Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 Feb 15;12(4):514-26.
doi: 10.1101/gad.12.4.514.

eIF2 independently binds two distinct eIF2B subcomplexes that catalyze and regulate guanine-nucleotide exchange

G D Pavitt  1 , K V Ramaiah, S R Kimball, A G Hinnebusch
Affiliations

eIF2 independently binds two distinct eIF2B subcomplexes that catalyze and regulate guanine-nucleotide exchange

G D Pavitt et al. Genes Dev. .

Abstract

eIF2B is a heteropentameric guanine-nucleotide exchange factor essential for protein synthesis initiation in eukaryotes. Its activity is inhibited in response to starvation or stress by phosphorylation of the alpha subunit of its substrate, translation initiation factor eIF2, resulting in reduced rates of translation and cell growth. We have used an in vitro nucleotide-exchange assay to show that wild-type yeast eIF2B is inhibited by phosphorylated eIF2 [eIF2(alphaP)] and to characterize eIF2B regulatory mutations that render translation initiation insensitive to eIF2 phosphorylation in vivo. Unlike wild-type eIF2B, eIF2B complexes with mutated GCN3 or GCD7 subunits efficiently catalyzed GDP exchange using eIF2(alphaP) as a substrate. Using an affinity-binding assay, we show that an eIF2B subcomplex of the GCN3, GCD7, and GCD2 subunits binds to eIF2 and has a higher affinity for eIF2(alphaP), but it lacks nucleotide-exchange activity. In contrast, the GCD1 and GCD6 subunits form an eIF2B subcomplex that binds equally to eIF2 and eIF2(alphaP). Remarkably, this second subcomplex has higher nucleotide-exchange activity than wild-type eIF2B that is not inhibited by eIF2(alphaP). The identification of regulatory and catalytic eIF2B subcomplexes leads us to propose that binding of eIF2(alphaP) to the regulatory subcomplex prevents a productive interaction with the catalytic subcomplex, thereby inhibiting nucleotide exchange.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Guanine–nucleotide exchange catalyzed by wild-type and mutant eIF2B, and its inhibition by phosphorylation of eIF2. eIF2 ⋅ [3H]GDP binary complexes were preformed, with or without prior phosphorylation of eIF2 by HCR, and challenged with nonradiolabeled GDP (see Materials and Methods). (A–D) Level of eIF2 ⋅ 3H]GDP (filled symbols connected with solid lines) and eIF2(αP) ⋅ [3H]GDP (open symbols connected with broken lines) binary complexes remaining with time, following incubation with cell extracts (150 μg) from yeast strains bearing high-copy plasmids encoding the form of eIF2B indicated in each inset: (A) Wild-type five-subunit eIF2B (h.c. eIF2B); (B) eIF2B*4s, regulatory mutant eIF2B containing the four essential eIF2B subunits, but lacking GCN3; (C) eIF2B*5s-M1, eIF2B containing five eIF2B subunits with a regulatory mutation in GCD7 (GCD7-S119P); and (D) eIF2B*5s-M2, eIF2B containing five eIF2B subunits with a regulatory mutation in GCD7 (GCD7-I118T,D178Y). Control reactions with cell extracts from yeast strains containing empty vectors (filled circles linked with solid lines) are shown in A–D, and a reaction with extract buffer substituted for yeast cell extract (crosses linked by dotted lines) is shown in A.
Figure 2
Figure 2
Phosphorylation of purified eIF2 in vitro. (A) Purified eIF2 was phosphorylated for the indicated times (lanes 2–4), after which samples were resolved by IEF PAGE and eIF2α detected by Western blotting. (Lane 1) Unphosphorylated purified eIF2. (B) Guanine–nucleotide exchange reactions were performed exactly as described in Fig. 1, except that unlabeled GDP was used throughout. Samples were taken at the indicated times following the addition of cell extracts (150 μg) from yeast strains bearing high-copy plasmids encoding the indicated form of eIF2B (as in Fig. 1). eIF2α was resolved and detected as in A. (Lanes 1,2) Samples from reactions with unphosphorylated eIF2 ⋅ GDP binary complexes; (lanes 3–14) binary complexes prephosphorylated with HCR kinase; (lane 15) buffer and wild-type eIF2B extract only with no added eIF2 ⋅ GDP binary complexes. The positions of phosphorylated and unphosphorylated eIF2α are indicated.
