Tag‐mediated fractionation of yeast ribosome populations proves the monomeric organization of the eukaryotic ribosomal stalk structure

Guarinos, Esther; Santos, Cruz; Sánchez, Arancha; Qiu, Deyi; Remacha, Miguel; Ballesta, Juan P. G.

doi:10.1046/j.1365-2958.2003.03733.x

Cited by 37 publications

(47 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The difference in mass between the sum of the four P1/P2 proteins and the pentameric stalk is consistent with the presence of P0. Although it is not possible from these experiments to rule out the possibility of different combinations of P1/P2 proteins coexisting with the same stalk complex, recent data are consistent with homogeneous populations of ribosomes carrying a single copy of each of the P proteins (27). Taken together these results therefore establish that a population of ribosomes with the pentameric stalk complex is present in solution and demonstrate that the interactions between these proteins can be maintained in the mass spectrometer, despite their reduced stability when compared with their prokaryotic counterparts.…”

Section: Resultsmentioning

confidence: 72%

Mass Spectrometry of Ribosomes from Saccharomyces cerevisiae

Hanson

Videler

Santos

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The acidic ribosomal P proteins form a distinct protuberance on the 60 S subunit of eukaryotic ribosomes. In yeast this structure is composed of two heterodimers (P1␣-P2␤ and P1␤-P2␣) attached to the ribosome via P0. Although for prokaryotic ribosomes the isolation of a pentameric stalk complex comprising the analogous proteins is well established, its observation has not been reported for eukaryotic ribosomes. We used mass spectrometry to examine the composition of the stalk proteins on ribosomes from Saccharomyces cerevisiae. The resulting mass spectra reveal a noncovalent complex of mass 77,291 ؎ 7 Da assigned to the pentameric stalk. Tandem mass spectrometry confirms this assignment and is consistent with the location of the P2 proteins on the periphery of the stalk complex, shielding the P1 proteins, which in turn interact with P0. No other oligomers are observed, confirming the specificity of the pentameric complex. At lower m/z values the spectra are dominated by individual proteins, largely from the stalk complex, giving rise to many overlapping peaks. To define the composition of the stalk proteins in detail we compared spectra of ribosomes from strains in which genes encoding either or both of the interacting stalk proteins P1␣ or P2␤ are deleted. This enables us to define novel post-translational modifications at very low levels, including a population of P2␣ molecules with both phosphorylation and trimethylation. The deletion mutants also reveal interactions within the heterodimers, specifically that the absence of P1␣ or P2␤ destabilizes binding of the partner protein on the ribosome. This implies that assembly of the stalk complex is not governed solely by interactions with P0 but is a cooperative process involving binding to partner proteins for additional stability on the ribosome.Ribosomes are the universal translators of messenger RNA into proteins. X-ray analysis of the 30 S subunit from Thermus thermophilus, the 50 S subunit from Haloarcula marismortui, and the intact 70 S particle from T. thermophilus have revealed fascinating insights into the structures of these macromolecular machines (1-3). There are currently, however, no high resolution structures of eukaryotic ribosomes. These 80 S particles consist of two subunits, the 60 and 40 S subunits. Low resolution structures obtained by electron microscopy indicate an overall topology similar to that of the Escherichia coli 70 S ribosome but indicate that the eukaryotic particle is larger and more complex. In general, the core of the ribosome is highly conserved, but in S. cerevisiae there are large differences in the periphery of the particle. This is attributed to the presence of additional ribosomal proteins and ribosomal RNA (rRNA) regions of variable size, interrupting the universal core secondary structure of eukaryotic rRNA (4). The eukaryotic 60 S subunit is larger than the prokaryotic 50 S subunit, but the greatest differences are observed between the 30 S subunit and the eukaryotic 40 S subunit .A common feature of all ribosomes is ...

show abstract

Section: Resultsmentioning

confidence: 72%

Mass Spectrometry of Ribosomes from Saccharomyces cerevisiae

Hanson

Videler

Santos

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…P2 is a member of a group of acidic phosphoproteins that form a universally conserved lateral stalk on the large ribosomal subunit (Mö ller, 1990). P0 (approximately 35 kD) interacts with the 23S-like rRNA to form the base of the stalk, which is extended by the binding of 2 molecules of P1 and P2 (approximately 12 kD; Tsurugi and Ogata, 1985;Uchiumi et al, 1987;Guarinos et al, 2003). Mammals possess one type of P1 and P2, whereas S. cerevisae possesses 2 types of P1 and P2 (Nakao et al, 2004).…”

Section: Products Of Evolutionarily Distinct Gene Family Members Are mentioning

confidence: 99%

“…The mouse, human, and Drosophila melanogaster genomes encode all 79 proteins, whereas some r-proteins are absent in the genomes of Baker's yeast (L28), and Caenorhabditis elegans and Schizosaccharomyces pombe (S27a; Nakao et al, 2004). Based on biochemical analyses of ribosomes of mammals and yeast, each r-protein is present in unimolar amounts with the exception of P1 and P2 of the 60S subunit, which can be either entirely absent or present as 2 or 4 monomers in yeast and 2 dimers in animals (Tsurugi and Ogata, 1985;Guarinos et al, 2003).…”

mentioning

confidence: 99%

Proteomic Characterization of Evolutionarily Conserved and Variable Proteins of Arabidopsis Cytosolic Ribosomes

