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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

eEF1A Isoforms Change in Abundance and Actin-Binding Activity during Maize Endosperm Development¹

Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721 (J.A.L.-V., B.C.G., P.A.H., B.A.L.); and Department of Agricultural Chemistry, Oregon State University, Corvallis, Oregon 97331–7301 (T.W.D.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Eukaryotic elongation factor 1A (eEF1A) appears to be a multifunctional protein because several biochemical activities have been described for this protein, in addition to its role in protein synthesis. In maize (Zea mays) endosperm, the synthesis of eEF1A is increased in o2 (opaque2) mutants, and its concentration is highly correlated with the protein-bound lysine content. To understand the basis of this relationship, we purified eEF1A isoforms from developing endosperm and investigated their accumulation and their functional and structural properties. Formation of three isoforms appears to be developmentally regulated and independent of the o2 mutation, although one isoform predominated in one high lysine o2 inbred. The purified proteins differ in their ability to bind F-actin in vitro, suggesting that they are functionally distinct. However, they share similar aminoacyl-tRNA-binding activities. Tandem mass spectrometry revealed that each isoform is composed of the four same gene products, which are modified posttranslationally by methylation and phosphorylation. The chemical differences that account for their different actin-binding activities could not be determined.

eEF1A contains 11% (w/v) Lys, and it is subject to several types of posttranslational modifications, including methylation of Lys residues (Hiatt et al., 1982), addition of glycerylphosphorylethanolamine to glutamic or Asp residues (Whiteheart et al., 1989; Ransom et al., 1998), phosphorylation (Venema et al., 1991), and methylesterification at the C terminus (Zobel-Thropp et al., 2000). In higher eukaryotes, eEF1A is typically encoded by a multigene family: There are 18 genes in humans (Homo sapiens; Lund et al., 1996), four genes in Arabidopsis (Axelos et al., 1989), and 10 to15 genes in maize (Zea mays; Carneiro et al., 1999). The amino acid sequences encoded by these genes are highly conserved, even between different species (Browning, 1996). Several studies have provided evidence for tissue-specific expression of members of the eEF1A gene family (Dje et al., 1990; Aguilar et al., 1991; Lund et al., 1996; Kahns et al., 1998), but the importance of this has not been investigated widely. Thus, the different biological activities of eEF1A could result from isoforms of the protein generated by posttranslational modifications and/or expression of different eEF1A genes.

eEF1A is an abundant protein in maize endosperm, where its concentration is highly correlated (r² = 0.9) with the Lys content (Habben et al., 1995). Moro et al. (1996) documented a 5-fold variation in the concentration of eEF1A in the endosperm of several maize genotypes and found that eEF1A levels were consistently increased on a dry weight basis in o2 (opaque2) mutants, which normally contain a higher than average Lys content. In W64Ao2, eEF1A accounts for about 1% of the total endosperm protein and 2.3% of the Lys content (Sun et al., 1997). Thus, there appears to be a stoichiometric relationship between eEF1A and the other major Lys-containing proteins in the endosperm.

Immunocytochemistry studies showed eEF1A co-localizes with a network of F-actin surrounding the rough endoplasmic reticulum at sites where protein bodies are forming in maize endosperm cells (Clore et al., 1996). This localization was disrupted with cytochalasin D, indicating that eEF1A interacts with F-actin. eEF1A purified from developing endosperm was shown to bundle actin in vitro (Sun et al., 1997). Wang et al. (2001) found a quantitative trait locus associated with eEF1A content genetically linked to a cluster of 22-kD -zein genes and proposed a positive correlation between eEF1A content and the surface area of protein bodies; hence, the network of cytoskeletal proteins in the endosperm. Thus, the relationship between eEF1A concentration and Lys content may arise from eEF1A, providing an index of the amount of cytoskeletal proteins.

Previous work from our lab suggested there are different isoforms of eEF1A in maize endosperm (Sun et al., 1997), which may reflect different physiological functions of the protein. In the present study, we purified three eEF1A isoforms from developing endosperm and compared their F-actin- and aminoacyl-tRNA-binding activities in vitro. We also investigated their amino acid sequences and posttranslational modifications by tandem mass spectrometry (MS/MS). Our results showed the purified eEF1A isoforms differ in their ability to bind F-actin but share similar aminoacyl-tRNA-binding activities. They consist of the same four gene products modified posttranslationally by methylation and phosphorylation.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Purification of eEF1A Isoforms

eEF1A was purified from 16- to 20-d after pollination (DAP) frozen endosperm of several maize inbred lines. After comparing different approaches for extraction and separation, the following procedure was found to yield highly purified preparations of eEF1A. Proteins in endosperm extract were initially fractionated by ammonium sulfate (40%–80% [w/v]) precipitation, followed by liquid chromatography (LC) using anion exchange on Q Sepharose, cation exchange on SP Sepharose, hydrophobic interaction on Phenyl PHE, and cation exchange on SB, as described in "Materials and Methods." Because eEF1A is a very basic protein (pI of approximately 9.2), the Q Sepharose column was used only to clarify and reduce the complexity of the extract before binding to the SP Sepharose column. The SP Sepharose column was used to enrich the protein extract for eEF1A and prevent saturation of the phenyl column with protein when using more than 20 g of frozen endosperm tissue.

Three major eEF1A isoforms (eEF1A22, eEF1A24, and eEF1A26) were separated by SB cation-exchange chromatography and identified based on their elution profile. Figure 1 shows SDS-PAGE analysis of 2 µg of proteins recovered after each step of purification; proteins were visualized by staining with Coomassie Blue R (Fig. 1A) and by probing with eEF1A polyclonal antibodies (Habben et al., 1995) after transfer to nitrocellulose membrane (Fig. 1B). Based on this analysis, the different eEF1A isoforms were more than 95% pure. Typically, 600 µg of eEF1A22, 700 µg of eEF1A24, and 650 µg of eEF1A26 were purified from 70 g of 16-DAP W64Ao2 maize kernels. After purification, the proteins were used for comparison of their functional and chemical properties.

