PHI+], a novel Sup35-prion variant propagated with non-Gln/Asn oligopeptide repeats in the absence of the chaperone protein Hsp104

BACKGROUND: The [PSI+] element of the budding yeast is an aggregated form of the translation release factor Sup35 that is propagated and transmitted cytoplasmically in a manner analogous to that of mammalian prions. The N-terminal of Sup35, necessary for [PSI+], contains oligopeptide repeats and multiple Gln/Asn residues. RESULTS: We replaced the Gln/Asn-rich prion repeats of Sup35 with non-Gln/Asn repeats from heterologous yeast strains. These non-Gln/Asn repeat Sup35s propagated a novel [PSI+] variant, [PHI+], that appeared de novo 103 times more frequent than [PSI+]. [PHI+] was stably inherited in a non-Mendelian fashion, but not eliminated upon the inactivation of Hsp104, unlike known [PSI+] elements. In vitro, non-Gln/Asn repeat domains formed amyloid fibres that were shorter and grew more slowly than did Gln/Asn-rich prion domains, while [PHI+] aggregates were smaller than [PSI+] aggregates in vivo. CONCLUSIONS: These findings suggest the existence of an alternative, Hsp104-independent pathway to replicate non-Gln/Asn variant Sup35 prion seeds.     

An antiprion effect of the anticytoskeletal drug latrunculin A in yeast

Prions are infectious aggregation-prone isoforms of the normal proteins, supposedly able to seed aggregation of the normal cellular counterparts. In vitro, prion proteins form amyloid fibers, resembling cytoskeletal structures. Yeast prion [PSI], which is a cytoplasmically inherited aggregated isoform of the translation termination factor Sup35p (eRF3), serves as a useful model for studying mechanisms of prion diseases and other amyloidoses. The previously described interaction between Sup35p and cytoskeletal assembly protein Sla1p points to the possible relationships between prions and cytoskeletal networks. Although the Sup35PSI+ aggregates do not colocalize with actin patches, we have shown that yeast cells are efficiently cured of the [PSI] prion by prolonged incubation with latrunculin A, a drug disrupting the actin cytoskeleton. On the other hand, treatments with sodium azide or cycloheximide, agents blocking yeast protein synthesis and cell proliferation but not disrupting the cytoskeleton, do not cause a significant loss of [PSI]. Moreover, simultaneous treatment with sodium azide or cycloheximide blocks [PSI] curing by latrunculin A, indicating that prion loss in the presence of latrunculin A requires a continuation of protein synthesis during cytoskeleton disruption. The sodium azide treatment also decreases the toxic effect of latrunculin A. Latrunculin A influences neither the levels of total cellular Sup35p nor the levels of chaperone proteins, such as Hsp104 and Hsp70, which were previously shown to affect [PSI]. This makes an indirect effect of latrunculin A on [PSI] via induction of Hsps unlikely. Fluorescence microscopy detects changes in the structure and/or localization of the Sup35PSI+ aggregates in latrunculin A-treated cells. We conclude that the stable maintenance of the [PSI] prion aggregates in the protein-synthesizing yeast cells partly depends on an intact actin cytoskeleton, suggesting that anticytoskeletal treatments could be used to counteract some aggregation-related disorders.     

Yeast prion protein New1 can break Sup35 amyloid fibrils into fragments in an ATP-dependent manner

We report a new type of interaction between two yeast prion proteins, Sup35 and New1. New1 consists of an N-terminal prion region (New1N) and a C-terminal region homologous to a translation elongation factor with two ATP-binding motifs. Amyloid formation of the Sup35 prion region (Sup35NM) was accelerated by a small amount of sonicated New1N amyloid (New1N-seeds) to produce Sup35NM[New1] amyloid. New1N amyloid formation was accelerated by Sup35NM[New1]-seeds but not by spontaneously generated Sup35NM-seeds, indicating that the structural features of the New1N amyloid were transmitted via the Sup35NM amyloid. Surprisingly, full-length New1 broke the Sup35NM amyloid fibrils in an ATP-dependent manner. This activity of New1 was independent from Hsp104. It was lost by a mutation in the second ATP-binding motif, by the truncation of the N-terminal prion region of New1 and by the pre-incubation of New1 with New1N-seeds. When New1 was overproduced in yeast [PSI(+)] cells carrying GFP-fused Sup35NM, diverse morphological changes in fluorescent foci occurred. Thus, New1 potentially has a regulatory role in prion state in yeast, working independently of the Hsp104 system.     

