The characteristics of eukaryotic ribosomal proteins PO, P1, and P2 (P proteins) and their antigenic determinants were studied using the sera of patients with systemic lupus erythematosus (SLE). PO, P1, and P2 were isolated as a macromolecular complex by preparative isoelectric focusing and anion-exchange chromatography in the-presence of 6 M urea. The apparent molecular size of the complex was 140 kDa as determined by gel fitration on a Sephadex G-200 column. PO may, therefore, be the eukaryotic equivalent ofEscherichia coli ribosomal protein L10. In addition, all three P proteins were detected in the postribosomal supernatant of HeLa cells, and PO and P1 were found to be more acidic than their ribosome-bound counterparts. Partial proteolysis experiments revealed that SLE anti-P sera recognized one or both ends of the P2 equivalent protein from Artemia saUna (eL12). Sixteen SLE sera containing antibodies to PO, P1, and P2 reacted with a carboxyl-terminal peptide 22 amino acids in length of eL12 and not with an amino-terminal peptide of 20 anmno acids. Even though the carboxyl-terminal peptide completely inhibited the ability of the antiserum to react with all three proteins on an immunological blot, the same peptide produced only small decreases in binding of the SLE antibody to the native, nondenatured P proteins. These findings indicate that SLE anti-P antibodies react with a single sequential (linear) antigenic determinant on all three P proteins, but that additional antibodies recognize a conformational determinant(s).Systemic lupus erythematosus (SLE) is an autoimmune disease generally characterized by serum antibodies directed against nuclear proteins and nucleic acids (1). That some patients' sera contain antibodies against ribosomal constituents (2, 3) has long been known, but the identity of these ribosomal antigens has only recently been determined (4). SLE anti-ribosome antibodies show almost exclusive reactivity against three 60S ribosomal subunit phosphoproteins called PO, P1, and P2 (4, 5). These same three proteins are also recognized by a mouse monoclonal antibody raised against chicken ribosomes (6). These "P" proteins have molecular sizes of -38, 19, and 17 kDa, respectively. P1 and P2 are believed to be the eukaryotic equivalent of the Escherichia coli ribosomal protein L12 and have been shown to contain sequences that are highly conserved among eukaryotes (6). Thus, P2 (7) from rat liver shows a high degree of amino acid sequence homology with Artemia salina ribosomal protein eL12 (8) and yeast ribosomal protein YPA1 (9). P1 and P2 also appear to be the functional counterparts of Artemia ribosomal proteins eL12' and eL12 and yeast ribosomal proteins YPA1/YPA2 (10-12). In order to further evaluate the remarkable specificity of the SLE anti-PO, -P1, and -P2 (anti-P) antibodies, we have mapped the antigenic determinant on the P proteins. This determinant is present on all three proteins and is contained within a common sequence of 22 amino acids at the carboxyl terminus of the Artemia r...

In the three-dimensional model of adenylate kinase, the phosphate-binding site for AMP and ATP has been identified [Pai, E.F. et al. (1977) J. Mol. Biol. 114, 37451. In this region one can distinguish a sequence glycine XXXX glycinelysine. The same sequence is found in many other mononucleotide-binding proteins including elongation factors and oncogenic P2 I proteins. Dinucleotide-binding proteins display a pyrophosphate-binding unit with a glycine pattern different from that of mononucleotide-binding proteins. It has been found that P2l ras protein possesses a strand motif typical for (pyro)phosphate binding of a mononucleotide. A single mutation at position 12 can confer oncogenic activity on the protein. Based on the assumption that amino acid residues which are critical for function are preferentially conserved, we predict from the sequence that glycine residue I5 rather than residue I2 is important for (pyro)phosphate binding.

The acidic L7/L12 (prokaryotes) and P1/P2 (eukaryotes) proteins are the only ribosomal components that occur in more than one, specifically four, copies in the translational machinery. These ribosomal proteins are the only ones that do not directly interact with ribosomal RNA but bind to the particles via a protein, L10 and P0, respectively. They constitute a morphologically distinct feature on the large subunit, the stalk protuberance. Since a long time proteins L7/L12 have been implicated in translation factor binding and in the stimulation of the factor-dependent GTP-hydrolysis. Recent studies reproduced such activities with the isolated components and L7/L12 can therefore in retrospect be regarded as the first GTPase activating proteins identified. GTP-hydrolysis induces a drastic conformational change in elongation factor (EF) Tu, which enables it to dissociate from the ribosome after having successfully delivered aminoacylated tRNA into the A-site. It is also used as a driving force for translocation, mediated by EF-G. The in vitro stimulation of translation-uncoupled EF-G-dependent GTP-hydrolysis seems to be an intrinsic property of the ribosome that is dependent on L7/L12, reaches a maximum with four copies of the proteins per particle, and reflects the in vivo hydrolysis rate during translation. It is much larger than the analogous activity observed for EF-Tu, which is correlated with the in vitro polypeptide synthesis rate. Therefore, at least certain stimulatory activities of L7/L12 are controlled by the ribosomal environment, which in the case of EF-Tu senses the successful codon-anticodon pairing. Present knowledge is consistent with a picture in which proteins L7/L12 constitute a "landing platform" for the factors and after rearrangements induce GTP-hydrolysis. The molecular mechanism of the GTPase activation is unknown. While sequence comparisons show a large diversity in the stalk proteins across the kingdoms, a conserved functional domain organization and conserved designs of their genetic units are discernible. Consistently, stalk transplantation experiments suggest that coevolution took place to maintain functional L7/L12 EF-G and P-protein EF-2 couples. The acidic proteins are organized into three distinct functional parts: An N-terminal domain is responsible for oligomerization and ribosome association, a C-terminal domain is implicated in translation factor interactions, and a hinge region allows a flexible relative orientation of the latter two portions. The bacterial L7/L12 proteins have long been portrayed as highly elongated dimers displaying globular C-terminal domains, helical N-termini, and unstructured hinges. Conversely, recent crystal structures depict a compact hetero-tetrameric assembly with the hinge region adopting either an alpha-helical or an open conformation. Two different dimerization modes can be discerned in these structures. Models suggest that dimerization via one association mode can lead to elongated dimeric complexes with one helical and one unstructured hinge. The ...

The primary structures of the ribosomal proteins A, (= L,) and A, (= L,,) have been elucidated. Comparison of the two amino acid sequences confirms earlier studies by us (1972) which indicated that the two proteins are identical except that A, possesses an N-terminal acetyl group.Sequencing of tryptic and chymotryptic peptides was accomplished primarily by automatic solid-phase Edman degradation of 50-to 70-nanomole peptide samples. A large tryptic peptide T,Phe, which could not be sequenced by this method, was fragmented with elastase and its sequence mainly derived by conventional methods. The sequence of the first 51 residues of the nonacetylated A,-protein was obtained by Edman degradation in the Beckman sequencer.A-protein comprises three distinct regions : I (residues 1 -55), which is negatively-charged and hydrophobic, I1 (56-81), which is positively charged, and I11 (82-120), which is negatively charged and hydrophilic. &-Helix promoting residues are located primarily in regions I and 111. e-N-Monomethyllysine, which occurs in 50°/, of the A, and A, chains is located in the more flexible region I1 at position 81.The distinctive clustering of hydrophobic and charged residues is quite remarkable and suggests that in situ certain of these regions may contain a binding site for components involved in peptide chain elongation.

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