PMC

Interaction of eIF4GII with eIF4A and eIF3. HA-tagged protein expression plasmids pcDNA3-HA-La (lane 1), pcDNA3-HA-eIF4GI (lane 2), pcDNA3-HA-eIF4GII (lane 3), and pcDNA3-HA (lane 4) were transfected into HeLa cells after infection with vTF7-3 as described in Materials and Methods. Proteins were immunoprecipitated (IP) with the 12CA5 anti-HA monoclonal antibody, and immunoprecipitates were resolved by SDS-PAGE (10% polyacrylamide). Western blotting was performed with anti-eIF3 (upper panel), anti-eIF4A (middle panel), or anti-HA (lower panel) antibody (α) as described in Materials and Methods. Mock-infected cell extracts (60 μg of protein) are shown to the left of lane 1. Positions of molecular mass standards (in kilodaltons) are indicated to the right.

eIF4GII is a substrate for rhinovirus 2A^pro. Recombinant eIF4GI (0.5 μg) and eIF4GII (0.5 μg) were incubated in the absence (lanes 1 and 3) or presence (0.1 μg [lanes 2, 4, and 5]) of rhinovirus 2A^pro in buffer containing 100 mM KOAc–20 mM Tris-HCl (pH 7.6)–2.5 mM MgOAc–10% glycerol for 30 min at 30°C in a final volume of 10 μl. In lanes 2 and 5, samples were supplemented with 0.1 μg of eIF4E. Laemmli buffer was added to stop the reaction. Samples were resolved by SDS-PAGE (8% polyacrylamide), and proteins were transferred onto a nitrocellulose membrane which was blotted with anti-Xpress antibody (Invitrogen), a monoclonal antibody which recognizes an epitope located in the baculovirus vector. Protein size markers (Bio-Rad) are indicated (in kilodaltons) to the left.

Expression of eIF4GII protein. (A) Coomassie blue staining of recombinant proteins. His-eIF4GII and His-eIF4GI were produced in Sf9 cells, and equal amounts of each protein were resolved by SDS-PAGE (8% polyacrylamide) as described in Materials and Methods. Molecular masses (in kilodaltons) of protein standards (Bio-Rad) are indicated to the left. (B) Immunological identification of eIF4GI and eIF4GII proteins. Recombinant eIF4GI (0.5 μg [lanes 1 and 3]) and eIF4GII (0.5 μg [lanes 2 and 4]) were resolved on an SDS–5 to 10% gradient polyacrylamide gel, and proteins were transferred onto a nitrocellulose membrane, which was probed with anti-eIF4GI (lanes 1 and 2) or anti-eIF4GII (lanes 3 and 4) antibodies (α). Positions of molecular mass standards (in kilodaltons) are indicated to the left. (C) Cytoplasmic extracts were prepared from mock-infected (lanes 1 and 2) or poliovirus-infected (lanes 3 and 4) HeLa cells as described in Materials and Methods. Extracts (75 μg) were resolved on an SDS–5 to 10% gradient polyacrylamide gel, and proteins were transferred onto a nitrocellulose membrane, which was probed with anti-eIF4GI (lanes 1 and 3) or anti-eIF4GII (lanes 2 and 4) antibodies. N-terminal fragments are bracketed, and the C-terminal fragment of eIF4GI is indicated by a dot. Protein size markers (Bio-Rad) are indicated (in kilodaltons) to the left.

eIF4GII co-purifies with eIF4E by m⁷GDP affinity chromatography. RSW was prepared from HeLa R19 cells according to the method of Merrick (). An m⁷GDP-coupled Sepharose resin () was incubated with 1 mg of RSW for 60 min at 4°C, washed three times with 1 ml of buffer A containing 20 mM Tris-HCl (pH 7.5)–100 mM KCl–2 mM DTT–2 mM EDTA–0.5% Triton X-100, and further incubated for 30 min at 4°C with 100 μl of GDP (200 μM). After being washed once with 1 ml of buffer A, the resin was further incubated for 20 min at 4°C with 100 μl of m⁷GDP (200 μM). Aliquots of the eluted fractions (25 μl) were resolved by SDS-PAGE (12% polyacrylamide) (with 1/10 of the input RSW [10 μg] loaded in lanes 1, 4, and 7). Proteins were transferred to a nitrocellulose membrane, which was cut and probed with anti-eIF4GI, anti-eIF4GII, anti-p97, anti-eIF4A, or anti-eIF4E antibodies (α) as indicated to the left of the blots.

eIF4GII restores cap-dependent translation to an eIF4G-deficient extract. Rabbit reticulocyte lysate (90 μl) was mock treated (lane 1) or treated with 3.6 μg of rhinovirus 2A^pro () (lanes 2 to 8) for 5 min at 30°C, followed by a 10-min incubation on ice in the presence of 0.8 mM elastatinal (Sigma). Aliquots (12.5 μl) were programmed for translation with 0.1 μg of capped bicistronic pGEMCAT/EMC/LUC mRNA (shown schematically at the top of the figure) in the presence of [³⁵S]methionine and supplemented with the indicated initiation factors as follows: lanes 1 and 2, buffer alone; lane 3, 0.05 μg of eIF4E; lane 4, 0.45 μg of eIF4GI; lane 5, 0.05 μg of eIF4E and 0.45 μg of eIF4GI; lane 6, 0.5 μg of eIF4GII; lane 7, 0.05 μg of eIF4E and 0.5 μg of eIF4GII; lane 8, 0.5 μg of rabbit reticulocyte eIF4F. Translation and processing for electrophoresis were conducted as described in Materials and Methods. Following autoradiography, the bands corresponding to luciferase (luc) and CAT were quantified densitometrically. The efficiency of translation of the luciferase and CAT products is given as a percentage of that of the control (lane 1). The positions of the luciferase and CAT proteins are indicated to the left by arrows.

