Spectrin domain of eukaryotic initiation factor 3a is the docking site for formation of the a:b:i:g subcomplex

Abstract

eIF3a (eukaryotic translation initiation factor 3a), one of the core subunits of the eIF3 complex, has been implicated in regulating translation of different mRNAs and in tumorigenesis. A subcomplex consisting of eIF3a, eIF3b, eIF3g, and eIF3i (eIF3(a:b:i:g)) has also been identified. However, how eIF3a participates in translational regulation and in formation of the eIF3(a:b:i:g) subcomplex remain to be solved. In this study, we used the tandem affinity purification approach in combination with tandem MS/MS and identified the spectrin domain of eIF3a as the docking site for the formation of eIF3(a:b:i:g) subcomplex. Although eIF3b and eIF3i bind concurrently to the spectrin domain of eIF3a within ∼10-15 amino acids apart, eIF3g binds to eIF3a indirectly via binding to the carboxyl-terminal domain of eIF3b. The binding of eIF3b to the spectrin domain of eIF3a occurs in its RNA recognition motif domain where eIF3j also binds in a mutually exclusive manner. Together, we conclude that the spectrin domain of eIF3a is responsible for the formation of eIF3(a:b:i:g) subcomplex and, because of mutually exclusive nature of bindings of eIF3a and eIF3j to eIF3b, different subcomplexes of eIF3 likely exist and may perform noncanonical functions in translational regulation.

Keywords: Mass Spectrometry (MS); Protein-protein Interactions; Proteomics; TAP Purification; Translation Control; Translation Initiation Factors.

FIGURE 1.

TAP analysis. A, schematic structure of TAP constructs. The eIF3a domain represents PCI, SPT, or 10-amino acid RP domain. B and C, TAP. HeLa cells transiently transfected with TAP constructs without or with different domains (PCI, SPT, and RP) of eIF3a were harvested and subjected to TAP. Purified proteins were separated by SDS-PAGE and stained with silver (B) or analyzed using Western blot probed with antibodies against eIF3b, -c, -d, -g, -i, and -j (C). Vec., vector.

FIGURE 2.

In vitro pulldown assay. Newly synthesized and [³⁵S]methionine-labeled eIF3b, eIF3g, eIF3i, and control luciferase (Luc) protein products from a cell-free expression system were subjected to pulldown assay using GST-SPT or control GST as bait (A) or GST-PCI and GST-RP as bait (B). The pulldown and input materials were separated by SDS-PAGE followed by autoradiography.

FIGURE 3.

Co-immunoprecipitation analysis of wild type and mutant eIF3a. A, schematic map of FLAG-tagged wild type and deletion mutant eIF3a. B and C, expression and co-immunoprecipitation analysis of wild type and mutant eIF3a. HeLa cells transiently transfected with FLAG-tagged wild type or deletion mutant eIF3a constructs were harvested for Western blot analysis of FLAG-tagged ectopic eIF3a and endogenous eIF3b, eIF3g, and eIF3i (B) or subjected to co-immunoprecipitation using FLAG antibody followed by Western blot analysis of co-precipitated eIF3b, eIF3g, and eIF3i (C). Vec, vector.

FIGURE 4.

Analysis of direct or indirect binding using pulldown assay. A and B, pulldown assay using GST-SPT fusion protein or control GST as bait and purified His-tagged eIF3b, eIF3g, and eIF3i as targets individually (A) or together (B). C, pulldown assay using GST-eIF3b as bait and His-tagged eIF3g or eIF3i as targets. D, pulldown (PD) assay using GST, GST-eIF3i, or GST-eIF3b as bait and His-tagged eIF3g as target. The pulldown and input materials were separated by SDS-PAGE and followed by Western blot analysis of eIF3g.

FIGURE 5.

Deletion mapping of eIF3b and eIF3i binding sites in the SPT domain of eIF3a. A, schematic presentation of deletions in the SPT domain of eIF3a and the GST fusion protein constructs. B, purified GST-fusion proteins from constructs shown in A. C, pulldown assay. Newly synthesized and [³⁵S]methionine-labeled eIF3b and eIF3i from a cell-free expression system were subjected to pulldown assay using different GST-SPT constructs as bait. The pulldown and input materials were subjected to separation by SDS-PAGE and autoradiography analysis. Luc, luciferase.

FIGURE 6.

Ala-scanning mutagenesis analysis of eIF3b binding site in the SPT domain of eIF3a. A, schematic presentation of the Ala-scanning (As) mutations and the GST fusion protein constructs. B–D, pulldown assay. Newly synthesized and [³⁵S]methionine-labeled eIF3b from a cell-free expression system (B and C) or purified His-tagged eIF3b (D) was subjected to pulldown (PD) assay using different GST-SPT mutant constructs as bait. The WT and the SPT-ΔC26 (C26) constructs were used as positive and negative controls, respectively. The pulldown and input materials were subjected to separation by SDS-PAGE and autoradiography or Western blot analysis. E, competitive analysis. Newly synthesized and [³⁵S]methionine-labeled eIF3i from a cell-free expression system was subjected to pulldown assay using GST-SPT wild type fusion protein or control GST as bait in the absence (lane 1) or presence of increasing concentration of purified His-eIF3b. The pulldown and input materials were separated by SDS-PAGE and detected by Western blot analysis of eIF3i.

FIGURE 7.

Mapping eIF3a, eIF3g, and eIF3j binding sites in eIF3b. A, schematic presentation of wild type and mutant GST-eIF3b fusion protein constructs. B and C, pulldown assays. Pulldown assays were performed using different GST-eIF3b mutant or wild type fusion proteins as bait and purified His-tagged eIF3g and eIF3j, and the SPT domain of eIF3a as targets. The pulldown and input materials were separated by SDS-PAGE and detected by Western blot analysis.

FIGURE 8.

Competitive analysis and schematic model of interactions in the eIF3(a:b:i:g) subcomplex. A, competitive pulldown assay. Pulldown of the SPT domain of eIF3a using GST-eIF3b RRM as bait was performed in the presence of excess amount of purified His-tagged eIF3j or GST control proteins. The pulldown (PD) materials were then separated by SDS-PAGE followed by Western blot analysis using anti-His-tagged antibody. B, effect of eIF3a knockdown on co-immunoprecipitation between endogenous eIF3b and eIF3j. HeLa cells were transfected with scrambled (Scr) or eIF3a siRNAs (Si) followed by co-immunoprecipitation using eIF3b antibody or a normal IgG (nIgG) control. The input or precipitated materials were separated by SDS-PAGE followed by Western blot analysis of eIF3b and eIF3j.

FIGURE 9.

A, schematic model of interactions in the eIF3(a:b:i:g) subcomplex. CTD, carboxyl-terminal domain. B, alignment of the SPT domain of eIF3a in different species. Identical residues are highlighted with Phe⁷⁶⁰ shown in red.