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Gorkovskiy et al. observed that many [PSI+ ] prion isolates, obtained in yeast with the mutant Hsp104T160M chaperone, propagate poorly in wild-type cells and suggested that Hsp104 is part of the cellular anti-prion system, curing many nascent [PSI+ ] variants. Here, we argue that the concept may require reassessment. We induced [PSI+ ] variants in both the wild-type and the mutant background. Three new variants were isolated in the T160M background. They exhibited lower thermostability, possessed novel structural features, and were inherently mutable, changing to well-characterized VH, VK, and VL variants in wild-type cells. In contrast, VH, VK, and VL of the wild-type background, could not change freely and were lost in the mutant, due to insufficient chaperone activity. Thus, mutant Hsp104 can impose as much restriction against emerging prion variants as the wild-type protein. Such restriction conserved the transmutable variants in the T160M background, since new structures mis-templated from them could not gain a foothold. We further demonstrate excess Hsp104T160M or Hsp104∆2-147 can eliminate nearly all of the [PSI+ ] variants in their native background. This finding contradicts the generally held belief that Hsp104-induced [PSI+ ] curing requires its N-terminal domain, and may help settling the current contention regarding how excess Hsp104 cures [PSI+ ].

Amyloids and their infectious subset, prions, represent fibrillary aggregates with regular structure. They are formed by proteins that are soluble in their normal state. In amyloid form, all or part of the polypeptide sequence of the protein is resistant to treatment with proteinase K (PK). Amyloids can have structural variants, which can be distinguished by the patterns of their digestion by PK. In this review, we describe and compare studies of the resistant cores of various amyloids from different organisms. These data provide insight into the fine structure of amyloids and their variants as well as raise interesting questions, such as those concerning the differences between amyloids obtained ex vivo and in vitro, as well as the manner in which folding of one region of the amyloid can affect other regions.

Cell death plays a major role in development, pathology and aging and can be triggered by various types of acute external and internal stimuli, such as chemicals or mutations. However, little is known about chronic cell death in the context of continuing cell division. Here, we performed a genome-wide search for mutants with this type of death in dividing bakers yeast by assaying the accumulation of phloxine B, which stains dead cells. We identified 83 essential and 43 non-essential gene mutants. Surprisingly, three distinct types of spatial distribution of dead cells in colonies were observed which corresponded to gene ontology enrichments for (i) DNA replication and repair, RNA processing, chromatin organization, and nuclear transport; (ii) mitosis and cytokinesis; and (iii) vesicular transport and glycosylation/cell wall homeostasis. We further developed methods for analyzing the death of newborn cells (DON) and cell death in real time using microfluidics-based microscopy which revealed rapid stochastic necrosis during bud generation or cytokinesis without prior division arrest. This coincided with commonality of sensitivity to some plasma membrane and cell-wall perturbing agents, as well as mitigating effects of increasing external pH for most of the tested mutants. Our results suggest that rapid stochastic necrosis during cell division is a common type of cell death resulting from the dysfunction of different genes, and that this type of death seems to have a common proximal cause which might be related to the properties of the cell wall and/or plasma membrane.

Proteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci, revealed by super-resolution microscopy, indicated that the foci were compressed between other entities. Based on our findings, we suggest a new model of cytosol architecture as a collection of numerous gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions., Summary: We describe a novel cellular response to hyperosmotic shock – rapid reversible formation of foci by various proteins. Data on foci behavior suggest a novel ‘beads in liquid’ cytosolic structure.