(A) Protein sequence alignment of human eIF4GI and eIF4GII. The pattern-induced multisequence alignment program () was used to align eIF4GI and eIF4GII amino acid sequences. Identical and conserved amino acid residues are in solid and shaded boxes, respectively. The eIF4E binding site () is indicated by dashed lines above and below the sequences. The rhinovirus 2A^pro cleavage site () is indicated by arrows. The published ATG initiation codon of eIF4GI is marked with an asterisk. (The sequence of eIF4GI is revised from the original entry of reference [accession no. AF012088], but it does not contain the newly discovered extension at the N terminus [see panel C].) (B) Alignment of nucleotide and deduced amino acid sequences of eIF4GI and eIF4GII flanking the eIF4GI assigned initiator AUG (). The published ATG initiation codon and the first methionine of eIF4GI are marked as +1. EcoRI is a conserved restriction site in both eIF4GI and eIF4GII cDNA. A putative splice acceptor site (SA) for an intron in eIF4GI is indicated by an arrow. The putative intron sequence is in lowercase letters. (C) Protein alignment of human eIF4GI and eIF4GII N-terminal regions (accession no. AF012088 and AF012072). Identical and conserved amino acid residues are in solid and shaded boxes, respectively. The published ATG initiation codon of eIF4GI is marked with an asterisk.

Northern analysis. (A) A Northern blot of human fetal brain poly(A)⁺ RNA (2 μg [Clontech]) was probed with a labeled PCR product derived from the 3′ UTR of eIF4GI cDNA (lane 1), as described in Materials and Methods. The membrane was then stripped and reprobed with a random-labeled PCR probe derived from the 3′ UTR of the eIF4GII cDNA (lane 2). Autoradiograms of lanes 1 and 2 were superimposed (lane 3). RNA size markers (in kilobases) are indicated to the left. (B) Tissue distribution. Membranes containing poly(A)⁺ RNA from human adult or fetal tissues or human cancer cell lines (2 μg [Clontech]) were probed with PCR probes derived from the 3′ UTRs of eIF4GII (upper panel), eIF4GI (middle panel), or a human β-actin cDNA (lower panel). RNA samples were used as indicated in the figure, and the cell lines were as follows: lane 20, peripheral blood leukocytes (PBL); lane 21, promyelocytic leukemia HL-60; lane 22, HeLa S3; lane 23, chronic myelogenous leukemia K-562; lane 24, lymphoblastic leukemia MOLT-4; lane 25, Burkitt’s lymphoma Raji; lane 26, colorectal adenocarcinoma SW480; lane 27, lung carcinoma A549; lane 28, melanoma G361. RNA size markers (in kilobases) are indicated to the left of the figure.

eIF4GII interacts with eIF4E. (A) Alignment of eIF4E-binding regions of eIF4G proteins from several species and human 4E-binding proteins (, , , ). (B) Schematic representation of GST-HMK-eIF4GII fusion constructs. (C) eIF4E interaction with eIF4GII. GST-eIF4GII fusion fragments (positions 445 to 604 and 445 to 718) were expressed in E. coli and resolved by SDS-PAGE (10% polyacrylamide). Proteins were blotted onto nitrocellulose membranes as described in Materials and Methods. Immunoblot analysis (left panel) was performed with an anti-GST polyclonal antibody (1:1,000), and far-Western analysis of an identical membrane (right panel) was conducted with ³²P-labeled FLAG-HMK-eIF4E. E. coli-purified FLAG-HMK-eIF4E fusion protein () (3 μg) was ³²P-labeled with the catalytic subunit of bovine HMK (Sigma) (). Processing of the nitrocellulose filters through a denaturation-renaturation cycle and hybridization (3 to 4 h, 4°C) with the probe (10⁵ cpm/ml) were performed as described previously (). Membranes were washed twice with the hybridization buffer and processed for autoradiography. (D) Coimmunoprecipitation (IP) of eIF4E with eIF4GII. HA-tagged protein expression plasmids pcDNA3-HA-luciferase (lane 1), pcDNA3-HA-eIF4GI (lane 2), pcDNA3-HA-eIF4GII (lane 3), and pcDNA3-HA (lane 4) were transfected into HeLa cells after infection with vTF7-3 as described in Materials and Methods. Proteins were immunoprecipitated with the 16B12 anti-HA monoclonal antibody (Babco), and immunoprecipitates were resolved by SDS-PAGE (12% polyacrylamide). Western blotting was performed with anti-eIF4E (upper panel) or anti-HA (lower panel) antibody (α) as described in Materials and Methods.