P1 and P2 protein heterodimer binding to the P0 protein of Saccharomyces cerevisiae is relatively non-specific and a source of ribosomal heterogeneity (original) (raw)
Related papers
Molecular Microbiology, 2006
The stalk is an essential domain of the large ribosomal subunit formed by a complex of a set of very acidic proteins bound to a core rRNA binding component. While in prokaryotes there is only one type acidic protein, L7/12, two protein families are found in eukaryotes, phosphoproteins P1 and P2, which presumably have different roles. To search for differences zero-length cross-linking by S-S bridge formation was applied using Saccharomyces cerevisiae mutant P1 and P2 proteins carrying single cysteine residues at various positions. The results show a more exposed location of the N-terminal domain of the P2 proteins, which in contrast to P1, can be found as dimers when the Cys is introduced in this domain. Similarly, the Cys containing C-terminal domain of mutant P2 proteins shows a notable capacity to form cross-links with other proteins, which is considerably lower in the P1 type. On the other hand, mutation at the conserved C-domain of protein P0, the eukaryotic stalk rRNA binding component, results in removal of about 14 terminal amino acids. Protein P2, but not P1, protects mutant P0 from this truncation. These results support a eukaryotic stalk structure in which P1 proteins are internally located with their C-terminals having a restricted reactivity while P2 proteins are more external and accessible to interact with other cellular components.
Biochemistry, 2000
The eukaryotic acidic P1 and P2 proteins modulate the activity of the ribosomal stalk but playing distinct roles. The aim of this work was to analyze the structural features that are behind their different function. A structural characterization of Saccharomyces cereVisaie P1R and P2 proteins was performed by circular dichroism, nuclear magnetic resonance, fluorescence spectroscopy, thermal denaturation, and protease sensitivity. The results confirm the low structure present in both proteins but reveal clear differences between them. P1R shows a virtually unordered secondary structure with a residual helical content that disappears below 30°C and a clear tendency to acquire secondary structure at low pH and in the presence of trifluoroethanol. In agreement with this higher disorder P1R has a fully solventaccessible tryptophan residue and, in contrast to P2 , is highly sensitive to protease degradation. An interaction between both proteins was observed, which induces an increase in the global secondary structure content of both proteins. Moreover, mixing of both proteins causes a shift of the P1R tryptophan 40 signal, pointing to an involvement of this region in the interaction. This evidence directly proves an interaction between P1R and P2 before ribosome binding and suggests a functional complementation between them. On a whole, the results provide structural support for the different functional roles played by the proteins of the two groups showing, at the same time, that relatively small structural differences between the two stalk acidic protein types can result in significant functional changes.
Phosphorylation of the Acidic Ribosomal P Proteins in Saccharomyces cerevisiae: A Reappraisal
Biochemistry, 1997
Previous reports had pointed to serines 62 and 71/79 as possible phosphorylation sites in the yeast acidic ribosomal proteins YP1R and YP2R, respectively. However, it has been found that mutation of these serine residues did not affect the phosphorylation level of the proteins. A detailed examination of the YP2R tryptic digest from the in ViVo labeled protein demonstrates the existence of a totally trypsininsensitive site at lysine 88 that led to a misinterpretation of previous results. The unique YP2R tryptic phosphopeptide obtained contains, in addition to serines 71 and 79, a serine at position 96 near the carboxyl end, which automatic Edman degradation confirmed as the phosphorylated residue. In addition, by using Staphyloccocus protease V8, it was possible to obtain phosphopeptides containing only serine 96, whose phosphorylation has likewise been confirmed by radioactive labeling as well as by chemical methods. A similar analysis of the other 12 kDa acidic proteins, YP1R, YP1 , and YP2 , has shown the presence of equivalent phosphorylation sites in the four P proteins, which correspond to position 96 in proteins YP1R, YP1 , and YP2R and position 100 in YP2. This conclusion has been confirmed by the fact that mutation of serine 96 in proteins YP1R and YP2R abolishes their capacity to be phosphorylated in ViVo. The mutation of the phosphorylation site of the individual acidic proteins seems not to alter their interaction with the ribosome. However, it has been found that the level of phosphorylation of the stalk proteins has an effect on the response of the cells to some specific metabolic conditions, indicating that it may modulate the translation of specific proteins.
1995
Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1␣, YP1, YP2␣, and YP2, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23؇C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.
Phosphorylation of the yeast ribosomal stalk. Functional effects and enzymes involved in the process
FEMS Microbiology Reviews, 1999
The ribosomal stalk is directly involved in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes the acidic components correspond to the 12-kDa P1 and P2 proteins, and the RNA binding component is the P0 protein. All these proteins are found phosphorylated in eukaryotic organisms, and previous in vitro data suggested this modification was involved in the activity of this structure. Results from mutational studies have shown that phosphorylation takes place at a serine residue close to the carboxy end of the P proteins. Modification of this serine residue does not affect the formation of the stalk and the activity of the ribosome in standard conditions but induces an osmoregulation-related phenotype at 37³C. The phosphorylatable serine is part of a consensus casein kinase II phosphorylation site. However, although CKII seems to be responsible for part of the stalk phosphorylation in vivo, it is probably not the only enzyme in the cell able to perform this modification. Five protein kinases, RAPI, RAPII and RAPIII, in addition to the previously reported CKII and PK60 kinases, are able to phosphorylate the stalk proteins. A comparison of the five enzymes shows differences among them that suggest some specificity regarding the phosphorylation of the four yeast acidic proteins. It has been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins. All the data suggest that although phosphorylation is not involved in the interaction of the acidic P proteins with the ribosome, it can affect the ribosome activity and might participate in a possible ribosome regulatory mechanism.
