Phosphorylation of the Acidic Ribosomal P Proteins in <i>Saccharomyces cerevisiae</i>:  A Reappraisal

Zambrano, Reina; Briones, Elisa; Remacha, Miguel; Ballesta, Juan P. G.

doi:10.1021/bi971494o

Cited by 68 publications

(91 citation statements)

References 25 publications

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“…CK2 is a pleiotropic protein kinase that has crucial roles in cell differentiation, proliferation, and survival (for review, see Ahmed et al 2002). It is able to phosphorylate over 300 proteins; between them are proteins affecting protein synthesis and ribosomal proteins of the large subunit (Zambrano et al 1997;Meggio and Pinna 2003). This almost universal kinase CK2 has a quaternary structure composed of two catalytic (CKA1 and CKA2) and two regulatory subunits (CKB1 and CKB2).…”

Section: Discussionmentioning

confidence: 99%

Rrp15p, a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation

Marchis¹,

Giorgi²,

Schininà³

et al. 2005

RNA

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In eukaryotes ribosome biogenesis required that rRNAs primary transcripts are assembled in pre-ribosomal particles and processed. Protein factors and pre-ribosomal complexes involved in this complex pathway are not completely depicted. The essential ORF YPR143W encodes in yeast for an uncharacterized protein product, named here Rrp15p. Cellular function of Rrp15p has not so far defined even if nucleolar location was referred. With the aim to define the possible role of this orphan gene, we performed TAP-tagging of Rrp15p and investigated its molecular association with known pre-ribosomal complexes. Comparative sucrose gradient sedimentation analyses of yeast lysates expressing the TAP-tagged Rrp15p, strongly indicated that this protein is a component of the pre-60S particles. Northern hybridization, primer extension and functional proteomics on TAP-affinity isolated complexes proved that Rrp15p predominately associated with pre-rRNAs and proteins previously characterized as components of early pre-60S ribosomal particles. Finally, depletion of Rrp15p inhibited the accumulation of 27S and 7S pre-rRNAs and 5.8S and 25S mature rRNA. These results provide the first indication that Rrp15p is a novel factor involved in the early maturation steps of the 60S subunits. Moreover, the identification of the protein kinase CK2 in the Rrp15p-containing pre-ribosomal particles here reported, sustains the link between ribosome synthesis and cell cycle progression.

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Section: Discussionmentioning

confidence: 99%

Rrp15p, a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation

Marchis¹,

Giorgi²,

Schininà³

et al. 2005

RNA

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“…In addition to P-proteins P0, P1, and P2, plants possess an additional P1/P2 type protein termed P3 (56). The P-proteins were initially identified as phosphoproteins in the yeast ribosome (57), and their phosphorylation states likely play a direct role in protein synthesis and translational responses to external stimuli (26,58). Phosphorylation sites in the yeast P-proteins occur in the C-terminal region of the proteins (58), a feature that has now been confirmed for all members of the Arabidopsis family of ribosomal P-proteins.…”

Section: Conservation Of Covalent Modifications Between Eukaryoticmentioning

confidence: 99%

Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

Carroll¹,

Heazlewood²,

Ito³

et al. 2008

Molecular & Cellular Proteomics

185

250

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Finding gene-specific peptides by mass spectrometry analysis to pinpoint gene loci responsible for particular protein products is a major challenge in proteomics especially in highly conserved gene families in higher eukaryotes. We used a combination of in silico approaches coupled to mass spectrometry analysis to advance the proteomics insight into Arabidopsis cytosolic ribosomal composition and its post-translational modifications. In silico digestion of all 409 ribosomal protein sequences in Arabidopsis defined the proportion of theoretical genespecific peptides for each gene family and highlighted the need for low m/z cutoffs of MS ion selection for MS/MS to characterize low molecular weight, highly basic ribosomal proteins. We undertook an extensive MS/MS survey of the cytosolic ribosome using trypsin and, when required, chymotrypsin and pepsin. We then used custom software to extract and filter peptide match information from Mascot result files and implement high confidence criteria for calling gene-specific identifications based on the highest quality unambiguous spectra matching exclusively to certain in silico predicted gene-or gene family-specific peptides. This provided an in-depth analysis of the protein composition based on 1446 high quality MS/MS spectra matching to 795 peptide sequences from ribosomal proteins. These identified peptides from five gene families of ribosomal proteins not identified previously, providing experimental data on 79 of the 80 different types of ribosomal subunits. We provide strong evidence for gene-specific identification of 87 different ribosomal proteins from these 79 families. We also provide new information on 30 specific sites of co-and post-translational modification of ribosomal proteins in Arabidopsis by initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, and phosphorylation. These sitespecific modification data provide a wealth of resources for further assessment of the role of ribosome modification in influencing translation in Arabidopsis. Molecular & Cellular Proteomics 7:347-369, 2008.Ribosomes are large ribonucleoprotein complexes that catalyze the peptidyltransferase reaction in polypeptide synthesis and are thus responsible for the translation of transcripts encoded in cellular genomes. These complexes play the most fundamental role of any protein complex in the generation of the cellular proteome as a whole. Ribosomes consist of two subunits, large and small, but the internal composition of these subunits and their macromolecular size varies between bacteria, animals, fungi, and plants. Both these subunits are composed on rRNA and protein (r-protein) 1 components. Among eukaryotes the 80 S cytosolic ribosomes of the yeast (Saccharomyces cerevisiae), rat (Rattus norvegicus), and human (Homo sapiens) have been the most extensively investigated. These studies have revealed four distinct rRNAs, the 18 S rRNA of the 40 S subunit and 5, 5.8, and 23 S rRNAs of the 60 S subunit. Up to 79 distinct proteins are part ...

