A, binding of partially trypsinized HeLa eIF3 to S-eIF4G(1015–1118). eIF3 was incubated with trypsin as described under ”Experimental Procedures.“ Untrypsinized eIF3 (lane 1), aliquots of the eIF3 tryptic digest from 2 min (lane 2), 5 min (lane 3), 15 min (lane 4), 30 min (lane 5), and 60 min (lane 6), as well as an equivalent reaction without eIF3 (60 min; lane 7) were incubated with S-eIF4G(1015–1118) as described under ”Experimental Procedures“ and analyzed by SDS-PAGE with Coomassie Blue staining. B–J, MALDI-TOF-MS analysis of lane 1 (C–J) and lane 6 (B). Each lane was cut into ~2-mm slices, digested to completion with trypsin, and peptides analyzed by MALDI-TOF-MS as described under ”Experimental Procedures.“ Matched peptides (black bars) are shown below the polypeptides (gray bars) from which they are derived. The x-axis identifies the amino acid location in the sequence. The peptide mapping outputs of selected gel slices (A) are shown in B–J. GenBank^TM accession numbers for amino acid sequences corresponding to eIF3 subunits are as follows: eIF3a (Q14152), eIF3b (P55884), eIF3c (Q99613), eIF3d (O15371), eIF3e (P60228), eIF3f (O00303), eIF3g (O75821), eIF3h (O15372), eIF3i (Q13347), eIF3j (O75822), eIF3k (NP_037366), eIF3l (AF077207), and eIF3m (NP_006351). The amino acid sequence for recombinant S-eIF4G(1015–1118) used in this experiment in B–J (4G) was generated in Vector NTI, Suite 9.0.0 (Informax; North Bethesda, MD).

A, SDS-PAGE of purified FLAG-eIF3e, FLAG-eIF3i, and FLAG-eIF3j stained with Coomassie Blue. B, reactions contained 0.1 μM S-eIF4G(653–1118) and 0.4 μM FLAG-eIF3e in the absence (lane 1) or presence (lane 2) of 20 μg/μl RNase A. A control reaction contained 0.4 μM FLAG-eIF3e alone (lane 3). C, reactions contained equimolar concentrations of S-eIF4G(1015–1118) and HeLa eIF3 (0.2 μM) and increasing concentrations (0.2, 0.4, and 0.8 μM) of either FLAG-eIF3e (lanes 2– 4), FLAG-eIF3j (lanes 6 – 8), or FLAG-eIF3i (lanes 11–13). Separate reactions contained only 0.2 μM S-eIF4G(1015–1118) with either 0.8 μM FLAG-eIF3e (lane 5), FLAG-eIF3j (lane 9), or FLAG-eIF3i (lane 14). Lanes 1 and 10 contained 0.2 μM S-eIF4G(1015–1118) with 0.2 μM HeLa eIF3. The molar ratio of FLAG-eIF3 subunit to HeLa eIF3 is indicated above the lanes. The amount of eIF3 bound is displayed below the lanes as a percentage of the reaction lacking added FLAG-eIF3 subunits (lane 1 for FLAG-eIF3e and FLAG-eIF3j and lane 10 for FLAG-eIF3i). All binding reactions in B and C were performed and analyzed by Western blotting as described under ”Experimental Procedures.“ Native eIF3 subunits (eIF3a, eIF3b/c) and FLAG-tagged eIF3 subunits (FLAG-eIF3e, -eIF3i, and -eIF3j) were detected with anti-eIF3 and anti-FLAG M2 primary antibodies, respectively, and secondary antibodies coupled to alkaline phos-phatase. S-eIF4G(653–1118) and S-eIF4G(1015–1118) were detected using S-protein-coupled alkaline phosphatase.