et al. 2005

View full text Add to dashboard Cite

Analysis of 80S ribosomes of Arabidopsis (Arabidopsis thaliana) by use of high-speed centrifugation, sucrose gradient fractionation, one-and two-dimensional gel electrophoresis, liquid chromatography purification, and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization) identified 74 ribosomal proteins (r-proteins), of which 73 are orthologs of rat r-proteins and one is the plant-specific r-protein P3. Thirty small (40S) subunit and 44 large (60S) subunit r-proteins were confirmed. In addition, an ortholog of the mammalian receptor for activated protein kinase C, a tryptophan-aspartic acid-domain repeat protein, was found to be associated with the 40S subunit and polysomes. Based on the prediction that each r-protein is present in a single copy, the mass of the Arabidopsis 80S ribosome was estimated as 3.2 MD (1,159 kD 40S; 2,010 kD 60S), with the 4 single-copy rRNAs (18S, 26S, 5.8S, and 5S) contributing 53% of the mass. Despite strong evolutionary conservation in r-protein composition among eukaryotes, Arabidopsis 80S ribosomes are variable in composition due to distinctions in mass or charge of approximately 25% of the r-proteins. This is a consequence of amino acid sequence divergence within r-protein gene families and posttranslational modification of individual r-proteins (e.g. amino-terminal acetylation, phosphorylation). For example, distinct types of r-proteins S15a and P2 accumulate in ribosomes due to evolutionarily divergence of r-protein genes. Ribosome variation is also due to amino acid sequence divergence and differential phosphorylation of the carboxy terminus of r-protein S6. The role of ribosome heterogeneity in differential mRNA translation is discussed.The ribosome is a two-subunit ribonucleoprotein complex that catalyzes the peptidyl transferase reaction of polypeptide synthesis, an absolute requirement for cellular growth and differentiation. The structure and function of both prokaryotic and eukaryotic ribosomes have been investigated, with the eukaryotic emphasis on ribosomes of Baker's yeast (Saccharomyces cerevisiae) and rat (Rattus rattus and Rattus norvegicus). The cytosolic ribosomes of eukaryotes are composed of a large number of ribosomal proteins (r-proteins) and four distinct rRNAs, the 18S rRNA of the 40S subunit, and the 5S, 5.8S, and 23S-like (25-28S) rRNAs of the 60S subunit (Bielka, 1982). Early evaluation of the buoyant density and sedimentation coefficients of eukaryotic ribosomes predicted that the rat 80S ribosome has a higher mass (4.2-4.6 MD) than that of pea (Pisum sativum; 3.9 MD) due to distinctions in the 60S subunit (Cammarano et al., 1972). Further evidence that the plant ribosome is smaller than that of mammals was provided by the three-dimensional reconstruction of wheat (Triticum aestivum) and rabbit (Oryctolagus cuniculus) ribosomes by use of cryoelectron microscopy (Verschoor et al., 1996). Despite an overall similarity in architecture, the 60S subunit of wheat appeared approximately 20% smaller than that...

show abstract

“…In eukaryotes, the stalk occurs in a pentameric configuration P0-(P1/P2) 2 (14,15), where P0 constitutes the base of the stalk and anchors two P1/P2 dimers (16). The N-terminal domain of the P0 protein is responsible for attachment of the stalk to the large rRNA, and the P-domain anchors the P1/P2 proteins at two separate binding sites (17)(18)(19).…”

mentioning

confidence: 99%

Pentameric Organization of the Ribosomal Stalk Accelerates Recruitment of Ricin A Chain to the Ribosome for Depurination

Grela

Krokowski

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Ribosome inactivating proteins (RIPs) depurinate a universally conserved adenine in the ␣-sarcin/ricin loop (SRL) and inhibit protein synthesis at the translation elongation step. We previously showed that ribosomal stalk is required for depurination of the SRL by ricin toxin A chain (RTA). The interaction between RTA and ribosomes was characterized by a twostep binding model, where the stalk structure could be considered as an important interacting element. Here, using purified yeast ribosomal stalk complexes assembled in vivo, we show a direct interaction between RTA and the isolated stalk complex. Detailed kinetic analysis of these interactions in real time using surface plasmon resonance (SPR) indicated that there is only one type of interaction between RTA and the ribosomal stalk, which represents one of the two binding steps of the interaction with ribosomes. Interactions of RTA with the isolated stalk were relatively insensitive to salt, indicating that nonelectrostatic interactions were dominant. We compared the interaction of RTA with the full pentameric stalk complex containing two pairs of P1/P2 proteins with its interaction with the trimeric stalk complexes containing only one pair of P1/P2 and found that the rate of association of RTA with the pentamer was higher than with either trimer. These results demonstrate that the stalk is the main landing platform for RTA on the ribosome and that pentameric organization of the stalk accelerates recruitment of RTA to the ribosome for depurination. Our results suggest that multiple copies of the stalk proteins might also increase the scavenging ability of the ribosome for the translational GTPases.

show abstract

Tag‐mediated fractionation of yeast ribosome populations proves the monomeric organization of the eukaryotic ribosomal stalk structure

Abstract: SummaryThe analysis of the not well understood composition of the stalk, a key ribosomal structure, in eukaryotes having multiple 12 kDa P1/P2 acidic protein components has been approached using these proteins tagged with a histidine tail at the C-terminus. Tagged

Cited by 37 publications

References 37 publications

Mass Spectrometry of Ribosomes from Saccharomyces cerevisiae

Mass Spectrometry of Ribosomes from Saccharomyces cerevisiae

Proteomic Characterization of Evolutionarily Conserved and Variable Proteins of Arabidopsis Cytosolic Ribosomes

Pentameric Organization of the Ribosomal Stalk Accelerates Recruitment of Ricin A Chain to the Ribosome for Depurination

Contact Info

Product

Resources

About