Partial reassembly of active 60S ribosomal subunits from rat liver following treatment with dimethylmaleic anhydride

Conquet, François; Lavergne, Jean‐Pierre; Paleologue, Anne; Reboud, Jean‐Paul; Reboud, Anne-Marie

doi:10.1111/j.1432-1033.1987.tb10730.x

Cited by 30 publications

(11 citation statements)

References 33 publications

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“…Reconstitution of Active Particles-DMMA core particles were prepared from 60 S ribosomal subunits as described previously (9). The selective extraction of proteins P1-P2 from 60 S subunits by treatment with ethanol and 0.08 M KCl has been also described (11).…”

Section: Methodsmentioning

confidence: 99%

“…Reconstitution of Active 60 S Ribosomal Subunits Using Phosphorylated Recombinant P1-P2-Core particles were prepared from 60 S ribosomal subunits treated with DMMA as described previously (9). The absence of P1-P2 proteins from these particles was confirmed using a monoclonal antibody prepared against recombinant P1 and which reacted with P0, P1, and P2 in the 60 S subunits (Fig.…”

Section: Overexpression and Purification Of Ribosomal Proteins P1mentioning

confidence: 99%

“…We have shown previously that active rat liver 60 S ribosomal subunits could be reconstituted from inactive core particles prepared with 2,3-dimethylmaleic anhydride (DMMA) 1 (9), by adapting a method previously used with yeast ribosomes (10). Reactivation of the rat liver core particles was obtained not only with DMMA-split proteins containing several proteins including P1-P2 but also with a 50% ethanol, 0.08 M KCl extract containing P1-P2 exclusively.…”

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confidence: 99%

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A Specific Role for the Phosphorylation of Mammalian Acidic Ribosomal Protein P2

Vard

Guillot

Bargis

et al. 1997

Journal of Biological Chemistry

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The acidic ribosomal proteins P1-P2 from rat liver were overproduced for the first time by expression of their cDNA in Escherichia coli. They were tested for their ability to reactivate inactive P1-P2-deficient core particles derived from 60 S ribosomal subunits treated with dimethylmaleic anhydride, in poly(U)-directed poly(Phe) synthesis. The recombinant P1-P2 were unable to reactivate these core particles although they could bind to them. When recombinant P1-P2 had been phosphorylated first with casein kinase II, they were as efficient in the reactivation process as P1-P2 extracted with ethanol/KCl from the 60 S subunits. Reconstitution experiments were carried out using all possible combinations of the two recombinant proteins phosphorylated or not. Reactivation of the core particles required the presence of both P1 and P2 with the latter in its phosphorylated form. These experiments reveal a distinct role for P1 and P2 in protein synthesis. Phosphorylated P2 produced a partial quenching of the intrinsic fluorescence of eukaryotic elongation factor 2, which was not observed with the unphosphorylated protein. This result demonstrates the existence of an interaction between phosphorylated P2 and eukaryotic elongation factor 2. P2 also quenched part of the intrinsic fluorescence of P1, due to the interaction between the two proteins.The large subunit of eukaryotic ribosomes contains 12-kDa acidic P proteins, which seem more numerous in lower eukaryotes than in higher organisms (1). In these, two types of P proteins are found, designated as P1 and P2 (2, 3). They share similar properties with prokaryotic proteins L7-L12, which form a pentamer with protein L10, (L7/L12) 4 -L10, constituting the lateral stalk of the 50 S ribosomal subunits (4). The eukaryotic P proteins, as their prokaryotic counterparts, seem to play an essential role in the interaction with elongation factors and in factor-dependent GTPase activity (5). However, some specific properties are observed with eukaryotic P proteins. First, these proteins exist on the ribosome as phosphorylated derivatives. They can be phosphorylated in vitro by either casein kinase II or by an endogenous ribosome-bound enzyme (6). Second, proteins P1-P2 present on the ribosome can exchange with a cytoplasmic pool of these unphosphorylated proteins (7). Phosphorylation of P1-P2, which appears to be necessary for ribosome activity, was originally suggested to be a requirement for the binding of these proteins to ribosomes (5), but recently this hypothesis has been challenged by new results obtained in yeast (8).We have shown previously that active rat liver 60 S ribosomal subunits could be reconstituted from inactive core particles prepared with 2,3-dimethylmaleic anhydride (DMMA) 1 (9), by adapting a method previously used with yeast ribosomes (10). Reactivation of the rat liver core particles was obtained not only with DMMA-split proteins containing several proteins including P1-P2 but also with a 50% ethanol, 0.08 M KCl extract containing P1-P2 exclusively. Dephospho...