View larger version (78K): [in this window] [in a new window]   Figure 1. SDS-PAGE and immunoblot analysis of maize endosperm eEF1A obtained during steps of purification. eEF1A from crude endosperm extract was purified by ammonium sulfate precipitation (40%–80% [w/v]), cation-exchange chromatography on SP Sepharose, hydrophobic interaction on phenyl PHE, and cation exchange on SB. The major isoforms (eEF1A22, eEF1A24, and eEF1A26) separated by SB cation exchange chromatography were identified according to their approximate retention time. Two micrograms of protein from each step of purification were separated by 12.5% (w/v) SDS-PAGE and stained with Coomassie Blue R (A) or transferred to nitrocellulose membrane and probed with polyclonal antibodies against maize eEF1A (B). The purified eEF1A proteins were the only bands detected in the immunoblot. Mr estimates were based on migration of prestained protein markers.

The Relative Abundance of eEF1A Isoforms Changes during Endosperm Development

Because differences in the accumulation of the eEF1A isoforms were observed in W64A+ and o2, we compared their relative levels at early and middle stages of endosperm development. Figure 2 shows a comparison of the eEF1A isoforms obtained from W64Ao2 and wild-type endosperm at 11, 16, and 20 DAP. In W64Ao2 (Fig. 2A) at 11 DAP, eEF1A26 contributed about 70% of the protein, eEF1A24 contributed 30%, and eEF1A22 was barely detectable. At 16 DAP, the proportions of eEF1A26, eEF1A24, and eEF1A22 were about 30%, 40%, and 30%, respectively. By 20 DAP, eEF1A26 contributed about 10% of the protein, eEF1A24 contributed 30%, and eEF1A22 contributed 60%. Thus, the isoform with the longest retention time (eEF1A26) was most abundant at 11 DAP, and by 20 DAP, the isoform with the shortest retention time (eEF1A22) was the most abundant. Similar qualitative changes in the accumulation of the eEF1A isoforms, although not exactly in the same proportion, were observed during development of W64A+ endosperm (Fig. 2B), suggesting that their accumulation is developmentally regulated and independent of the o2 mutation.

View larger version (22K): [in this window] [in a new window]   Figure 2. Accumulation of eEF1A isoforms during development of W64Ao2 and W64A+ endosperm. eEF1A was purified from W64Ao2 (A) and W64A+ (B) endosperm at 11, 16, and 20 DAP. Three eEF1A isoforms (eEF1A22, eEF1A24, and eEF1A26), measured by A280 and identified by immunoblot analysis, were separated by SB cation exchange chromatography and labeled according to their retention time.

Previous studies showed a broad range of eEF1A concentration among various maize inbreds (Moro et al., 1996). The amount of eEF1A in mature endosperm of Oh51Ao2 is twice that in Oh545o2 (Wang et al., 2001), and the levels in the reciprocal F₁s are intermediate between the parental inbreds. Recombinant inbred lines (RILs) derived from the F₂ progeny of this cross show continuous variation in eEF1A concentration that ranges between the parents. To investigate the abundance of the three isoforms in these genotypes, eEF1A was purified from 10-, 15-, and 20-DAP endosperms (Fig. 3) as previously described. Accumulation of the isoforms during development of Oh545o2 endosperm was similar to W64Ao2 (Fig. 3A). At 10 DAP, eEF1A26 contributed about 80% of the protein and eEF1A24 contributed 20%; there was no eEF1A22 detectable. At 15 DAP, the proportions of eEF1A26, eEF1A24, and eEF1A22 were about 50%, 10%, and 40%, respectively. At 20 DAP, eEF1A26 contributed about 30% of the protein, eEF1A24 contributed 10%, and eEF1A22 contributed 60%. Thus, eEF1A26 was the most abundant isoform at 10 DAP, whereas eEF1A22 was the most abundant at 20 DAP. However, only eEF1A26 (with possibly very low levels of eEF1A24) was observed during development of the Oh51Ao2 endosperm (Fig. 3B). Accumulation of eEF1A isoforms in RILs 31-5 (low eEF1A content) and 34-3 (high eEF1A content) was analyzed only at 20 DAP. In RIL 31-5 endosperm, the proportions of eEF1A26, eEF1A24, and eEF1A22 were about 30%, 20%, and 50%, respectively (Fig. 3C); in RIL 34-3, eEF1A26 contributed about 85% of the protein, whereas eEF1A24 and eEF1A22 contributed only about 10% and 5% of the protein, respectively (Fig. 3D). The eEF1A profiles of the two RILs resembled those of their phenotypically similar parents at 20 DAP.

View larger version (36K): [in this window] [in a new window]   Figure 3. Comparison of protein isoforms in low- (Oh545o2) and high- (Oh51Ao2) eEF1A maize inbreds. eEF1A was purified from endosperm of Oh545o2 (A), a low-eEF1A inbred and Oh51Ao2(B), a high-eEF1A inbred, at 10, 15, and 20 DAP. eEF1A was purified from 20-DAP endosperm of the RILs 31-5 (C, low eEF1A content) and 34-3 (D, high eEF1A content) derived from a cross between Oh545o2 and Oh51Ao2.

eEF1A Isoforms Differ in Their Ability to Bind F-Actin in Vitro

eEF1A appears to be a multifunctional protein because several biochemical activities have been reported for this protein in addition to its classic role in the elongation cycle of protein synthesis (Durso and Cyr, 1994a; Gonen et al., 1994; Hotokezaka et al., 2002). The existence of a variety of posttranslational modifications of the protein raises the possibility that some of eEF1A's activities are associated with different modifications.

