Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

Uchiumi, Toshio; Kikuchi, M; Terao, Kazuo; Iwasaki, Kentaro; Ogata, Kazuhiro

doi:10.1111/j.1432-1033.1986.tb09545.x

Cited by 69 publications

(44 citation statements)

References 47 publications

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“…Moreover the ionic conditions are not identical in the two situations. It is clear that, in the cell, eEF-2 undergoes several other interactions with ribosomal proteins and rRNA [11,12]. The fact that we were unable to measure any interaction with eEF-2 when the P proteins were covalently linked to the sensor chip is possibly due to the fact that in the ribosome these proteins are flexible and at least partly mobile, as shown for their prokaryotic equivalents L7/L12 [13], whereas they were fixed on the sensor chip and could not move in our assay.…”

Section: Discussionmentioning

confidence: 99%

Interaction of elongation factor eEF‐2 with ribosomal P proteins

Bargis-Surgey

Lavergne

Gonzalo

et al. 1999

European Journal of Biochemistry

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The eukaryotic P1 and P2 ribosomal proteins which constitute, with P0, a pentamer forming the lateral stalk of the 60 S ribosomal subunit, exhibit several differences from their prokaryotic equivalents L7 and L12; in particular, P1 does not have the same primary structure as P2 and both of them are phosphorylated, the significance of the latter remaining unclear. Rat liver P1 and P2 were overproduced in Escherichia coli cells and their interaction with elongation factor eEF-2 was studied. Both recombinant proteins were found to be required for the ribosomedependent GTPase activity of eEF-2, with P2 in the phosphorylated form. The surface plasmon resonance technique revealed that, in vitro, both proteins interact specifically with eEF-2, with a higher affinity for P1 (K d = 3.8 Â 10 28 m) than for P2 (K d = 2.2 Â 10 26 m). Phosphorylation resulted in a moderate increase (two-to four-fold) in these affinities. The interaction of both P1 and P2 (phosphorylated or not) with eEF-2 resulted in a conformational change in the factor, revealed by an increase in the accessibility of Glu554 to proteinase Glu-C. This increase was observed in both the presence and absence of GTP and GDP, which themselves produced marked opposite effects on the conformation of eEF-2. Our results suggest that the two proteins P1 and P2 both interact with eEF-2 inducing a conformational transition of the factor, but have acquired some specific properties during evolution.Keywords: acidic ribosomal proteins; ribosomal; elongation factor 2; phosphorylation.P proteins are present in the 60 S ribosomal subunits of all eukaryotic cells. In mammalian cells, there are three different proteins called P0, P1 and P2. P1 and P2 are 12-kDa acidic proteins which exhibit some similarities to and some differences from the prokaryotic ribosomal proteins L7 and L12. Among the similarities are their localization on the large ribosomal subunit forming a lateral stalk, their molecular mass and physicochemical properties, and their requirement for translational factor activity. Thus, the GTPase activity of the eukaryotic elongation factor eEF-2 which catalyses the translocation of peptidyl-tRNA from the A to the P site of the ribosome is dependent on the presence of P1 and P2 on the large ribosomal subunit [1], just as the GTPase activity of the corresponding prokaryotic factor EF-G is dependent on the presence of L7/L12 on this subunit. A direct interaction between L7/L12 and EF-G has been recently visualized directly by cryoelectron microscopy [2]. The differences between P1/P2 and L7/L12 are numerous and interesting to study in terms of evolution. First, sequence homologies between eukaryotic and prokaryotic proteins are not obvious [3]. Secondly, P1 and P2 do not have the same primary structure [3], whereas L7 and L12 do, with the only exception being an N-acetyl group. Thirdly, in contrast with L7 and L12, P1 and P2 are phosphorylated on serine residues when present on the ribosome [4], and this phosphorylation seems to be important for their function, althoug...

