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Comparative Study
. 2005 Sep 12;170(6):925-34.
doi: 10.1083/jcb.200503162.

Dynamic cycling of eIF2 through a large eIF2B-containing cytoplasmic body: implications for translation control

Susan G Campbell  1 , Nathaniel P Hoyle, Mark P Ashe
Affiliations
Comparative Study

Dynamic cycling of eIF2 through a large eIF2B-containing cytoplasmic body: implications for translation control

Susan G Campbell et al. J Cell Biol. .

Abstract

The eukaryotic translation initiation factor 2B (eIF2B) provides a fundamental controlled point in the pathway of protein synthesis. eIF2B is the heteropentameric guanine nucleotide exchange factor that converts eIF2, from an inactive guanosine diphosphate-bound complex to eIF2-guanosine triphosphate. This reaction is controlled in response to a variety of cellular stresses to allow the rapid reprogramming of cellular gene expression. Here we demonstrate that in contrast to other translation initiation factors, eIF2B and eIF2 colocalize to a specific cytoplasmic locus. The dynamic nature of this locus is revealed through fluorescence recovery after photobleaching analysis. Indeed eIF2 shuttles into these foci whereas eIF2B remains largely resident. Three different strategies to decrease the guanine nucleotide exchange function of eIF2B all inhibit eIF2 shuttling into the foci. These results implicate a defined cytoplasmic center of eIF2B in the exchange of guanine nucleotides on the eIF2 translation initiation factor. A focused core of eIF2B guanine nucleotide exchange might allow either greater activity or control of this elementary conserved step in the translation pathway.

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Figures

Figure 1.
Figure 1.
Localization of eIFs in S. cerevisiae . (A) Diagram representing the eukaryotic translation initiation pathway. (B) Live cell confocal microscopic images of strains YMK1170, 1171, 1172, 885, 881, 883, 1211, 880, and 882 bearing chromosomally integrated COOH-terminal eGFP tags, (i) TIF1-GFP (eIF4AI-GFP), (ii) TIF5-GFP (eIF5-GFP), (iii) TIF4631-GFP (eIF4GI-GFP), (iv) CDC33-GFP (eIF4E-GFP), (v) PRT1-GFP (eIF3b-GFP), (vi) SUI2-GFP (eIF2α-GFP), (vii) GCD11-GFP (eIF2γ-GFP), (viii) GCD1-GFP (eIF2Bγ-GFP), and (ix) GCD6-GFP (eIF2Bɛ-GFP). (C) Colocalization (left) GCD1-CFP (eIF2Bγ-GFP), (middle) SUI2-YFP (eIF2α-GFP), and (right, overlay) using strain YMK1144. (D) Immunofluorescence of fixed YMK467 cells with anti-eIF2Bɛ antibodies. Four defined images from the same field of view are shown.
Figure 2.
Figure 2.
The eIF2–eIF2B foci are not sites of eIF2B regulation or TC formation however they do require active translation. (A) The localization of (i) initiator and (ii) elongator Met-tRNAMet was analyzed in the eIF2Bγ-GFP–containing strain YMK880 by FISH using end-labeled oligonucleotides. FISH images were compared with eIF2Bγ-GFP localization in the overlay images. (B) GCN2 and gcn2-null strains bearing eIF2Bγ-GFP, YMK880 (i) and YMK1087 (ii), and eIF2α-GFP, YMK883 (iii), and YMK1088 (iv) were grown in media containing (+AA) or lacking amino acids (−AA) for 15 min. Cells were visualized by live cell confocal microscopy. (C) Strains YMK880 (i, eIF2Bγ-GFP) and YMK883 (ii, eIF2α-GFP) were incubated at room temperature for 10 min in the presence or absence of cycloheximide (100 μg/ml). (D) The strains (i) YMK1123 (eIF2Bγ-GFP, prt1-1), (ii) YMK880 (eIF2Bγ-GFP), (iii) YMK1124 (eIF2α-GFP, prt1-1), and (iv) YMK883 (eIF2α-GFP) were incubated at the permissive (26°C) and nonpermissive (37°C) temperature for 15 min. Cells were visualized by live cell confocal microscopy. (E) Protein extracts from eIF2Bγ-GFP strains YMK880 (GCN2, lanes 1–3), YMK1087 (gcn2Δ, lanes 4–6), and eIF2α-GFP strains, YMK883 (GCN2, lanes 8–10), and YMK1088 (gcn2Δ, lanes 11–13) were blotted and probed with antibodies to eIF2α and phosphospecific antibodies to phophoserine 51 on eIF2α.
Figure 3.
Figure 3.
eIF2 cycles rapidly through the foci whereas eIF2B is less dynamic. (A) eIF2α-GFP FRAP analysis. Panels show representative prebleach (pb), bleach (b), and recovery (r) images from FRAP experiment on strain YMK883. The bleached focus is marked with a white arrowhead and the asterisk on the recovery image corresponds to time point on the graph when the image was taken. (B) Graph show quantitation of eIF2α-GFP FRAP experiments. Control represents the FRAP results from YMK883 fixed cells. (C) eIF2Bγ-GFP FRAP analysis. Panels show representative prebleach (pb), bleach (b), and recovery (r) images from FRAP experiment on strain YMK880. The bleached focus is marked with a white arrowhead and the asterisk on the recovery image corresponds to time point on the graph when the image was taken. (D) Graph show quantitation of eIF2Bγ-GFP FRAP experiments. Control represents the FRAP results from YMK880-fixed cells.
Figure 4.
Figure 4.
eIF2α-GFP shuttling is altered in the absence of amino acids. Figure shows FRAP experiments on eIF2α-GFP–bearing strains as described in Fig. 3. (A) YMK883 FRAP after (i) 15-min control incubation and (ii) 15-min starvation for amino acids. (B) Graph showing quantitation of eIF2α-GFP amino acid starvation FRAP experiments. (C) YMK1088 (i, gcn2Δ) and (ii) YMK883 strains after 1 h starvation for amino acids. (D) Graph showing quantitation of eIF2α-GFP FRAP experiments after a 1-h amino acid starvation in the presence and absence of Gcn2p. pb, Prebleach; b, bleach; and r, recovery.
Figure 5.
Figure 5.
The eIF2–eIF2B foci represent sites of guanine nucleotide exchange. Figure shows FRAP experiments on eIF2α-GFP bearing strains as described in Fig. 3. (A) YMK883 strains transformed with (i) control plasmid, pRS316, (ii) pAV1245 (GCN2 cM788V-E1606G), and (iii) pAV1248 (GCN2 cM788-E1591K), respectively. (B) Graph showing quantitation of eIF2α-GFP FRAP experiments with GCN2 c mutants. (C) Protein extracts from strains YMK883 pRS316, YMK883 pAV1245[GCN2 cM788V-E1606G], and YMK883 pAV1248[GCN2 cM788V-E1591K] were blotted and probed with antibodies to eIF2α and phosphospecific antibodies to phophoserine 51 on eIF2α. (D) FRAP analysis of eIF2α-GFP in strains (i) YMK1168 (GCD6) and (ii) YMK1169 (gcd6-F250L). (E) Graph showing quantitation of eIF2α-GFP FRAP experiments with wt and eIF2Bɛ catalytic mutant. pb, Prebleach; b, bleach; r, recovery.
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