Phosphorylation of elongation factor 1 in polyribosome fraction of rabbit reticulocytes

A single protein, Mr approximately 50000, is shown to be phosphorylated during incubation of a mono- and polyribosome fraction of rabbit reticulocytes with [gamma-32P]ATP at a low ionic strength. This protein has been identified as the elongation factor 1 alpha (EF-1 alpha). The phosphorylated EF-1 alpha, in contrast to the unmodified factor, is not detected in complexes with mono- and polyribosomes. It is suggested that the phosphorylation of EF-1 alpha can result in its decompartmentation from polyribosomes and thus affect the rate of protein synthesis.     

cAMP-dependent activation of protein synthesis correlates with dephosphorylation of elongation factor 2

The addition of 5 mM cAMP to a cell-free translation system from rabbit reticulocytes increases the rate of protein synthesis 3 5-fold. Lower concentrations of cAMP (0.005, 0.05 and 0.5 mM) have no effect on translation in this system. cAMP at all the concentrations tested stimulates the phosphorylation of the same pattern of polypeptides, while 5 mM cAMP additionally stimulates dephosphorylation of the 95 kDa polypeptide identified as elongation factor 2 (EF-2). Testing of the preparations of EF-2 with a different content of the phosphorylated form in poly(U)-directed poly(Phe) synthesis reveals that the EF-2 activity correlates with the fraction of non-phosphorylated EF-2. Thus cAMP-dependent activation of protein synthesis seems to be due to dephosphorylation of EF-2.     

Phosphorylation of elongation factor 1 beta by an endogenous kinase affects its catalytic nucleotide exchange activity

Elongation factor 1 beta (EF-1 beta) from Artemia is phosphorylated to a high percentage at serine 89 by an endogenous kinase present in EF-1 beta gamma. Protein sequencing of EF-1 beta revealed that this serine residue is located N-terminally of an acidic cluster of amino acids, (formula; see text) which is critical for casein kinase II-type substrate recognition. A number of compounds known to influence casein kinases were studied, revealing that the kinase activity as present in EF-1 beta gamma belongs to the class of casein kinase II. The rate of nucleotide exchange on EF-1 alpha as catalyzed by EF-1 beta was found to be affected reversibly by the state of phosphorylation of EF-1 beta. In the presence of dephosphorylated EF-1 beta, the exchange rate is almost twice as large compared to the rate in the presence of phosphorylated EF-1 beta. Rephosphorylation of dephosphorylated EF-1 beta diminishes the activity of the protein again. The role of casein kinase II-type enzymes in modulating the function of proteins involved in polypeptide synthesis is discussed.     

Inhibition of protein synthesis by didemnin B: how EF-1alpha mediates inhibition of translocation

The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1alpha; however, the dependence upon EF-1alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1alpha/GTP in solution, but binds to ribosome. EF-1alpha complexes with a dissociation constant K(d) = 4 microM. Thus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome.EF-1alpha complexes, thus preventing ribosomal translocation. Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.     

On the nature of cellular ADP-ribosyltransferase from rat liver specific for elongation factor 2

A cellular ADP-ribosyltransferase, specific for elongation factor 2 (EF-2), is found in extracts from rat liver. Co-migrating with EF-2 throughout purification, this activity is, moreover, located in the protein bands corresponding to EF-2 after native or sodium dodecyl sulfate polyacrylamide gel electrophoresis. The observed activity is thus implicated to be an inherent property of EF-2. Preincubation of EF-2 with GuoPPCH2Pox inhibits endogenous, but not diphtheria toxin catalyzed ADP-ribosylation.     

Dissimilarity in protein chain elongation factor requirements between yeast and rat liver ribosomes.

Factor requirements for yeast and rat liver ribosomes were determined in several different reactions using either yeast or liver factors. In polymerization assays yeast ribosomes required a factor in addition to elongation factor 1 (EF-1) and elongation factor 2 (EP-2). The third factor (EF-3) requirement was observed with EFs from either yeast or liver for both poly(U)-directed polyphenylalanine synthesis and elongation of endogenous peptidyl-tRNA. No significant effect of EF-3 was observed with liver risomes in either assay. In contrast to results with polypeptide synthesis EF-3 was not required for EF-1 dependent binding of [3H]Phe-tRNA or the translocation-dependent formation of N-acetylphenylalanylpuromycin. Up to 2-fold stimulation of the binding reaction was observed with saturating levels of either yeast or liver EF-1. No effect of EF-3 was observed on ribosome-EF-2-GDP-fusidic acid complex formation. The data suggest that the yeast EF-3 may be a loosely bound ribosomal protein which is not required for a specific step in the elongation cycle but is involved in the coordination of the partial reactions required for polymerization.     

