1. Volume 36, Issue 1, Pages 141-152 (October 2009)
The eIF3 Interactome Reveals the Translasome, a Supercomplex Linking Protein Synthesis and Degradation Machineries 
Zhe Sha, Laurence M. Brill, Rodrigo Cabrera, Oded Kleifeld, Judith S. Scheliga, Michael H. Glickman, Eric C. Chang, Dieter A. Wolf 
Molecular Cell 
Volume 36, Issue 1, Pages (October 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 Purification of eIF3 Binding Proteins from Fission Yeast
(A) Flow chart showing the procedure for affinity protein purification of eIF3 complexes from eIF3e-ProA, eIF3m-ProA, or untagged parental cells (strains C648, C617/1, and DS448/1, respectively).
(B) Protein samples collected at various steps throughout the purification as indicated in (A) were separated by SDS-PAGE and stained with Coomassie blue.
(C) The same samples as in (B) were analyzed by immunoblot using an antibody against Rpn1p. Asterisks mark eIF3e-ProA and eIF3m-ProA, whose protein A (ProA) tags were recognized by the secondary antibody. The positions of the IgG heavy chain and the cleaved ProA tag are marked by arrows.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Summary of the eIF3 Interactome and Distinct eIF3 Complexes
(A) Intensity map of the spectrum counts of the 230 proteins that were identified, using LC-MS/MS, as specific and reproducible interactors with the eIF3e-ProA and eIF3m-ProA bait proteins. The identified proteins were grouped manually into protein complexes and pathways. Individual proteins are listed in Table S2 and Supplemental Data. Data from three independent experiments are shown (indicated as #1, #2, and #3). In experiment #3, the cell lysate was treated with RNase prior to affinity capture. The overall signal intensity appears higher in experiments #2 and #3, because the data represent the spectrum count sums of four consecutive mass spectrometry runs, whereas samples of experiment #1 were run only three times.
(B and C) Relative abundance of all 11 S. pombe eIF3 subunits. The left panel (B) shows an intensity map based on raw spectrum counts. The right panel (C) shows the same data after adjustment to the molecular weight of the subunits and normalization to eIF3a. Only experiments #2 and #3 are shown, because their spectral count intensities are directly comparable (see [A]).
(D) Models of fission yeast eIF3 complexes with proposed functions in global and mRNA-specific translation. The topology of the complexes is based on results from top-down mass spectrometry and was adapted from Zhou et al. (Zhou et al., 2008).
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4. Figure 3 Protein Components of the Translasome
(A–E) Spectrum count intensity maps of eIFs (A), eEFs (B), tRNA synthetases (C), 19S proteasome (D), and ribosome biogenesis proteins (E) from purifications 2 and 3 (#2 and #3, respectively).
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5. Figure 4 Genetic Interactions between kap123Δ and sal3Δ with eif3eΔ
(A) Spectrum count intensity map of eIF3-interacting proteins involved in cellular transport.
(B) Strain YIN6A (yin6/eif3eΔ) was crossed with strain KAP123U (kap123Δ) (Chen et al., 2004), and the diploid cells were induced to sporulate to form tetrads. A tetra-type tetrad was dissected to show spore viability of the indicated genotypes (left). A microcolony derived from a kap123Δ eif3eΔ spore was scraped off the plate and visualized by DIC microscopy (right).
(C) Cells with the indicated genotypes were obtained from a tetra-type tetrad produced by eif3eΔ/+ sal3Δ/+ diploid cells, which were obtained by crossing strains YIN6A and SAL3U (Chen et al., 2004). These cells were spotted onto YEAU plates and grown at the indicated temperatures.
(D) Cells with the indicated genotypes were obtained from a tetra-type tetrad produced by sal3Δ/+ kap123Δ/+ diploid cells, which were generated by crossing strains SAL3U and KAP123C. The cells were spotted onto YEAU plates and grown at 30°C.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Efficient Nuclear Accumulation of the Proteasome Requires eIF3e and Sal3p
(A) The indicated strains were serially diluted, spotted on minimal media plates with or without canavanine, and grown at 32°C.
(B) Cells carrying Rpn7p-GFP were optically sectioned through the middle of the cell body by confocal microscopy. Representative confocal images are shown. While Rpn7p-GFP can be readily seen in the nucleoplasm (except for an area that is presumed to be the nucleolus) of WT cells as well as the single-mutant cells, there was very little Rpn7p-GFP signal inside the nucleus of ∼30% of sal3Δ eif3eΔ cells (example marked by an arrow).
(C) Individual nuclei of the indicated cells were photobleached with a laser (marked by dotted green circles), and reaccumulation of nuclear Rpn7p-GFP was recorded in 1 min intervals over 60 min. To quantify nuclear accumulation, the nucleus of an unbleached cell (arrowhead) was measured to correct for spontaneous photobleaching. The relative intensities (RI) corrected for spontaneous photobleaching at each time point after photobleaching were measured, and the ratio of nuclear versus cytoplasmic RI (RIN/C) was plotted over time. The data in the graph represent values averaged from measuring ten cells with error bars (SEM). Unpaired Student's t tests confirmed that the difference between WT and sal3Δ eif3eΔ cells was highly significant at 38/60 time points with p ≤ 0.01 and at 52/60 time points with p ≤ 0.05 (Table S4). Scale bar, 5 μm.
(D) Cells were obtained as described in Figure 4C, spotted onto YEAU plates with or without phleomycin, and grown at 32°C.
(E) WT, sal3Δ, and kap123Δ cells (strains MBY1270, SAL3U, and KAP123U, respectively) (Chen et al., 2004) were spotted onto YEAU plates with or without phleomycin and grown at 30°C.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Model of the eIF3-Associated Translasome
Light green circles represent eEFs 1, 2, and 3; initiation factors are in dark blue; eIF3 is in light blue. An actin filament is depicted in purple and a nascent polypeptide chain as black balls. MSC, multisynthetase complex.
Molecular Cell  , DOI: ( /j.molcel )
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