Characterization of protein synthesis initiation factor 2 from Xenopus laevis oocytes

The protein synthesis initiation factor 2 (eIF2) from Xenopus laevis oocytes has been extensively purified and characterized. Depending upon the purification scheme, eIF2 containing three subunits (alpha, beta and gamma) with Mr of 160,000, or two subunits (alpha and gamma) with Mr 90,000 can be obtained. The key step for obtaining the three subunit factor is the addition of 30 mM benzamidine to the initial homogenization, since this compound protects the highly sensitive beta subunit from proteolytic degradation. Subunit alpha of the oocyte eIF2 can be phosphorylated by the specific kinase from rabbit reticulocytes, whereas subunit beta is phosphorylated by oocyte casein kinase II. The oocyte eIF2 has a KD of 7.2 X 10(-8) M for GDP and 3.8 X 10(-6) M for GTP. The purified three subunit eIF2 has 0.4 mol of GDP bound/mol of factor. The crude preparations of eIF2 are not affected by Mg2+ in their exchange of guanine nucleotides or in the formation of ternary complexes with GTP and methionyl-tRNA, but these reactions are strongly inhibited by Mg2+ when the highly purified preparations are used.     

Eukaryotic initiation factor 6 (eif6) overexpression affects eye development in Xenopus laevis

The translation initiation factor eif6 has been implicated as a regulator of ribosome assembly, selective mRNA translation and apoptosis. Many of these activities depend upon the phosphorylation of eif6 serine 235 by PKC. Previous data showed that eif6 binds to the 60S ribosomal subunit when unphosphorylated, inhibiting assembly with the 40S subunit. Phosphorylation of Ser235 releases eif6 from the 60S subunit and allows assembly. eif6 acts as an anti-apoptotic factor via regulation of the bcl2/bax balance and acts selectively upstream of bcl2. This activity also depends upon phosphorylation of eif6 Ser235. One of the consequences of eif6 overexpression in Xenopus embryos is aberrant eye development. Here we evaluate the eye phenotype and show that it is transient. We show that the whole eye, particularly the retina layers, of the embryos injected with eif6-encoding mRNA recover by stage 42. Embryos over-expressing eif6 have normal expression of anterior- and brain-specific markers, indicating that outside the eye field, other neural regions appear unaffected by the eif6 injection. No eye defect was detected when morpholinos were used to reduce eif6 protein synthesis. We tested how two known pathways of eif6 function with respect to alteration of eye development. We found that injection of bcl2 did not produce the eye phenotype and eif6-bax co-injection did not rescue the eye defect, suggesting that the eye phenotype is not bearing on the anti-apoptotic role played by eif6 is not linked to its role as an anti-apoptotic factor. We also determined that PKC-dependant phosphorylation of Ser235 in eif6 is not required to produce defective eye development. These results indicate that the aberrant eye phenotype, produced by eif6 overexpression, is not directly linked to the PKC-regulated effects of eif6 on translation and ribosomal subunit interaction or on eif6 anti-apoptotic properties.     

A function for dystroglycan in pronephros development in Xenopus laevis

Dystroglycan (Dg) is a laminin receptor that is expressed at the interface between the basement membrane and the cell membrane. Dg has been reported to play a role in skeletal muscle cell stability, morphogenesis of neuroepithelial tissues, and in regulating cytoskeletal organization, cell polarization, and cell signalling. In this study, we have focused our analysis on the expression of Dg-mRNA and protein at different developmental stages in the pronephros of Xenopus laevis. In order to study its role, we performed loss-of-function experiments mediated by Dg antisense morpholinos and dominant negative mutant. We show that Dg expression is first detectable when epithelialization begins in the pronephric anlage and persists later during tubulogenesis. Loss-of-function experiments induced a disorganization of the basement membrane, a drastic reduction of pronephric tubules and duct that can lead to a renal agenesis. A diminished proliferation of pronephric cell progenitors was also observed in Dg depleted embryos. Together, these data indicate that Dg plays a key role for laminin-1 assembly and pronephric cell anchoring to the basement membrane during early development of the pronephros. They also indicate that Dg may induce a signal transduction pathway controlling cell proliferation needed for the formation of tubules and their growth.     