To investigate the functional activities of the isoforms from developing endosperm, we analyzed their ability to interact with rabbit (Oryctolagus cuniculus) muscle F-actin in vitro, using an assay based on that described by Demma et al. (1990). Equimolar concentrations of actin and each of the eEF1A isoforms were incubated in actin polymerization buffer for 1 h at room temperature. After centrifugation at 200,000g, the eEF1A associated with F-actin (pellet) was compared with that remaining soluble (supernatant) by SDS-PAGE (Fig. 4). Monomeric actin (G-actin), used as a control, was found mainly in the supernatant (Fig. 4, lane 2), whereas filamentous actin (F-actin) was recovered in the pellet (Fig. 4, lane 3). When eEF1A22 was incubated with actin in polymerization buffer, the amount of eEF1A22 in the pellet (lane 5) did not increase when compared with eEF1A22 alone (lane 7). The amount of eEF1A24 in the pellet with F-actin (lane 9) was 34% ± 5% more than that of eEF1A24 alone (lane 11), whereas the amount of eEF1A26 in the pellet with F-actin (lane 13) increased 64% ± 5% when compared with eEF1A26 alone (lane 15). These results suggested the isoforms differ in their ability to associate with F-actin. eEF1A22, the isoform most predominant at later stages of endosperm development (20 DAP), had no apparent F-actin-binding activity, whereas about 64% of eEF1A26, the isoform most predominant at the early stages of endosperm development (11-DAP), binds F-actin. Similar results were obtained when the analysis was conducted using actin purified from maize pollen (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 4. F-actin-binding activities of eEF1A isoforms. Equimolar amounts of rabbit muscle actin and each of the eEF1A isoforms were incubated in actin polymerization buffer for 1 h at room temperature. After centrifugation at 200,000g, the pellet (P) and supernatant (S) were analyzed by 12.5% (w/v) SDS-PAGE and stained with Commassie Blue R. As controls, actin and each of the eEF1A isoforms were incubated alone. The eEF1A associated with polymerized actin (P) was compared with that not bound to polymerized actin (S).

eEF1A Isoforms Share Similar Participation in Ternary Complex Formation with Aminoacylated tRNA (aa-tRNA)

To investigate whether the purified eEF1A isoforms differ in their interaction with aminoacyl tRNA during protein synthesis, the formation of aa-tRNAeEF1AGTP ternary complexes was studied and compared against a reference wheat germ eEF1A preparation. The fraction of protein capable of participating in ternary complex formation was assayed in eEF1AGTP-binding experiments conducted in the presence of excess ³H-Gly-tRNA^Gly, whereas binding affinities were measured by determining dissociation constants (K_d) from binding experiments in which ³H-aminoacyl-tRNA was present at 2 to 5 nM and eEF1AGTP concentrations varied from 0 to 300 nM (Dreher et al., 1999). All three maize endosperm eEF1A fractions were active for ternary complex formation. There were no large differences between the three preparations (8%–12% active; Table I), although rather small fractions of the maize eEF1As were active in ternary complex formation. For unknown reasons, eEF1A preparations commonly are only partially active in these assays; although this wheat germ eEF1A preparation was 26% active, a previous one was only 14% active.

View this table: [in this window] [in a new window]   Table I. Formation of aa-tRNAeEF1AGTP ternary complex by various preparations of eEF1A

The aa-tRNA-binding affinities of the active eEF1A molecules present in each of the maize endosperm preparations were similar and equivalent to the binding affinity of wheat germ eEF1A (Fig. 5; Table I). Dissociation constants of about 1.5 and 3 nM were measured for binding to bovine Gly-tRNA^Gly and wheat germ Val-tRNA^Val, respectively, indicating tight interactions.

View larger version (33K): [in this window] [in a new window]   Figure 5. Interaction of wheat germ Val-tRNAVal with eEF1A from maize endosperm and wheat germ. Ternary complex formation was estimated by liquid scintillation counting of 3H-Val-tRNAVal bound to paper filters after ribonuclease protection assay (Dreher et al., 1999). The curve fits (KaleidaGraph) were used to generate the Kd values reported in Table I.

MS Analysis

To investigate the nature of the eEF1A isoforms as a possible explanation for their chromatographic and functional differences, we used LC/MS/MS to determine their amino acid sequences and posttranslational modifications. Each of the isoforms was digested with trypsin, Glu-C, and Asp-N proteases, as described in "Materials and Methods." The resultant peptides mixtures were separated by LC and characterized by two consecutive mass analyzers. Multiple analyses were conducted for each isoform/protease combination.

The peptide patterns obtained for the three isoforms were very similar. Because eEF1A contains about 11% (w/v) Lys, trypsin generated a complex mixture of peptides, most of which were between 500 and 3,000 D. As a consequence, trypsin was the most informative protease. Glu-C was the second most informative protease; its coverage overlapped extensively with trypsin, although it generated some additional peptide sequences. AspN produced a smaller number of peptides, most of which corresponded to peptide sequences covered by the other two proteases. Each of the peptides was identified by comparison of the fragmentation pattern obtained in the second mass analyzer with that predicted from eEF1A protein sequences using the SEQUEST program (Yates, 1998).

