The core of human eIF3. (A) Complexes comprising five subunits (eIF3a, eIF3b, eIF3e, eIF3f, and eIF3h) or six subunits (eIF3a, eIF3b, eIF3c, eIF3e, eIF3f, and eIF3h) were resolved by SDS–PAGE and analyzed by Western blotting with relevant antisera. (B) Toeprint analysis of the five-or six-subunit complex was performed as in Figure 3B.

Analysis of recombinant eIF3 lacking eIF3d, eIF3l, or eIF3k subunit. (A) r-eIF3(11), r-eIF3(del-d), and r-eIF3(del-l) (0.5 μg each) were resolved by SDS–PAGE and stained by CBB. The asterisk indicates an unidentified contaminant. (B) Western blot analysis of r-eIF3(11), r-eIF3(del-d), and r-eIF3(del-l) with anti-eIF3l or anti-eIF3d antiserum. (C) Toeprint analysis of r-eIF3(11), r-eIF3(del-d), and r-eIF3(del-l) was performed as in Figure 3B. (D) r-eIF3(11) and r-eIF3(del-k) (0.5 μg each) were resolved by SDS–PAGE and stained by CBB. The asterisk indicates the position of eIF3k. (E) Western blot analysis of r-eIF3(11) and r-eIF3(del-k) with anti-eIF3k antiserum. (F) Toeprint analysis of r-eIF3(11) and r-eIF3(del-k) was performed as in Figure 3B.

Identification of mammalian eIF3 subunits. (A) n-eIF3 (10 μg) was resolved by SDS–PAGE (10%) and stained with CBB. Each band was identified by MALDI-TOF/MS analysis. (B) Immunological identification of eIF3 subunits. n-eIF3 (10 μg) and HeLa cell extracts (75 μg protein) were resolved by SDS–PAGE and analyzed by Western blotting using specific antiserum against each subunit.

Analysis of recombinant eIF3 lacking eIF3c, eIF3i, or eIF3g subunit. (A) r-eIF3(11) and r-eIF3(del-c) (1 μg each) were resolved by SDS–PAGE and stained by CBB. The asterisk indicates a C-terminal fragment of eIF3c. (B) Western blotting analysis of r-eIF3(11) and r-eIF3(del-c) with anti-eIF3b or anti-eIF3c antiserum. (C) Toeprint analysis of r-eIF3(11) and r-eIF3(del-c) was performed as in Figure 3B. (D) r-eIF3(11), r-eIF3(del-i), and r-eIF3(del-g) (1 μg each) were resolved by SDS–PAGE and stained by CBB. (E) Western blotting analysis of r-eIF3(11), r-eIF3(del-g), and r-eIF3(del-i) with anti-eIF3g, anti-eIF3i, or anti-eIF3a antiserum. (F) Toeprint analysis of r-eIF3(11), r-eIF3(del-i), and r-eIF3(del-g) was performed as in Figure 3B.

Recombinant eIF3 activity. (A) Native-eIF3 (n-eIF3) and recombinant eIF3, r-eIF3(11), (1 μg each) were resolved by SDS–PAGE (10%) and stained by CBB. The asterisk indicates a C-terminal fragment of eIF3c. (B) Functional assay of r-eIF3(11). Toeprint analysis of 48S ribosomal complex formation on β-globin mRNA was performed with n-eIF3 or r-eIF3(11). Control: no eIF3. The efficiency (%) of the ribosomal complex formation on the initiator AUG is shown below each lane. C: toeprint complex. E: full-length cDNA.

Depletion of eIF3g and eIF3i from n-eIF3 in HeLa cells. (A) HeLa cells were transfected with siRNA against eIF3g (lane 2) or control siRNA (lane 1). Cell lysate was analyzed by Western blotting with anti-eIF3c, anti-eIF3g, or anti-eIF3i antiserum. (B) HeLa cells transfected with siRNA as in (A) were pulse-labeled with [³⁵S] methionine for 30 min. Cell extracts were resolved by SDS–PAGE, and the incorporated radioactivity of the protein bands was quantified in a combined manner. Data are the mean values of three experiments±s.d. (C) Purification of n-eIF3 depleted of eIF3g and eIF3i. HeLa cells were transfected with siRNA against eIF3g or control siRNA. Ribosomal salt-washed fractions from the transfected cells were resolved by sucrose-density gradient centrifugation. Fractions 7–23 (the bottom of the gradient) were analyzed by Western blotting for eIF3c, eIF3f, eIF3g, and eIF3i.

Coexpression system for recombinant eIF3 complex formation. (A) Three recombinant baculoviruses were used to express the eIF3 subunits. Arrows indicate the direction of transcription. (B) Expression of eIF3 subunits in Sf9 cells. Sf9 cells were infected with baculovirus-1, -2, or -3. Extracts (75 μg protein) from infected cells (inf.) and uninfected cells (uninf.) were resolved by SDS–PAGE and analyzed by Western blotting. (C) Diagram of recombinant eIF3 expression and purification protocol. (D) Gel filtration of eIF3a-His and associated subunits. Proteins eluted from the Ni-NTA resin were resolved by Sephacryl S-300 as described in Materials and methods. Aliquots of each fraction were analyzed by Western blotting with antiserum against each subunit. Approximate positions of the molecular weight reference proteins (thyroglobulin: 669 kDa; ferritin: 440 kDa; catalase: 232 kDa; aldolase: 158 kDa) on the chromatography are indicated above the panels. The fractions used for further purification are underlined. Bottom panel: n-eIF3 was fractionated in the same manner and analyzed with anti-eIF3c. Vo: void volume.