribosomal subunit and is necessary for protein synthesis in all cells. Phosphorylation of eIF2 [eIF2(alphaP)] is critical for translational control in diverse settings including nutrient deprivation, viral infection and memory formation. eIF5 functions in start site selection as a GTPase accelerating protein (GAP) for the eIF2.GTP.tRNA(i)(Met) ternary complex within the ribosome-bound pre-initiation complex. Here we define new regulatory functions of eIF5 in the recycling of eIF2 from its inactive eIF2.GDP state between successive rounds of translation initiation. First we show that eIF5 stabilizes the binding of GDP to eIF2 and is therefore a bi-functional protein that acts as a GDP dissociation inhibitor (GDI). We find that this activity is independent of the GAP function and identify conserved residues within eIF5 that are necessary for this role. Second we show that eIF5 is a critical component of the eIF2(alphaP) regulatory complex that inhibits the activity of the guanine-nucleotide exchange factor (GEF) eIF2B. Together our studies define a new step in the translation initiation pathway, one that is critical for normal translational controls.

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The CTD of eIF5 is critical for interaction with eIF2Affinity chromatography assay between eIF2 (110 pmol) and the indicated immobilized GST-eIF5 constructs. eIF2 was detected by immunoblotting using antibodies specific for a) eIF2γ or b) eIF2α. Representative blots are shown. Signal intensity was quantified (Adobe Photoshop) and the mean ± standard deviation (n=3) are shown below. c) Total protein in each sample stained with Ponceau S. Inputs (lanes 1) represent 10% of total.
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Figure 2: The CTD of eIF5 is critical for interaction with eIF2Affinity chromatography assay between eIF2 (110 pmol) and the indicated immobilized GST-eIF5 constructs. eIF2 was detected by immunoblotting using antibodies specific for a) eIF2γ or b) eIF2α. Representative blots are shown. Signal intensity was quantified (Adobe Photoshop) and the mean ± standard deviation (n=3) are shown below. c) Total protein in each sample stained with Ponceau S. Inputs (lanes 1) represent 10% of total.

Mentions: The invariant Arg15 residue in eIF5 is critical for eIF5-dependent GAP activity and the eIF5R15M mutation eliminates this function6-8. The R15M mutant retains full eIF5 GDI activity (Fig.1c, #3) demonstrating that GAP and GDI activities are independent. The tif5-7A allele is a well-characterised eIF5 mutant in which seven evolutionarily-conserved residues in the carboxy-terminal domain (CTD) are mutated to alanines15. These amino acid substitutions impair eIF5-eIF2 interactions11,15,16. We found that eIF57A-FLAG eliminated GDI function, as did a single conservative substitution within this motif at Trp391 (eIF5W391F; Fig.1c, #11& #4). GST-protein affinity chromatography was performed to examine interactions between purified eIF5 and eIF2. This confirmed that GST-eIF5W391F reduces the affinity of eIF5 for purified eIF2, similar to that described for the 7A mutant (Fig.2, lane 5)15 and shows that the eIF2 binding domain provided by the eIF5-CTD is necessary for GDI activity.


eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation.

Jennings MD, Pavitt GD - Nature (2010)

The CTD of eIF5 is critical for interaction with eIF2Affinity chromatography assay between eIF2 (110 pmol) and the indicated immobilized GST-eIF5 constructs. eIF2 was detected by immunoblotting using antibodies specific for a) eIF2γ or b) eIF2α. Representative blots are shown. Signal intensity was quantified (Adobe Photoshop) and the mean ± standard deviation (n=3) are shown below. c) Total protein in each sample stained with Ponceau S. Inputs (lanes 1) represent 10% of total.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875157&req=5

Figure 2: The CTD of eIF5 is critical for interaction with eIF2Affinity chromatography assay between eIF2 (110 pmol) and the indicated immobilized GST-eIF5 constructs. eIF2 was detected by immunoblotting using antibodies specific for a) eIF2γ or b) eIF2α. Representative blots are shown. Signal intensity was quantified (Adobe Photoshop) and the mean ± standard deviation (n=3) are shown below. c) Total protein in each sample stained with Ponceau S. Inputs (lanes 1) represent 10% of total.
Mentions: The invariant Arg15 residue in eIF5 is critical for eIF5-dependent GAP activity and the eIF5R15M mutation eliminates this function6-8. The R15M mutant retains full eIF5 GDI activity (Fig.1c, #3) demonstrating that GAP and GDI activities are independent. The tif5-7A allele is a well-characterised eIF5 mutant in which seven evolutionarily-conserved residues in the carboxy-terminal domain (CTD) are mutated to alanines15. These amino acid substitutions impair eIF5-eIF2 interactions11,15,16. We found that eIF57A-FLAG eliminated GDI function, as did a single conservative substitution within this motif at Trp391 (eIF5W391F; Fig.1c, #11& #4). GST-protein affinity chromatography was performed to examine interactions between purified eIF5 and eIF2. This confirmed that GST-eIF5W391F reduces the affinity of eIF5 for purified eIF2, similar to that described for the 7A mutant (Fig.2, lane 5)15 and shows that the eIF2 binding domain provided by the eIF5-CTD is necessary for GDI activity.

Bottom Line: We find that this activity is independent of the GAP function and identify conserved residues within eIF5 that are necessary for this role.Second we show that eIF5 is a critical component of the eIF2(alphaP) regulatory complex that inhibits the activity of the guanine-nucleotide exchange factor (GEF) eIF2B.Together our studies define a new step in the translation initiation pathway, one that is critical for normal translational controls.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.

ABSTRACT
In protein synthesis initiation, the eukaryotic translation initiation factor (eIF) 2 (a G protein) functions in its GTP-bound state to deliver initiator methionyl-tRNA (tRNA(i)(Met)) to the small ribosomal subunit and is necessary for protein synthesis in all cells. Phosphorylation of eIF2 [eIF2(alphaP)] is critical for translational control in diverse settings including nutrient deprivation, viral infection and memory formation. eIF5 functions in start site selection as a GTPase accelerating protein (GAP) for the eIF2.GTP.tRNA(i)(Met) ternary complex within the ribosome-bound pre-initiation complex. Here we define new regulatory functions of eIF5 in the recycling of eIF2 from its inactive eIF2.GDP state between successive rounds of translation initiation. First we show that eIF5 stabilizes the binding of GDP to eIF2 and is therefore a bi-functional protein that acts as a GDP dissociation inhibitor (GDI). We find that this activity is independent of the GAP function and identify conserved residues within eIF5 that are necessary for this role. Second we show that eIF5 is a critical component of the eIF2(alphaP) regulatory complex that inhibits the activity of the guanine-nucleotide exchange factor (GEF) eIF2B. Together our studies define a new step in the translation initiation pathway, one that is critical for normal translational controls.

Show MeSH
Related in: MedlinePlus