Dynamics of yeast prion aggregates in single living cells

Prions are propagating proteins that are ordered protein aggregates, in which the phenotypic trait is retained in the altered protein conformers. To understand the dynamics of the prion aggregates in living cells, we directly monitored the fate of the aggregates using an on-chip single-cell cultivation system as well as fluorescence correlation spectroscopy (FCS). Single-cell imaging revealed that the visible foci of yeast prion Sup35 fused with GFP are dispersed throughout the cytoplasm during cell growth, but retain the prion phenotype. FCS showed that [PSI+] cells, irrespective of the presence of foci, contain diffuse oligomers, which are transmitted to their daughter cells. Single-cell observations of the oligomer-based transmission provide a link between previous in vivo and in vitro analyses of the prion and shed light on the relationship between the protein conformation and the phenotype.     

The role of pre-existing aggregates in Hsp104-dependent polyglutamine aggregate formation and epigenetic change of yeast prions

Amyloid-like protein aggregates have been implicated in various diseases and in the protein-based inheritance of yeast prions. The molecular chaperone Hsp104 has been shown to be necessary for the aggregate formation of polyglutamine in yeast, and for the maintenance of several yeast prion phenotypes through the formation of self-propagating aggregates. In this paper, we show that the polyglutamine aggregates that are formed independently of Hsp104, are required for Hsp104 to efficiently produce more aggregates. Similarly, in the yeast prion [PSI+] system, Hsp104-dependent epigenetic changes to the [PSI+] prion phenotype require the presence of prion aggregates in the normal [psi-] state. We also show that the co-localization of different prion aggregates suggests that cross-seeding by different yeast prions increases the probability of Hsp104-dependent epigenetic change. These findings highlight the role of pre-existing aggregates in chaperone-dependent establishment of the epigenetic trait in yeast prions, and possibly in the pathology of several neurodegenerative diseases.     

Single mother–daughter pair analysis to clarify the diffusion properties of yeast prion Sup35 in guanidine-HCl-treated [PSI+] cells

The yeast prion [ PSI ⁺ ] is a protein-based heritable element, in which aggregates of Sup35 protein are transmitted to daughter cells in a non-Mendelian manner. To elucidate the mechanism of the transmission, we have developed methods to directly analyse the dynamics of Sup35 fused with GFP in single mother–daughter pairs. As it is known that the treatment of yeast cells with guanidine hydrochloride (GuHCl) cures [ PSI ⁺ ] by perturbing Hsp104, a prion-remodelling factor, we analysed the diffusion profiles of Sup35–GFP in GuHCl-treated [ PSI ⁺ ] cells using fluorescence correlation spectroscopy (FCS). FCS analyses revealed that Sup35–GFP diffusion in the daughter cells was faster; that is, the Sup35–GFP particle was smaller, than that in the mother [ PSI ⁺ ] cells, and it eventually reached the diffusion profiles in [ psi ⁻ ] cells. We then analysed the flux of Sup35–GFP oligomers from mother to daughter [ PSI ⁺ ] cells in the presence of GuHCl, using a modified fluorescent recovery after photobleaching technique, and found that the flux of the diffuse oligomers was completely inhibited. The noninvasive methods described here can be applied to other protein-based transmissible systems inside living cells.     

酵母朊蛋白Sup35NM体外淀粉样纤维的形成动态研究

目的 研究酵母朊蛋白Sup35在近似于生理环境的体外条件下淀粉样纤维形成的动态过程，为阐明淀粉样纤维的形成机制提供材料和线索。方法 在E．coli中表达Sup35蛋白的NM段，并用Ni^2＋螫合层析对重组蛋白在变性条件下进行纯化，在不同的时间点用透射电子显微镜观察、圆二色谱检测以及蛋白酶K消化试验，同时利用硫黄素（thioflavin T，ThT）结合试验对淀粉样纤维形成的动态过程进行检测。结果 在变性条件下成功纯化出Sup35NM重组蛋白。利用透射电子显微镜观察到了Sup35NM蛋白在PBS（pH7．4）缓冲液中发生聚集时的形态变化，圆二色谱检测显示该过程伴随蛋白结构由α-螺旋到β-折叠的转变。纤维具有较强的抗蛋白酶K消化的特性。ThT结合试验提示淀粉样纤维的形成在经过了一个快速的上升阶段后达到平台期，纤维形成的速率会随着蛋白浓度的提高而加快。结论酵母朊蛋白Sup35NM在接近生理环境的体外条件下极易发生聚集，聚集物具有淀粉样纤维性质，Sup35NM淀粉样纤维形成的动态过程为淀粉样变成核聚集模型的研究提供了依据。     

Estimating the number of prions in yeast cells.