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Section: Discussionmentioning

confidence: 99%

Interaction of elongation factor eEF‐2 with ribosomal P proteins

Bargis-Surgey

Lavergne

Gonzalo

et al. 1999

European Journal of Biochemistry

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“…Ribosomal Protein L10e Confers Sordarin Resistance-Identification of the large ribosomal subunit as the determinant of sordarin resistance, in addition to the biochemical and genetic associations of the stalk proteins with EF-G or eEF2 (11,13,(15)(16)(17)(18)(19), focused our efforts on the ribosomal stalk proteins. Because both fusidic acid and sordarin stabilize the eEF2-GDPribosome complex, we hypothesized that proteins in the stalk were candidate biochemical targets of sordarin.…”

Section: L10e Mutants and Eef2 Functionmentioning

confidence: 99%

“…The prokaryotic L7/L12 ribosomal proteins have been studied extensively by many physical and biological techniques, but much less is known about the structure and function of prokaryotic or eukaryotic L10. L10 is among the proteins reported to be cross-linked to eEF2 in 80S ribosomes by bifunctional reagents (13). In yeast, mutational analysis of the L10e ribosomal protein gene has shown that only L10e is essential, and that the carboxyl-terminal 132 amino acids of the L10e protein are required for viability.…”

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confidence: 99%

Mutations in Ribosomal Protein L10e Confer Resistance to the Fungal-specific Eukaryotic Elongation Factor 2 Inhibitor Sordarin

Justice

Hsu

et al. 1999

Journal of Biological Chemistry

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The natural product sordarin, a tetracyclic diterpene glycoside, selectively inhibits fungal protein synthesis by impairing the function of eukaryotic elongation factor 2 (eEF2). Sordarin and its derivatives bind to the eEF2-ribosome-nucleotide complex in sensitive fungi, stabilizing the post-translocational GDP form. We have previously described a class of Saccharomyces cerevisiae mutants that exhibit resistance to varying levels of sordarin and have identified amino acid substitutions in yeast eEF2 that confer sordarin resistance. We now report on a second class of sordarin-resistant mutants. Biochemical and molecular genetic analysis of these mutants demonstrates that sordarin resistance is dependent on the essential large ribosomal subunit protein L10e in S. cerevisiae. Five unique L10e alleles were characterized and sequenced, and several nucleotide changes that differ from the wild-type sequence were identified. Changes that result in the resistance phenotype map to 4 amino acid substitutions and 1 amino acid deletion clustered in a conserved 10-amino acid region of L10e. Like the previously identified eEF2 mutations, the mutant ribosomes show reduced sordarin-conferred stabilization of the eEF2-nucleotide-ribosome complex. To our knowledge, this report provides the first description of ribosomal protein mutations affecting translocation. These results and our previous observations with eEF2 suggest a functional linkage between L10e and eEF2.Eukaryotic elongation factor 2 (eEF2) 1 and its prokaryotic counterpart, elongation factor G (EF-G), promote the translocation of the ribosome along messenger RNA during the elongation phase of protein synthesis. Hydrolysis of GTP to GDP drives translocation and is associated with a presumed conformational change in eEF2. Sordarin (1) and its analogs are fungal-specific translation inhibitors (2, 3) that bind to the eEF2-ribosome-GDP complex in Saccharomyces cerevisiae, stabilizing the post-translocational GDP form in a manner similar to that of fusidic acid (3). However, in contrast to fusidic acid, which binds both EF-G and eEF2 and is a general translocation inhibitor, sordarin inhibits translation only in susceptible fungi, deriving its unique specificity from the source of eEF2 (3-5). The observation that eEF2 is the major determinant of sordarin specificity was confirmed by the identification of 15 unique sordarin-resistant alleles of EFT1 and EFT2 that encode eEF2 in S. cerevisiae. In our original characterization of 21 sordarin-resistant mutants, five mutations were not linked to the EFT1 or EFT2 genes. In this work, we show that these five mutations map to the essential ribosomal protein L10e.The ribosome, although not contributing significantly to the fungal specificity of sordarin, is a critical partner in forming the stabilized post-translocational complex (3). Detection of a complex between fungal eEF2 and a labeled sordarin analog is strongly dependent upon the presence of ribosomes. L10, the prokaryotic counterpart of S. cerevisiae L10e, has been localiz...