Figure 3
Figure 3
Guanine–nucleotide exchange catalyzed by eIF2B subcomplexes. Experiments were performed exactly as in Fig. 1 except with yeast cell extracts (150 μg) containing the overexpressed eIF2B subunits indicated in each inset. (A) Wild-type GCN3, GCD7, and GCD2 (h.c. GCN3/GCD2/GCD7); (B) wild-type GCN3 and GCD2 with regulatory mutant GCD7-S119P (GCD7*M1); (C) wild-type GCN3 and GCD2 with regulatory mutant GCD7-I118T,D178Y (GCD7*M2); (D) wild-type GCD7 and GCD2 only; (E) wild-type GCD1 and GCD6 (h.c. GCD1/GCD6) and all five subunits of wild-type eIF2B (h.c. eIF2B); (F) wild-type GCD1 alone (h.c. GCD1) and wild-type GCD6 alone (h.c. GCD6) in addition to the extracts shown in E. Control reactions with cell extracts from yeast cells containing empty vectors (filled circles linked with solid lines) are shown in all panels.
Figure 4
Figure 4
Binding of wild-type eIF2B and mutant eIF2B lacking the GCN3 subunit to His-tagged eIF2. Yeast whole cell extracts (100 μg) from strains overexpressing wild-type eIF2B (lanes 1–4) or eIF2B lacking the GCN3 subunit (eIF2B*4s, lanes 5–8) were incubated with 2.5 μg of prephosphorylated purified His-tagged eIF2 (lanes 2,6), unphosphorylated His-tagged eIF2 (lanes 3,7), or no eIF2 (lanes 4,8); proteins bound to eIF2 were recovered by Ni-NTA affinity chromatography. One third of each reaction was separated by SDS-PAGE and proteins identified by Western blotting. (A) Western blot analysis with specific polyclonal antisera to eIF2α and eIF2B subunits. (Input) Ten micrograms of each cell extract used (lanes 1,5). For GCN3, only the lower band represents the GCN3 signal, the upper diffuse band in each lane is a nonspecific cross-reacting band (Pavitt et al. 1997). (B) Histograms showing densitometry of signals for each eIF2B antiserum used in pellet lanes (lanes 2–4,6–8) from A relative to the density of the signal in lane 4, which was assigned an arbitrary value of 1. Mean densitometry for all subunits is shown.
Figure 4
Figure 4
Binding of wild-type eIF2B and mutant eIF2B lacking the GCN3 subunit to His-tagged eIF2. Yeast whole cell extracts (100 μg) from strains overexpressing wild-type eIF2B (lanes 1–4) or eIF2B lacking the GCN3 subunit (eIF2B*4s, lanes 5–8) were incubated with 2.5 μg of prephosphorylated purified His-tagged eIF2 (lanes 2,6), unphosphorylated His-tagged eIF2 (lanes 3,7), or no eIF2 (lanes 4,8); proteins bound to eIF2 were recovered by Ni-NTA affinity chromatography. One third of each reaction was separated by SDS-PAGE and proteins identified by Western blotting. (A) Western blot analysis with specific polyclonal antisera to eIF2α and eIF2B subunits. (Input) Ten micrograms of each cell extract used (lanes 1,5). For GCN3, only the lower band represents the GCN3 signal, the upper diffuse band in each lane is a nonspecific cross-reacting band (Pavitt et al. 1997). (B) Histograms showing densitometry of signals for each eIF2B antiserum used in pellet lanes (lanes 2–4,6–8) from A relative to the density of the signal in lane 4, which was assigned an arbitrary value of 1. Mean densitometry for all subunits is shown.
Figure 5
Figure 5
Binding of the regulatory GCN3/GCD7/GCD2 subcomplex to His-tagged eIF2. Overexpressed trimeric GCN3/GCD7/GCD2 subcomplex (lanes 1–4) or overexpressed single eIF2B subunits GCN3 (lanes 5–8), GCD2 (lanes 9–12), or GCD7 (lanes 13–16) were bound to eIF2 as in Fig. 4. (A) Western blot analysis of the binding eIF2B subunits to Ni–NTA–agarose in the presence of prephosphorylated purified His-tagged eIF2 (lanes 2,6,10,14), unphosphorylated purified His-tagged eIF2 (lanes 3,7,11,15), or without purified eIF2 (lanes 4,8,12,16). (Input) 10 μg of each cell extract used (lanes 1,5,9,13). (B) Histograms showing densitometry of signals for eIF2B antisera shown in pellet lanes (lanes 2–4,6–8,10–12,14–16) from A relative to the density of the signal in lanes 4,8,12, and 16, which were assigned an arbitrary value of 1. Mean densitometry for the three subunits of the trimeric regulatory complex is shown.
