L. N. Mironova

Non-Mendelian determinant [ISP+] in yeast is a nuclear-residing prion form of the global transcriptional regulator Sfp1

Four protein-based genetic determinants or prions—[SWI+], [MCA], [OCT+], and [MOT3+]—are recent additions to the list of well-known Saccharomyces cerevisiae prions, [PSI+], [URE3], and [PIN+]. A rapid expansion of this list may indicate that many yeast proteins can convert into heritable prion forms and underscores a problem of prion input into cellular physiology. Here, we prove that the global transcriptional regulator Sfp1 can become a prion corresponding to the prion-like determinant [ISP+] described earlier. We show that SFP1 deletion causes an irreversible [ISP+] loss, whereas increased SFP1 expression induces [ISP+] appearance. Cells that display the [ISP+] phenotype contain the aggregated form of Sfp1. Indeed, these aggregates demonstrate a nuclear location. We also show that the phenotypic manifestation of Sfp1 prionization differs from the manifestation of SFP1 deletion. These properties and others distinguish [ISP+] from yeast prions described to date.

SUP35 expression is enhanced in yeast containing (ISP + ), a prion form of the transcriptional regulator Sfp1

[ISP+] is a prion form of the global transcriptional regulator Sfp1 in Saccharomyces cerevisiae that manifests phenotypically as an antisuppressor of specific sup35 nonsense suppressor mutations. Although SUP35 is a Sfp1 target, the mechanism of antisuppression is unclear. Here we show that the level of SUP35 transcription in [ISP+] cells containing the sup35 mutation is increased relative to [isp-] cells and cells with a SFP1 deletion. As a result, [ISP+] cells have increased amounts of Sup35 encoded by the mutant allele. Indeed, additional experiments showed that increased amounts of mutant Sup35 may cause antisuppression. Remarkably, [ISP+] effects are not equivalent to those produced by SFP1 deletion, so [ISP+] represents an obvious example of a functionally active prion form of a protein. This feature distinguishes [ISP+] from other yeast prions, where prion switch often has the same effect as inactivation of a prion host gene. We suggest that enhancement of SUP35 expression in [ISP+] cells is caused by specific interaction of Sfp1 in its prion form with some negative SUP35 regulator. We also demonstrate that the advantage of [ISP+] strains over [isp-] strains described in our earlier work is specific for certain genetic background and growth conditions.

The HAL3-PPZ1 dependent regulation of nonsense suppression efficiency in yeast and its influence on manifestation of the yeast prion-like determinant [ISP(+)].

The efficiency of stop codons read‐through in yeast is controlled by multiple interactions of genetic and epigenetic factors. In this study, we demonstrate the participation of the Hal3‐Ppz1 protein complex in regulation of read‐through efficiency and manifestation of non‐Mendelian anti‐suppressor determinant [ISP+]. Over‐expression of HAL3 in [ISP+] strain causes nonsense suppression, whereas its inactivation displays as anti‐suppression of sup35 mutation in [isp−] strain. [ISP+] strains carrying hal3Δ deletion cannot be cured from [ISP+] in the presence of GuHCl. Since Hal3p is a negative regulatory subunit of Ppz1 protein phosphatase, consequences of PPZ1 over‐expression and deletion are opposite to those of HAL3. The observed effects are mediated by the catalytic function of Ppz1 and are probably related to the participation of Ppz1 in regulation of eEF1Bα elongation factor activity. Importantly, [ISP+] status of yeast strains is determined by fluctuation in Hal3p level, since [ISP+] strains have less Hal3p than their [isp−] derivatives obtained by GuHCl treatment. A model considering epigenetic (possibly prion) regulation of Hal3p amount as a mechanism underlying [ISP+] status of yeast cell is suggested.

Phenotypic expression of epigenetic determinant [ISP+] in Saccharomyces cerevisiae depends on the combination of sup35 and sup45 mutations

Translation fidelity in Saccharomyces yeasts is determined by genetic and epigenetic (prion) factors. A study was made of S. cerevisiae strains containing the nonchromosomal determinant [ISP+], described earlier. Some of its properties suggest that [ISP+] is a prion. [ISP+] is expressed phenotypically as an antisuppressor of two sup35 mutations and can be cured with guanidine chloride (GuHCl). It was shown that sup35 mutants containing [ISP+] carried additional sup45 mutations. These mutations caused amino acid substitutions in different regions of translation termination factor eRF1, encoded by SUP45. Strains bearing the sup35-25 mutation contained the sup45 mutation that caused amino acid substitution at position 400 of eRF1; strains bearing sup35-10 contained the mutation that altered eRF1 at position 75. Thus, the antisuppressor phenotype of the [ISP+] strains proved to depend on the interaction of sup35 and sup45 mutations, as well as on the GuHCl-curable epigenetic determinant.