Analysis of the data with SEQUEST, as well as manually, revealed the presence of peptide sequences corresponding to the same four gene products in each eEF1A isoform. This complexity made it difficult to identify chemical differences unique to the purified proteins. Analysis of the data revealed the purified proteins were modified by methylation and phosphorylation, although the same pattern of modifications was found in each of them. Figure 6 shows selected LC/MS/MS spectra of peptides found in the eEF1A26 isoform; however, similar results were obtained for eEF1A22 and eEF1A24. Figure 6A shows the spectrum of a Glu-C-derived peptide of apparent (M + H)⁺ mass-to-charge ratio (m/z) = 948.5. This value corresponds to the addition of 42 D (trimethylation) to the unmodified peptide "IALWKFE" (calculated [M + H]⁺ m/z = 906.5), which corresponds to amino acid residues 75 to 81 (e.g. GenBank accession no. AAB64207.1). The product ions are consistent with the predicted amino acid sequence of eEF1A, provided that the 42-D increment is located on the Lys residue at position 79. The mass difference of 170 D between ions y3 and y2 and between ions b5 and b4 corresponds to the mass of Lys plus 42 D. Figure 6B shows the spectrum of a tryptic peptide with apparent (M + H)⁺ m/z = 2592.2. This value corresponds to the addition of 80 D to the unmodified peptide "SVEMQHEALQEALPGDNVGFNVK" (calculated [M + H]⁺ m/z = 2512.2), which is predicted by accession number AAF42978.1 and corresponds to amino acid residues 279 to 301. Although the product ion corresponding to the phosphorylated Ser residue was not observed, this is perhaps because the peptide is rather long; there is a consistent shift of 80 D in the mass of the b ion series. For example, the predicted (M + H)⁺ m/z value for the unmodified ion (b13) is 1,466.6, whereas that observed in Figure 6B is 1,546.6. Figure 6C shows the spectrum of a tryptic peptide of apparent (M + H)⁺ m/z = 2291.1. This value corresponds to the addition of 14 D (monomethylation) to the unmodified peptide "MVPTKPMVVETFSQYPPLGR" (calculated [M + H]⁺ m/z = 2,277.1), which corresponds to amino acid residues 392 to 411 (e.g. accession no. AAB64207.1). The product ions are consistent with the predicted amino acid sequence of eEF1A, provided that the 14-D increment is located on the Lys residue at position 396. The mass difference of 142 D between ions b5 and b4 corresponds to the mass of Lys plus 14 D. On the other hand, ion y16 was not observed and, therefore, cannot be compared with y15. However, the m/z of ion y17 corresponds to an addition of 14 D to the calculated m/z of ion y17 in the unmodified peptide. Analyses with eEF1A22 and eEF1A24 produced results similar to these. Although it was possible to identify specific peptides and modifications, none were found to be unique for a given eEF1A isoform.

View larger version (29K): [in this window] [in a new window]   Figure 6. LC/MS/MS spectra of peptides identified in the eEF1A26 isoform. eEF1A26 was purified by HPLC and digested with trypsin, Glu-C, and Asp-N proteases, as described in "Materials and Methods," and the digestion products were analyzed by LC/MS/MS. Identification of the peptides and posttranslational modifications were conducted by comparison of the fragmentation patterns of individual peptides with those predicted from protein databases using SEQUEST. A, LC/MS/MS spectrum of a Glu-C derived peptide trimethylated at a Lys residue. B, LC/MS/MS spectrum of a tryptic peptide phosphorylated at a Ser residue. C, LC/MS/MS spectrum of a tryptic peptide monomethylated at a Lys residue.

Figure 7 shows the amino acid sequences of the eEF1A proteins identified in each of the isoforms and their posttranslational modifications. Highlighted sequences correspond to peptides differing between the gene products, which in combination with the posttranslational modifications allowed us to distinguish between the protein sequences. For example, the amino acid sequence of the phosphorylated peptide spanning residues 279 to 301 is unique to gene product AAF42978.1. Similarly, the amino acid sequence of the monomethylated peptide spanning amino acids 392 to 411 is specific for AAB64207.1 and the peptide spanning amino acids 6 to 20. AAF42976.1 contains a Glu residue at position 168, whereas the other gene products contain Asp at this position, and both amino acid sequences were identified in the analysis. This is the only amino acid difference between AAF42976.1 and AAF42977.1; therefore, AAF42977.1 was also included. The amino acid sequence coverage obtained with trypsin (solid line) was about 60%, followed by Glu-C (dashed line) with 50% and AspN (dotted line) with about 40%. The combined amino acid sequence coverage obtained with the three proteases was about 80%.

View larger version (58K): [in this window] [in a new window]   Figure 7. MS/MS analysis of endosperm eEF1A isoforms. eEF1A isoforms purified by HPLC were digested with trypsin, Glu-C, and Asp-N proteases, as described in "Materials and Methods." The digestion products were analyzed by LC/MS/MS. Four eEF1A gene products were identified, all present in each of the three isoforms. Solid lines, Amino acid sequences obtained by trypsin digestion; dashed and dotted lines, sequences obtained by Glu-C and AspN digestion. Peptides distinguishing the four proteins are highlighted; the posttranslational modifications identified are designated as follows: °, monomethylation; *, trimethylation; and #, phosphorylation.

The eEF1A isoforms were highly modified (Fig. 7). Lys residues at positions 36, 79, 187, 227, and 306 were trimethylated, whereas Lys at position 396 was monomethylated. Because trimethylated Lys were found in peptides of identical sequence for at least three of the four proteins, they could not be assigned to a specific gene product. Trimethylated lysines at positions 36 and 79 were not found in AAF42978.1. Monomethylated Lys appeared to be specific for AAB64207.1. Ser at position 279 was phosphorylated specifically in AAF42978.1. We did not identify glycerylphosphorylethanolamine modifications in Glu at position 285, the site of modification reported in carrot (Daucus carota) eEF1A (Ransom et al., 1998). From the amino acid sequence coverage obtained in our LC/MS/MS analysis (approximately 80%), it was not possible to identify isoform-specific sequences or posttranslational modifications that could be correlated with or explain the functional differences of the eEF1A isoforms.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The ability to separate maize endosperm eEF1A into three chromatographic forms (Figs. 2 and 3) suggests the existence of chemical diversity in amino acid sequence, posttranslational modification, or both. At least five eEF1A genes producing proteins with minor sequence differences are expressed in maize endosperm (Carneiro et al., 1999), and diversity is also documented at the posttranslational level. The functional significance of these modifications is poorly understood. Phosphorylation of eEF1A has been associated with increased translational activity (Venema et al., 1991), activation of phosphatidylinositol-4 kinase (PI-4K; Yang and Boss, 1994), and decreased F-actin binding in vitro (Izawa et al., 2000). In the fungus Mucor racemosus, methylation of eEF1A was associated with an increase in protein synthetic activity after sporulation (Fonzi et al., 1985). This methylation did not affect the affinity of eEF1A for GTP, aminoacyl-tRNA, and ribosomes, although it may affect the ability of eEF1A to form complexes with other EF1 subunits (Sherman and Sypherd, 1989). In yeast (Saccharomyces cerevisiae), mutation of methylation sites in eEF1A did not affect its ability to promote protein synthesis in vitro (Cavallius et al., 1997). Two isoforms from rabbit, eEF1A-1 and eEF1A-2, showed similar patterns of glycerylphosphorylethanolamine, but two of the Lys residues dimethylated in eEF1A-1 were trimethylated in eEF1A-2 (Kahns et al., 1998; Kristensen et al., 1998). These forms were indistinguishable with regard to in vitro protein synthesis activity, although the GDP dissociation rate constant was higher for eEF1A-1 than eEF1A-2. Nevertheless, the functional significance of these isoforms of eEF1A is unclear.