Binding analysis of Xenopus laevis translation initiation factor 4E (eIF4E) in initiation complex formation.

A translation initiation factor, eIF4E, of Xenopus laevis was purified by affinity column chromatography after the gene expression as a full-length protein in a baculovirus-insect cell system. Interaction between X. laevis eIF4E and 4E-BP2 was analyzed by affinity column chromatography, gel permeation chromatography (GPC), and surface plasmon resonance (SPR). It was found that the interaction of eIF4E with an mRNA cap-analogue enhanced the binding activity of eIF4E with 4E-BP2. Furthermore, the SPR analysis showed that the eIF4E-cap-analogue interaction was very weak regardless of complex formation of 4E-BP2 with eIF4E; the dissociation constant of eIF4E for the cap-analogue was estimated to be 10(-2)-10(-4) M. These results suggest that the participation of another initiation factor is required for eIF4E to recognize the cap structure in vivo. The results reported in this paper support "the performed complex model" of Lee et al., in which eIF4E binds to the mRNA cap structure after the initiation factors have formed the initiation complex eIF4F.     

A role for Xlim-1 in pronephros development in Xenopus laevis

Xlim-1, a LIM class homeobox gene expressed in Xenopus laevis, is one of the earliest known marker genes of pronephros development and is expressed in pronephros rudiment. In this study, we examined the role of Xlim-1 in pronephros development. Temporal expression of Xlim-1 in explants was analyzed in a series of induction assays using RT-PCR analysis. Xlim-1 was expressed 9 to 15 h after activin/retinoic acid treatment, corresponding to pronephros differentiation in explants. We further examined the role of Xlim-1 using a series of microinjection experiments. Presumptive pronephric anlagen of embryos were injected with various Xlim-1 mutants, and effects of these Xlim-1 mutants on pronephrogenesis in embryos and in explants were analyzed by RT-PCR and immunohistochemistry. Dominant-negative Xlim-1 inhibited differentiation of pronephros in activin/retinoic acid-treated animal caps. In embryos injected with a dominant-negative form of Xlim-1, development of pronephric tubules was inhibited at the late tail-bud stage. Our results suggest that Xlim-1 may not initiate differentiation of the pronephros, but that it is necessary for growth and elongation in the development of pronephric tubules.     

Formation and release of eukaryotic initiation factor 2 X GDP complex during eukaryotic ribosomal polypeptide chain initiation complex formation

The formation and release of an eukaryotic initiation factor (eIF)-2 X GDP binary complex during eIF-5-mediated assembly of an 80 S ribosomal polypeptide chain initiation complex have been studied by sucrose gradient centrifugation analysis. Isolated 40 S initiation complex reacts with eIF-5 and 60 S ribosomal subunits to form an 80 S ribosomal initiation complex with concomitant hydrolysis of an equimolar amount of bound GTP to GDP and Pi. Sucrose gradient analysis of reaction products revealed that GDP was released from ribosomes as an eIF-2 X GDP complex. Evidence is presented that eIF-5-mediated hydrolysis releases the GTP bound to the 40 S initiation complex as an intact eIF-2 X GDP complex rather than as free GDP and eIF-2 which subsequently recombine to form the binary complex. Furthermore, formation and release of eIF-2 X GDP from the ribosomal complex do not require concomitant formation of an 80 S initiation complex since both reactions occur efficiently when the 40 S initiation complex reacts with eIF-5 in the absence of 60 S ribosomal subunits. These results, along with the observation that the 40 S initiation complex formed with the nonhydrolyzable analogue of GTP, 5'-guanylylmethylene diphosphonate, can neither join a 60 S ribosomal subunit nor releases ribosome-bound eIF-2, suggest that following eIF-5-mediated hydrolysis of GTP bound to the 40 S initiation complex, both Pi and eIF-2 X GDP complex are released from ribosomes prior to the joining of 60 S ribosomal subunits to the 40 S initiation complex.     

