Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. The central region of eIF4G binds the ATP-dependent RNA helicase eIF4A, the 40 S binding factor eIF3, and RNA. In the present work, we have further characterized the binding properties of the central region of human eIF4G. Both titration and competition experiments were consistent with a 1:1 stoichiometry for eIF3 binding. Surface plasmon resonance studies showed that three recombinant eIF4G fragments corresponding to amino acids 642-1560, 613-1078, and 975-1078 bound eIF3 with similar kinetics. A dissociation equilibrium constant of approximately 42 nm was derived from an association rate constant of 3.9 x 10(4) m(-1) s(-1) and dissociation rate constant of 1.5 x 10(-3) s(-1). Thus, the eIF3-binding region is included within amino acid residues 975-1078. This region does not overlap with the RNA-binding site, which suggests that eIF3 binds eIF4G directly and not through an RNA bridge, or the central eIF4A-binding site. Surprisingly, the binding of eIF3 and eIF4A to the central region was mutually cooperative; eIF3 binding to eIF4G increased 4-fold in the presence of eIF4A, and conversely, eIF4A binding to the central (but not COOH-terminal) region of eIF4G increased 2.4-fold in the presence of eIF3.     

Translation initiation factor eIF4G-1 binds to eIF3 through the eIF3e subunit

eIF3 in mammals is the largest translation initiation factor ( approximately 800 kDa) and is composed of 13 nonidentical subunits designated eIF3a-m. The role of mammalian eIF3 in assembly of the 48 S complex occurs through high affinity binding to eIF4G. Interactions of eIF4G with eIF4E, eIF4A, eIF3, poly(A)-binding protein, and Mnk1/2 have been mapped to discrete domains on eIF4G, and conversely, the eIF4G-binding sites on all but one of these ligands have been determined. The only eIF4G ligand for which this has not been determined is eIF3. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact(s) with eIF4G. Established procedures for detecting protein-protein interactions gave ambiguous results. However, binding of partially proteolyzed HeLa eIF3 to the eIF3-binding domain of human eIF4G-1, followed by high throughput analysis of mass spectrometric data with a novel peptide matching algorithm, identified a single subunit, eIF3e (p48/Int-6). In addition, recombinant FLAG-eIF3e specifically competed with HeLa eIF3 for binding to eIF4G in vitro. Adding FLAG-eIF3e to a cell-free translation system (i) inhibited protein synthesis, (ii) caused a shift of mRNA from heavy to light polysomes, (iii) inhibited cap-dependent translation more severely than translation dependent on the HCV or CSFV internal ribosome entry sites, which do not require eIF4G, and (iv) caused a dramatic loss of eIF4G and eIF2alpha from complexes sedimenting at approximately 40 S. These data suggest a specific, direct, and functional interaction of eIF3e with eIF4G during the process of cap-dependent translation initiation, although they do not rule out participation of other eIF3 subunits.     

Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A.

Mammalian translation initiation factor 4F (eIF4F) consists of three subunits, eIF4A, eIF4E, and eIF4G. eIF4G interacts directly with both eIF4A and eIF4E. The binding site for eIF4E is contained in the amino-terminal third of eIF4G, while the binding site for eIF4A was mapped to the carboxy-terminal third of the molecule. Here we show that human eIF4G possesses two separate eIF4A binding domains in the middle third (amino acids [aa] 478 to 883) and carboxy-terminal third (aa 884 to 1404) of the molecule. The amino acid sequence of the middle portion of eIF4G is well conserved between yeasts and humans. We show that mutations of conserved amino acid stretches in the middle domain abolish or reduce eIF4A binding as well as eIF3 binding. In addition, a separate and nonoverlapping eIF4A binding domain exists in the carboxy-terminal third (aa 1045 to 1404) of eIF4G, which is not present in yeast. The C-terminal two-thirds region (aa 457 to 1404) of eIF4G, containing both eIF4A binding sites, is required for stimulating translation. Neither one of the eIF4A binding domains alone activates translation. In contrast to eIF4G, human p97, a translation inhibitor with homology to eIF4G, binds eIF4A only through the amino-terminal proximal region, which is homologous to the middle domain of eIF4G.     