Certain yeast cells contain proteins that behave like the mammalian prion PrP and are called yeast prions. The yeast prion protein Sup35p can exist in one of two stable forms, giving rise to phenotypes [PSI(+)] and [psi(-)]. If the chemical guanidine hydrochloride (GdnHCl) is added to a culture of growing [PSI(+)] cells, the proportion of [PSI(+)] cells decreases over time. This process is called curing and is due to a failure to propagate the prion form of Sup35p. We describe how curing can be modelled, and improve upon previous models for the underlying processes of cell division and prion segregation; the new model allows for asymmetric cell division and unequal prion segregation. We conclude by outlining plans for future experimentation and modelling.     

Prion model in yeast

Two unusual phenotypes in Saccharomyces cerevisiae, [PSI+] and [URE3], have been suggested to be due to prion proteins. Various research groups have shown that this is indeed the case and have characterized these yeast prions in more detail. The factors involved in prion formation, such as chaperone protein, and the intramolecular determinants of prion formation have been investigated. The ability of these yeast proteins to form prion is due to modular domains conserved throughout evolution.     

Sup35p yeast prion-like protein as an adapter for production of the Gag-p55 antigen of HIV-1 and the L-chain of botulinum neurotoxin in Saccharomyces cerevisiae

Effective expression of the HIV-1 core protein Gag-p55 was obtained in Saccharomyces cerevisiae under control of the inducible UASgal/CYC1 promoter as a translational fusion with the prion-forming NM domain of the translation terminator Sup35p (eRF3) of S. cerevisiae. where only poor expression of the original-type Gag-p55 was observed. A deletion within the Sup35NM prion-forming domain altering Sup35-associated [PSI] inheritance did not compromise expression of the Sup35NM Gag-p55 fusion protein. Therefore, either the mechanism of this phenomenon is not directly related to the effect of Sup35p prion-formation or the modified protein maintains residual prion-forming abilities. The recombinant Sup35p-Gag-p55 protein was quite stable under boiling in an alkali/sodium dodecyl sulfate (SDS) solution and completely retained its antigenic properties. Moreover, 10-min boiling of the native yeast cells in this solution allowed immediate inhibition of lysosomal and other yeast proteases, responsible for autolysis of many natural and recombinant proteins. The use of this method of preliminary enrichment for the recombinant fusion protein Sup35p-Gag-p55 with the SDS-alkaline extraction could be useful for yeast heterologous expression and purification of other of insoluble and unstable proteins. A translational fusion with the NM domain of Sup35p was also used to produce another poorly soluble protein, the L-chain of botulinum exotoxin A, in S. cerevisiae. When the Sup35p fragment was removed from the recombinant construct encoding a fused Sup35/BoNT protein, a dramatic drop in both transformation efficiency and growth rate of transformants was shown.     

Selfish prion of Rnq1 mutant in yeast

[ PIN ⁺] is a prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [ PSI ⁺]. We previously isolated a truncated form of Rnq1, named Rnq1Δ100, as a [ PSI ⁺]-eliminating factor in S. cerevisiae . Rnq1Δ100 deletes the N-terminal non-prion domain of Rnq1, and eliminates [ PSI ⁺] in [ PIN ⁺] yeast. Here we found that [ PIN ⁺] is transmissible to Rnq1Δ100 in the absence of full-length Rnq1, forming a novel prion variant [ RNQ1Δ100 ⁺]. [ RNQ1Δ100 ⁺] has similar [ PIN ⁺] properties as it stimulates the de novo induction of [ PSI ⁺] and is eliminated by the null hsp104Δ mutation, but not by Hsp104 overproduction. In contrast, [ RNQ1Δ100 ⁺] inherits the inhibitory activity and hampers the maintenance of [ PSI ⁺] though less efficiently than [ PIN ⁺] made of Rnq1–Rnq1Δ100 co-aggregates. Interestingly, [ RNQ1Δ100 ⁺] prion was eliminated by de novo [ PSI ⁺] induction. Thus, the [ RNQ1Δ100 ⁺] prion demonstrates selfish activity to eliminate a heterologous prion in S. cerevisiae , showing the first instance of a selfish prion variant in living organisms.     