Localization of the sites of ADP-ribosylation and GTP binding in the eukaryotic elongation factor EF-2

Tryptic cleavage of EF-2, molecular mass 93 kDa, produced an 82-kDa polypeptide and a 10-kDa fragment, which was further degraded. By a slower reaction the 82-kDa polypeptide was gradually split into a 48-kDa and a 34-kDa fragment. Similarly, treatment with chymotrypsin resulted in the formation of an 82-kDa polypeptide and a small fragment. In contrast to the tryptic 82-kDa polypeptide the corresponding chymotryptic cleavage product was relatively resistant to further attack. The degradation of the 82-kDa polypeptide with either trypsin or chymotrypsin was facilitated by the presence of guanosine nucleotides, indicating a conformational shift in native EF-2 upon nucleotide binding. No effect was observed in the presence of ATP, indicating that the effect was specific for guanosine nucleotides. After affinity labelling of native EF-2 with oxidized [3H]GTP and subsequent trypsin treatment the radioactivity was recovered in the 48-kDa polypeptide showing that the GTP-binding site was located within this part of the factor. Correspondingly, tryptic degradation of EF-2 labelled with [14C]NAD+ in the presence of diphtheria toxin showed that the site of ADP-ribosylation was within the 34-kDa polypeptide. By cleavage with the tryptophan-specific reagent N-chlorosuccinimide the site of ADP-ribosylation could be located at a distance of 40-60 kDa from the GTP-binding site and about 4-11 kDa from the nearest terminus.     

Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2

The major substrate for Ca2+/calmodulin-dependent protein kinase III in mammalian cells is a species of Mr 100,000 that has a primarily cytoplasmic localization. This substrate has now been identified as elongation factor-2 (EF-2), a protein that catalyzes the translocation of peptidyl-tRNA on the ribosome. The amino acid sequence of 18 residues from the N-terminal of the Mr 100,000 CaM-dependent protein kinase III substrate purified from rat pancreas was found to be identical to the N-terminal sequence of authentic rat EF-2 as previously deduced from nucleic acid sequencing of a cDNA (Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui, T., Ohtsuka, E., Ikehara, M., and Okada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4978-4982). CaM-dependent protein kinase III phosphorylated EF-2 in vitro with a stoichiometry of approximately 1 mol/mol on a threonine residue. Amino acid sequencing of the purified tryptic phosphopeptide revealed that this threonine residue lies within the sequence: Ala-Gly-Glu-Thr-Arg-Phe-Thr-Asp-Thr-Arg (residues 51-60 of EF-2). The Mr 100,000 protein was stoichiometrically ADP-ribosylated in vitro by the addition of diphtheria toxin and NAD. The Mr 100,000 protein was photoaffinity labeled with a GTP analog and the protein had an endogenous GTPase activity that could be stimulated by the addition of salt-washed ribosomes. These properties are all characteristic of EF-2. Dephospho-EF-2 could support poly(U)-directed polyphenylalanine synthesis in a reconstituted elongation system when combined with EF-1. In the same system, phospho-EF-2 was virtually inactive in supporting polypeptide synthesis; this effect could be reversed by dephosphorylation of phospho-EF-2. These results suggest that intracellular Ca2+ inhibits protein synthesis in mammalian cells via CaM-dependent protein kinase III-catalyzed phosphorylation of EF-2.     

Isolation and properties of elongation factor 1 from Saccharomyces cerevisiae

Polypeptide elongation factor 1 was isolated from yeast postribosomal supernatant. The highly purified factor was resolved on Ultrogel AcA-44 into two complementary fractions. One of these fractions contained two different polypeptide chains corresponding to a Ts-like elongation factor EF-1 beta gamma. The other fraction represented the light form of the factor, designated EF-1 alpha, with a molecular weight of approximately 50,000. The obtained results indicate that EF-1 from lower eukaryotes is also composed of three distinct polypeptides.     

The mitotic apparatus-associated 51-kDa protein from sea urchin eggs is a GTP-binding protein and is immunologically related to yeast polypeptide elongation factor 1 alpha