The changes we observed in the relative abundance of the three different eEF1A isoforms in W64Ao2 and W64A+ during endosperm development (Fig. 2) suggest that their accumulation is developmentally regulated. Although the total amount of eEF1A is higher in W64Ao2, this does not appear to be a consequence of the accumulation of a specific isoform. Together with the qualitative differences observed between high and low eEF1A genotypes (Fig. 3), these results also suggest the isoforms have different functions, which is supported by their different actin-binding activities (Fig. 4).

To explain why the purified eEF1A isoforms differ in their affinity for F-actin, we investigated their chemical properties by MS. LC/MS/MS analysis identified the same four gene products in each of the purified isoforms (Fig. 7), suggesting they have different patterns of posttranslational modifications. We found that the isoforms are modified by methylation and phosphorylation (Fig. 7). Only trimethylation of lysines at positions 36, 79, and 306 have been reported in other organisms, although only that at position 79 appears to be conserved (Cavallius et al., 1993). Monomethylated Lys at position 396 and phosphorylation of Ser at position 279 have not been reported before. In contrast to the amino acid sequence of eEF1A, it is well documented that posttranslational modifications of the protein are not highly conserved.

The presence of multiple gene products in each of the isoforms made the peptide analysis complex and made it difficult to identify isoform-specific modifications that could distinguish between them. In addition to conducting the protease digestions multiple times, these experiments were also done in the presence of 2 to 4 M urea. However, these treatments did not allow us to recover any new peptides that increased the protein sequence coverage, which was about 80%. Peptide fingerprinting by matrix-assisted laser-desorption ionization time of flight also failed to distinguish between the isoforms. It is unlikely this was due to cross contamination of the isoform peaks during purification because products similar to those obtained from eEF1A26 of W64Ao2 were recovered when eEF1A26 was purified from Oh51Ao2. eEF1A26 was the only isoform observed during development of Oh51Ao2 endosperm (Fig. 3).

Phosphorylation of eEF1A has been suggested to interfere with its actin-binding activity in vitro (Izawa et al., 2000). eEF1A26, the isoform that binds actin most efficiently, could be phosphorylated in vitro by a calcium-dependent protein kinase 4 times more efficiently than eEF1A24 and eEF122, but this phosphorylation did not appear to affect the in vitro actin-binding activity of the protein (data not shown). Phosphorylation has also been associated with the ability of eEF1A to be a functional activator of PI-4K (Yang et al., 1993; Yang and Boss, 1994). In collaboration with Dr. Wendy Boss (North Carolina State University, Raleigh, NC), we analyzed the ability of the isoforms to activate PI-4K and found no significant differences between them, even though they differ in their phosphorylation levels when treated with a calcium-dependent protein kinase (data not shown).

As a further characterization of the eEF1A isoforms, they were compared with wheat germ eEF1A for the formation of aa-tRNAeEF1AGTP ternary complexes. The isoforms had similar aa-tRNA-binding affinities (Fig. 5; Table I), suggesting the nature of their chemical differences had no significant effect on their interaction with aminoacyl tRNA during protein synthesis. Although we did not measure protein synthesis activity, these results are in agreement with other studies reporting similar in vitro protein synthesis activities for eEF1A proteins from yeast and rabbit, even though they are only about 80% identical and have different patterns of methylation and glycerylphosphorylethanolamine modifications (Merrick et al., 1990; Cavallius et al., 1993).

At present, it is not clear why there are multiple eEF1A isoforms in maize endosperm. In addition to the qualitative and quantitative differences in their accumulation during endosperm development, the only apparent biological difference we found is their F-actin-binding activity. eEF1A from rat (Rattus norvegicus) metastatic cells had a 10% to 20% reduction in the binding affinity to F-actin in vitro when compared with eEF1A from non-metastatic cells (Edmonds et al., 1996). Both eEF1A proteins had similar protein synthesis activities, and the different eEF1A activities with F-actin were proposed to be important for cytoskeletal organization and differential translation of mRNAs associated with the cytoskeleton. A similar situation may occur in maize endosperm. The isoform that binds actin most efficiently was predominantly accumulated in endosperm of high-eEF1A genotypes (Fig. 3). This may be related to enhanced cytoskeleton formation and, therefore, increased synthesis of cytoskeleton-associated proteins, which are presumably Lys rich (Habben et al., 1995).

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials

The maize (Zea mays) inbreds W64A+ and W64Ao2 were grown at the University of Arizona West Agricultural Center (Tucson). The maize inbreds Oh545o2 (low eEF1A) and Oh51Ao2 (high eEF1A) and the RILs developed from their cross, 31-5 and 34-3, were grown in the greenhouse at the University of Arizona Campus Agricultural Center. The 31-5 (low eEF1A) and 34-3 (high eEF1A) RILs were developed by single seed descent and were chosen because of their extreme eEF1A content. Developing kernels from W64A+ and o2 were harvested at 11, 16, and 20 DAP. Developing kernels from Oh545o2, Oh51Ao2, and the RILs were harvested at 10, 15, and 20 DAP. Harvested kernels were frozen in liquid nitrogen and stored at –80°C.