Transfer-RNA, magnesium and the formation of ternary complexes by eukaryotic initiation factor eIF-2

The likely concentrations of free magnesium ions in assay systems measuring ternary complex formation with the eukaryotic initiation factor eIF-2 and the exchange of bound GDP have been calculated. Contrary to the suggestion of Roy et al. (Biochem. Biophys. Res. Commun. 146, 114-120) amounts of added tRNA are unlikely to sequester sufficient magnesium ions to affect significantly their role in the assays. There seems little correlation between methionyl-tRNA added and the extent of ternary complex formation in published data other than that expected from mass action. In vivo the concentration of methionyl-tRNA is probably greater than or equal to that of eIF-2 which is shown to be necessary for efficient functioning of the initiation factor.     

Role of eukaryotic initiation factor 5 in the formation of 80 S initiation complexes.

The function of eukaryotic initiation factor 5 (eIF-5) from rabbit reticulocyte lysate has been studied by sucrose gradient preparation of 40 S and 80 S initiation complexes. eIF-5 is required for transfer of initiator tRNA from 40 S preinitiation complexes to puromycin-reactive 80 S complexes. The transfer is dependent upon GTP hydrolysis and is associated with release of eIF-2 and eIF-3 from the 40 S subunit. The GTP-dependent loss of eIF-2 and eIF-3 is catalyzed by eIF-5 in the absence of 60 S subunits or when subunit joining is prevented by edeine, but not when GTP is replaced by GuoPP(NH)P. Unstable 40 S subunit . Met-tRNAf complexes generated by eIF-5 can form puromycin-reactive 80 S complexes when 60 S subunits are added in the absence of added GTP. In addition, kinetic evidence is presented that indicates GTP hydrolysis occurs prior to 80 S complex formation.     

A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation

Formation of the ternary complex Met-tRNAi X eukaryotic initiation factor (eIF) 2 X GTP from eIF-2 X GDP requires exchange of GDP for GTP. However, at physiological Mg2+ concentrations, GDP is released from eIF-2 exceedingly slowly (Clemens, M.J., Pain, V.M., Wong, S.T., and Henshaw, E.C. (1982) Nature (Lond.) 296, 93-95). However, GDP is released rapidly from impure eIF-2 preparations, indicating the presence of a GDP/GTP exchange factor. We have now purified this factor from Ehrlich cells and refer to it as GEF. CM-Sephadex chromatography of ribosomal salt wash separated two peaks of eIF-2 activity. GEF was found in association with eIF-2 in the first peak and co-purified with eIF-2 under low salt conditions. It was separated from eIF-2 in high salt buffers and further purified on hydroxylapatite and phosphocellulose. Gel electrophoresis of our purest preparations showed major bands at 85, 67, 52, 37, 27, and 21 kDa. Purified GEF increased the rate of exchange of [32P] GDP for unlabeled GDP 25-fold but did not function with phosphorylated eIF-2 (alpha subunit). The factor also stimulated markedly the rate of ternary complex formation using eIF-2 X GDP as substrate with GTP and Met-tRNAi but not using phosphorylated eIF-2 X GDP as substrate. eIF-2 is released from the 80 S initiation complex with hydrolysis of GTP. If eIF-2 X GDP is actually the complex released, then GEF is absolutely required for eIF-2 to cycle and it is therefore a new eukaryotic initiation factor. Furthermore, the inability of GEF to utilize eIF-2 (alpha P) X GDP explains how phosphorylation of eIF-2 can inhibit polypeptide chain initiation.     

Translational regulation of the mRNA encoding the eukaryotic translation initiation factor 4E in Xenopus

We have cloned the cDNA for Xenopus eukaryotic translation initiation factor 4E (eIF4E). Here we show that translation of a luciferase mRNA that contains the 5' untranslated region derived from Xenopus eIF4E is active in fertilized eggs, but is repressed in oocytes. The results suggest that the expression of Xenopus eIF4E is regulated at the translation level.     