The yeast [PSI+] prion, formed by the Sup35 (eRF3) protein, can exist as multiple structural variants exhibiting phenotypic variation in the strength of nonsense suppression and mitotic stability. Structure of [PSI+] and its variation is only partly characterized. Here, we mapped the Sup35 proteinase K-resistant amyloid cores of 26 [PSI+] prions of different origin, isolated from yeast cells. In all cases the Sup35 amino acid residues 2-32 were fully resistant and the region up to residue 72 was partially resistant. Proteinase K-resistant structures were also found within regions 73-124, 125-153 and 154-221, but their presence differed between [PSI+] isolates. The [PSI+] phenotype depended mainly, if not solely, on the structure in region 2-72. Structures in region 73-221 were in some cases mitotically unstable and heterogenous. Two distinct digestion patterns were observed for the 2-72 fragment, which correlated with the “strong” and “weak” [PSI+] nonsense-suppressor phenotypes. All [PSI+] with a weak pattern were eliminated by multicopyHSP104gene and were not toxic when combined with multicopySUP35.[PSI+] with a strong pattern showed opposite properties, being resistant to multicopyHSP104and lethal in the presence of multicopySUP35. Thus, our data suggest existence of two distinct and reliably distinguishable structural classes of [PSI+] rather than a continuum of prions with gradually altering phenotype.ImportancePrions and amyloids are relatively novel and incompletely characterized structures. To understand them better, we mapped amyloid cores of 26 isolates of the Sup35 yeast prion using proteinase K digestion and mass spectrometry. We found that these cores are composed of up to four proteinase K-resistant elements spanning almost the whole length of Sup35 region inessential for viability. However, only the N-terminal element was present in all structures. There are many variants of the Sup35 prion, and these are usually roughly combined into two groups, “strong” and “weak”, based on the strength of their nonsense-suppressor phenotype. However, it was not clear whether such groups could be distinguished by any reliable qualitative criteria. Our data indicate that these groups do exist and can be reliably distinguished based on the N-terminal element digestion pattern and the effects of the multicopySUP35andHSP104genes on these prion variants.

Proteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci revealed by super-resolution microscopy indicated that the foci were compressed between other entities. Based on our findings, we propose a new model of the cytosol architecture as a collection of numerous of gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions.

This commentary describes scientific path and accomplishments of our late colleague, Prof. Michael D. Ter-Avanesyan, who made several seminal contributions into prion research.

Proteins with expanded polyglutamine (polyQ) regions are prone to form amyloids, which can cause diseases in humans and toxicity in yeast. Recently, we showed that in yeast non-toxic amyloids of Q-rich proteins can induce aggregation and toxicity of wild type huntingtin (Htt) with a short non-pathogenic polyglutamine tract. Similarly to mutant Htt with an elongated N-terminal polyQ sequence, toxicity of its wild type counterpart was mediated by induced aggregation of the essential Sup35 protein, which contains a Q-rich region. Notably, polymerization of Sup35 was not caused by the initial benign amyloids and, therefore, aggregates of wild type Htt acted as intermediaries in seeding Sup35 polymerization. This exemplifies a protein polymerization cascade which can generate a network of interdependent polymers. Here we discuss cross-seeded protein polymerization as a possible mechanism underlying known interrelations between different polyQ diseases. We hypothesize that similar mechanisms may enable proteins, which possess expanded Q-rich tracts but are not associated with diseases, to promote the development of polyQ diseases.

Mammalian prions are infectious agents of proteinaceous nature that cause several incurable neurodegenerative diseases. Interspecies transmission of prions is usually impeded or impossible. Barriers in prion transmission are caused by small interspecies differences in the primary structure of prion proteins. The barriers can also depend on the strain (variant) of a transmitted prion. Interspecies barriers were also shown for yeast prions, which define some heritable phenotypes. Yeast prions reproduce all the main traits of prion transmission barriers observed for mammals. This allowed to show that the barrier in prion transmission can be observed even upon copolymerization of two prionogenic proteins. Available data allow elucidation of the mechanisms that impede prion transmission or make it impossible.

Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI(+)] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI(+)] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI(+)] cells often resulted in frequent loss of the native [PSI(+)] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI(+)] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI(+)] variant. The observed loss of S. cerevisiae [PSI(+)] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.

