Characterization of maize elongation factor 1A and its relationship to protein quality in the endosperm.

The protein synthesis elongation factor 1A (eEF1A) is a multifunctional protein in eukaryotic cells. In maize (Zea mays L.) endosperm eEF1A co-localizes with actin around protein bodies, and its accumulation is highly correlated with the protein-bound lysine (Lys) content. We purified eEF1A from maize kernels by ammonium sulfate precipitation, ion-exchange, and chromatofocusing. The identify of the purified protein was confirmed by microsequencing of an endoproteinase glutamic acid-C fragment and by its ability to bundle actin. Using purified eEF1A as a standard, we found that this protein contributes 0.4% of the total protein in W64A+ endosperm and approximately 1% of the protein in W64Ao2. Because eEF1A contains 10% Lys, it accounts for 2.2% of the total Lys in W64A+ and 2.3% of the Lys in W64Ao2. However, its concentration predicts 90% of the Lys found in endosperm proteins of both genotypes, indicating that eEF1A is a key component of the group of proteins that determines the nutritional quality of the grain. This notion is further supported by the fact that in floury2, another high-Lys mutant, the content of eEF1A increases with the dosage of the floury2 gene. These data provide the biochemical basis for further investigation of the relationship between eEF1A content and the nutritional quality of cereals.     

Quantitative trait locus mapping of loci influencing elongation factor 1alpha content in maize endosperm

The nutritional value of maize (Zea mays) seed is most limited by its protein quality because its storage proteins are devoid of the essential amino acid lysine (Lys). The Lys content of the kernel can be significantly increased by the opaque-2 mutation, which reduces zein synthesis and increases accumulation of proteins that contain Lys. Elongation factor 1alpha (eEF1A) is one of these proteins, and its concentration is highly correlated with the Lys content of the endosperm. We investigated the genetic regulation of eEF1A and the basis for its relationship with other Lys-containing proteins by analyzing the progeny of a cross between a high (Oh51Ao2) and a low (Oh545o2) eEF1A maize inbred. We identified 83 simple sequence repeat loci that are polymorphic between these inbreds; the markers are broadly distributed over the genome (1,402 cM) with an average interval of 17 cM. Genotypic analysis of the F(2) progeny revealed two significant quantitative trait loci that account for 25% of the variance for eEF1A content. One of these is on the short arm of chromosome 4 and is linked with a cluster of 22-kD alpha-zein coding sequences; the other quantitative trait locus is on the long arm of chromosome 7. The content of alpha-zein and gamma-zein was measured in pools of high- and low-eEF1A individuals obtained from this cross, and a higher level of alpha-zein was found to cosegregate with high eEF1A content. Allelic variation at the 22-kD alpha-zein locus may contribute to the difference of eEF1A content between Oh51Ao2 and Oh545o2 by increasing the surface area of protein bodies in the endosperm and creating a more extensive network of cytoskeletal proteins.     

Identification of a new isoform of eEF2 whose phosphorylation is required for completion of cell division in sea urchin embryos

Elongation factor 2 (eEF2) is the main regulator of peptide chain elongation in eukaryotic cells. Using sea urchin eggs and early embryos, two isoforms of eEF2 of respectively 80 and 83 kDa apparent molecular weight have been discovered. Both isoforms were identified by immunological analysis as well as mass spectrometry, and appeared to originate from a unique post-translationally modified protein. Accompanying the net increase in protein synthesis that occurs in early development, both eEF2 isoforms underwent dephosphorylation in the 15 min period following fertilization, in accordance with the active role of dephosphorylated eEF2 in regulation of protein synthesis. After initial dephosphorylation, the major 83 kDa isoform remained dephosphorylated while the 80 kDa isoform was progressively re-phosphorylated in a cell-cycle dependent fashion. In vivo inhibition of phosphorylation of the 80 kDa isoform impaired the completion of the first cell cycle of early development implicating the involvement of eEF2 phosphorylation in the exit from mitosis.     