Purification of eEF1A Isoforms

Seventy grams of maize kernels was homogenized in 140 mL of buffer A (50 mM Tris-HCl [pH 7.5], 50 mM KCl, 1 mM dithiothreitol [DTT], 10% [v/v] glycerol, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride) using a Polytron (Kinematica Gmbh, Littau-Lucern, Switzerland). The homogenate was centrifuged at 10,000g, filtered though two layers of Miracloth (Calbiochem, La Jolla, CA), and adjusted to 40% (w/v) and then 80% (w/v) ammonium sulfate. The protein precipitated between 40% (w/v) and 80% (w/v) ammonium sulfate was collected by centrifugation at 20,000g for 30 min with a Sorvall RC-5B centrifuge, resuspended in 10 mL of buffer A, and desalted by gel filtration (G25, Pharmacia Biotech, Piscataway, NJ). The protein was loaded onto a Q Sepharose (Pharmacia Biotech) column connected to a SP Sepharose (Pharmacia Biotech) column, which were both pre-equilibrated with buffer A. The SP column was eluted with a 40-min linear gradient (1.5 mL min^–1) of 0.05 to 0.5 M KCl in buffer A, and the fractions containing eEF1A were monitored by immunoblot analysis using a rabbit polyclonal antibody raised against recombinant maize eEF1A (Habben et al., 1995). Pooled fractions containing eEF1A were dialyzed against buffer B (20 mM HEPES [pH 7.5], 2 M ammonium sulfate, 1 mM DTT, and 1 mM EDTA) and loaded onto a phenyl (GMB 1000 PHE, Biospher, Labio, Prague, Czech Republic) column pre-equilibrated with the same buffer. The column was eluted with a 40-min linear gradient (1 mL min^–1) of 2 to 0 M ammonium sulfate. Fractions containing eEF1A were dialyzed against buffer C (20 mM HEPES [pH 7.5], 10% [v/v] glycerol, 1 mM DTT, and 1 mM EDTA) and loaded onto a cation exchange (GMB 1000 SB, Biospher) column pre-equilibrated with the same buffer. The column was eluted with a 40-min linear gradient (1 mL min^–1) of 0 to 0.5 M NaCl in buffer C. The amount of protein eluting from the column was measured by A₂₈₀.

For analysis of the eEF1A isoforms from different genotypes and at different developmental stages, 15 g of frozen, developing maize kernels were homogenized with 30 mL of buffer C, and the homogenate was adjusted to 40% (w/v) and then 80% (w/v) ammonium sulfate. The protein precipitated between 40% (w/v) and 80% (w/v) ammonium sulfate was collected by centrifugation, resuspended in 5 mL of buffer B, and loaded directly onto the phenyl column. The remainder of the procedure was as described above. Purification of eEF1A was conducted at least two times for each genotype/developmental stage combination. To estimate the relative abundance of each isoform, the area of the corresponding peak, as measured by A₂₈₀, was divided by the total of all eEF1A peaks in the chromatogram.

Actin-Binding Assay

The ability of each eEF1A isoform to associate with F-actin (rabbit (Oryctolagus cuniculus) skeletal muscle, Cytoskeleton Inc., Denver) was tested in a cosedimentation assay according to Demma et al. (1990) with some modifications. Actin and each of the eEF1A isoforms were combined in a final equimolar concentration of 2.5 µM and then incubated for 1 h at room temperature in 200 µL of actin polymerization buffer (20 mM HEPES [pH 7.5], 50 mM KCl, 2 mM MgCl₂, 1 mM ATP, and 0.5 mM EDTA). As controls, actin and each of the isoforms were incubated alone in polymerization buffer. The samples were then centrifuged at 200,000g for 30 min with the TLA 100.3 rotor of a Beckman TL-100 ultracentrifuge (Beckman Instruments, Fullerton, CA). The supernatant was recovered and adjusted to 1x SDS-PAGE buffer (Laemmli, 1970). The pellet was dissolved in 1x SDS-PAGE buffer to a final volume equal to the supernatant. In both cases, a 30-µL aliquot was separated by 12.5% (w/v) SDS-PAGE and stained with Comassie Blue R. The relative amount of protein in the pellets and supernatants was estimated by densitometry. The proportion of eEF1A binding to actin was calculated as the eEF1A in the pellet minus eEF1A in the pellet of the control, divided by the total (pellet + supernatant).

Analysis of Ternary Complex Formation by eEF1A

Purified eEF1A isoforms were compared for their ability to form ternary complexes with aa-tRNAeEF1AGTP. tRNA^Gly purified from bovine liver was a gift to Theo W. Dreher from Dr. Boris Negrutskii (Institute of Molecular Biology and Genetics, Kiev, Ukraine). tRNA^Val was purified from wheat germ by hybridization affinity selection as described by Dreher et al. (1999); eEF1A was also purified from wheat germ.

tRNA^Gly and tRNA^Val were aminoacylated with ³H-labeled Gly and Val (specific activities of 41.1 and 40.9 Ci mmol^–1, respectively; New England Nuclear, Boston) using aminoacyl-tRNA synthetase activities present in a partially purified extract made from wheat germ (Dreher et al., 1992). The formation of ternary complex (aa-tRNAeEF1AGTP) was assayed by a ribonuclease protection assay as described by Dreher et al. (1999). The molar concentrations of eEF1A were based on total protein concentrations determined by Coomassie Blue R staining assays (Pierce, Rockford, IL).

MS Analysis

The purified eEF1A isoforms were digested with the proteases Trypsin (Boehringer Mannheim/Roche, Basel, Switzerland), Glu-C (V8, Sigma, St. Louis), and Asp-N (Sigma) according to the manufacturer. For trypsin digestion, 50 µg of eEF1A protein was digested with 1 µg of protease in 25 mM NH₄HCO₃ (pH 7.8) for 20 h at 37°C in a 100-µL reaction. For Glu-C digestion, 50 µg of eEF1A protein was digested with 1 µg of protease in 25 mM of NH₄HCO₃ (pH 7.8) for 20 h at room temperature in a 100-µL reaction. For Asp-N digestion, 50 µg of eEF1A protein was digested with 1 µg of protease in 50 mM sodium phosphate buffer (pH 8.0) for 20 h at 37°C in a 100-µL reaction. In all cases, the reactions were stopped by freezing and then used for LC/MS/MS analysis. The instrument used was a Finnigan LCQ HPLC/MS combined with an electrospray ionization source available at the University of Arizona Chemistry Department MS facilities. A 20-µL aliquot of the peptide mixture was first separated by reverse-phase HPLC using a C18 column and a 0.1% (v/v) trifluoroacetic acid/acetonitrile gradient. Peptides eluting from the column were ionized, and their masses were determined by an ion trapping mass analyzer. Individual ions were selected, subjected to fragmentation, and the ion products were characterized in a second mass analyzer. The SEQUEST program (Yates, 1998) was used to identify the protein(s). The experimental fragmentation patterns produced for each peptide were compared with those predicted from eEF1A protein sequences using the same protease. Posttranslational modifications were investigated by introducing a mass shift in a specific amino acid, such as 14 for methylation in Lys residues, to generate the predicted fragmentation patterns. In addition to SEQUEST identification, the nature of selected peptides was confirmed manually by analysis of the fragmentation pattern.