A, FLAG-eIF3e inhibits luciferase synthesis from m⁷G-Luc-A₆₀. Translation reactions were programmed with m⁷G-Luc-A₆₀ at a final concentration of 0.5 μg/ml in the presence of the indicated concentrations of FLAG-eIF3e. Luciferase synthesis was measured after 60 min as described under ”Experimental Procedures“ and is expressed as a percentage of a control reaction without FLAG-eIF3e. The average of three independent experiments is plotted and curve fitting performed as described under ”Experimental Procedures,“ yielding a K_I value of 0.018 ± 0.002 μM with an R² value of 0.996. B, FLAG-eIF3e decreases the rate of luciferase synthesis in an RRL translational system. Translation reactions (75 μl) were programmed with 15 μg/ml m⁷G-Luc-A₃₁ in the absence (black diamonds) or presence (red squares) of 0.03 μM FLAG-eIF3e. Aliquots of 10 μl were removed at the indicated times. ³⁵S radioactivity was analyzed as described under ”Experimental Procedures.“ C, FLAG-eIF3e decreases the amount of m⁷G-Luc-A₆₀ mRNA present in heavy polysomes. Translation reactions were preincubated for 15 min with no added eIF3 subunit (black diamonds), 0.24 μM FLAG-eIF3e (red squares), or 0.24 μM FLAG-eIF3j (blue triangles). Then ³²P-labeled m⁷G-Luc-A₆₀ (specific activity ~2.75 μCi/μg) was added at a final concentration of 2.0 μg/ml and incubation continued for 60 min. Polysomes were fractionated by ultracentrifugation as described under ”Experimental Procedures.“ The presence of ³²P-labeled mRNA in each fraction was detected as Cerenkov radiation and is displayed as % total mRNA. D, similar experiments were performed as in C with FLAG-eIF3e, except that incubation times were for either 0, 5, 10, or 60 min following mRNA addition. The ratio of mRNA present in polysomes (disomes, trisomes, and heavy polysomes) to that in monosomes (80 S) is shown. The results for 60 min represents the average of three independent experiments.

A, schematic representation of the bicistronic mRNA used in this experiment. B, in vitro translation from m⁷G-CycB2-HCV-NS′ in the presence of FLAG-eIF3e or FLAG-eIF3j. In vitro translation reactions (25 μl) contained the indicated concentrations of FLAG-eIF3e (lanes 3–7) or FLAG-eIF3j (lanes 8 –12) and m⁷G-CycB2-HCV-NS′ at 15 μg/ml. Control reactions contained no added mRNA (lane 1) and no added FLAG-tagged eIF3 subunits (lane 2). After a 90-min incubation, radiolabeled translation products were resolved by SDS-PAGE and detected by Phospho-rImager. The percent of product synthesized in each reaction is displayed below each lane as a percentage of the reaction without added FLAG-eIF3 subunits (lane 2). C, the average values from three independent experiments similar to B are plotted as described under ”Experimental Procedures.“ A K_I of 0.035 ± 0.002 μM was determined for inhibition of cap-dependent translation by FLAG-eIF3e with an R² value of 0.998. Squares, CycB2 with FLAG-eIF3e; circles, NS′ with FLAG-eIF3e; diamonds, CycB2 with FLAG-eIF3j; and triangles, NS′ with FLAG-eIF3j.

The experiment was performed as in Fig. 4 except m⁷G-CycB2-CSFV-NS′ was used. An estimated K_I of 0.0396 ± 0.0039 μM was determined for inhibition of cap-dependent translation by FLAG-eIF3e with an R² value of 0.992.

In vitro translation reactions (100 μl) with or without 0.05 μM FLAG-eIF3e were incubated for 15 min, layered on 10 –35% sucrose gradients, and centrifuged at 38,000 rpm for 5 h at 4 °C. Fractions (500 μl) were collected, and absorbance at 260 nm was monitored. A, typical absorbance profile indicating distribution of 40 S, 60 S, and 80 S complexes. Inset, plot of S values versus fraction number. B, real time PCR of 18 S rRNA present in fractions 1–14 with (filled bars) or without (open bars) FLAG-eIF3e. Data are plotted as the percent total 18 S rRNA present in each gradient. C, representative Western blots of trichloroacetic acid-precipitated protein from fractions 1 to 14 for eIF4G, eIF3, FLAG-eIF3e, and eIF2α in reactions lacking (left panel) or containing (right panel) FLAG-eIF3e. eIF4G and eIF3 blots were probed with secondary antibodies coupled to alkaline phosphatase, whereas FLAG-eIF3e and eIF2α were probed with secondary antibodies coupled to horseradish peroxidase and developed with ECL+. D, experiments similar to that in C were performed in triplicate, immunological signals quantitated using ImageQuant software, and average values plotted as the percent of total protein in fractions 1–14. The eIF3b band was used for quantitation of eIF3. Peak fractions of the 40 S ribosomal subunit, as determined in B, are indicated in C and D.