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“…Yeast strains in which the phosphorylated C-terminal serine of P1␣ was mutated to an alanine were less sensitive to osmotic stress than wild-type or mutant strains in which this serine was mutated to threonine, leading to the prediction that dephosphorylation of P1 is required for an adaptive response to stress (25). Dephosphorylation is plausibly a global down-regulator of protein synthesis because dephosphorylation of the C-terminal serine of rat P2 reduced translational activity in vitro (17).…”

Section: Discussionmentioning

confidence: 99%

“…Ears were hand pollinated and harvested at 10,15,20,25,30, and 40 days postpollination (DPP) and after complete desiccation to isolate embryos (including the scutellum and embryonic axis), aleurone (tissue included the aleurone layer and attached pericarp), and endosperm (refers to tissue within the pericarp and aleurone, excluding the embryo).…”

Section: Methodsmentioning

confidence: 99%

“…In yeast, the presence of P1 was required for the assembly of P2 into the ribosome (23,24), and the ability to dephosphorylate one or more of these proteins was necessary for an adaptive response to osmotic stress (25). Studies with recombinant P1 and P2 from rat suggested that phosphorylation of P2 more effectively stimulates eEF2 activity in vitro than phosphorylation of P1 (17).…”

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confidence: 99%

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Regulated Heterogeneity in 12-kDa P-protein Phosphorylation and Composition of Ribosomes in Maize (Zea mays L.)

Szick-Miranda

Bailey‐Serres

2001

Journal of Biological Chemistry

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Maize (Zea mays L.) possesses four distinct ϳ12-kDa P-proteins (P1, P2a, P2b, P3) that form the tip of a lateral stalk on the 60 S ribosomal subunit. RNA blot analyses suggested that the expression of these proteins was developmentally regulated. Western blot analysis of ribosomal proteins isolated from various organs, kernel tissues during seed development, and root tips deprived of oxygen (anoxia) revealed significant heterogeneity in the levels of these proteins. P1 and P3 were detected in ribosomes of all samples at similar levels relative to ribosomal protein S6, whereas P2a and P2b levels showed considerable developmental regulation. Both forms of P2 were present in ribosomes of some organs, whereas only one form was detected in other organs. Considerable tissue-specific variation was observed in levels of monomeric and multimeric forms of P2a. P2b was not detected in root tips, accumulated late in seed embryo and endosperm development, and was detected in soluble ribosomes but not in membrane-associated ribosomes that copurified with zein protein bodies of the kernel endosperm. The phosphorylation of the 12-kDa P-proteins was also developmentally and environmentally regulated. The potential role of P2 heterogeneity in P-protein composition in the regulation of translation is discussed.A complex of acidic ribosomal proteins (r-proteins) 1 forms a universally conserved lateral stalk on the large ribosomal subunit that facilitates the translocation phase of protein synthesis (1). In eukaryotes the structure is formed by a complex of acidic phosphoproteins. P0 (ϳ35 kDa), homologous to prokaryotic L10, interacts with 28 S rRNA to form the base of the stalk, and P1 and P2 (ϳ12 kDa), homologous to prokayotic L7/L12, are tethered as dimers to the stalk (2-5). P1 and P2 are structurally similar; each protein has three domains that include an ␣-helical N-terminal region and a central, flexible acidic hinge region followed by a highly conserved C terminus (E/KSD/ EDMGFG/SLD). The C-terminal region of P0 is structurally similar to 12-kDa P-proteins because it possesses the three domains of P1 and P2 (for review, see Ref. 6).The 12-kDa P-proteins are the only r-proteins found in multiple copies within the ribosome. They do not assemble onto preribosomes in the nucleolus but cycle between ribosomes and a cytosolic pool in numerous species including, Artemia salina, Saccharomyces cerevisiae (yeast), humans, and rats (7-11). demonstrated quantitatively that exponentially growing yeast cells contain more 12-kDa P-proteins/ribosome than cells in the stationary phase of growth. This suggests that the level of P-proteins in yeast ribosomes is affected by the metabolic state of the cell and possibly reflects the translational activity of the ribosome. The presence of these proteins in ribosomes has been shown to stimulate the eEF2-dependent GTPase activity of ribosomes (13-17), poly(U)-directed phenylalanine synthesis (14, 18), and eEF1A binding (19). Hence, modulation of the 12-kDa P-protein component of ribosomes may impar...

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Phosphorylation of the Acidic Ribosomal P Proteins in Saccharomyces cerevisiae: A Reappraisal

Cited by 68 publications

References 25 publications

Rrp15p, a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation

Rrp15p, a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation

Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

Regulated Heterogeneity in 12-kDa P-protein Phosphorylation and Composition of Ribosomes in Maize (Zea mays L.)

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