Nucleic Acids Research, 2000
The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expressed in the conditional P0-null strains W303dGP0 and D67dGP0 of Saccharomyces cerevisiae, the ribosomes of which contain either standard amounts or are totally deprived of the P1/P2 proteins, respectively. The presence of the CcP0 protein restored cell viability but reduced the growth rate. In the W303CcP0 strain, all four acidic yeast proteins were found on the ribosomes, but in notably less quantity, while a preferable binding of the YP1α/YP2β pair was established. In the absence of the endogenous P1/P2 proteins in the D67CcP0 strain, the complementation capacity of the CcP0 protein was considerably reduced. The simultaneous expression of the three medfly genes resulted in alterations of the stalk composition: both the CcP1 and CcP2 proteins were found on the particles substituting the YP1α and YP2α proteins, respectively, but their presence did not alter the growth rate, except in the case of the YP1α/β defective strain, where a helping effect on the binding of the YP2α and YP2β proteins on the ribosomes was confirmed. Therefore, the medfly ribosomal P-proteins complement the yeast P-protein deficient strains forming an heterogeneous ribosomal stalk, which, however, is not functionally equivalent to the endogenous one.
Eukaryotic acidic phosphoproteins interact with the ribosome through their amino-terminal domain
Biochemistry, 1995
Variable-size fragments of the four yeast acidic ribosomal protein genes rpYPla, rpYPlP, rpYP2a and rpYP2P were fused to the LacZ gene in the vector series YEp356-358. The constructs were used to transform wild-type Saccharomyces cerevisiae and several gene-disrupted strains lacking different acidic ribosomal protein genes. The distribution of the chimeric proteins between the cytoplasm and the ribosomes, tested as P-galactosidase activity, was estimated. Hybrid proteins containing around a minimum of 65-75 amino acids from their amino-terminal domain are able to bind to the ribosomes in the presence of the complete native proteins. Hybrid proteins containing no more than 36 amino terminal amino acids bind to the ribosomes in the absence of a competing native protein. The fused YPl-P-galactosidase proteins are also able to form a complex with the native YP2 type proteins, promoting their binding to the ribosome. The stability of the hybrid polypeptides seems to be inversely proportional to the size of their P protein fragment. These results indicate that only the amino-terminal domain of the eukaryotic P proteins is needed for the Pl-P2 complex formation required for interaction with the ribosome. The highly conserved P protein carboxyl end is not implicated in the binding to the particles and is exposed to the medium.
Functional characterization of ribosomal P1/P2 proteins in human cells
Biochemical Journal, 2008
The ‘stalk’ is a large ribosomal subunit domain that regulates translation. In the present study the role of the ribosomal stalk P proteins in modulating ribosomal activity has been investigated in human cells using RNA interference. A strong down-regulation of P2 mRNA and a drastic decrease in P2 protein in a stable human cell line was achieved using a doxycycline-inducible system. Interestingly, the amount of P1 protein was similarly decreased in these cells, in contrast with the expression of P1 mRNA. The loss of P1/P2 proteins produced a decrease in the growth rate of these cells, as well as an altered polysome pattern with reduced translation efficiency, but without affecting the free 40 S/60 S subunit ratio. A decrease in the ribosomal-subunit joining capacity was also observed. These data indicate that P1/P2 proteins modulate cytoplasmic translation by influencing the interaction between subunits, thereby regulating the rate of cell proliferation.
The acidic ribosomal proteins as regulators of the eukaryotic ribosomal activity
… et Biophysica Acta (BBA …, 1990
The acidic proteins, A-proteins, from the large ribosomal subunit of Saccharomyces cerevisiae grown under different conditions have been quantitatively estimated by ELISA tests using rabbit sera speclfic for these polypeptides. It has been found that the amount of A-protein present in the ribosome is not constant and depends on the metabolic state of the cell. Ribosomes from exponentially growing cultures have about 40% more of these proteins than those from stationary phase. Similarly, the particles forming part of the polysomes are enriched in A-proteins as compared with the free 80 S ribosomes. The cytoplasmic pool of A-protein is considerably high, containing as a whole as much protein as the total ribosome population. These results are compatible with an exchanging process of the acidic proteins during protein synthesis that can regulate the activity of the ribosome. On the other hand, cells inhibited with different metabolic inhibitors produce a very low yield of ribosomes that contain, however, a surprisingly high amount of acidic proteins while the cytoplasmic pool is considerably reduced, suggesting that under stress conditions the ribosome and the A-protein may aggregate, forming complex structures that are not recovered by the standard preparation methods.
Biochimie, 1991
m The covalent structures of rat ribosomal proteins P0, Pl, and P2 were deduced from the sequences of nucleotides in recombinant cDNAs. P0 contains 316 amino acids and has a molecular weight of 34 178; Pl has 114 residues and a molecular weight of 11 490: and P2 has 115 amino acids and a molecular weight of 11 684. The rat P-proteins have a near identical (16 of 17 residues) Sequence of amino acids at their carboxyl termini and are related to analogous proteins in other eukaryotic species. A proposal is made for a uniform nomenclature for rat and yeast ribosomal proteins. recombinant DNA / nucleotide sequences / amino acid sequence comparisons / number of genes / mRNA size / uniform nomenclature