We investigated the biochemical characteristics of the 51-kDa protein that is a major mitotic apparatus-associated basic protein of sea urchin eggs (Toriyama, M., Ohta, K., Endo, S., and Sakai, H. (1988) Cell Motil. Cytoskeleton 9, 117-128). The amino acid composition of the 51-kDa protein was apparently different from those of tubulin, actin, histones, and myelin basic protein; yet it was similar to those of polypeptide elongation factors 1 alpha (EF-1 alpha). In addition, antibody to EF-1 alpha from yeast cross-reacted with the 51-kDa protein. [3H] GTP binding activity was detected in the phosphocellulose-purified fraction (PC fraction) which predominantly contained the 51-kDa protein and was shown to be specific to GTP, GDP, guanylyl imidodiphosphate, and ITP. Photo-affinity labeling using [alpha-32P]8-azidoguanosine triphosphate (8-azido-GTP) demonstrated that a 51-kDa polypeptide in the PC fraction specifically bound 8-azido-GTP. This GTP-binding polypeptide was bound to a GTP affinity column, could be eluted by the addition of GTP, and was immunoreactive with anti-51-kDa protein antibodies. When the PC fraction was applied to a gel filtration chromatography column, GTP binding activity was completely coeluted with the 51-kDa protein. Furthermore, the PC fraction and the gel filtration-purified fraction had EF-1 alpha activity: [14C]Phe-tRNA transferring activity to ribosomes in the presence of poly(U) and ribosome-dependent GTPase activity. The results indicate that the mitotic apparatus-associated 51-kDa protein is a GTP-binding protein and suggest that it is structurally and functionally related to yeast EF-1 alpha.     

A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha.

The microtubules (MTs) of higher plant cells are organized into arrays with essential functions in plant cell growth and differentiation; however, molecular mechanisms underlying the organization and regulation of these arrays remain largely unknown. We have approached this problem using tubulin affinity chromatography to isolate carrot proteins that interact with MTs. From these proteins, a 50-kD polypeptide was selectively purified by exploiting its Ca(2+)-dependent binding to calmodulin (CaM). This polypeptide was identified as a homolog of elongation factor-1 alpha (EF-1 alpha)--a highly conserved and ubiquitous protein translation factor. The carrot EF-1 alpha homolog bundles MTs in vitro, and moreover, this bundling is modulated by the addition of Ca2+ and CaM together (Ca2+/CaM). A direct binding between the EF-1 alpha homolog and MTs was demonstrated, providing novel evidence for such an interaction. Based on these findings, and others discussed herein, we propose that an EF-1 alpha homolog mediates the lateral association of MTs in plant cells by a Ca2+/CaM-sensitive mechanism.     

Inhibition of polypeptide synthesis by tRNA fractions in rat liver cell-free systems.

The mechanism of inhibition of polypeptide synthesis by the addition of a tRNA fraction in a rat liver cell-free system was studied. The inhibition was found to occur at the step of aminoacyl-tRNA binding to ribosomes, in which aminoacyl-tRNA's were mainly responsible for the inhibition. The addition of EF-1 decreased the inhibition by the tRNA fraction. The tRNA fraction inhibited polypeptide synthesis in a polysome-S100 system under conditions in which poly U- and poly A-dependent polypeptide syntheses were not inhibited. The possibility that the aminoacyl-tRNA inhibitory activity functions through improper binding to the ribosomes in the polysome-S100 system is discussed.     

Transport of messenger RNA into different classes of membrane-associated polyribosomes in Ehrlich-ascites-tumor cells.

The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.     

Elongation factor 2 as the major substrate for Ca2+/calmodulin-dependent protein kinase in rat adrenal glomerulosa cells

We have previously shown the existence of the major substrate protein of Mr 100,000 (substrate 100 K protein) for Ca2+/calmodulin (CaM)-dependent protein kinase in rat adrenal glomerulosa cells. In the present study, the identity of the substrate 100 K protein to elongation factor 2 (EF-2) was investigated. In a 105,000 g-supernatant fraction (cytosol), the protein of Mr 100,000 with the pI (isoelectric point) value of 6.7 was phosphorylated in the presence of calcium and CaM. The optical densities of this phosphorylated band were greatly enhanced in the presence of the EF-2 purified from pig liver (1 microgram) [20-23-fold, n = 5] when compared with those in the absence of the component. In the presence of the purified EF-2, the phosphorylation of Mr 100,000 was detected only in the presence of calcium alone or calcium plus CaM. This phosphorylation in the presence of calcium alone was completely inhibited in the presence of the CaM antagonist pimozide (500 microM), showing the existence of endogenous CaM in the cytosol. In the same fraction, the ADP-ribosylated protein of Mr 100,000 was detected in the presence of diphtheria toxin (fragment A) and (adenylate-32P) NAD, indicating the presence of EF-2 in the cytosol from rat adrenal glomerulosa cells. These results suggest that the substrate 100 K protein may be identical to EF-2 in rat adrenal glomerulosa cells.     