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“…Ears were hand pollinated and harvested at 10,15,20,25,30, and 40 days postpollination (DPP) and after complete desiccation to isolate embryos (including the scutellum and embryonic axis), aleurone (tissue included the aleurone layer and attached pericarp), and endosperm (refers to tissue within the pericarp and aleurone, excluding the embryo).…”

Section: Methodsmentioning

confidence: 99%

“…This suggests that the level of P-proteins in yeast ribosomes is affected by the metabolic state of the cell and possibly reflects the translational activity of the ribosome. The presence of these proteins in ribosomes has been shown to stimulate the eEF2-dependent GTPase activity of ribosomes (13)(14)(15)(16)(17), poly(U)-directed phenylalanine synthesis (14,18), and eEF1A binding (19). Hence, modulation of the 12-kDa P-protein component of ribosomes may impart eukaryotes with a means of ribosomeregulated translational control.…”

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confidence: 99%

Regulated Heterogeneity in 12-kDa P-protein Phosphorylation and Composition of Ribosomes in Maize (Zea mays L.)

Szick-Miranda

Bailey‐Serres

2001

Journal of Biological Chemistry

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Maize (Zea mays L.) possesses four distinct ϳ12-kDa P-proteins (P1, P2a, P2b, P3) that form the tip of a lateral stalk on the 60 S ribosomal subunit. RNA blot analyses suggested that the expression of these proteins was developmentally regulated. Western blot analysis of ribosomal proteins isolated from various organs, kernel tissues during seed development, and root tips deprived of oxygen (anoxia) revealed significant heterogeneity in the levels of these proteins. P1 and P3 were detected in ribosomes of all samples at similar levels relative to ribosomal protein S6, whereas P2a and P2b levels showed considerable developmental regulation. Both forms of P2 were present in ribosomes of some organs, whereas only one form was detected in other organs. Considerable tissue-specific variation was observed in levels of monomeric and multimeric forms of P2a. P2b was not detected in root tips, accumulated late in seed embryo and endosperm development, and was detected in soluble ribosomes but not in membrane-associated ribosomes that copurified with zein protein bodies of the kernel endosperm. The phosphorylation of the 12-kDa P-proteins was also developmentally and environmentally regulated. The potential role of P2 heterogeneity in P-protein composition in the regulation of translation is discussed.A complex of acidic ribosomal proteins (r-proteins) 1 forms a universally conserved lateral stalk on the large ribosomal subunit that facilitates the translocation phase of protein synthesis (1). In eukaryotes the structure is formed by a complex of acidic phosphoproteins. P0 (ϳ35 kDa), homologous to prokaryotic L10, interacts with 28 S rRNA to form the base of the stalk, and P1 and P2 (ϳ12 kDa), homologous to prokayotic L7/L12, are tethered as dimers to the stalk (2-5). P1 and P2 are structurally similar; each protein has three domains that include an ␣-helical N-terminal region and a central, flexible acidic hinge region followed by a highly conserved C terminus (E/KSD/ EDMGFG/SLD). The C-terminal region of P0 is structurally similar to 12-kDa P-proteins because it possesses the three domains of P1 and P2 (for review, see Ref. 6).The 12-kDa P-proteins are the only r-proteins found in multiple copies within the ribosome. They do not assemble onto preribosomes in the nucleolus but cycle between ribosomes and a cytosolic pool in numerous species including, Artemia salina, Saccharomyces cerevisiae (yeast), humans, and rats (7-11). demonstrated quantitatively that exponentially growing yeast cells contain more 12-kDa P-proteins/ribosome than cells in the stationary phase of growth. This suggests that the level of P-proteins in yeast ribosomes is affected by the metabolic state of the cell and possibly reflects the translational activity of the ribosome. The presence of these proteins in ribosomes has been shown to stimulate the eEF2-dependent GTPase activity of ribosomes (13-17), poly(U)-directed phenylalanine synthesis (14, 18), and eEF1A binding (19). Hence, modulation of the 12-kDa P-protein component of ribosomes may impar...

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Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

Cited by 69 publications

References 47 publications

Interaction of elongation factor eEF‐2 with ribosomal P proteins

Interaction of elongation factor eEF‐2 with ribosomal P proteins

Mutations in Ribosomal Protein L10e Confer Resistance to the Fungal-specific Eukaryotic Elongation Factor 2 Inhibitor Sordarin

Regulated Heterogeneity in 12-kDa P-protein Phosphorylation and Composition of Ribosomes in Maize (Zea mays L.)

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