Structure and phosphorylation of an acidic protein from 60S ribosomes and its involvement in elongation factor-2 dependent GTP hydrolysis

Agthoven, A.J. van; Maassen, J. Antonie; Möller, W.

doi:10.1016/s0006-291x(77)80075-2

Cited by 67 publications

(33 citation statements)

References 16 publications

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“…Amino acid sequence analysis of acidic proteins from ribosomes of archaebacterial and eukaryotic organisms indicates structural similarity with the E. coli acidic proteins L7/L12 (12,34,35). Preliminary experiments suggest their presence in multiple copies and functional assays have pointed to their involvement in GTP-related processes (13)(14)(15). Our experiments were directed toward determining whether or not eukaryotic acidic ribosomal proteins are, like L7/L12, present in multiple copies, and the other ribosomal proteins to which they are proximal in the large ribosomal subunit.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by crosslinking.

Uchiumi¹,

Wahba²,

Traut³

1987

Proc. Natl. Acad. Sci. U.S.A.

127

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The 60S subunits isolated fromArtemia salina ribosomes were treated with the crosslinking reagent 2-iminothiolane under mild conditions. Proteins were extracted and fractions containing crosslinked acidic proteins were obtained by stepwise elution from CM-cellulose. Each fraction was analyzed by "diagonal" (two-dimensional nonreducing-reducing) NaDodSO4/polyacrylamide gel electrophoresis. Crosslinked proteins below the diagonal were radioiodinated and identified by two-dimensional acidic urea-NaDodSO4 gel electrophoresis. Each of the acidic proteins P1 and P2 was crosslinked individually to the same third protein, P0. The fractions containing acidic proteins were also analyzed by two-dimensional nonequilibrium isoelectric focusing-NaDod-S04/polyacrylamide gel electrophoresis. Two crosslinked complexes were observed that coincide in isoelectric positions with monomeric P1 and P2, respectively. Both P1 and P2 appear to form crosslinked homodimers. These results suggest the presence in the 60S subunit of (P1)2 and (P2)2 dimers, each of which is anchored to P0. Protein PO appears to play the same role as L10 in Escherichia coli ribosomes and may form a pentameric complex with the two dimers in the 60S subunits.The large ribosomal subunits of Escherichia coli contain multiple copies of a protein, L7/L12, that is present as two dimers, each of which is anchored to the ribosome through interaction with a common protein, L10 (1-4). The four copies of L7/L12 are easily and selectively removed from the ribosome and can be reconstituted (5). The proteins are involved in the GTP-dependent reactions of protein synthesis-in particular, translocation (6-8). A model has been proposed recently, showing different orientation for the two L7/L12 dimers on the ribosome surface (9).The large subunits of eukaryotic ribosomes also contain multicopy acidic proteins (10, 11) that are structurally and functionally related to L7/L12 (12-15). These proteins exist in phosphorylated states and have been designated P proteins (16-18). Two different P proteins having slightly different amino acid compositions, electrophoretic mobilities, and molecular weights have been designated P1 and P2 (16). Anti-ribosome antibodies from certain patients with systemic lupus erythematosus react exclusively with P1 and P2 and, in addition, with a third acidic protein designated PO (19,20). These three proteins also react with a mouse monoclonal antibody raised against chicken ribosomes (17).The spatial arrangement of the P proteins in the eukaryotic ribosome is not yet well established. We have investigated the topography of P1 and P2 [designated eL12' and eL12 in Artemia ribosomes (10) Elution of the proteins from CM-cellulose at pH 5.0 (6 M urea/10 mM sodium acetate/0.1% methylamine/3 mM iodoacetamide, adjusted to pH 5.0 with acetic acid) instead of pH 7.2 was also used for some experiments.Abbreviations: P proteins, eukaryotic ribosomal proteins PO, P1, and P2; NEPHGE, nonequilibrium pH gradient electrophoresis.tPresent address:

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Section: Discussionmentioning

confidence: 99%

“…The large subunits of eukaryotic ribosomes also contain multicopy acidic proteins (10,11) that are structurally and functionally related to L7/L12 (12)(13)(14)(15). These proteins exist in phosphorylated states and have been designated P proteins (16)(17)(18).…”

mentioning

confidence: 99%

Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by crosslinking.

