Characterization of the yeast acidic ribosomal phosphoproteins using monoclonal antibodies

Vilella, Maria Dolores; Remacha, Miguel; Ortiz, Blanca L.; Méndez, Enrique; Ballesta, Juan P. G.

doi:10.1111/j.1432-1033.1991.tb15831.x

Cited by 76 publications

(82 citation statements)

References 35 publications

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“…Western blots were performed by using Immobilon-P membranes (21). The specific antibodies to stalk proteins used in the present study were described in previous publications (17,23).…”

Section: Methodsmentioning

confidence: 99%

Ribosomal P0 Protein Domain Involved in Selectivity of Antifungal Sordarin Derivatives

Santos

Gabriel

Remacha

et al. 2004

Antimicrob Agents Chemother

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The ribosomal stalk protein P0 is involved in the susceptibility to the antifungal sordarin derivatives, as reported for a number of Saccharomyces cerevisiae resistant mutants. Mammals and some lower eukaryotes are naturally resistant to these compounds. It is shown here that expression in S. cerevisiae of the ribosomal protein P0 from Homo sapiens and from other sordarin-resistant organisms results in a decrease in the sensitivity of the cells to an agent of this class. To further characterize the P0 region responsible for inducing sordarin resistance, a series of protein chimeras containing complementary regions of the human and yeast P0 proteins were constructed and expressed in yeast. The chimeras complement the absence of the native yeast P0 except in chimeras containing the human P0 carboxyl-terminal domain. Resistance to sordarins was found to be associated with the presence of an HsP0 amino acid sequence comprising P118 to F138, which unexpectedly led to higher resistance than the presence of the complete human P0. A comparison of the corresponding region in P0 from yeast and sordarin-insensitive organisms, followed by site-directed mutagenesis, indicates that residues in positions 119, 124, and 126 have an important role in determining resistance to sordarins. Moreover, since sordarins block the eukaryotic elongation factor 2 (EF2) function, the P0 region affecting sordarin susceptibility must correspond to EF2-interacting domains of the ribosomal stalk protein, which affects the drug-binding site in the elongation factor.

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“…Western blots were performed by using Immobilon-P membranes (21). The specific antibodies to stalk proteins used in the present study were described in previous publications (17,23).…”

Section: Methodsmentioning

confidence: 99%

Ribosomal P0 Protein Domain Involved in Selectivity of Antifungal Sordarin Derivatives

Santos

Gabriel

Remacha

et al. 2004

Antimicrob Agents Chemother

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“…The presence of the acidic proteins in cell fractions was estimated by using specific monoclonal antibodies (33) in inhibition or indirect enzyme-linked immunosorbent assays (ELISAs) as previously described (33).…”

Section: Methodsmentioning

confidence: 99%

Ribosomal Acidic Phosphoproteins P1 and P2 Are Not Required for Cell Viability but Regulate the Pattern of Protein Expression in Saccharomyces cerevisiae

Remacha

Jiménez-Díaz

Bermejo

et al. 1995

Molecular and Cellular Biology

131

148

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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.The presence of a set of strongly acidic proteins is a universal feature of ribosomes from all organisms. These proteins have been involved in interactions with different supernatant factors during protein synthesis in prokaryotic as well as eukaryotic systems (see reference 16 for a review).In eukaryotic organisms, the acidic ribosomal proteins, called generically P proteins, are capable of being phosphorylated, and this modification drastically affects the interaction of the polypeptides with the ribosome in vitro (11,14). In fact, this process might be part of a control mechanism that, by regulating the amount of bound protein, can affect the activity of the particles in relation to the metabolic requirements of the cell (24).In Saccharomyces cerevisiae, four acidic ribosomal proteins have been reported and characterized; according to structural and functional considerations, they can be classified in two groups, YP1␣/␤ and YP2␣/␤, corresponding to the two types of acidic polypeptides present in mammalian ribosomes (35). Using gene disruption techniques, we previously found that the individual elimination of each polypeptide, and even the double disruption of any two of the four proteins, is not a lethal event for the cell, although the generation time is affected to different extents (20, 21). The effect is especially important when the genes encoding the two members of the same group are disrupted, since in this case the remaining acidic proteins present in the c...

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“…The RTA-stalk complexes pulled down with anti-RTA were separated by SDS-PAGE, transferred to nitrocellulose, and probed with monoclonal antibodies against the conserved C termini of P proteins (41). As shown in Fig.…”

Section: Rta Interacts With Stalkmentioning

confidence: 99%

“…A, stalk complexes (50 pmol) were incubated with N-His-RTA (500 pmol) and immunoprecipitated using polyclonal anti-RTA. Immunoprecipitated proteins (10 l) were separated on a 15% SDS-PAGE, transferred to nitrocellulose, and probed with monoclonal antibody against the C termini of P proteins (3BH5, 1:4 dilution) (41). B, the blot was stripped using 8 M guanidine-HCl and reprobed with monoclonal anti-RTA antibody (MAB3, 1:1000 dilution).…”

Section: Rta and Stalk Pentamer Th199 (P0 200 -312 -P1␣/p2␤-p1␤/ P2␣)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

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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.

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Characterization of the yeast acidic ribosomal phosphoproteins using monoclonal antibodies

Cited by 76 publications

References 35 publications

Ribosomal P0 Protein Domain Involved in Selectivity of Antifungal Sordarin Derivatives

Ribosomal P0 Protein Domain Involved in Selectivity of Antifungal Sordarin Derivatives

Ribosomal Acidic Phosphoproteins P1 and P2 Are Not Required for Cell Viability but Regulate the Pattern of Protein Expression in Saccharomyces cerevisiae

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

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