Characterization of Missense Mutations in the SUP45 Gene of Saccharomyces cerevisiae Encoding Translation Termination Factor eRF1

Collection of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1 has been obtained by different approaches. It has been shown that most of isolated mutations cause amino acid substitutions in the N-terminal part of eRF1 and do not decrease the eRF1 amount. Most of mutations studied do not abolish eRF1–eRF3 interaction. The role of the N-terminal part of eRF1 in stop codon recognition is discussed.

The effect of paromomycin on the expression of ribosomal suppressors in yeast

SummaryMutants of the yeast Saccharomyces cerevisiae carrying ribosomal suppressor mutations in either sup1 or sup2 genes express a higher sensitivity to paromomycin — aminoglycoside antibiotic known to induce translational errors in eukaryotes. Paromomycin also induces a phenotypic suppression of all three types of nonsense mutations (ochre, amber and opal), missense mutations and frame-shift mutations. The influence of paromomycin on the activity of ribosomal suppressors has at least two aspects: (1) the drug increases translational ambiguity in sup1 and sup2 mutants in vitro and (2) it induces the alteration (extension or restriction) of sup 1 or sup2 suppression spectra in vivo. A modification of selectivity of the mutant ribosomes towards different tRNAs in the presence of paromomycin is proposed.

Further characterization of recessive suppression in yeast: Isolation of the low-temperature sensitive mutant of Saccharomyces cerevisiae defective in the assembly of 60 S ribosomal subunit

It has been shown that recessive suppressor mutations in the yeast Saccharomyces cerevisiae may cause sensitivity towards low temperatures (very slow growth or lack of growth at 10 degrees C). One of the sup 1 low temperature sensitive (Lts-) mutants, 26-125A-P-2156, was studied in detail. After a prolonged period of incubation (70 h) under restrictive conditions the protein synthesis apparatus in the mutant cells was irreversibly damaged. In addition, Lts- cells incubated under restrictive conditions synthesize unequal amounts of ribosomal subunits, the level of 60 S subunit being reduced. It has been suggested that the recessive suppression is mediated by a mutation in the gene coding for 60 S subunit component, probably a ribosomal protein. The mutation leads simultaneously to a defect in the assembly of 60 S subunit and to low-temperature sensitive growth of the mutant.

[Yeast prions, mammalian amyloidoses, and the problem of proteomic networks].

Prion proteins are infective amyloids and cause several neurodegenerative diseases in humans and animals. In yeasts, prions are detected as the cytoplasmic heritable determinants of a protein nature. Yeast prion [PSI], which results from a conformational rearrangement and oligomerization of translation termination factor eRF3, is used as an example to consider the structural-functional relationships in a potentially prion molecule, specifics of its evolution, and interactions with other prions, which form so-called prion networks. In addition, the review considers the results of modeling mammalian prion diseases and other amyloidoses in yeast cells. A hypothesis of proteomic networks is proposed by analogy with prion networks, involving interactions of different amyloids in mammals.

Reversions to respiratory competence of omnipotent sup45 suppressor mutants may be caused by secondary sup45 mutations

The molecular nature of the sup45 respiratory deficient omnipotent suppressor, and of three reversions to respiratory competence which removed the suppressor effect of the initial mutation, was examined. All reversions were caused by secondary sup45 mutations which indicates a direct connection between sup45 “respiratory” and “translational” functions. Computer analysis showed the local changes of Sup45 protein characteristics in the suppressor strain and revertants in comparison to the wild-type protein. The distribution of mutant sites in relation to evolutionary conserved, and tentatively functional, regions in the Sup45 protein is discussed.

Transcriptional response to the [ISP+] prion of Saccharomyces cerevisiae differs from that induced by the deletion of its structural gene, SFP1

Currently, several protein-based genetic determinants, or prions, are described in yeast, and several hundred prion candidates have been predicted. Importantly, many known and potential prion proteins regulate transcription; therefore, prion induction should affect gene expression. While it is generally believed that the prion phenotype should mimic the deletion phenotype, this rule has exceptions. Formed by the transcription factor Sfp1p, [ISP(+) ] is one such exception as the [ISP(+) ] and sfp1Δ strains differ in many phenotypic traits. These data suggest that effects of prion formation by a transcription factor and its absence may affect gene expression in a different way. However, studies addressing this issue are practically absent. Here, we explore how [ISP(+) ] affects gene expression and how these changes correspond to the effect of SFP1 deletion. Our data indicate that the [ISP(+) ]-related expression changes cannot be explained by the inactivation of Sfp1p. Remarkably, most Sfp1p targets are not affected in the [ISP(+) ] strain; instead, the genes upregulated in the [ISP(+) ] strain are enriched in Gcn4p and Aft1p targets. We propose that Sfp1p serves as a part of a regulatory complex, and the activity of this complex may be modulated differently by the absence or prionization of Sfp1p.