Mass spectrometric analysis of the N terminus of translational initiation factor eIF4G-1 reveals novel isoforms

In eukaryotes, translation initiation factor 4G (eIF4G) acts as the central binding protein for an unusually large number of proteins involved in mRNA metabolism. Several gene products homologous to eIF4G have been described, the most studied being eIF4G-1. By its association with other initiation factors, eIF4G-1 effects mRNA cap and poly(A) recognition, unwinding of secondary structure, and binding to the 43S initiation complex. Multiple electrophoretic isoforms of eIF4G-1 are observed, and multiple cDNAs have been reported, yet the relationship between the two is not known. We report here a new cDNA for eIF4G-1, present as a previously unidentified human expressed sequence tag, that extends the long open reading frame, provides a new in-frame initiation codon, and predicts a longer form of eIF4G-1 than reported previously. eIF4G isoforms from human K562 cells were cleaved with recombinant Coxsackievirus 2A protease and the N- terminal domains purified by m(7)GTP-Sepharose chromatography and polyacrylamide gel electrophoresis. Proteins were digested with proteolytic enzymes and peptides masses determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. In selected cases, peptides were sequenced by electrospray-mass spectrometry fragmentation. This identified the N termini of the three most abundant eIF4G-1 isoforms, two of which had not previously been proposed. These proteins appear to have been initiated from three different AUG codons.     

Characterization of mammalian eIF4E-family members.

The translational factor eukaryotic initiation factor 4E (eIF4E) is a central component in the initiation and regulation of translation in eukaryotic cells. Through its interaction with the 5' cap structure of mRNA, eIF4E functions to recruit mRNAs to the ribosome. The accumulation of expressed sequence tag sequences has allowed the identification of three different eIF4E-family members in mammals termed eIF4E-1, eIF4E-2 (4EHP, 4E-LP) and eIF4E-3, which differ in their structural signatures, functional characteristics and expression patterns. Unlike eIF4E-1, which is found in all eukaryotes, orthologues for eIF4E-2 appear to be restricted to metazoans, while those for eIF4E-3 have been found only in chordates. Like prototypical eIF4E-1, eIF4E-2 was found to be ubiquitously expressed, with the highest levels in the testis. Expression of eIF4E-3 was detected only in heart, skeletal muscle, lung and spleen. Similarly to eIF4E-1, both eIF4E-2 and eIF4E-3 can bind to the mRNA cap-structure. However, in contrast to eIF4E-1 which interacts with both the scaffold protein, eIF4G and the translational repressor proteins, the eIF4E-binding proteins (4E-BPs), eIF4E-2 and eIF4E-3 each possesses a range of partial activities. eIF4E-2 does not interact with eIF4G, but does interact with 4E-BPs. Conversely, eIF4E-3 interacts with eIF4G, but not with 4E-BPs. Neither eIF4E-2 nor eIF4E-3 is able to rescue the lethality of eIF4E gene deletion in yeast. It is hypothesized that each eIF4E-family member fills a specialized niche in the recruitment of mRNAs by the ribosome through differences in their abilities to bind cap and/or to interact with eIF4G and the 4E-BPs.     

Ultrastructural localization of concanavalin A-binding sites in satellite cells of human skeletal muscle

Summary Electron-microscopic cytochemical studies on satellite cells of normal human skeletal muscle were carried out using the concanavalin Aperoxidase (Con A-HRP) coupling method. Con A-binding sites, which probably correspond to glycoproteins, were found to be associated with the cell surface, smooth surfaced vesicles, nuclear envelope and endoplasmic reticulum of the satellite cells and were also identified at the cell surface of the adjacent muscle fiber. The possible relationships of these observations to the functions of satellite cells are discussed.     

Characterization of nucleotide-binding sites on the chloroplast coupling factor 1 using two photolabile analogs

Nucleotide-binding sites on the chloroplast coupling factor 1 (CF1) have been probed using two photoreactive ADP analogs: 2-azido-ADP (2-N3-ADP) and 2',3'-O-(4-benzoyl)benzoyl-ADP (Bz-ADP). Photolabeling of the isolated CF1 with 2-N3-ADP results in incorporation of the analog exclusively into the beta-subunit of the enzyme. The location of the nucleotide-binding site(s) within the beta-subunit of the CF1 was investigated using peptide mapping. Within the discrimination limits of this technique, it is concluded that the azido- and benzoyl-modified analogs both bind to the same conformation of the nucleotide-binding site(s) of soluble CF1. Bz-ADP, however, labels the binding site(s) on membrane-bound CF1 in a slightly different manner.     