Prion variant maintained only at high levels of the Hsp104 disaggregase

The yeast prion [PSI ⁺ ] is a self-perpetuating aggregated isoform of the translation termination factor Sup35. [PSI ⁺ ] propagation is promoted by moderate levels and antagonized by high levels of the chaperone Hsp104. In agreement with the model postulating that excess Hsp104 acts on [PSI ⁺ ] by disaggregating prion polymers, we show that an increase in Sup35 levels, accompanied by an increase in size of prion aggregates, also partially protects [PSI ⁺ ] from elimination by excess Hsp104. Despite retention of [PSI ⁺ ], excess Hsp104 decreases toxicity of overproduced Sup35 in [PSI ⁺ ] strains. A heritable variant of [PSI ⁺ ], which has been isolated and is maintained only in the presence of increased levels of Hsp104, is characterized by an abnormally large aggregate size, and exhibits an altered response to overproduction of the Hsp70 chaperone Ssa1. These features resemble the previously described prion generated by a deletion derivative of Sup35, but are not associated with any sequence alteration and are controlled exclusively at the protein level. Our data provide a proof of the existence of conditionally stable prion variants maintained only at altered levels of Hsps, that could in principle be beneficial if the normal cellular function of a prion protein becomes detrimental to the cell in such conditions. A. S. Borchsenius and S. Müller contributed equally to this study.     

PSI(+)] aggregate enlargement in rnq1 nonprion domain mutants, leading to a loss of prion in yeast

[PIN(+)] is the prion form of the Rnq1 protein of unknown function in Saccharomyces cerevisiae. A glutamine/asparagine (Q/N)-rich C-terminal domain is necessary for the propagation of [PIN(+)], whereas the N-terminal region is non-Q/N-rich and considered the nonprion domain. Here, we isolated numerous single-amino-acid mutations in Rnq1, phenotypically similar to Rnq1Δ100, which inhibit [PSI(+)] propagation in the [PIN(+)] state, but not in the [pin(-)] state, when overproduced. The dynamics of the prion aggregates was analyzed by semi-denaturing detergent-agarose gel electrophoresis and fluorescence correlation spectroscopy. The results indicated that [PSI(+)] aggregates were enlarged in mother cells and, instead, not apparently transmitted into daughter cells. Under these conditions, the activity of Hsp104, a known prion disaggregase, was not affected when monitored for the thermotolerance of the rnq1 mutants. These [PSI(+)]-inhibitory rnq1 mutations did not affect [PIN(+)] propagation itself when over-expressed from a strong promoter, but instead destabilized [PIN(+)] when expressed from the weak authentic RNQ1 promoter. The majority of these mutated residues are mapped to the surface, and on one side, of contiguous α-helices of the nonprion domain of Rnq1, suggesting its involvement in interactions with a prion or a factor necessary for prion development.     

C-terminal truncation of the Sup35 protein increases the frequency of de novo generation of a prion-based [PSI+] determinant in Saccharomyces cerevisiae

The yeast non-Mendelian [PSI ⁺ ] determinant is presumed to be the manifestation of the aggregated prion-like state of the Sup35 protein. Plasmid-mediated amplification of the SUP35 gene greatly increases the frequency of Sup35p transition to this prion-like state. Here we show that the 3′-deletions of plasmid SUP35, leading to the C-terminal truncation of Sup35p, further increase the frequency of [PSI ⁺ ] induction despite a marked decrease in Sup35p expression levels. The data suggest that the presence of Sup35p N-terminal proteolytic fragments can cause [PSI ⁺ ] appearance in wild-type yeast cells. Received: 2 February / 24 June 1998     

Candida albicans Als proteins mediate aggregation with bacteria and yeasts.

Candida albicans occupies a microniche on mucosal surfaces where diverse microbial populations interact within a biofilm. Because C. albicans is intimately involved with other microbes in this environment we studied the interactions of C. albicans with other fungi and bacteria that form mixed microbial aggregates. Once aggregation is initiated, aggregates form rapidly and incorporate fungal as well as bacterial cells. The fungus formed mixed microbial aggregates with homotypic cells (i.e., self to self, e.g., C. albicans or Als1p-expressing yeast cells aggregating with cells bearing Als1p); with heterotypic cells (i.e., self to non-self, e.g., C. albicans or Alsp-expressing yeast cells aggregating with other Candida species); and with xenotypic cells (e.g., C. albicans or Alsp-expressing yeast cells forming aggregates with bacteria). When either of the C. albicans adhesins Als1p or Als5p was displayed on the surface of non-adherent Saccharomyces cerevisiae cells, the S. cerevisiae also mediated these mixed microbial interactions. Thus the Als adhesins are potentially important for the co-adhesion of mixed microbial communities in biofilms and on mucus surfaces.     

