Phosphorylation of tobacco eukaryotic translation initiation factor 4A upon pollen tube germination.

Eukaryotic translation initiation factor eIF-4A is a member of the DEAD box family of RNA helicases and RNA-dependent ATPases. In tobacco, eIF-4A is encoded by a gene family with one isoform, eIF-4A8, being exclusively expressed in pollen. This pollen-specific isoform is a candidate for mediating translational control in the developing gametophyte. Here we show that eIF-4A is barely phosphorylated in mature pollen, but during pollen tube germination, two isoforms of eIF-4A become phosphorylated. Phosphoamino acid analysis indicated phosphorylation of threonine. In order to determine whether pollen-specific eIF-4A8 is among the phosphorylated isoforms, we raised transgenic tobacco plants overexpressing eIF-4A8 containing a histidine tag. Hereby, we could show that indeed eIF-4A8 is modified through phosphorylation. The biological relevance of the phosphorylation of eIF-4A is discussed.     

Functional characterization of the Arabidopsis eukaryotic translation initiation factor 5A-2 that plays a crucial role in plant growth and development by regulating cell division, cell growth, and cell death

The eukaryotic translation initiation factor 5A (eIF-5A) is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies in mammalian and yeast (Saccharomyces cerevisiae) cells suggest that eIF-5A is mainly involved in RNA metabolism and trafficking, thereby regulating cell proliferation, cell growth, and programmed cell death. In higher plants, the physiological function of eIF-5A remains largely unknown. Here, we report the identification and characterization of an Arabidopsis (Arabidopsis thaliana) mutant fumonisin B(1)-resistant12 (fbr12). The fbr12 mutant shows an antiapoptotic phenotype and has reduced dark-induced leaf senescence. Moreover, fbr12 displays severe defects in plant growth and development. The fbr12 mutant plant is extreme dwarf with substantially reduced size and number of all adult organs. During reproductive development, fbr12 causes abnormal development of floral organs and defective sporogenesis, leading to the abortion of both female and male germline cells. Microscopic studies revealed that these developmental defects are associated with abnormal cell division and cell growth. Genetic and molecular analyses indicated that FBR12 encodes a putative eIF-5A-2 protein. When expressed in a yeast mutant strain carrying a mutation in the eIF-5A gene, FBR12 cDNA is able to rescue the lethal phenotype of the yeast mutant, indicating that FBR12 is a functional eIF-5A. We propose that FBR12/eIF-5A-2 is fundamental for plant growth and development by regulating cell division, cell growth, and cell death.     

Isolation and characterization of senescence-induced cDNAs encoding deoxyhypusine synthase and eucaryotic translation initiation factor 5A from tomato

Full-length cDNA clones encoding deoxyhypusine synthase (DHS) and eucaryotic initiation factor 5A (eIF-5A) have been isolated from a cDNA expression library prepared from tomato leaves (Lycopersicon esculentum, cv. Match) exposed to environmental stress. DHS mediates the first of two enzymatic reactions that activate eIF-5A by converting a conserved lysine to the unusual amino acid, deoxyhypusine. Recombinant protein obtained by expressing tomato DHS cDNA in Escherichia coli proved capable of carrying out the deoxyhypusine synthase reaction in vitro in the presence of eIF-5A. Of particular interest is the finding that DHS mRNA and eIF-5A mRNA show a parallel increase in abundance in senescing tomato flowers, senescing tomato fruit, and environmentally stressed tomato leaves exhibiting programmed cell death. Western blot analyses indicated that DHS protein also increases at the onset of senescence. It is apparent from previous studies with yeast and mammalian cells that hypusine-modified eIF-5A facilitates the translation of a subset of mRNAs mediating cell division. The present study provides evidence for senescence-induced DHS and eIF-5A in tomato tissues that may facilitate the translation of mRNA species required for programmed cell death.     

The Subcellular Distribution of Eukaryotic Translation Initiation Factor, eIF-5A, in Cultured Cells

To gain insight into the role of the eukaryotic translation initiation factor, eIF-5A, we investigated the subcellular distribution of this protein in several cultured cell types and at different stages of the cell cycle using a highly potent monospecific polyclonal antibody to eIF-5A. Studies using indirect immunofluorescence and confocal microscopy in conjunction with subcellular fractionation demonstrate that eIF-5A is primarily localized in the cytoplasm of cells. This cytoplasmic location of eIF-5A is not significantly altered in different stages of the cell cycle and the subcellular distribution pattern of eIF-5A is not changed by viral oncogene transformation. Cell fractionation experiments identified two populations of eIF-5A in the cytoplasm, a soluble fraction and a fraction bound to internal membranes. By double immunofluorescence staining with an antibody against calnexin, a resident protein of the endoplasmic reticulum (ER), we demonstrate that the membrane-bound fraction of eIF-5A colocalizes with the ER and not with the cytoskeleton. Expression of Rev, a regulatory protein of human immunodeficiency virus type 1 (HIV-1), does not alter the subcellular distribution of endogenous eIF-5A in these cells. eIF-5A is detected in all tissues and cells examined including extracts prepared fromXenopusoocytes. Our results indicate that eIF-5A is a ubiquitous cytoplasmic protein and suggest that a site of eIF-5A function is likely to be in association with the ER.     