Uchiumi¹,

Wahba²,

Traut³

1987

Proc. Natl. Acad. Sci. U.S.A.

127

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“…The involvement of P1/P2 proteins in translation elongation has been suggested previously by immunochemical inhibition assays (40) and partial reconstitution studies with rat and yeast ribosomes (23,41). An essential role of P0 for cell viability has been demonstrated in yeast (23).…”

Section: Figmentioning

confidence: 65%

Translation Elongation by a Hybrid Ribosome in Which Proteins at the GTPase Center of the Escherichia coli Ribosome Are Replaced with Rat Counterparts

Uchiumi¹,

Honma²,

Nomura³

et al. 2002

Journal of Biological Chemistry

105

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Ribosomal L10⅐L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0⅐P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1␣ (eEF-1␣) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10⅐L7/L12 complex or L11 with rat protein showed that the P0⅐P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.The "GTPase center" of the ribosome is a region involved in interaction with GTP-bound translation factors, GTP hydrolysis (1), and post-GTPase events including tRNA movements on the ribosome (2, 3). Translation elongation is markedly stimulated by the interaction of this region with two elongation factors in a GTP-dependent manner. The GTPase center includes two essential RNA regions around positions 1070 and 2660 (Escherichia coli numbering) of the 23 S/28 S rRNA (4), which appear to bind to the elongation factors (5, 6). Despite the highly conserved structure of the 1070 and 2660 RNA regions, ribosomes show a kingdom-dependent accessibility for translation factors, i.e., prokaryotic ribosomes do not engage in translation elongation with the eukaryotic factors instead of the prokaryotic factors (7-9). Furthermore, there are differences in the rate of GTPase turnover between the two systems; in vitro eukaryotic eEF-2 1 /80 S ribosomedependent GTP hydrolysis is 10-fold slower than the prokaryotic EF-G/70 S ribosome system (10). This may reflect, in part, the elaborate regulation of eukaryotic translation.The other important component of the GTPase center is the acidic stalk protein, termed L7/L12 in prokaryotes (11-15). Four copies of this proteins bind to protein L10 and form a stable complex (16), designated here as L10⅐L7/L12. This protein complex and another protein, L11, are assembled on the 1070 RNA domain (17). Flexible property of L7/L12 protein in the ribosome (16, 18 -20) seems to be correlated with the fast turnover of EF-G-dependent GTPase. The eukaryotic counterparts of the prokaryotic L7/L12 and L10 are P1/P2 and P0, respectively (21,22)....

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“…It is more difficult to explain why no crossreaction was found between E. coli proteins L7 and L12 and A. salina proteins EL7 and EL12 (van Angthoven et al, 1977), although negative immunochemical results need not be regarded as conclusive. Perhaps differences between the amino acid sequences of the A. salina and the rodent proteins are a key factor.…”

Section: Resultsmentioning

confidence: 97%

The acidic ribosomal phosphoprotein of eukaryotes and its relationship to ribosomal proteins L7 and L12 of Escherichia coli

Leader¹,

Coia²

1978

Biochemical Journal

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The acidic ribosomal phosphoprotein, L,, of Krebs II ascites cells was further characterized and compared with proteins L7 and L12 of Escherichia coli. Ribosomal protein L, was selectively removed from 60S ribosomal subunits by 50% ethanol and 1 M-NH4CI, and antibodies raised against protein L, cross-reacted with E. coli protein L12 in immunodiffusion experiments. These and other, previously reported, data support the proposal that the eukaryotic counterpart of E. coli proteins L7 and L12 is phosphorylated.

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Structure and phosphorylation of an acidic protein from 60S ribosomes and its involvement in elongation factor-2 dependent GTP hydrolysis

Cited by 67 publications

References 16 publications

Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by crosslinking.

Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by crosslinking.

Translation Elongation by a Hybrid Ribosome in Which Proteins at the GTPase Center of the Escherichia coli Ribosome Are Replaced with Rat Counterparts

The acidic ribosomal phosphoprotein of eukaryotes and its relationship to ribosomal proteins L7 and L12 of Escherichia coli

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