In yeast, fragmentation of amyloid polymers by the Hsp104 chaperone allows them to propagate as prions. The prion-forming domain of the yeast Sup35 protein is rich in glutamine, asparagine, tyrosine, and glycine residues, which may define its prion properties. Long polyglutamine stretches can also drive amyloid polymerization in yeast, but these polymers are unable to propagate because of poor fragmentation and exist through constant seeding with the Rnq1 prion polymers. We proposed that fragmentation of polyglutamine amyloids may be improved by incorporation of hydrophobic amino acid residues into polyglutamine stretches. To investigate this, we constructed sets of polyglutamine with or without tyrosine stretches fused to the non-prion domains of Sup35. Polymerization of these chimeras started rapidly, and its efficiency increased with stretch size. Polymerization of proteins with polyglutamine stretches shorter than 70 residues required Rnq1 prion seeds. Proteins with longer stretches polymerized independently of Rnq1 and thus could propagate. The presence of tyrosines within polyglutamine stretches dramatically enhanced polymer fragmentation and allowed polymer propagation in the absence of Rnq1 and, in some cases, of Hsp104.

The yeast [PSI+] determinant is related to formation of large prion-like aggregates of the conformationally altered Sup35 protein. Here, we show that these aggregates are composed of small Sup35 prion polymers and associated proteins. In contrast to other protein complexes of yeast lysates, but similarly to amyloid fibers, these polymers are insoluble in SDS at room temperature. The polymers on average are about 30-fold smaller than the aggregates and comprise from 8 to 50 Sup35 monomers. The size of polymers is characteristic of a given [PSI+] variant and differs between the variants. Blocked expression of Hsp104 chaperone causes gradual increase in the size of prion polymers, while inactivation of Hsp104 by guanidine HCl completely stops their fragmentation, which shows indispensability of Hsp104 for this process.

The Sup35 (eRF3) translation termination factor of Saccharomyces cerevisiae can undergo a prion-like conformational conversion, thus resulting in the [PSI(+)] nonsense-suppressor determinant. In vivo this process depends critically on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)]. The use of artificial prion [PSI(+)PS] based on a hybrid Sup35PS with prion domain from the yeast Pichia methanolica allowed us to uncover three more chaperones, Ssb1, Ssa1, and Ydj1, whose overexpression can cure prion determinants. Here, we used the [PSI(+)PS] to search a multicopy yeast genomic library for novel factors able to cure prions. It was found that overexpression of the Hsp40 family chaperones Sis1 and Ynl077w, chaperone Sti1, transcriptional factors Sfl1 and Ssn8, and acidic ribosomal protein Rpp0 can interfere with propagation and manifestation of [PSI(+)PS] in a prion strain-specific manner. Some of these factors also affected the manifestation and propagation of conventional [PSI(+)]. Excess of Sfl1, Ssn8, and Rpp0 influenced at least one of the tested chaperone-specific promoters, SSA4, HSP104, and model promoters, with either the heat shock or stress response elements. Thus, the induction of chaperone expression by these proteins could explain their prion-curing effects.

The yeast cytoplasmically-inherited nonsense suppressor [PSI(+)] determinant is presumed to be a manifestation of the aggregated prion-like state of the Sup35 protein. Overexpression of the Sup35 protein induces generation of [PSI(+)] determinants with various suppressor efficiency and mitotic stabilities. Here, we demonstrate that the relative frequency of appearance of [PSI(+)] with different properties depends on the SUP35 allele used to induce their generation. The difference in properties of [PSI(+)] determinants was preserved after their transmission from one yeast strain to another. This difference correlated with variation in properties of the Sup35 protein. A novel type of prion instability was observed: some [PSI(+)] with weak suppressor efficiency could convert spontaneously into strong suppressor determinants.

The [PSI(+)] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI(+)] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI(+)] against Hsp104 [3,4]. We used an artificial [PSI(+)(PS)] based on the Sup35 prion-forming domain from yeast Pichia methanolica [5] to find other prion-curing factors. Both [PSI(+)(PS)] and [PSI(+)] have prion 'strains', differing in their suppressor efficiency and mitotic stability. We show that [PSI(+)(PS)] and a 'weak' strain of [PSI(+)] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI(+)(PS)] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.

