Article Figures & Data
Figures
- FIG. 1.
Intragenic suppressors of the eIF5B G479A mutation. (A) Amino acid sequences of the Switch I (G-2 sequence motif), Switch II (G-3 sequence motif), and helix H8 elements in yeast eIF5B. The G479A mutation and the A444V and D740R suppressor mutations are shown, and the position numbers in yeast eIF5B are indicated. The invariable residues Thr439 in Switch I and Asp476 and Gly479 in Switch II are shown in boldface. (B) Growth rate analysis of yeast expressing WT and mutant forms of eIF5B. The ΔeIF5B strain J111 was transformed with the empty vector YCplac33 (ΔeIF5B) or the same plasmid containing the indicated WT or mutant eIF5B genes. Transformants were grown to saturation, and 4 μl of serial dilutions (at optical densities at 600 nm of 1.0, 0.1, 0.01, 0.001, and 0.0001) was spotted onto synthetic dextrose medium supplemented with the required nutrients and incubated at 30°C for 4 days. (C) Western blot analysis of eIF5B expression. Whole-cell extracts prepared from transformants described in the legend for panel B were subjected to immunoblot analysis using anti-eIF5B or anti-eIF2α antiserum as described previously (7). Immune complexes were visualized using enhanced chemiluminescence. (D) Analysis of polysome profiles for strains expressing eIF5B, eIF5B-G479A, eIF5B-A444V,G479A, and eIF5B-G479A,D740R. Whole-cell extracts from yeast strain J111 expressing the indicated eIF5B WT or mutant protein were resolved by velocity sedimentation in 7 to 47% sucrose gradients. Gradients were fractionated while scanning at A254, and the positions of the 40S and 60S subunits, 80S ribosomes, and polysomes are indicated. P/M ratios were calculated by measuring the areas under the peaks representing the polysome fractions and the 80S peak.
- FIG. 2.
Biochemical analysis of eIF5B-G479A Switch II mutant and eIF5B-A444V,G479A and eIF5B-G479A,D740R suppressor mutants. (A) Summary of guanine nucleotide Kd values for eIF5B mutants. Values shown in parentheses are standard errors. (B) Results from a ribosome-dependent GTPase assay. Reaction mixtures containing 1 μM eIF5B, 0.4 μM 40S and 60S ribosomal subunits, and 50 nM [γ-33P]GTP were incubated at 30°C, and samples were quenched at the indicated times. Data were fit to the single exponential expression A[1 − exp(−kt)], in which A is the amplitude, k is the rate constant, and t is time. Fits were performed using KaleidaGraph (Synergy Software). The data presented are representative of results from at least three independent experiments. (C) Trypsin cleavage analysis of eIF5B. Recombinant WT or mutant forms of eIF5B396-1002 (3.5 μM) were digested with trypsin (100 nM), and reactions were stopped at the indicated times. Digestion products were analyzed by 4 to 20% SDS-PAGE (Tris-glycine buffer). Molecular mass markers are shown on the right. No, intact eIF5B396-1002 prior to trypsin cleavage; FL, full-length eIF5B396-1002. Results shown are representative of results from three independent experiments. (D) Trypsin cleavage analysis of eIF5B. Reactions were performed for 25 min as described for panel C, and products were analyzed by SDS-PAGE using 10% NuPAGE bis-Tris gels with MOPS buffer. N-terminal sequencing revealed that the peptide fragments marked by the triangles (closed or open) start at residue L689 (located in domain II), fragments marked by the asterisks start at residue L721 (located at the C-terminal end of domain II), and fragments marked by the circles (closed or open) start at the N terminus of eIF5B396-1002. Refer to Fig. 4 (upper panel) for the locations of the cleavage sites. (E) Results from the 80S complex formation assay. (Upper panel) Phosphorimage of a native gel for examining the ability of eIF5B to stimulate 80S complex formation. The progress of 80S complex formation was monitored in reactions with mixtures containing WT eIF5B, eIF5B-G479A, eIF5B-A444V,G479A, or no eIF5B by stopping the reactions at 15 and 30 min. The staggering of the bands is due to the fact that the samples were loaded at different times onto a running gel. The positions of 80S and 48S complexes and free [35S]Met-tRNAiMet are indicated. The data presented are representative of results from at least three independent experiments. (Lower panel) The amount of [35S]Met-tRNAiMet that was free or bound to 48S or 80S complexes was quantified, and the fraction of Met-tRNAiMet present in the 80S complexes relative to the total Met-tRNAiMet (80S + 48S + free) was calculated. (F) Analysis of GCN4-lacZ expression. The GCN4-lacZ plasmid p180 (10) was introduced into derivatives of strain J111 expressing WT eIF5B, eIF5B-G479A, eIF5B-A444V,G479A, or no eIF5B (ΔeIF5B). Cells were grown and β-galactosidase activity was determined as described previously (10), except that longer growth periods were required for sufficient quantities of cells from the slow-growing ΔeIF5B- and eIF5B-G479A-expressing strains. R, cells were grown under nonstarvation conditions in SD minimal medium where GCN4 expression is repressed; DR, cells were grown under amino acid starvation conditions (SD medium + 10 mM 3-aminotriazole) where GCN4 expression is normally derepressed. The β-galactosidase activities are the averages from three independent transformants and have standard errors of 30% or less.