Characterization of human beta-interferon-binding sites on human cells

Radioiodinated recombinant human beta-interferon (rHuIFN beta Ser), with almost full (greater than 90%) biological activity, was used to study the binding of human beta-interferon to Daudi cells. Specific binding was not observed with less biologically active (less than or equal to 10%) radioiodinated interferon. The bound radioiodinated interferon was shown to compete with human beta-interferon (HuIFN beta), rHuIFN beta Ser, human alpha-interferon (HuIFN alpha) and with human gamma-interferon (HuIFN gamma). Scatchard plot analyses suggest the presence of about 10,000 binding sites for HuIFN beta/Daudi cell. About 6,600 of these sites can be blocked by HuIFN alpha and 3,700 sites can be blocked by HuIFN gamma. The apparent Kd for HuIFN beta is 2.7 nM. The apparent Kd values for HuIFN alpha and HuIFN gamma are 3.7 and 1.1 nM, respectively. It was possible to demonstrate the cross-linking of HuIFN beta to two macromolecular components of Mr = 128,000 and 103,000. We propose the existence of at least two binding sites for HuIFN beta in Daudi cells, one site recognizing both HuIFN beta and HuIFN gamma, the other site recognizing both HuIFN beta and HuIFN alpha. Each site is capable of recognizing only HuIFN gamma or HuIFN alpha.     

Characterization and comparison of two binding sites on obscurin for small ankyrin 1

Obscurin A, an ～720 kDa modular protein of striated muscles, binds to small ankyrin 1 (sAnk1, Ank 1.5), an integral protein of the sarcoplasmic reticulum, through two distinct carboxy-terminal sequences, Obsc(6316-6436) and Obsc(6236-6260). We hypothesized that these sequences differ in affinity but that they compete for the same binding site on sAnk1. We show that the sequence within Obsc(6316-6436) that binds to sAnk1 is limited to residues 6316-6345. Comparison of Obsc(6231-6260) to Obsc(6316-6345) reveals that Obsc(6316-6345) binds sAnk1 with an affinity (133 ± 43 nM) comparable to that of the Obsc(6316-6436) fusion protein, whereas Obsc(6231-6260) binds with lower affinity (384 ± 53 nM). Oligopeptides of each sequence compete for binding with both sites at half-maximal inhibitory concentrations consistent with the affinities measured directly. Five of six site-directed mutants of sAnk1 showed similar reductions in binding to each binding site on obscurin, suggesting that they dock to many of the same residues of sAnk1. Circular dichroism (CD) analysis of the synthetic oligopeptides revealed a 2-fold greater α-helical content in Obsc(6316-6346), ～35%, than Obsc(6231-6260,) ～17%. Using these data, structural prediction algorithms, and homology modeling, we predict that Obsc(6316-6345) contains a bent α-helix of 12 amino acids, flanked by short disordered regions, and that Obsc(6231-6260) has a short, N-terminal α-helix of 4-5 residues followed by a long disordered region. Our results are consistent with a model in which both sequences of obscurin differ significantly in structure but bind to the ankyrin-like repeat motifs of sAnk1 in a similar though not identical manner.     

Two structurally atypical HEAT domains in the C-terminal portion of human eIF4G support binding to eIF4A and Mnk1

The X-ray structure of the C-terminal region of human eukaryotic translation initiation factor 4G (eIF4G) has been determined at 2.2 A resolution, revealing two atypical HEAT-repeat domains. eIF4G recruits various translation factors and the 40S ribosomal subunit to the mRNA 5' end. In higher eukaryotes, the C terminus of eIF4G (4G/C) supports translational regulation by recruiting eIF4A, an RNA helicase, and Mnk1, the kinase responsible for phosphorylating eIF4E. Structure-guided surface mutagenesis and protein-protein interaction assays were used to identify binding sites for eIF4A and Mnk1 within the HEAT-repeats of 4G/C. p97/DAP5, a translational modulator homologous to eIF4G, lacks an eIF4A binding site in the corresponding region. The second atypical HEAT domain of the 4G/C binds Mnk1 using two conserved aromatic/acidic-box (AA-box) motifs. Within the first AA-box, the aromatic residues contribute to the hydrophobic core of the domain, while the acidic residues form a negatively charged surface feature suitable for electrostatic interactions with basic residues in Mnk1.     

Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.

Eukaryotic initiation factor (eIF) 4A is a DEAD box RNA helicase that works in conjunction with eIF4B, eIF4H, or as a subunit of eIF4F to unwind secondary structure in the 5'-untranslated region of mRNA, which facilitates binding of the mRNA to the 40 S ribosomal subunit. This study demonstrates how the helicase activity of eIF4A is modulated by eIF4B, eIF4H, or as a subunit of eIF4F. Results indicate that a linear relationship exists between the initial rate or amplitude of unwinding and duplex stability for all factor combinations tested. eIF4F, like eIF4A, behaves as a non-processive helicase. Either eIF4B or eIF4H stimulated the initial rate and amplitude of eIF4A-dependent duplex unwinding, and the magnitude of stimulation is dependent on duplex stability. Furthermore, eIF4A (or eIF4F) becomes a slightly processive helicase in the presence of eIF4B or eIF4H. All combinations of factors tested indicate that the rate of duplex unwinding is equivalent in the 5' --> 3' and 3' --> 5' directions. However, the optimal rate of unwinding was dependent on the length of the single-stranded region of the substrate when different combinations of factors were used. The combinations of eIF4A, eIF4A + eIF4B, eIF4A + eIF4H, and eIF4F showed differences in their ability to unwind chemically modified duplexes. A simple model of how eIF4B or eIF4H affects the duplex unwinding mechanism of eIF4A is proposed.     

Synergistic activation of eIF4A by eIF4B and eIF4G

eIF4A is a key component in eukaryotic translation initiation; however, it has not been clear how auxiliary factors like eIF4B and eIF4G stimulate eIF4A and how this contributes to the initiation process. Based on results from isothermal titration calorimetry, we propose a two-site model for eIF4A binding to an 83.5 kDa eIF4G fragment (eIF4G-MC), with a high- and a low-affinity site, having binding constants K_D of ∼50 and ∼1000 nM, respectively. Small angle X-ray scattering analysis shows that the eIF4G-MC fragment adopts an elongated, well-defined structure with a maximum dimension of 220 Å, able to span the width of the 40S ribosomal subunit. We establish a stable eIF4A–eIF4B complex requiring RNA, nucleotide and the eIF4G-MC fragment, using an in vitro RNA pull-down assay. The eIF4G-MC fragment does not stably associate with the eIF4A–eIF4B–RNA-nucleotide complex but acts catalytically in its formation. Furthermore, we demonstrate that eIF4B and eIF4G-MC act synergistically in stimulating the ATPase activity of eIF4A.     

Characterization of serotonin binding sites on human platelets

A high affinity, saturable 3H-spiroperidol binding site was identified for the first time on the intact human platelet, with drug affinities comparable to the serotonin-2 (S-2) receptor in human frontal cortex. The site was characterized by a KD of 2.7 +/- 0.3nM and a Bmax of 1.4 +/- 0.2 pmoles/10(8) platelets. A 3H-serotonin binding site was also found, with a KD of 42 +/- 8 nM, which appeared to represent the serotonin uptake site. No 3H-serotonin binding site with features of the serotonin-1 (S-1) receptor in brain was found on the platelet. Assay of 3H-spiroperidol binding to platelets may serve as an easily applied model for studying S-2 receptor function in man, and its relationship to age, hormonal, drug, and disease effects.     

eIF4B and eIF4G Jointly Stimulate eIF4A ATPase and Unwinding Activities by Modulation of the eIF4A Conformational Cycle