The Sup35 protein (Sup35p) of Saccharomyces cerevisiae is a translation termination factor of the eRF3 family. The proteins of this family possess a conservative C-terminal domain responsible for translation termination and N-terminal extensions of different structure. The N-terminal domain of Sup35p defines its ability to undergo a heritable prion-like conformational switch, which is manifested as the cytoplasmically inherited [PSI(+)] determinant. Here, we replaced the N-terminal domain of S.cerevisiae Sup35p with an analogous domain from Pichia methanolica. Overexpression of hybrid Sup35p induced the de novo appearance of cytoplasmically inherited suppressor determinants manifesting key genetic and biochemical traits of [PSI(+)]. In contrast to the conventional [PSI(+)], 'hybrid' [PSI(+)] showed lower mitotic stability and preserved their suppressor phenotype upon overexpression of the Hsp104 chaperone protein. The lack of Hsp104 eliminated both types of [PSI(+)]. No transfer of prion state between the two Sup35p variants was observed, which reveals a 'species barrier' for the [PSI(+)] prions. The data obtained show that prion properties are conserved within at least a part of this protein family.

The methods currently used for protein extraction from yeast are either laborious or insufficiently reliable. Here I report a method for protein extraction for electrophoretic analysis that is both easy and reliable. In this method, yeast cells are subjected to mild alkali treatment and then boiled in a standard electrophoresis loading buffer. The method was tested for different strains of Saccharomyces cerevisiae and for yeast Hansenula polymorpha DL-1. It yields virtually complete extraction independently of the strain, growth conditions and protein molecular weight and allows working with small amounts of yeast cells grown on agar plates. Copyright © 2000 John Wiley & Sons, Ltd.

We have previously shown that multicopy plasmids containing the complete SUP35 gene are able to induce the appearance of the non-Mendelian factor [PSI]. This result was later interpreted by others as a crucial piece of evidence for a model postulating that [PSI] is a self-modified, prion-like conformational derivative of the Sup35 protein. Here we support this interpretation by proving that it is the overproduction of Sup35 protein, and not the excess of SUP35 DNA or mRNA that causes the appearance of [PSI]. We also show that the “prion-inducing domain” of Sup35p is in the N-terminal region, which, like the “prion-inducing domain” of another yeast prion, Ure2p, was previously shown to be distinct from the functional domain of the protein. This suggests that such a chimeric organization may be a common pattern of some prion elements. Finally, we find that [PSI] factors of different efficiencies and different mitotic stabilities are induced in the same yeast strain by overproduction of the identical Sup35 protein. We suggest that the different [PSI]-containing derivatives are analogous to the mysterious mammalian prion strains and result from different conformational variants of Sup35p.

The Sup35p protein of yeast Saccharomyces cerevisiae is a homologue of the polypeptide chain release factor 3 (eRF3) of higher eukaryotes. It has been suggested that this protein may adopt a specific self-propagating conformation, similar to mammalian prions, giving rise to the [psi+] nonsense suppressor determinant, inherited in a non-Mendelian fashion. Here we present data confirming the prion-like nature of [psi+]. We show that Sup35p molecules interact with each other through their N-terminal domains in [psi+], but not [psi-] cells. This interaction is critical for [psi+] propagation, since its disruption leads to a loss of [psi+]. Similarly to mammalian prions, in [psi+] cells Sup35p forms high molecular weight aggregates, accumulating most of this protein. The aggregation inhibits Sup35p activity leading to a [psi+] nonsense-suppressor phenotype. N-terminally altered Sup35p molecules are unable to interact with the [psi+] Sup35p isoform, remain soluble and improve the translation termination in [psi+] strains, thus causing an antisuppressor phenotype. The overexpression of Hsp104p chaperone protein partially solubilizes Sup35P aggregates in the [psi+] strain, also causing an antisuppressor phenotype. We propose that Hsp104p plays a role in establishing stable [psi+] inheritance by splitting up Sup35p aggregates and thus ensuring equidistribution of the prion-like Sup35p isoform to daughter cells at cell divisions.

The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro. We show, using the two-hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo. The ability of Sup45p C-terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro. Although overexpression of either the SUP45 or SUP35 genes alone did not reduce the efficiency of codon-specific tRNA nonsense suppression, the simultaneous overexpression of both the SUP35 and SUP45 genes in nonsense suppressor tRNA-containing strains produced an antisuppressor phenotype. These data are consistent with Sup35p and Sup45p forming a complex with release factor properties. Furthermore, overexpression of either Xenopus or human eRF1 (SUP45) genes also resulted in anti-suppression only if that strain was also overexpressing the yeast SUP35 gene. Antisuppression is a characteristic phenotype associated with overexpression of both prokaryote and mitochondrial release factors. We propose that Sup45p and Sup35p interact to form a release factor complex in yeast and that Sup35p, which has GTP binding sequence motifs in its C-terminal domain, provides the GTP hydrolytic activity which is a demonstrated requirement of the eukaryote translation termination reaction.