Influence of 5' proximal secondary structure on the translational efficiency of eukaryotic mRNAs and on their interaction with initiation factors

The effects of 5' proximal secondary structure in mRNA molecules on their translation and on their interaction with the eukaryotic initiation factors (eIF)-4F, eIF-4A, and eIF-4B have been examined. Secondary structures were generated in the 5' noncoding region of rabbit globin and reovirus mRNAs by means of hybridization with cDNA molecules. cDNAs hybridized to the first 15 bases downstream from the cap inhibited the translation of the mRNAs in both reticulocyte and wheat germ lysates. The degree of inhibition was directly related to the monovalent ion concentration and inversely related to reaction temperature. These hybrid structures also reduced the competitive ability of the messages. Hybrid structures beginning downstream from the first 15 bases did not inhibit the translation of beta-globin mRNA or reovirus s3 mRNA. None of the hybrid structures were detrimental to the interaction of the mRNAs with the 26-kDa cap binding protein of eIF-4F, as determined by chemical cross-linking assays. However, in the presence of ATP, hybrid structures immediately adjacent to the cap severely inhibited the cross-linking to the p46 subunit of eIF-4F or to additional eIF-4A or eIF-4B. In order to account for these observations, a two-step mechanism is proposed for the interaction of eIF-4F with the 5' end of an mRNA molecule. The first step involves a weak initial interaction of the p26 subunit with the cap. The second step requires the hydrolysis of ATP and results in the formation of a stable initiation factor-mRNA complex, which may involve eIF-4A and eIF-4B. This second step is inhibited by the presence of 5' proximal secondary structure. In any event, our results demonstrate that the effect of mRNA structure on translation rate depends strongly on its position with respect to the 5' end and that this effect is due at least in part to an inhibition of the action of initiation factors normally required for the unwinding of structure.     

结构生物学研究中的eIF-5A的表达与纯化

真核细胞翻译官始因子eIF-5A（eukaryotic initiation factor 5A）是迄今发现的惟一含有特殊氨基酸hypusine残基的蛋白质,其具体生物学功能仍不明确。为了推进对其功能的研究,拟从结构生物学入手,对其结构进行核磁共振（NMR）结构解析。利用GST融合蛋白原核表达系统,将eIF-5A进行原核表达,经过优化表达与纯化条件,得到了高产率与高纯度的可溶性eIF-5A用以进行NMR测试：经过!1H-^15N HSQC NMR实验,发现其适合应用NMR方法进行结构解析,从而为溶液中eIF-5A三维构象的研究奠定了基础.     

eIF5A binds to translational machinery components and affects translation in yeast

The putative translation factor eIF5A is essential for cell viability and is highly conserved from archebacteria to mammals. Although this protein was originally identified as a translation initiation factor, subsequent experiments did not support a role for eIF5A in general translation. In this work, we demonstrate that eIF-5A interacts with structural components of the 80S ribosome, as well as with the translation elongation factor 2 (eEF2). Moreover, eIF5A is further shown to cofractionate with monosomes in a translation-dependent manner. Finally, eIF5A mutants show altered polysome profiles and are sensitive to translation inhibitors. Our results re-establish a function for eIF5A in translation and suggest a role for this factor in translation elongation instead of translation initiation.     

Hypoxia enhances phosphorylation of eukaryotic initiation factor 4A in maize root tips.

We have identified two isoforms of initiation factor 4A (eIF-4A) in maize root tips, with distinct isoelectric points and similar molecular mass (approximately 50 kDa). Both isoforms of maize eIF-4A cross-react with antibodies raised against wheat germ eIF-4A, and one of the maize proteins (higher pI isoform) comigrates with purified wheat germ eIF-4A on two-dimensional gels. The two maize eIF-4As were indistinguishable by comparative peptide fingerprint analysis, which also showed a very strong similarity between eIF-4A in maize roots and wheat germ. Maize eIF-4As copurify with eIF-4F and eIF-(iso)4F on a 7-methyl-GTP-Sepharose affinity column, indicating that they are part of the 5'-cap-binding complex. Two-dimensional gel electrophoresis and immunoblotting of proteins from 32P-labeled maize root tips revealed that the lower pI isoform of eIF-4A is phosphorylated. Two-dimensional phosphopeptide maps of trypsin-digested eIF-4A contained one principal phosphorylated fragment; phosphoamino acid analysis indicated phosphorylation of threonine. In oxygenated maize root tips, the ratio of phosphorylated to nonphosphorylated eIF-4A is approximately 0.2. This ratio increases to approximately 1 within 20 min following the onset of hypoxia, due to interconversion between the two maize eIF-4A isoforms. The hypoxia-induced phosphorylation of eIF-4A is discussed with respect to metabolic responses, and the translational control of gene expression, in hypoxic plant tissues.     

ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors

Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with 32P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or "unwind" mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. Our results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A.     

Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.     

The mammalian hypusine-containing protein, eukaryotic initiation factor 4D. Structural homology of this protein from several species

A single cellular protein of Mr approximately 18,000 and pI near 5.1, recently identified as eukaryotic translation initiation factor eIF-4D, contains the unusual amino acid hypusine [N epsilon-(4-amino--2-hydroxybutyl)lysine] formed post-translationally from lysine with a structural contribution from the polyamine spermidine. When the 3H-labeled hypusine-containing protein isolated from Chinese hamster ovary (CHO) cells that were grown with radioactive polyamine is digested with trypsin and the digest is subjected to two-dimensional separation, a single radioactive peptide is seen. A labeled peptide that occupies this same position is found in a digest of the [3H]hypusine protein from human lymphocytes and the single hypusine-containing tryptic peptide from purified rabbit reticulocyte eIF-4D also moves to this identical position. Stepwise Edman degradation of the tryptic digest of CHO cell hypusine-protein releases the radioactivity as a single peak in accordance with our earlier evidence for a single hypusine residue per molecule of eIF-4D. The similar patterns of radioactive peptides obtained from tryptic digests of radioiodinated eIF-4D from CHO cells, human lymphocytes, and rabbit reticulocytes suggest a highly conserved primary structure for this protein.     

Catalytic utilization of eIF-2 and mRNA binding proteins are limiting in lysates from vesicular stomatitis virus infected L cells

Infection of mouse L cells by vesicular stomatitis virus results in the inhibition of cellular protein synthesis. Lysates prepared from these infected cells are impaired in their ability to translate endogenous or exogenous cellular and viral mRNAs. The ability of initiation factors from rabbit reticulocytes to stimulate protein synthesis in these lysates was examined. Preparations of eukaryotic initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) stimulated protein synthesis strongly in L cell lysates from infected cells but only slightly in lysates from mock-infected cells. Maximal stimulation was obtained when a fraction containing eukaryotic initiation factors 4B (eIF-4B) and 4F (eIF-4F) was also present. In lysates from infected cells, these initiation factors increased endogenous cellular mRNA translation on the average 2-fold. In contrast, endogenous viral mRNA translation was increased to a much greater extent: the M protein was stimulated 8-fold, NS 5-fold, N 2.5-fold, and G 12-fold. When fractions containing eIF-4B, eIF-4F, or eIF-4A were added to these lysates in the presence of eIF-2, all three stimulated translation. Fractions containing rabbit reticulocyte initiation factors eIF-3 and eIF-6 had no effect on translation in either lysate. The results suggest that lysates from infected L cells are defective in the catalytic utilization of eIF-2 and deficient in mRNA binding protein activity.     

Cell Cycle Progression and Proliferation Despite 4BP-1 Dephosphorylation

Proliferation and cell cycle progression in response to growth factors require de novo protein synthesis. It has been proposed that binding of the eukaryotic translation initiation factor 4E (eIF-4E) to the inhibitory protein 4BP-1 blocks translation by preventing access of eIF-4G to the 5' cap of the mRNA. The signal for translation initiation is thought to involve phosphorylation of 4BP-1, which causes it to dissociate from eIF-4E and allows eIF-4G to localize to the 5' cap. It has been suggested that the ability of the macrolide antibiotic rapamycin to inhibit 4BP-1 phosphorylation is responsible for the potent antiproliferative property of this drug. We now show that rapamycin-resistant cells exhibited normal proliferation despite dephosphorylation of 4BP-1 that allows it to bind to eIF-4E. Moreover, despite rapamycin-induced dephosphorylation of 4BP-1, eIF-4E-eIF-4G complexes (eIF-4F) were still detected. In contrast, amino acid withdrawal, which caused a similar degree of 4BP-1 dephosphorylation, resulted in dissociation of the eIF-4E-eIF-4G complex. Thus, 4BP-1 dephosphorylation is not equivalent to eIF-4E inactivation and does not explain the antiproliferative property of rapamycin.     