Human rhinovirus 2 2Apro recognition of eukaryotic initiation factor 4GI. Involvement of an exosite

The 2A proteinase (2Apro) of human rhinovirus 2 is a cysteine proteinase with a unique chymotrypsin-like fold. During viral replication, 2Apro performs self-processing by cleaving between its own N terminus and the C terminus of the preceding protein, VP1. Subsequently, 2Apro cleaves the two isoforms of the cellular protein, eukaryotic initiation factor (eIF) 4G. We have previously shown that HRV2 2Apro can directly bind to eIF4G isoforms. Here we demonstrate using deletion mutants of eIF4GI that HRV2 2Apro requires eIF4GI amino acids 600-674 for binding; however, the amino acids at the cleavage site, Arg681 downward arrow Gly, are not required. The HRV2 2Apro binding domain for eIF4GI was identified by site-directed mutagenesis. Specifically, mutations Leu17 --> Arg and Asp35 --> Glu severely impaired HRV2 2Apro binding and thus processing of eIF4GI in rabbit reticulocyte lysates; self-processing, however, was not affected. Alanine scanning analysis further identified the loop containing residues Tyr32, Ser33, and Ser34 as important for eIF4GI binding. Although Asp35 is part of the catalytic triad, most of the eIF4GI binding domain lies in a unique exosite structure absent from other chymotrypsin-like enzymes and is distinct from the substrate binding cleft. The exosite represents a novel virulence determinant that may allow the development of specific inhibitors for HRV2 2Apro.     

Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2.GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNA(f) remains bound. The released eIF-2.GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNA(f).eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2.GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2.GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNA(f)l.eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2.eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2.GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2.GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2.GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2.GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2.eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2.GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.     

Structure and formation of ankylosis in Xenopus laevis

The structure of ankylotic teeth in Xenopus laevis was studied by light, transmission, and scanning electron microscopy as well as by microradiography in decalcified and undecalcified specimens. The mature teeth of Xenopus laevis are calcified from the crown to the base, fused to the jaw bone, and have no uncalcified area, such as a fibrous ring separating the tooth into the crown and pedicle. Microradiography shows that the mature tooth and jaw bone appear as an X-ray opaque area, except for the basal region of the dentine. This region is composed of an X-ray translucent area and an X-ray opaque thin layer on the lingual side of the translucent area. The mature tooth is composed of two differently calcified areas: (1) a highly calcified area, which makes up almost all of the tooth and contains a thin layer of the basal dentine on the lingual side, and (2) a lowly calcified basal dentine, which is fused to the jaw bone. Therefore, the lowly calcified area does not completely separate the dentine and jaw bone. Repeating banding patterns among the collagen fibrils differ among the dentine-forming area and the matrices of dentine and jaw bone. During the formation of ankylosis of the tooth germ, collagen bundles in the dentine-forming area accumulate directly on the surface of the jaw bone. Consequently, the mature teeth of Xenopus laevis fuse to the jaw bone directly without the mediation of the other structures.     

A ubiquitin-conjugating enzyme, ube2d3.2, regulates xMLK2 and pronephros formation in Xenopus

Abstract The Jun N-terminal kinase kinase kinase MLK2 is required for the formation of the pronephros during early Xenopus development. Here, we have used a yeast 2-hybrid screen to identify proteins that interact with and regulate xMLK2. Using an N-terminal polypeptide encompassing the SH3 and kinase domains of xMLK2 as bait, five independent cDNAs were identified, all of which encoded a Xenopus ubiquitin conjugating enzyme, ube2d3.2. Ube2d3.2 is a functional E2 enzyme expressed maternally and in a tissue-restricted fashion during development. Ectopic expression of ube2d3.2 inhibits formation of the pronephric tubules, resulting in a phenotype very similar to the loss of xMLK2 function. Because ube2d3.2 is also shown to limit accumulation of xMLK2, it is likely that this effect is direct, although other explanations are possible. Ube2d3.2 is thus probably an endogenous regulator of xMLK2, and hence of JNK activity.     

Postribosomal complexes containing eukaryotic initiation factor eIF-2

Eukaryotic initiation factors are found in the postribosomal supernatant as well as bound to the 40S ribosomal subunits. We have analyzed the factor activities from the supernatant by means of zonal centrifugation followed by Sepharose-heparin affinity chromatography. They exist both as free factors, sedimenting in a broad range from 4 to 7S, and complexed with other protein(s) with a sedimentation value of 16–20S. This complexed fraction contains besides eIF-2 another activity which exhibits a profound stimulation on amino acid incorporation in crude lysates and appears to counteract the heme-regulated inhibitor.Abbreviations eIF-2, eIF-3, eIF-4A and eIF-4B are eukaryotic initiation factors, see FEBS Letters 76, 1-10 (1977).