Amyloids are fibrillar protein aggregates resulting from non-covalent autocatalytic polymerization of various structurally and functionally unrelated proteins. Previously we have selected DNA aptamers, which bind specifically to the in vitro assembled amyloid fibrils of the yeast prionogenic protein Sup35. Here we show that such DNA aptamers can be used to detect SDS-insoluble amyloid aggregates of the Sup35 protein, and of some other amyloidogenic proteins, including mouse PrP, formed in yeast cells. The obtained data suggest that these aggregates and the Sup35 amyloid fibrils assembled in vitro possess common conformational epitopes recognizable by aptamers. The described DNA aptamers may be used for detection of various amyloid aggregates in yeast and, presumably, other organisms.

Fragmentation of amyloid polymers by the chaperone Hsp104 allows them to propagate as prions in yeast. The factors which determine the frequency of fragmentation are unclear, though it is often presumed to depend on the physical strength of prion polymers. Proteins with long polyglutamine stretches represent a tractable model for revealing sequence elements required for polymer fragmentation in yeast, since they form poorly fragmented amyloids. Here we show that interspersion of polyglutamine stretches with various amino acid residues differentially affects the in vivo formation and fragmentation of the respective amyloids. Aromatic residues tyrosine, tryptophan and phenylalanine strongly stimulated polymer fragmentation, leading to the appearance of oligomers as small as dimers. Alanine, methionine, cysteine, serine, threonine and histidine also enhanced fragmentation, while charged residues, proline, glycine and leucine inhibited polymerization. Our data indicate that fragmentation frequency primarily depends on the recognition of fragmentation-promoting residues by Hsp104 and/or its co-chaperones, rather than on the physical stability of polymers. This suggests that differential exposure of such residues to chaperones defines prion variant-specific differences in polymer fragmentation efficiency.

Prions are not uniquely associated with rare fatal neurodegenerative diseases in the animal kingdom; prions are also found in fungi and in particular the yeast Saccharomyces cerevisiae. As with animal prions, fungal prions are proteins able to exist in one or more self-propagating alternative conformations, but show little primary sequence relationship with the mammalian prion protein PrP. Rather, fungal prions represent a relatively diverse collection of proteins that participate in key cellular processes such as transcription and translation. Upon switching to their prion form, these proteins can generate stable, sometimes beneficial, changes in the host cell phenotype. Much has already been learnt about prion structure, and propagation and de novo generation of the prion state through studies in yeast and these findings are reviewed here.

In eukaryotic cells amyloid aggregates may incorporate various functionally unrelated proteins. In mammalian diseases this may cause amyloid toxicity, while in yeast this could contribute to prion phenotypes. Insolubility of amyloids in the presence of strong ionic detergents, such as SDS or sarcosyl, allows discrimination between amorphous and amyloid aggregates. Here, we used this property of amyloids to study the interdependence of their formation in yeast. We observed that SDS-resistant polymers of proteins with extended polyglutamine domains caused the appearance of SDS or sarcosyl-insoluble polymers of three tested chromosomally-encoded Q/N-rich proteins, Sup35, Rnq1 and Pub1. These polymers were non-heritable, since they could not propagate in the absence of polyglutamine polymers. Sup35 prion polymers caused the appearance of non-heritable sarcosyl-resistant polymers of Pub1. Since eukaryotic genomes encode hundreds of proteins with long Q/N-rich regions, polymer interdependence suggests that conversion of a single protein into polymer form may significantly affect cell physiology by causing partial transfer of other Q/N-rich proteins into a non-functional polymer state.