Interactions of the eIF-4F subunits in the yeast Saccharomyces cerevisiae.

Recognition of the cap structure at the 5' end of mRNA is one of the first events in initiation of eukaryotic translation. This step is mediated by the translation initiation factor 4F (eIF-4F). In mammalian cells this factor is composed of the cap-binding protein eIF-4E, eIF-4A, and a 220-kDa polypeptide. In yeast Saccharomyces cerevisiae, eIF-4E is found associated with a 150-kDa protein (p150) and a 20-kDa protein (p20). The resulting protein complex is proposed to represent yeast eIF-4F. To study the functions of p150 and p20 and their interaction with eIF-4E, we disrupted the genes encoding p150 and p20 and analyzed the effects on protein complex formation and cell viability. Yeast cells with single and double disruptions of the genes encoding p150 and p20 are viable, but p150 single and p150/p20 double disruptions show a slow growth phenotype. Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E.     

Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation.

Eukaryotic translation initiation factor-4A (eIF-4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA-dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA-ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF-4F. To gain further understanding into the mechanism of action of eIF-4A in mRNA-ribosome binding, defective eIF-4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF-4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap-independent internal initiation mechanism. Addition of eIF-4A or eIF-4F relieved inhibition of translation, but eIF-4F was six times more effective than eIF-4A, whereas eIF-4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF-4A is defective in recycling through eIF-4F, thus explaining the dramatic inhibition of translation. Mutant eIF-4A proteins also inhibited eIF-4F-dependent, but not eIF-4A-dependent RNA helicase activity. Taken together these results suggest that eIF-4A functions primarily as a subunit of eIF-4F, and that singular eIF-4A is required to recycle through the complex during translation. Surprisingly, eIF-4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.     

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.     

The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins.

Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of cap-dependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4 gamma, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors.     

Evidence that the 5'-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4G

The efficiency of translation of alfalfa mosaic virus (AMV) RNA 4, barley alpha-amylase (B alpha A) mRNA, and two chimeric mRNAs, AMV 4-B alpha A and B alpha A-AMV 4 (in which the 5' leader sequences of the two mRNAs were interchanged), was measured in an S30 extract from wheat germ and a fractionated system from wheat germ in which translation could be made dependent upon initiation factor (eIF) 3, 4A, 4F, or 4G. In the S30 system, AMV RNA 4 and the chimeric mRNA AMV 4-B alpha A are translated much more efficiently than B alpha A mRNA and the chimeric mRNA B alpha A-AMV 4. When the S30 system was supplemented with high amounts of purified eIF-3, eIF-4A, eIF-4F, and eIF-4G, B alpha A and B alpha A-AMV 4 mRNAs were translated as efficiently as AMV RNA 4 and AMV 4-B alpha A mRNA. These findings indicated that the mRNAs containing the B alpha A leader sequence required higher amounts of one or more of the initiation factors (eIF-3, eIF-4A, eIF-4F, and eIF-4G) for efficient translation. Determination of the amounts of the initiation factors required for translation in the fractionated system showed that AMV RNA 4 required 2-4-fold lower amounts of eIF-3, eIF-4A, eIF-4F, and eIF-4G than did B alpha A mRNA. Replacement of the B alpha A leader sequence with that of AMV RNA 4 decreased the amounts of eIF-4A, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4F required. Replacement of the AMV RNA 4 leader sequence with that of B alpha A mRNA increased the amounts of eIF-4F, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4A required. These data strongly suggest that the amounts of the factors required are affected not only by the 5' leader itself but also by interactions between the 5' leader and a region(s) of the mRNA 3' to the initiation codon.     

mRNAs containing extensive secondary structure in their 5' non-coding region translate efficiently in cells overexpressing initiation factor eIF-4E.

Cellular eukaryotic mRNAs (except organellar) contain at the 5' terminus the structure m7(5')Gppp(5')N (where N is any nucleotide), termed cap. Cap recognition by eukaryotic initiation factor eIF-4F plays an important role in regulating the overall rate of translation. eIF-4F is believed to mediate the melting of mRNA 5' end secondary structure and facilitate 43S ribosome binding to capped mRNAs. eIF-4E, the cap-binding subunit of eIF-4F, plays an important role in cell growth; its overexpression results in malignant transformation of rodent cells, and its phosphorylation is implicated in signal transduction pathways of mitogens and growth factors. The molecular mechanism by which eIF-4E transforms cells is not known. Here, we report that overexpression of eIF-4E facilitates the translation of mRNAs containing excessive secondary structure in their 5' non-coding region. This effect may represent one mechanism by which eIF-4E regulates cell growth and transforms cells in culture.