Kinetics of the in vivo synthesis of the polypeptide chain; the effect of translation inhibitors]

Effects of aurinetricarbonic acid and cycloheximide on kinetics of polypeptide chain synthesis and polyribosome protile in mouse liver cell in vivo are studied. Both compounds are found to decrease the absolute rate of protein synthesis and to increase the time of polypeptide synthesis. Cycloheximide changed the ratio of translating and non-translating ribosomes and different in size polyribosome types. Aurinetricarbonic acid exerts no effect on polyribosome profile. Effects of cycloheximide and aurinetricarbonic acid on kinetic parameters of translation and a mechanism of action of these compounds are studied.     

The mechanics of translocation: a molecular "spring-and-ratchet" system

The translation of genetic information into proteins is a fundamental process of life. Stepwise addition of amino acids to the growing polypeptide chain requires the coordinated movement of mRNA and tRNAs through the ribosome, a process known as translocation. Here, we review current understanding of the kinetics and mechanics of translocation, with particular emphasis on the structure of a functional mammalian ribosome stalled during translocation by an mRNA pseudoknot. In the context of a pseudoknot-stalled complex, the translocase EF-2 is seen to compress a hybrid-state tRNA into a strained conformation. We propose that this strain energy helps overcome the kinetic barrier to translocation and drives tRNA into the P-site, with EF-2 biasing this relaxation in one direction. The tRNA can thus be considered a molecular spring and EF-2 a Brownian ratchet in a "spring-and-ratchet" system within the translocation process.     

Purification and properties of the heterogeneous subunits of elongation factor EF-1 from Guerin epithelioma cells.

Elongation factor EF-1 from Guerin epithelioma was separated into two subunit forms EF-1A and EF-1B by chromatography in the presence of 25% glycerol, successively on CM-Sephadex and DEAE-Sephadex. It was shown that EF-1A is a thermolabile, single polypeptide which catalyses the binding of aminoacyl-tRNA to ribosomes, similarly as eukaryotic EF-1 alpha or prokaryotic EF-Tu. EF-1B was characterized as a complex composed of at least two polypeptides. One of them is EF-1A, the other EF-1C, which stimulates EF-1A activity and protects this elongation factor from thermal inactivation.     

Studies on the disaggregation of Elongation factor 1 by elastase

Incubation of Elongation factor (EF-1) with the protease elastase results in disaggregation of EF-1 with no loss of activity. Sodium dodecyl sulfate disc gel analysis shows that EF-1 is cleaved by elastase with the formation of two major polypeptides of 30,000 and 15,000 mol. wt. Evidence is also presented that under certain conditions, the polypeptide products formed after elastase treatment of EF-1 can rejoin in the presence of phenylmethanesulfonyl-fluoride. Phospholipase C preparations also cause disaggregation of EF-1 but the present results indicate that this is not due to a protease contamination in the phospholipase C.     

Effect of tumor promoting phorbol ester TPA on epidermal protein synthesis: stimulation of an elongation factor 2 phosphatase activity by TPA in vivo

Topical application of the phorbol ester TPA to mouse skin causes an increase in the amount of elongation factor 2 (EF-2), a factor in eukaryotic protein synthesis, in the epidermal cytosol (2- to 3-fold) and particulate fraction (7-fold). Furthermore, as a consequence of this TPA treatment the activity of an epidermal EF-2 phosphatase is stimulated. The EF-2 phosphatase has an apparent molecular weight of around 38,000 daltons. The enzyme activity is induced as early as 45 minutes after TPA treatment and remains at the elevated level for more than 17 hours. Both of the TPA-induced effects result in an increase in unphosphorylated, i.e. active EF-2 and can be suppressed by cyclosporine A.     

Cytokinin stimulates polyribosome loading of nuclear-encoded mRNAs for the plastid ATP synthase in etioplasts of Lupinus luteus: the complex accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space

Three of the nine subunits of the plastid ATP synthase, including the subunit of the CF(1) moiety (gene AtpC), are encoded in the nucleus. Application of cytokinin to etiolated lupine seedlings induces polyribosome association of their mRNAs. This appears to be specific as no such regulation was observed for messages for three ribosomal proteins. Cytokinin-mediated polyribosome loading was also observed for the spinach AtpC message in etiolated transgenic tobacco seedlings. Analysis of various spinach AtpC mRNA derivatives uncovered that the 5' untranslated region (5' UTR) of this message is sufficient to direct polyribosome loading, and that sequences at the 3' end of the AtpC 5' UTR, including an UC-rich motif, are crucial for this regulation. The increase in polyribosome loading of the AtpC message correlated with an increased synthesis of the polypeptide. The subunit, together with the ATP synthase complex, accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space of the etioplast. These results suggest that cytokinin promotes accumulation of the ATP synthase in the inner-envelope membrane of lupine etioplasts by stimulating the translation efficiency of their nuclear-encoded messages.