A2 isoform of mammalian translation factor eEF1A displays increased tyrosine phosphorylation and ability to interact with different signalling molecules

The eEF1A1 and eEF1A2 isoforms of translation elongation factor 1A have 98% similarity and perform the same protein synthesis function catalyzing codon-dependent binding of aminoacyl-tRNA to 80S ribosome. However, the isoforms apparently play different non-canonical roles in apoptosis and cancer development which are awaiting further investigations. We hypothesize that the difference in non-translational functions could be caused, in particular, by differential ability of the isoforms to be involved in phosphotyrosine-mediated signalling. The ability of eEF1A1 and eEF1A2 to interact with SH2 and SH3 domains of different signalling molecules in vitro was compared. Indeed, contrary to eEF1A1, eEF1A2 was able to interact with SH2 domains of Grb2, RasGAP, Shc and C-terminal part of Shp2 as well as with SH3 domains of Crk, Fgr, Fyn and phospholipase C-gamma1. Interestingly, the interaction of both isoforms with Shp2 in vivo was found using stable cell lines expressing eEF1A1-His or eEF1A2-His. The formation of a complex between endogenous eEF1A and Shp2 was also shown. Importantly, a higher level of tyrosine phosphorylation of eEF1A2 as compared to eEF1A1 was demonstrated in several independent experiments and its importance for interaction of eEF1A2 with Shp2 in vitro was revealed. Thus, despite the fact that both isoforms of eEF1A could be involved in the phosphotyrosine-mediated processes, eEF1A2 apparently has greater potential to participate in such signalling pathways. Since tyrosine kinases/phosphatases play a prominent role in human cancerogenesis, our observations may gave a basis for recently found oncogenicity of the eEF1A2 isoform.     

Characterisation of Translation Elongation Factor eEF1B Subunit Expression in Mammalian Cells and Tissues and Co-Localisation with eEF1A2

Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex.     

Cellular coexistence of two high molecular subsets of eEF1B complex

The elongation factor eEF1B involved in protein translation was found to contain two isoforms of the eEF1Bdelta subunit in sea urchin eggs. The eEF1Bdelta2 isoform differs from eEF1Bdelta1 by a specific insert of 26 amino acids. Both isoforms are co-expressed in the cell and likely originate from a unique gene. The feature appears universal in metazoans as judged from in silico analysis in EST-databanks. The eEF1B components were co-immunoprecipitated by specific eEF1Bdelta2 antibodies. Quantification of the proteins in immunoprecipitates and on immunoblots demonstrates that eEF1Bdelta1 and eEF1Bdelta2 proteins are present in two subsets of eEF1B complex. We discuss and propose a model for the different subsets of eEF1B complex concomitantly present in the cell.     

beta-Adrenergic agonists increase phosphorylation of elongation factor 2 in cardiomyocytes without eliciting calcium-independent eEF2 kinase activity

The beta-adrenergic agonist isoproterenol increased the phosphorylation of elongation factor eEF2 in ventricular cardiomyocytes from adult rats (ARVC). Phosphorylation of eEF2 inhibits its activity, and protein synthesis was inhibited in ARVC concomitantly with increased eEF2 phosphorylation. eEF2 kinase activity in ARVC extracts was completely dependent upon Ca(2+)/calmodulin. In contrast to other cell types, however, treatments designed to raise intracellular cAMP failed to induce Ca(2+)/calmodulin-independent activity. Instead, they increased maximal eEF2 kinase activity. Similar data were obtained when partially purified ARVC eEF2 kinase was treated with cAMP-dependent protein kinase in vitro. These data suggest that ARVC possess a distinct isoform of eEF2 kinase.     

Lysine-containing proteins in maize endosperm: a major contribution from cytoskeleton-associated carbohydrate-metabolizing enzymes