- FIG. 3.
The GTPase switch regulating ribosomal affinity is altered in the eIF5B-A444V,G479A suppressor mutant. (A) Results of a ribosome-binding assay. Purified WT eIF5B, eIF5B-G479A, or eIF5B-A444V,G479A was mixed with purified yeast 80S ribosomes in the presence of GTP, GDPNP, GDP, or no nucleotide as indicated and then loaded onto a 10% sucrose cushion. Following centrifugation, the supernatant and ribosomal pellet fractions were analyzed by SDS-PAGE. The amounts of eIF5B recovered in the supernatant and pellet fractions were determined by quantitative densitometry, and the fraction of total recovered eIF5B present in the ribosomal pellet was calculated. The data presented are the averages of results from at least three independent experiments. (B) Time course of ribosome-dependent GTPase assay. Equal amounts of purified WT eIF5B or eIF5B-A444V,G479A (0.4 μM) were incubated with 50 μM [γ-33P]GTP in the presence of purified yeast 80S ribosomes (0.1 μM). Aliquots from the reaction mixtures were analyzed at various time points by thin-layer chromatography, and the amount of phosphate released was quantified. The values were corrected by subtracting the GTPase activities observed for the proteins in the absence of ribosomes. To test whether the loss of activity by eIF5B-A444V,G479A after 10 min was due to protein instability, fresh [γ-33P]GTP (50 μM) was added at 18 min and the release of phosphate was quantified (dotted lines). Results shown are representative of results from three independent experiments. (C) GDP inhibition of eIF5B GTPase activity. Increasing amounts of GDP, as indicated, were added to GTPase reaction mixtures containing 1 μM GTP, 50 nM 80S ribosomes, and 100 nM eIF5B or eIF5B-A444V,G479A. Reaction mixtures were incubated at 30°C for 5 min, phosphate release was quantified, and the values were normalized to the amount of phosphate released in assays with mixtures lacking GDP. Results shown are from three independent experiments, and the data were fit with the following expression by nonlinear regression using KaleidaGraph: 1 − [GDP]/(Ki + [GDP]). Numbers in parentheses are errors of the fits.
- FIG. 4.
Model of the aIF5B active site depicting the interactions of Switch II with Switch I and helix H8. (Upper panels) Ribbon representation of aIF5B in complex with GTP (Protein Data Bank code, 1G7T [21]). The Switch II element is depicted in green, Switch I in blue, and helix H8 in purple. The four domains of the protein are labeled, and the N-terminal residues of trypsin cleavage fragments (L689 and L721) are indicated (Fig. 2D). The image on the right was rotated about the indicated axis to better visualize the interactions at the G domain active site. (Lower panel) Ribbon representation of the aIF5B active site in complex with GTP. As in the upper panel, the Switch II element is depicted in green, Switch I in blue, and helix H8 in purple, and GTP is depicted in gray. The side chains of key residues discussed in the text are shown, and the three residues mutated in this study (Gly79, Ala44, and Glu340) are depicted in red. The H bond or salt bridge interactions between R87 and E340, E81 and the γ-phosphate of GTP, G43 and D76, and T45 and F74 are depicted by dotted lines. The substitution of Val for A44 (A444V suppressor mutation) may create a clash (marked by the black double arrow) with I75. The image was generated using PyMOL software (8).
