Ribosomal proteins of yeast strains carrying mutations which affect the efficiency of nonsense suppression

Summary We have examined the ribosomal proteins of strains of Saccharomyces cerevisiae which differ in the efficiency with which ochre nonsense mutations are suppressed. The strains in which ochre suppression is poor were [psi]^- or carried antisuppressor mutations; those in which suppression was highly efficient were [psi]⁺ or carried allosuppressor mutations. The ribosomal proteins of these strains, as judged by two-dimensional polyacrylamide gel electrophoresis, were indistinguishable from those of wild-type.     

Quantitative effects of magnesium chloride stress on aggregation of Sup35p in [psi-] yeast cells

[PSI(+)] phenotype can be transiently induced when Magnesium chloride (MgCl(2)) was the selective pressure in SUP35 repeat-expansion mutant [psi(-)] yeast strains. We further investigated [PSI(+)] phenotype change under different MgCl(2) conditions with native Sup35p and quantified the Sup35p status changes with fluorescence recovery after photobleaching (FRAP) and semi-denaturing detergent-agarose gel electrophoresis (SDD-AGE) analysis. It was found that the [PSI(+)] phenotype presented a dose-dependent relationship with the concentrations of MgCl(2). Furthermore, Sup35p aggregated in MgCl(2) treated cells but did not form large aggregates as it does in [PSI(+)] cells, and the size of Sup35p aggregates showed a time-dependent relationship with the MgCl(2) application. The aggregation of Sup35p strictly depended on the presence of MgCl(2) stress in our strains.     

Variation of phenotypic suppression due to the psi+ and psi- extrachromosomal determinants in yeast.

Paromomycin, an aminoglycoside antibiotic, can phenotypically suppress nonsense mutations in the yeast Saccharomyces cerevisiae (Palmer et al., 1979). We report here that the extrachromosomal determinant, ψ⁺, enhances this phenotypic suppression of all three nonsense mutations. UAG. UAA and UGA, by three-to sevenfold.     

The ribosome-associated inhibitor A reduces translation errors

Recently we have reported about a novel stress response protein (pY or RaiA) associated with Escherichia coli ribosomes that inhibits translation at the aminoacyl-tRNA binding stage. Here we show that leucine misincorporation during in vitro poly(U) translation is inhibited by this protein much stronger than the incorporation of phenylalanine. The miscoding counteraction by RaiA is especially strong at the concentrations of magnesium ions close to those observed in vivo and diminishes at higher magnesium concentrations. The results obtained suggest that the anti-miscoding activity of RaiA could be the main function of the protein, rather than the inhibition of translation. The role of the protein in adaptation of cells to environmental stress is discussed.     

Induction of distinct [URE3] yeast prion strains

[URE3] is a non-Mendelian genetic element in Saccharomyces cerevisiae, which is caused by a prion-like, autocatalytic conversion of the Ure2 protein (Ure2p) into an inactive form. The presence of [URE3] allows yeast cells to take up ureidosuccinic acid in the presence of ammonia. This phenotype can be used to select for the prion state. We have developed a novel reporter, in which the ADE2 gene is controlled by the DAL5 regulatory region, which allows monitoring of Ure2p function by a colony color phenotype. Using this reporter, we observed induction of different [URE3] prion variants ("strains") following overexpression of the N-terminal Ure2p prion domain (UPD) or full-length Ure2p. Full-length Ure2p induced two types of [URE3]: type A corresponds to conventional [URE3], whereas the novel type B variant is characterized by relatively high residual Ure2p activity and efficient curing by coexpression of low amounts of a UPD-green fluorescent protein fusion protein. Overexpression of UPD induced type B [URE3] but not type A. Both type A and B [URE3] strains, as well as weak and strong isolates of type A, were shown to stably maintain different prion strain characteristics. We suggest that these strain variants result from different modes of aggregation of similar Ure2p monomers. We also demonstrate a procedure to counterselect against the [URE3] state.     

The ribosome-associated complex antagonizes prion formation in yeast

Abstract

The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI⁺] prion – an alternative conformer of Sup35 protein – and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Δzuo1 strains. Consistent with this finding, Δzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.     