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Section: Methodsmentioning

confidence: 99%

Section: Overexpression and Purification Of Ribosomal Proteins P1mentioning

confidence: 99%

mentioning

confidence: 99%

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A Specific Role for the Phosphorylation of Mammalian Acidic Ribosomal Protein P2

Vard

Guillot

Bargis

et al. 1997

Journal of Biological Chemistry

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“…Assay of GTPase activity. This activity was measured by means of the classical procedure using [y-"P]GTP under the conditions previously described (Conquet et al, 1987).…”

Section: Methodsmentioning

confidence: 99%

Photoaffinity Labeling of Elongation Factor‐2 with 8‐Azido Derivatives of GTP and ATP

Guillot

Yard

Reboud

1996

European Journal of Biochemistry

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Elongation factor 2 (eEF‐2) can interact not only with guanylic nucleotides but also with adenylic ones, as was shown by intrinsic fluorescence quenching studies [Sontag, B., Reboud, A. M., Divita, G., Di Pietro, A., Guillot, D. & Reboud, J. P. (1993) Biochemistry 32, 1976–1980]. Here we studied sites of these interactions by using photoactivable 8‐azido‐[γ‐32P]GTP and 8‐azido‐[γ‐32P]ATP. Photoincorporation of the radioactive GTP derivative into eEF‐2 was prevented by the previous addition of GTP and GDP. The addition of adenylic nucleotides (ATP, ADP) and some adenylic derivatives [NAD+, NADH, poly(A)] decreased the photoincorporation by only 40% at most. However, photoincorporation of the radioactive ATP derivative was prevented by the previous addition not only of adenylic compounds [ATP, ADP, NAD+, NADH, poly(A)] but also of GTP and GDP. Photoincorporation of radioactive nucleotide derivatives was not decreased by the addition of other nucleotidic compounds [UTP, poly(U), ITP, NADP+, NADPH]. ATP and GTP acted as non‐competitive inhibitors of the photoincorporation of 8‐azido‐[γ‐32P]GTP and 8‐azido‐[γ‐32P]ATP respectively. eEF‐2 photolabeled with these radioactive nucleotide derivatives was submitted to trypsin digestion under different conditions and the labeled peptidic fragments identified after HPLC purification and gel electrophoresis by N‐terminal sequencing. An octa‐peptide, Y264FDPANGK271, was the only peptide photolabeled with 8‐azido‐[γ‐32P]GTP whereas a N‐terminal fragment of about 7 kDa was the only one photolabeled with 8‐azido‐(γ‐32P]ATP. The different results support the hypothesis that guanylic and adenylic nucleotides do not interact with the same site of eEF‐2.

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“…Kinetic analysis of the GTPase has shown that the hydrolysis is triggered by ribosomes in the post-translocational state, even in the absence of tRNA and mRNA [37]. The reaction can take place in the presence of 60 S subunits alone [37,219], showing that the ribosome domain responsible for activating the GTPase is within the 60 S subunit. However, due to the low affinity of the factor for 60 S subunits the K, of the reaction is considerably higher than for hydrolysis in the presence of complete post-translocational ribosomes.…”

Section: Interaction Of Eef-2 With Ribosomesmentioning

confidence: 99%

Translational dynamics

Nygård

Nilsson

1990

European Journal of Biochemistry

156

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Partial reassembly of active 60S ribosomal subunits from rat liver following treatment with dimethylmaleic anhydride

Cited by 30 publications

References 33 publications

A Specific Role for the Phosphorylation of Mammalian Acidic Ribosomal Protein P2

A Specific Role for the Phosphorylation of Mammalian Acidic Ribosomal Protein P2

Photoaffinity Labeling of Elongation Factor‐2 with 8‐Azido Derivatives of GTP and ATP

Translational dynamics

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