Fig. 5. The TIF32-CTD interacts with eIF2 in vitro and this contact is important for efficient translation initiation in vivo. (A) The same as in Figure 4B. (B) Flag-tagged eIF2 was purified from WCEs using anti-FLAG affinity resin and used in pull-down assays with GST alone (lane 3) or GST fusions to TIF32-Δ4, TIF32-Δ6 or TIF5 (eIF5) (lanes 4–6). Binding of eIF2 was detected by western blotting with antibodies against eIF2γ (GCD11). Lane 2 contained 10% of the eIF2 added to each reaction. (C) Binding of ³⁵S-labeled eIF2 subunits to GST–TIF32-Δ4, GST–TIF32-Δ6 (lanes 3 and 4) or GST alone (lane 2), as described in Figure 3D. (D) Transformants of strain W303 containing empty vector (1), hc YEpTIF32-His (2) or hc YEpTIF32-Δ5-His-U (3–5) were co-transformed with empty vector (1–3), hc YEpNIP1-His (4) and hc pLPY-PRT1-His-TIF34-HA-TIF35-Flag (5). The resulting strains were streaked on SD medium and incubated at 30°C for 2 days. (E) The dominant-negative Slg^– phenotype of hc TIF32-Δ6-His is suppressed by high-copy eIF2 and tRNA_i^Met. Transformants of strain W303 containing empty vectors; hc YEpTIF32-Δ6-His and empty vector (YEp24); and hc YEpTIF32-Δ6-His and p1780-IMT were spotted in four serial dilutions on SD medium and incubated at 30°C for 2 days. (F) Overexpressing the CTD-less TIF32-Δ6-His protein severely exacerbates the translation initiation defect in tif5-7A Ts^– cells. Upper panels: strains H2898 (TIF5) and H2899 (tif5-7A) were transformed with empty vector or hc YEpTIF32-Δ6-His-U, grown in SD medium to an OD₆₀₀ of ∼1.5, and 50 µg/ml cycloheximide was added 5 min prior to harvesting. WCEs were prepared and resolved on 5–45% sucrose gradients. The positions of 40S and 60S subunits and 80S ribosomes are indicated. P/M, ratio of the A₂₅₄ in the combined polysome fractions to that in the 80S peak; d.t., cell doubling time in hours. Bottom panels: the latter strains were spotted in four serial dilutions on SD medium and incubated at 35°C for 2 days.