We measured fresh weight, dry weight, total protein, and the amounts of several individual proteins during endosperm development in three varieties of maize ( Zea mays L.): W64A wild-type (WT) and opaque-2 (o2), and sweet corn (SW). By 28 days after pollination (DAP), fresh weight was much higher in WT and SW than in o2, but o2 had a higher dry weight and thus a much lower water content. By 28 DAP, protein concentration [mg (g tissue(-1))] was highest in o2 and lowest in WT, while the protein content (microg seed(-1)) was lowest in o2. The storage proteins, alpha- and gamma-zeins, were low initially, but by 28 DAP they comprised over 50% of the total protein in WT and SW, but only about 30% in o2. In all varieties, the cytoskeleton proteins, actin, tubulin and eEF1alpha, sedimented with the protein bodies at 30 g to 27,000 g in tissue homogenized in cytoskeleton-stabilizing buffer. Other cytoskeleton-associated proteins increased during development, including UDP-glucose starch glucosyltransferase (UDP-GSGT, EC 2.4.1.11), sucrose synthase 1 (SuSy-1, EC 2.4.1.13) and fructose-1,6 bisphosphate aldolase (FBA, EC 4.1.2.13). At 28 DAP, these cytoskeleton-associated proteins combined make up 27% (WT), 23% (SW) and 33% (o2) of the total protein. These proteins are all rather high (5-11%) in lysine, and so they contribute about 75% (WT), 67% (o2), and 51% (SW) of the total endosperm lysine. We conclude that efforts to elevate the levels of these proteins could make a significant contribution to the nutritional value of corn.     

Mouse translation elongation factor eEF1A-2 interacts with Prdx-I to protect cells against apoptotic death induced by oxidative stress

eEF1A-1 and eEF1A-2 are two isoforms of translation elongation factor eEF1A. In adult mammalian tissues, isoform eEF1A-1 is present in all tissues except neurons, cardiomyocytes, and myotubes, where its isoform, eEF1A-2, is the only form expressed. Both forms of eEF1A have been characterized to function in the protein elongation step of translation, and eEF1A-1 is shown to possess additional non-canonical roles in actin binding/bundling, microtubule bundling/severing, and cellular transformation processes. To study whether eEF1A-2 has similar non-canonical functions, we carried out a yeast two-hybrid screening using a full sequence of mouse eEF1A-2 as bait. A total of 78 hits, representing 23 proteins, were identified and validated to be true positives. We have focused on the protein with the highest frequency of hits, peroxiredoxin I (Prdx-I), for in-depth study of its functional implication for eEF1A-2. Here we show that Prdx-I coimmunoprecipitates with eEF1A-2 from extracts of both cultured cells and mouse tissues expressing this protein, but it does not do so with its isoform, eEF1A-1, even though the latter is abundantly present. We also report that an eEF1A-2 and Prdx-I double transfectant increases resistance to peroxide-induced cell death as high as 1 mM peroxide treatment, significantly higher than do single transfectants with either gene alone; this protection is correlated with reduced activation of caspases 3 and 8, and with increased expression of pro-survival factor Akt. Thus, our results suggest that eEF1A-2 interacts with Prdx-I to functionally provide cells with extraordinary resistance to oxidative stress-induced cell death.     

Purification and some properties of starch branching enzyme (Q-enzyme) from tuberous root of sweet potato

The pattern of isoforms of starch branching enzyme II or Q-enzyme II in the tuberous root of sweet potato was distinct from those of other organs; altogether 7 isoforms of QEII were contained in the sweet potato plant. The QEIIf isoform, one of the two major QEII isoforms in the tuberous root, was purified to homogeneity by using a variety of HPLC columns. The purified QEIIf was a monomeric protein with a molecular mass of about 85 kDa. Western blot analysis showed that the polyclonal antibodies raised against the purified QEIIf was significantly reactive to the rice endosperm QEI, but not to the rice endosperm QEIIa. Furthermore, the sweet potato QEIIf reacted with the antiserum raised against the rice endosperm QEI, but not with that against the rice endosperm QEIIa. The results suggest that the sweet potato QEIIf is more similar to the rice endosperm QEI than to the rice endosperm QEIIa.     

Characterization of elongation factor-1A (eEF1A-1) and eEF1A-2/S1 protein expression in normal and wasted mice