Eukaryotic translation initiation factor 4A (eIF4A) is a DEAD-box protein that participates in translation initiation. As an ATP-dependent RNA helicase, it is thought to resolve secondary structure elements from the 5′-untranslated region of mRNAs to enable ribosome scanning. The RNA-stimulated ATPase and ATP-dependent helicase activities of eIF4A are enhanced by auxiliary proteins, but the underlying mechanisms are still largely unknown. Here, we have dissected the effect of eIF4B and eIF4G on eIF4A RNA-dependent ATPase- and RNA helicase activities and on eIF4A conformation. We show for the first time that yeast eIF4B, like its mammalian counterpart, can stimulate RNA unwinding by eIF4A, although it does not affect the eIF4A conformation. The eIF4G middle domain enhances this stimulatory effect and promotes the formation of a closed eIF4A conformation in the presence of ATP and RNA. The closed state of eIF4A has been inferred but has not been observed experimentally before. eIF4B and eIF4G jointly stimulate ATP hydrolysis and RNA unwinding by eIF4A and favor the formation of the closed eIF4A conformer. Our results reveal distinct functions of eIF4B and eIF4G in synergistically stimulating the eIF4A helicase activity in the mRNA scanning process.     

Characterization of concanavalin A-binding neuronal and glial surface glycoproteins from human foetal brain.

Neuronal and glial surface glycoproteins have been isolated from human foetal brains by affinity chromatography on 8 M urea or 6 M guanidine-treated Con A-Sepharose 4B at 4 degrees C and three groups of glycoproteins of molecular mass 65-73 kDa, 52-63 kDa and 43-48 kDa have been identified on SDS/PAGE. These glycoproteins exhibited anomalous behaviour on SDS/PAGE, indicating the existence of a gradation of mutually interconvertible protein-SDS aggregates in dynamic equilibrium with one another. Deglycosylation and deacylation did not alter the SDS/PAGE multiple band pattern. Purified glycoproteins contained 160 +/- 90 micrograms carbohydrate/mg protein, and a sialic acid content of 25 +/- 5 nmole/mg protein. The N-terminals were blocked. The glycoproteins moved preferentially on acid/urea/PAGE. Sepharose 6B gel filtration in the absence of lipid and detergents resolved the glycoproteins into an excluded peak I and a low molecular mass peak II. Peaks I and II were non-interconvertible on Sepharose 6B gel filtration or on reversed phase HPLC in an isopropanol/water/TFA gradient system. Both peaks rendered a single fast moving band of identical mobility on acid/urea/PAGE, suggesting that peak I was possibly a micellar aggregate of the monomeric peak II. The glycoproteins were refractory to digestion by trypsin or pronase and reacted identically towards various lectins.(ABSTRACT TRUNCATED AT 250 WORDS)     

A microtiter plate assay for assessing the interaction of eukaryotic initiation factor eIF4E with eIF4G and eIF4E binding protein-1

Modulation of interactions among proteins is an important mechanism for regulating both the subcellular location and the function of proteins. An example of the importance of protein-protein interaction is the reversible association of eukaryotic initiation factor eIF4E with the eIF4E binding proteins 4E-BP1 and eIF4G. When bound to 4E-BP1, eIF4E cannot bind to eIF4G to form the active eIF4F complex, an event that is required for the binding of mRNA to the ribosome. Thus, association of eIF4E with 4E-BP1 represses mRNA translation by preventing the binding of mRNA to the ribosome. Previous studies have measured the amount of 4E-BP1 or eIF4G bound to eIF4E by either affinity chromatography or immunoprecipitation of eIF4E followed by Western blot analysis for quantitation of 4E-BP1 and eIF4G. Both of these techniques have significant limitations. In the present study, we describe a microtiter plate-based assay for quantitation of the amount of 4E-BP1 and eIF4G bound to eIF4E that obviates many of the limitations of the earlier approaches. It also has the advantage that absolute amounts of the individual proteins can be easily estimated. The approach should be applicable to the study of a wide variety of protein-protein interactions.     

Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha2beta2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens. We converted 16 residues in the human alpha subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild-type beta subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the K(m) for Fe2+ 15-fold and that for 2-oxoglutarate 5-fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the K(m) for 2-oxoglutarate 15-fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85-95%; all these mutations increased the K(m) for 2-oxoglutarate 2- to 3-fold and enhanced the rate of uncoupled decarboxylation of 2-oxoglutarate as a percentage of the rate of the complete reaction up to 12-fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C-5 carboxyl group of 2-oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate.