Figure 6.
Disruption of the b/PRT1–i/TIF34 interaction prevents 40S-binding of the i/TIF34–g/TIF35 mini-module and dramatically increases leaky scanning over the AUG start site producing a severe Gcn- phenotype. (A) Physical detachment of i/TIF34 and g/TIF35 from the rest of eIF3 selectively affects stability of pre-initiation complexes in vivo. Transformants of H450 (tif34Δ) bearing wt or mutant i/TIF34-HA were grown in SD medium at 34 C to an OD600 of approximately 1.5 and cross-linked with 2% HCHO prior to harvesting. WCEs were prepared, separated on a 7.5–30% sucrose gradient by centrifugation at 41 000 rpm for 5 h and subjected to western blot analysis (note that the anti-RPS0A antibodies were generated in this study; see Supplementary Data). Fractions 1–4, 5–9, and 10–12 (43–48S) were pooled; lanes ‘In0’ and ‘In6’ show samples of the input WCEs (5%) that were processed immediately before (h0) or after (h6) incubation for 6 h on ice, mimicking the duration of the HCHO fractionation experiment to document the stability of the factors of interest in WCEs. Proportions of the 40S-bound proteins relative to the amount of 40S subunits were calculated using NIH ImageJ from three independent experiments. The resulting values obtained with the wt strain were set to 100% and those obtained with mutant strains were expressed as percentages of the wt (SDs are given). This experiment was repeated seven times with similar results. (B) tif34-DD/KK imparts the Gcn- phenotype. H417 (GCN2), H418 (gcn2Δ) and H450 (tif34Δ) bearing wt or mutant i/TIF34-HA were spotted in four serial 10-fold dilutions on SD (upper panel) or SD containing 30 mM 3-AT (lower panel) and then incubated at 34°C for 3 and 7 days, respectively. (C) tif34-DD/KK severely prevents derepression of GCN4-lacZ upon starvation. The H450 strains as in panel A were transformed with the GCN4-lacZ reporter p180 and grown in SD medium at 34°C to an OD600 of approximately 1. The β-galactosidase activities were measured in the WCEs and expressed in units of nmol of O-nitrophenyl-β-d-galactopyranoside hydrolyzed per minute per mg of protein. To induce GCN4-lacZ expression, strains were grown in minimal medium to an OD600 approximately 0.5 and then treated with 10 mM 3-AT for 6 h. The table gives mean values and standard deviations obtained from at least six independent measurements with three independent transformants, and activities with 3-AT-induction relative to those without induction. (D) Detachment of the i/TIF34–g/TIF35 mini-module from eIF3 provokes unusually severe leaky scanning defect. The H450 strains as in (A) were transformed with the GCN4-lacZ reporter plasmids pM226 (i) and plig102-3 (ii) and analyzed as in (C). The table gives activities in mutant relative to wt cells.