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

Abstract

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:

Binding of Mammalian Ribosomal Protein Complex P0·P1·P2 and Protein L12 to the GTPase-associated Domain of 28 S Ribosomal RNA and Effect on the Accessibility to Anti-28 S RNA Autoantibody

“…We have presented an in vitro binding system for studying interaction between rat ribosomal proteins and the GTPase domain of 28 S rRNA (Uchiumi and Kominami, 1992). Rat proteins P1, P2, and P0 are structurally and functionally related to E. coli proteins L12 and L10, respectively (reviewed by Wool et al, 1996), and the complex P0(P1) 2 (P2) 2 (P complex) 1 is believed to be a counterpart of E. coli L10(L12) 4 (Uchiumi et al, 1987). Furthermore, rat protein L12 is an equivalent of E. coli L11 (Suzuki et al, 1990).…”

Cross-linking of Selected Residues in the N- and C-terminal Domains of Escherichia coli Protein L7/L12 to Other Ribosomal Proteins and the Effect of Elongation Factor Tu

“…Additional evidence that a C-terminal domain can occupy a location not only extended across the body of the ribosome but also near the base of the stalk came from cross-linking between a predetermined location in the C-terminal domain, Cys-89, and Cys-70 of L10 (24,25) and also from hinge deletion studies (26,27). A different heterobifunctional reagent, APDP 1 showed a cross-link between Cys-89 and L11 and also with L10 in a lesser extent near the EF-G binding site near the base of the stalk (28). The site-specific cross-linking experiments led to the proposal of a possible "bent" conformation for one of the dimers in which the C-terminal domain could lie on the body of the subunit near the N-terminal domain at the base of the stalk.…”

“…The Escherichia coli ribosomal protein, L7/L12, is the most extensively investigated representative of the small, four-copy, dimeric acidic proteins that are found in large ribosomal subunits of all organisms and exist as a conserved quaternary structural element in which two dimers are integrated into the ribosome through binding to a common anchoring protein (1)(2)(3). One or both of the L7/L12 dimers form a conspicuous morphological feature on the ribosome known in E. coli as the L7/L12 stalk (4).…”

Caspase-dependent cleavage of nucleic acids

“…45 The P proteins are associated with the 60S ribosomal subunit as a pentameric complex (one copy of P0, and two copies of P1 and P2 each) via direct binding to the 28S rRNA molecule. 46 RNase protection studies indicated that the pentameric P protein complex protects nucleotides 1838 ± 1936 of the 28S rRNA molecule suggesting that this part of the RNA contains the protein binding site. 47 In addition to the anti-P antibodies, sera of SLE patients may also contain antibodies directed to the 28S rRNA molecule itself.…”