The eEF1Alpha-2 gene (S1) encodes a tissue-specific isoform of peptide elongation factor-1A (eEF1A-1); its mRNA is expressed only in brain, heart, and skeletal muscle, tissues dominated by terminally differentiated, long-lived cells. Homozygous mutant mice exhibit muscle wasting and neurodegeneration, resulting in death around postnatal day 28. eEF1Alpha-2/S1 protein shares 92% identity with eEF1A-1; because specific antibodies for each were not available previously, it was difficult to study the developmental expression patterns of these two peptide elongation factors 1A in wasted and wild-type mice. We generated a peptide-derived antiserum that recognizes the eEF1Alpha-2/S1 isoform and does not cross-react with eEF1A-1. We characterized the expression profiles of eEF1A-1 and eEF1A-2/S1 during development in wild-type (+/+), heterozygous (+/wst), and homozygous (wst/wst) mice. In wild-type and heterozygous animals, eEF1A-2/S1 protein is present only in brain, heart, and muscle; the onset of its expression coincides with a concomitant decrease in the eEF1A-1 protein level. In wasted mutant tissues, even though eEF1A-2/S1 protein is absent, the scheduled decline of eEF1A-1 occurs nonetheless during postnatal development, as it does in wild-type counterparts. In the brain of adult wild-type mice, the eEF1A-2/S1 isoform is localized in neurons, whereas eEF1A-1 is found in non-neuronal cells. In neurons prior to postnatal day 7, eEF1A-1 is the major isoform, but it is later replaced by eEF1A-2/S1, which by postnatal day 14 is the only isoform present. The postdevelopmental appearance of eEF1A-2/S1 protein and the decline in eEF1A-1 expression in brain, heart, and muscle suggest that eEF1A-2/S1 is the adult form of peptide elongation factor, whereas its sister is the embryonic isoform, in these tissues. The absence of eEF1A-2/S1, as well as the on-schedule development-dependent disappearance of its sister gene, eEF1A, in wst/wst mice may result in loss of protein synthesis ability, which may account for the numerous defects and ultimate fatality seen in these mice.     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.     

Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.     

.肽链延长因子eEF1A-1和eEF1A-2在小鼠神经元中的表达特征

研究2种肽链延长因子(eEF1A-1,eEF1A-2)在不同发育阶段的小鼠神经元中的表达特征,探 讨其调控机制.应用Western印迹和组织免疫荧光技术分析两蛋白质在不同基因型小鼠(n =10)神经细胞中的表达水平和分布.结果表明,在胚胎期和幼龄期的野生型小鼠神经元胞 质中,eEF1A-1呈高水平表达并随发育而下降,于出生后26 d时停止表达;而eEF1A-2蛋白于出生后7 d开始表达并呈上调趋势,出生后20 d时达到最高水平,其后一直保持稳定表达,2种肽链延长因子在野生型小鼠神经元中的表达随发育而呈相反变化.eEF1A_2基因突变小鼠无eEF1A-2蛋白,eEF1A_1蛋白的表达模式与野生型小鼠基本类似,但出生后26 d时仍有微量表达.2种肽链延长因子在野生型小鼠发育阶段的表达水平变化受内在机制调控,不直接受各自表达水平的影响;eEF1A_2蛋白与神经元生理功能的维持有密切关系.     

Regulation of maize lysine metabolism and endosperm protein synthesis by opaque and floury mutations.

The capacity of two maize opaque endosperm mutants (o1 and o2) and two floury (fl1 and fl2) to accumulate lysine in the seed in relation to their wild type counterparts Oh43+ was examined. The highest total lysine content was 3.78% in the o2 mutant and the lowest 1.87% in fl1, as compared with the wild type (1.49%). For soluble lysine, o2 exhibited over a 700% increase, whilst for fl3 a 28% decrease was encountered, as compared with the wild type. In order to understand the mechanisms causing these large variations in both total and soluble lysine content, a quantitative and qualitative study of the N constituents of the endosperm has been carried out and data obtained for the total protein, nonprotein N, soluble amino acids, albumins/globulins, zeins and glutelins present in the seed of the mutants. Following two-dimensional PAGE separation, a total of 35 different forms of zein polypeptides were detected and considerable differences were noted between the five different lines. In addition, two enzymes of the aspartate biosynthetic pathway, aspartate kinase and homoserine dehydrogenase were analyzed with respect to feedback inhibition by lysine and threonine. The activities of the enzymes lysine 2-oxoglutate reductase and saccharopine dehydrogenase, both involved in lysine degradation in the maize endosperm were also determined and shown to be reduced several fold with the introduction of the o2, fl1 and fl2 mutations in the Oh43+ inbred line, whereas wild-type activity levels were verified in the Oh43o1 mutant.