Yeast prion determinants are related to polymerization of some proteins into amyloid-like fibers. The [PSI(+)] determinant reflects polymerization of the Sup35 protein. Fragmentation of prion polymers by the Hsp104 chaperone represents a key step of the prion replication cycle. The frequency of fragmentation varies depending on the structure of the prion polymers and defines variation in the prion phenotypes, e.g., the suppressor strength of [PSI(+)] and stability of its inheritance. Besides [PSI(+)], overproduction of Sup35 can produce nonheritable phenotypically silent Sup35 amyloid-like polymers. These polymers are fragmented poorly and are present due to efficient seeding with the Rnq1 prion polymers, which occurs by several orders of magnitude more frequently than seeding of [PSI(+)] appearance. Such Sup35 polymers resemble human nonprion amyloids by their nonheritability, mode of appearance and increased size. Thus, a single protein, Sup35, can model both prion and nonprion amyloids. In yeast, these phenomena are distinguished by the frequency of polymer fragmentation. We argue that in mammals the fragmentation frequency also represents a key factor defining differing properties of prion and nonprion amyloids, including infectivity. By analogy with the Rnq1 seeding of nonheritable Sup35 polymers, the "species barrier" in prion transmission may be due to seeding by heterologous prion of nontransmissible type of amyloid, rather than due to the lack of seeding.

In yeast, fragmentation of amyloid polymers by the Hsp104 chaperone allows them to propagate as prions. The prion-forming domain of the yeast Sup35 protein is rich in glutamine, asparagine, tyrosine, and glycine residues, which may define its prion properties. Long polyglutamine stretches can also drive amyloid polymerization in yeast, but these polymers are unable to propagate because of poor fragmentation and exist through constant seeding with the Rnq1 prion polymers. We proposed that fragmentation of polyglutamine amyloids may be improved by incorporation of hydrophobic amino acid residues into polyglutamine stretches. To investigate this, we constructed sets of polyglutamine with or without tyrosine stretches fused to the non-prion domains of Sup35. Polymerization of these chimeras started rapidly, and its efficiency increased with stretch size. Polymerization of proteins with polyglutamine stretches shorter than 70 residues required Rnq1 prion seeds. Proteins with longer stretches polymerized independently of Rnq1 and thus could propagate. The presence of tyrosines within polyglutamine stretches dramatically enhanced polymer fragmentation and allowed polymer propagation in the absence of Rnq1 and, in some cases, of Hsp104.

SUP2 (SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part and a region homologous to EF-1 alpha. The comparison of amino acid sequences of the Sup2 proteins reveals high conservation (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae were found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.

Amyloid aggregates are associated with a number of mammalian neurodegenerative diseases. Infectious aggregates of the mammalian prion protein PrP(sc) are hallmarks of transmissible spongiform encephalopathies in humans and cattle (Griffith, 1967; Legname et al., 2004; Prusiner, 1982; Silveira et al., 2004). Likewise, SDS-stable aggregates and low-n oligomers of the Abeta peptide (Selkoe et al., 1982; Walsh et al., 2002) cause toxic effects associated with Alzheimer's disease (Selkoe, 2004). The discovery of prions in lower eukaryotes, for example, yeast prions [PSI(+)], [PIN(+)], and [URE3] suggested that prion phenomena may represent a fundamental process that is widespread among living organisms (Chernoff, 2004; Uptain and Lindquist, 2002; Wickner, 1994; Wickner et al., 2004). These protein structures are more stable than other cellular protein complexes, which generally dissolve in SDS at room temperature. In contrast, the prion polymers withstand these conditions, while losing their association with their non-prion partners. These bulky protein particles cannot be analyzed in polyacrylamide gels, because their pores are too small to allow the passage and acceptable resolution of the large complexes. This problem was first circumvented by Kryndushkin et al. (2003), who used Western blots of protein complexes separated on agarose gels to analyze the sizes of SDS-resistant protein complexes associated with the yeast prion [PSI(+)]. Further studies have used this approach to characterize [PSI(+)] (Allen et al., 2005; Bagriantsev and Liebman, 2004; Salnikova et al., 2005), and another yeast prion [PIN(+)] (Bagriantsev and Liebman, 2004). In this chapter, we use this method to assay amyloid aggregates of recombinant proteins Sup35NM and Abeta42 and present protocols for Western blot analysis of high molecular weight (5 MDa) amyloid aggregates resolved in agarose gels. The technique is suitable for the analysis of any large proteins or SDS-stable high molecular weight complexes.