Received May 31, 2003; returned for revision July 7, 2003; accepted July 27, 2003.

	FOOTNOTES

 
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.027854.

¹ This work was supported by the U.S. Department of Agriculture National Research Initiative (grant no. NRI 981427 to B.A.L.), by the Department of Energy (grant no. DE–96ER20242 to B.A.L.), and by the Consejo Nacional de Ciencia y Tecnologia, Mexico (graduate fellowship to J.A.L.-V.).

² Present address: Commonwealth Scientific and Industrial Research Organization, Plant Industry, P.O. Box 1600, Canberra ACT 2601, Australia.

^* Corresponding author; e-mail larkins{at}ag.arizona.edu; fax 520–621–3692.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Aguilar F, Montandon PE, Stutz E (1991) Two genes encoding the soybean translation elongation factor eEF-1 alpha are transcribed in seedling leaves. Plant Mol Biol 17: 351–360[Medline]

Axelos M, Bardet C, Liboz T, Le Van Thai A, Curie C, Lescure B (1989) The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression. Mol Gen Genet 219: 106–112[CrossRef][Web of Science][Medline]

Browning KS (1996) The plant translational apparatus. Plant Mol Biol 32: 107–144[CrossRef][Web of Science][Medline]

Carneiro NP, Hughes PA, Larkins BA (1999) The eEFIA gene family is differentially expressed in maize endosperm. Plant Mol Biol 41: 801–813[Medline]

Cavallius J, Popkie AP, Merrick WC (1997) Site-directed mutants of posttranslationally modified sites of yeast eEF1A using a shuttle vector containing a chromogenic switch. Biochim Biophys Acta Gene Struct Expr 1350: 345–358[Medline]

Cavallius J, Zoll W, Chakraburtty K, Merrick WC (1993) Characterization of yeast EF-1-Alpha: nonconservation of posttranslational modifications. Biochim Biophys Acta 1163: 75–80[CrossRef][Medline]

Clore AM, Dannenhoffer JM, Larkins BA (1996) EF-1 alpha is associated with a cytoskeletal network surrounding protein bodies in maize endosperm cells. Plant Cell 8: 2003–2014[Abstract]

Condeelis J (1995) Elongation factor 1 alpha, translation and the cytoskeleton. Trends Biochem Sci 20: 169–170[CrossRef][Web of Science][Medline]

Demma M, Warren V, Hock R, Dharmawardhane S, Condeelis J (1990) Isolation of an abundant 50,000-dalton actin filament bundling protein from Dictyostelium amoebae. J Biol Chem 265: 2286–2291[Abstract/Free Full Text]

Dje MK, Mazabraud A, Viel A, le Maire M, Denis H, Crawford E, Brown DD (1990) Three genes under different developmental control encode elongation factor 1-alpha in Xenopus laevis. Nucleic Acids Res 18: 3489–3493[Abstract/Free Full Text]

Dreher TW, Tsai CH, Florentz C, Giege R (1992) Specific valylation of turnip yellow mosaic virus RNA by wheat germ valyl-tRNA synthetase determined by three anticodon loop nucleotides. Biochemistry 31: 9183–9189[CrossRef][Medline]

Dreher TW, Uhlenbeck OC, Browning KS (1999) Quantitative assessment of EF-1alpha. GTP binding to aminoacyl-tRNAs, aminoacyl-viral RNA, and tRNA shows close correspondence to the RNA binding properties of EF-Tu. J Biol Chem 274: 666–672[Abstract/Free Full Text]

Durso NA, Cyr RJ (1994a) Beyond translation: elongation factor-1 alpha and the cytoskeleton. Protoplasma 180: 99–105[CrossRef][Web of Science]

Durso NA, Cyr RJ (1994b) A calmodulin-sensitive interaction between microtubules and a higher-plant homolog of elongation factor-1 alpha. Plant Cell 6: 893–905[Abstract]

Edmonds BT, Murray J, Condeelis J (1995) pH regulation of the F-actin binding properties of Dictyostelium elongation factor 1 alpha. J Biol Chem 270: 15222–15230[Abstract/Free Full Text]

Edmonds BT, Wyckoff J, Yeung YG, Wang Y, Stanley ER, Jones J, Segall J, Condeelis J (1996) Elongation factor-1 alpha is an overexpressed actin binding protein in metastatic rat mammary adenocarcinoma. J Cell Sci 109: 2705–2714[Abstract]

Fonzi WA, Katayama C, Leathers T, Sypherd PS (1985) Regulation of protein synthesis factor EF-1 alpha in Mucor racemosus. Mol Cell Biol 5: 1100–1103[Abstract/Free Full Text]

Gonen H, Smith CE, Siegel NR, Kahana C, Merrick WC, Chakraburtty K, Schwartz AL, Ciechanover A (1994) Protein synthesis elongation factor EF-1 alpha is essential for ubiquitin-dependent degradation of certain N alpha-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu. Proc Natl Acad Sci USA 91: 7648–7652[Abstract/Free Full Text]

Habben JE, Moro GL, Hunter BG, Hamaker BR, Larkins BA (1995) Elongation factor 1-alpha concentration is highly correlated with the lysine content of maize endosperm. Proc Natl Acad Sci USA 92: 8640–8644[Abstract/Free Full Text]