The PNM2 mutation in the prion protein domain of SUP35 has distinct effects on different variants of the [PSI+] prion in yeast

We have previously described different variants of the yeast prion [PSI ⁺ ] that can be obtained and maintained in the same genetic background. These [PSI ⁺ ] variants, which differ in the efficiency of nonsense suppression, mitotic stability and the efficiency of curing by GuHCl, may correspond to different [PSI ⁺ ] prion conformations of Sup35p or to different types of prion aggregates. Here we investigate the effects of overexpressing a mutant allele of SUP35 and find different effects on weak and strong [PSI ⁺ ] variants: the suppressor phenotype of weak [PSI ⁺ ] factors is increased, whereas the suppressor effect of strong [PSI ⁺ ] factors is reduced. The SUP35 mutation used was originally described as a “Psi no more” mutation (PNM2) because it caused loss of [PSI ⁺ ]. However, none of the [PSI ⁺ ] variants in the strains used in our study were cured by PNM2. Indeed, when overexpressed, PNM2 induced the de novo appearance of both weak and strong [PSI ⁺ ] variants with approximately the same efficiency as the overexpressed wild-type SUP35 allele. Our data suggest that the change in the region of oligopeptide repeats in the Sup35p N-terminus due to the PNM2 mutation modifies, but does not impair, the function of the prion domain of Sup35p. Received: 12 October / 15 December 1998     

NSI+] determinant has a pleiotropic phenotypic manifestation that is modulated by SUP35, SUP45, and VTS1 genes

We recently discovered the novel non-chromosomal determinant in Saccharomyces cerevisiae [NSI ⁺] (nonsense suppression inducer), which causes omnipotent nonsense suppression in strains where the Sup35 N-terminal domain is deleted. [NSI ⁺] possesses yeast prion features and does not correspond to previously identified yeast prion determinants. Here, we show that [NSI ⁺ ] enhances nonsense codon read-through and inhibits vegetative growth in S. cerevisiae. Using a large-scale overexpression screen to identify genes that impact the phenotypic effects of [NSI ⁺], we found that the SUP35 and SUP45 genes encoding the translation termination factors eRF3 and eRF1, respectively, modulate nonsense suppression in [NSI ⁺] strains. The VTS1 gene encodes an NQ-enriched RNA-binding protein that enhances nonsense suppression in [NSI ⁺] and [nsi ⁻] strains. We demonstrate that VTS1 overexpression, like [NSI ⁺] induction, causes translational read-through and growth defects in S. cerevisiae.     

Up-regulation of tRNA biosynthesis affects translational readthrough in maf1-delta mutant of Saccharomyces cerevisiae

Maf1p is a negative effector of RNA polymerase III in yeast. The maf1-delta mutation caused an increase in the level of cellular tRNAs, but a decrease of translational readthrough at nonsense codons. Using the lacZ- luc dual gene reporter system, we detected an almost twofold diminution of UAA and UAG readthrough in maf1-delta compared with the parental strain. The maf1-delta mutation did not affect the rate of protein biosynthesis and growth at standard conditions, but resulted in temperature-sensitive growth on non-fermentable carbon sources. We examined the correlation of the temperature sensitive and antisuppression phenotypes of maf1- Delta using a colour phenotype assay in the ade2-1 SUP11 strain. Antisuppression, but not the temperature-sensitive growth defect, was compensated either by increased dosage of SUP11or by [PSI(+)], the prion form of the translation termination factor Sup35p. Summarizing, the elevated tRNA levels in maf1- Delta increase translational fidelity and, independently, affect growth under special conditions.     

Mouse GSPT2, but not GSPT1, can substitute for yeast eRF3 in vivo

BACKGROUND: The termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs), eRF1 and eRF3. In mammals two genes encoding eRF3 structural homologues were identified and named GSPT1 and GSPT2. RESULTS: In the present study, we demonstrate that mouse mGSPT2 but not mGSPT1 could functionally substitute the essential yeast gene SUP35. However, we show that the complementation property of mGSPT1 protein is modified when NH2-tagged by GST. Since mGSPT1 and mGSPT2 differ mainly in their N-terminal regions, we developed a series of N-terminal deleted constructs and tested them for complementation in yeast. We found that at least amino acids spanning 84-120 of mGSPT1 prevent the complementation of sup35 mutation. The fact that chimeras between mGSPT1, mGSPT2 and yeast Sup35 complement the disruption of the SUP35 gene indicates that the N-terminal region of mGSPT1 is not sufficient by itself to prevent complementation. Complementation of the mutant with a double disruption of SUP35 and SUP45 genes is obtained when mGSPT2 and human eRF1 are co-expressed but not by co-expression of mGSPT1 and human eRF1. CONCLUSIONS: Our results strongly suggest that the two proteins (mGSPT1 and mGSPT2) are different. We hypothesize that the full length mGSPT1 does not have the properties expected for eRF3.