Figure 6
Figure 6
Subcomplex formation between His-tagged GCD1 and GCD6. Ni-NTA–silica affinity chromatography of whole-cell extracts from strains co-overexpressing His-tagged GCD1 and wild-type GCD6 (lanes 1,4), co-overexpressing wild-type GCD1 and GCD6 (lanes 2,5), or carrying only the plasmid vector (lanes 3,6). (A) Western blot analysis of the binding of eIF2B subunits from 120 μg of cell extracts to Ni–NTA–silica resin (lanes 4–6) and from 10 μg of each cell extract input (lanes 1–3). (B) Histograms showing densitometry of signals for GCD1 and GCD6 antiserum from A relative to the density of the signal in lane 3, which was assigned an arbitrary value of 1.
Figure 7
Figure 7
Binding of the catalytic GCD1/GCD6 subcomplex to His-tagged eIF2. As described for Fig. 4 except that the cell extracts used contained the overexpressed catalytic GCD6/GCD1 subcomplex (lanes 1–4) or overexpressed single subunits GCD6 (lanes 5–8) or GCD1 (lanes 9–12). (A) Western blot analysis of eIF2α and eIF2B subunits. The binding of 33 μg of cell extracts to Ni-NTA–agarose beads in the presence of purified His-tagged eIF2 prephosphorylated with HCR kinase (lanes 2,6,10), unphosphorylated purified His-tagged eIF2 (lanes 3,7,11), or without added purified His-tagged eIF2 (lanes 4,8,12). (Input) 10 μg of each cell extract used (lanes 1,5,9). (B) Histograms showing densitometry of signals for each eIF2B antibody shown in pellet lanes (lanes 2–4,6–8,10–12,14–16) from A relative to the density of the signal in lanes 4, 8, and 12, which were assigned an arbitrary value of 1.
Figure 8
Figure 8
A sequential binding model for eIF2B catalyzed guanine-nucleotide exchange and its inhibition by eIF2(αP). (A) Proposed two-binding surface scheme for GDP/GTP exchange with unphosphorylated eIF2. The eIF2 (unfilled oval labeled α, β, γ) in a binary complex with GDP (light-grey filled circle with a light center) makes initial contact with eIF2B via a surface on the regulatory GCN3/GCD7/GCD2 subcomplex (stippled shape labeled 2 3 7). A hypothetical conformational change in eIF2B (movement indicated by grey arrows) occurs to promote correct contact between eIF2γ and the eIF2B catalytic subcomplex (filled black with subunits labeled 6 1), permitting exchange of GDP for GTP (grey filled circle with dark center). (B) Inhibition of nucleotide exchange by phosphorylated eIF2. eIF2(αP) (as in A with an added filled circle, labeled ∼P) binds to the eIF2B regulatory subcomplex with high affinity (broad arrow), this inhibits the conformational change in eIF2B, preventing nucleotide exchange. (C) In the absence of the eIF2B regulatory subcomplex, direct binding of eIF2γ to the catalytic subcomplex allows nucleotide exchange even with eIF2(αP). (D) Regulatory mutant eIF2B can perform nucleotide exchange with eIF2(αP). eIF2(αP) binds to mutant eIF2B (eIF2B*), making contact with the altered regulatory subcomplex (altered shaped stippled box labeled 2 3 7). The regulatory defect allows the conformational change needed for productive interaction between eIF2γ and the catalytic subcomplex even when eIF2 is phosphorylated.
See this image and copyright information in PMC

References

    1. Boeke JD, LaCroute F, Fink GR. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-Fluoro-orotic acid resistance. Mol Gen Genet. 1984;197:345–346. - PubMed
    1. Boguski MS, McCormick F. Proteins regulating Ras and its relatives. Nature. 1993;366:643–654. - PubMed
    1. Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: Conserved structure and molecular mechanism. Nature. 1991;349:117–127. - PubMed
    1. Bushman JL, Asuru AI, Matts RL, Hinnebusch AG. Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae. Mol Cell Biol. 1993a;13:1920–1932. - PMC - PubMed
    1. Bushman JL, Foiani M, Cigan AM, Paddon CJ, Hinnebusch AG. Guanine nucleotide exchange factor for eIF-2 in yeast: Genetic and biochemical analysis of interactions between essential subunits GCD2, GCD6 and GCD7 and regulatory subunit GCN3. Mol Cell Biol. 1993b;13:4618–4631. - PMC - PubMed

Publication types

MeSH terms