The ribosome-associated complex antagonizes prion formation in yeast

The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI⁺] prion – an alternative conformer of Sup35 protein – and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Δzuo1 strains. Consistent with this finding, Δzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.     

In vitro suppression of a nonsense mutant of Drosophila melanogaster.

When RNA isolated from the Drosophila melanogaster alcohol dehydrogenase (ADH) negative mutant CyOnB was translated "in vitro" in the presence of yeast opal suppressor tRNA, a wild type size ADH protein was obtained in addition to the mutant gene product. This identifies the CyOnB mutant as an opal (UGA) nonsense mutant. From the molecular weight of the mutant protein, and from the known sequence of the ADH gene (Benyajati et al., Proc.Natl.Acad.Sci. USA 78, 2717-2721, 1981), we conclude that the tryptophan codon UGG in position 234 has been changed into a UGA nonsense codon in the CyOnB mutant. Furthermore, we show that the UAA stop codon of the wild type ADH gene is resistant to suppression by a yeast ochre suppressor tRNA. This is in contrast to the high efficiency of suppression of the CyOnB UGA nonsense codon, despite an almost identical codon context.     

Overexpression of gene PPZ1 in the yeast Saccharomyces cerevisiae affects the efficiency of nonsense suppression

The phenomenon of nonsense suppression, which leads to the stop codons reading-through, may be related to disturbances in the operation of various components of the translation apparatus and the proteins interacting with them. The phosphatase Ppzlp is one of the factors affecting the nonsense suppression efficiency in Saccharomyces yeast. In this work, the impact of the overexpression of gene PPZ1 and its mutant allele PPZ1-R451L on the phenotypic expression of various mutant alleles of genes SUP35 and SUP45 or the yeast prion [PSI ⁺] was analyzed. On the basis of the data obtained, a suggestion about the possible role of proteins Sup35p and Sup45p in the processes mediating the influence of gene PPZ1 overexpression on the efficiency of nonsense suppression is made.     

Sinergistic action of genetic and phenotypic suppression of nonsense mutations in yeast Saccharomyces cerevisiae

Summary It was found that the phenotypic suppression induced by the paromamine-containing antibiotic paromomycin could be significantly strengthened by a ribosomal suppressor mutation in yeast Saccharomyces cerevisiae. As a result the suppressor efficient towards ochre mutations in the presence of paromomycin acquired the ability to suppress both amber and opal mutations. It is suggested that phenotypic suppression by paromomycin and genotypic suppression by sup 1 both involve a similar mechanism of misreading.     

Overexpression of gene PPZ1 in the yeast Saccharomyces cerevisiae affects the efficiency of nonsense suppression

The phenomenon of nonsense suppression, which leads to the reading of stop codons as sense codons, may be related to disturbances in the operation of various components of the translation apparatus and the proteins interacting with them. The phosphatase Ppzlp is one of the factors affecting the nonsense suppression efficiency in the saccharomycete yeast. In this work, the impact of the overexpression of gene PPZ1 and its mutant allele PPZ1-R451L on the phenotypic expression of various mutant alleles of genes SUP35 and SUP45 or the yeast prion [PSI+] was analyzed. On the basis of the data obtained, a suggestion about the possible role of proteins Sup35p and Sup45p in the processes mediating the influence of gene PPZ1 overexpression on the efficiency of nonsense suppression is made.     

Lecithinase activity of various yeast strains]

The activity of lecithinase was studied among 50 yeast strains belonging to the genera Rhodotorula Harrison, Cryptococcus Kütz, and Lipomyces Lodder et Kreger van Rij. The maximum activity of lecithinase is typical of epiphyte yeast strains belonging to the genus Rhodotorula and is not manifested by species of the henus Lipomyces which inhabit soil. Strains of the genus Cryptococcus cannot be distinctly differentiated into soil and epiphyte cultures, and occupy an intermediate position by the activity of lecithinase.     

A comparison of small circular DNA molecules in psi+ and psi- strains of Saccharomyces cerevisiae.

The psi+ and psi- phenotypes, which affect the efficiency of ochre suppression in yeast, are inherited in a non-Mendelian fashion. There is no apparent difference in length or in length distribution of 2 micronm circular DNA molecules between psi+ and psi- strains. It seems that the psi genetic determinant is probably not connected with the presence or absence of these small circular DNA molecules.