Hesketh J (1994) Translation and the cytoskeleton: a mechanism for targeted protein synthesis. Mol Biol Rep 19: 233–243[CrossRef][Web of Science][Medline]

Hiatt WR, Garcia R, Merrick WC, Sypherd PS (1982) Methylation of elongation factor 1 alpha from the fungus Mucor. Proc Natl Acad Sci USA 79: 3433–3437[Abstract/Free Full Text]

Hotokezaka Y, Tobben U, Hotokezaka H, van Leyen K, Beatrix B, Smith DH, Nakamura T, Wiedmann M (2002) Interaction of the eukaryotic elongation factor 1A with newly synthesized polypeptides. J Biol Chem 277: 18545–18551[Abstract/Free Full Text]

Izawa T, Fukata Y, Kimura T, Iwamatsu A, Dohi K, Kaibuchi K (2000) Elongation factor-1 alpha is a novel substrate of Rho-associated kinase. Biochem Biophys Res Commun 278: 72–78[CrossRef][Web of Science][Medline]

Kahns S, Lund A, Kristensen P, Knudsen CR, Clark BF, Cavallius J, Merrick WC (1998) The elongation factor 1 A-2 isoform from rabbit: cloning of the cDNA and characterization of the protein. Nucleic Acids Res 26: 1884–1890[Abstract/Free Full Text]

Kaur KJ, Ruben L (1994) Protein translation elongation factor-1 alpha from Trypanosoma brucei binds calmodulin. J Biol Chem 269: 23045–23050[Abstract/Free Full Text]

Kristensen P, Lund A, Clark BF, Cavallius J, Merrick WC (1998) Purification and characterization of a tissue specific elongation factor 1 alpha (EF-1 alpha 2) from rabbit muscle. Biochem Biophys Res Commun 245: 810–814[CrossRef][Medline]

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline]

Liu G, Grant WM, Persky D, Latham VM, Singer RH, Condeelis J (2002) Interactions of elongation factor 1 alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions. Mol Biol Cell 13: 579–592[Abstract/Free Full Text]

Lund A, Knudsen SM, Vissing H, Clark B, Tommerup N (1996) Assignment of human elongation factor 1 alpha genes: eEF1A maps to chromosome 6q14 and eEF1A2 to 20q13.3. Genomics 36: 359–361[CrossRef][Web of Science][Medline]

Merrick WC, Dever TE, Kinzy TG, Conroy SC, Cavallius J, Owens CL (1990) Characterization of protein synthesis factors from rabbit reticulocytes. Biochim Biophys Acta 1050: 235–240[Medline]

Moore RC, Cyr RJ (2000) Association between elongation factor-1 alpha and microtubules in vivo is domain dependent and conditional. Cell Motil Cytoskelet 45: 279–292[CrossRef][Web of Science][Medline]

Moro GL, Habben JE, Hamaker BR, Larkins BA (1996) Characterization of the variability in lysine content for normal and opaque2 maize endosperm. Crop Sci 36: 1651–1659[Abstract/Free Full Text]

Motorin YA, Wolfson AD, Orlovsky AF, Gladilin KL (1988) Mammalian valyl-tRNA synthetase forms a complex with the first elongation factor. FEBS Lett 238: 262–264[CrossRef][Web of Science][Medline]

Ransom WD, Lao PC, Gage DA, Boss WF (1998) Phosphoglycerylethanolamine posttranslational modification of plant eukaryotic elongation factor 1 alpha. Plant Physiol 117: 949–960[Abstract/Free Full Text]

Sherman M, Sypherd PS (1989) Role of lysine methylation in the activities of elongation factor 1 alpha. Arch Biochem Biophys 275: 371–378[CrossRef][Medline]

Sun Y, Carneiro N, Clore AM, Moro GL, Habben JE, Larkins BA (1997) Characterization of maize elongation factor 1A and its relationship to protein quality in the endosperm. Plant Physiol 115: 1101–1107[Abstract]

Venema RC, Peters HI, Traugh JA (1991) Phosphorylation of valyl-tRNA synthetase and elongation factor 1 in response to phorbol esters is associated with stimulation of both activities. J Biol Chem 266: 11993–11998[Abstract/Free Full Text]

Wang W, Poovaiah BW (1999) Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha. J Biol Chem 274: 12001–12008[Abstract/Free Full Text]

Wang X, Woo YM, Kim CS, Larkins BA (2001) Quantitative trait locus mapping of loci influencing elongation factor 1alpha content in maize endosperm. Plant Physiol 125: 1271–1282[Abstract/Free Full Text]

Whiteheart SW, Shenbagamurthi P, Chen L, Cotter RJ, Hart GW (1989) Murine elongation factor 1 alpha (EF-1 alpha) is posttranslationally modified by novel amide-linked ethanolamine-phosphoglycerol moieties: addition of ethanolamine-phosphoglycerol to specific glutamic acid residues on EF-1 alpha. J Biol Chem 264: 14334–14341[Abstract/Free Full Text]

Yang F, Demma M, Warren V, Dharmawardhane S, Condeelis J (1990) Identification of an actin-binding protein from Dictyostelium as elongation factor-1A. Nature 347: 494–496[CrossRef][Medline]

Yang W, Boss WF (1994) Regulation of phosphatidylinositol 4-kinase by the protein activator PIK-A49: activation requires phosphorylation of PIK-A49. J Biol Chem 269: 3852–3857[Abstract/Free Full Text]

Yang WN, Burkhart W, Cavallius J, Merrick WC, Boss WF (1993) Purification and characterization of a phosphatidylinositol 4-kinase activator in carrot cells. J Biol Chem 268: 392–398[Abstract/Free Full Text]

Yates JR (1998) Mass spectrometry and the age of the proteome. J Mass Spectrom 33: 1–19[CrossRef][Web of Science][Medline]

Zobel-Thropp P, Yang MC, Machado L, Clarke S (2000) A novel posttranslational modification of yeast elongation factor 1A: methylesterification at the C terminus. J Biol Chem 275: 37150–37158[Abstract/Free Full Text]

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