Amyloids and prions represent aggregates of misfolded proteins, which consist of protein polymer fibrils with cross-beta sheet structure. Understanding of their occurrence and role is developing rapidly. Initially, they were found associated with mammalian diseases, mainly of neurodegenerative nature. Now they are known to relate to a range of non-disease phenomena in different species from mammals to lower eukaryotes. Uncovering new prion- and amyloid-related processes may be helped greatly by a procedure for purification of amyloid polymers. Studies of growth and propagation of these polymers require methods for determination of their size. Here, we describe such methods. They rely on the treatment with cold SDS or Sarcosyl detergents, which do not dissolve amyloids, but solubilize almost all non-amyloid complexes and associations between amyloid fibers. This allows purifying amyloids by centrifugation in the presence of these detergents. The size of amyloid polymers may be analyzed by electrophoresis in agarose gels containing SDS. Two procedures are described for determining the proportion between polymers and monomers of a particular protein using polyacrylamide gels.

The cytoplasmic [PSI+] determinant of Saccharomyces cerevisiae is the prion form of the Sup35 protein. Oligopeptide repeats within the Sup35 N-terminal domain (PrD) presumably are required for the stable [PSI+] inheritance that in turn involves fragmentation of Sup35 polymers by the chaperone Hsp104. The nonsense suppressor [PSI+] phenotype can vary in efficiency probably due to different inheritable Sup35 polymer structures. Here we study the ability of Sup35 mutants with various deletions of the oligopeptide repeats to support [PSI+] propagation. We define the minimal region of the Sup35–PrD necessary to support [PSI+] as amino acids 1–64, which include the first two repeats, although a longer fragment, 1–83, is required to maintain weak [PSI+] variants. Replacement of wild-type Sup35 with deletion mutants decreases the strength of the [PSI+] phenotype. However, with one exception, reintroducing the wild-type Sup35 restores the original phenotype. Thus, the specific prion fold defining the [PSI+] variant can be preserved by the mutant Sup35 protein despite the change of phenotype. Coexpression of wild-type and mutant Sup35 containing three, two, one, or no oligopeptide repeats causes variant-specific [PSI+] elimination. These data suggest that [PSI+] variability is primarily defined by differential folding of the Sup35–PrD oligopeptide-repeat region.

Amyloid aggregates are associated with a number of mammalian neurodegenerative diseases. Infectious aggregates of the mammalian prion protein PrPsc are hallmarks of transmissible spongiform encephalopathies in humans and cattle (Griffith, 1967; Legname et al., 2004; Prusiner, 1982; Silveira et al., 2004). Likewise, SDS‐stable aggregates and low‐n oligomers of the Aβ peptide (Selkoe et al., 1982; Walsh et al., 2002) cause toxic effects associated with Alzheimer's disease (Selkoe, 2004). The discovery of prions in lower eukaryotes, for example, yeast prions [PSI+], [PIN+], and [URE3] suggested that prion phenomena may represent a fundamental process that is widespread among living organisms (Chernoff, 2004; Uptain and Lindquist, 2002; Wickner, 1994; Wickner et al., 2004). These protein structures are more stable than other cellular protein complexes, which generally dissolve in SDS at room temperature. In contrast, the prion polymers withstand these conditions, while losing their association with their non‐prion partners. These bulky protein particles cannot be analyzed in polyacrylamide gels, because their pores are too small to allow the passage and acceptable resolution of the large complexes. This problem was first circumvented by Kryndushkin et al. (2003), who used Western blots of protein complexes separated on agarose gels to analyze the sizes of SDS‐resistant protein complexes associated with the yeast prion [PSI+]. Further studies have used this approach to characterize [PSI+] (Allen et al., 2005; Bagriantsev and Liebman, 2004; Salnikova et al., 2005), and another yeast prion [PIN+] (Bagriantsev and Liebman, 2004). In this chapter, we use this method to assay amyloid aggregates of recombinant proteins Sup35NM and Aβ42 and present protocols for Western blot analysis of high molecular weight (>5 MDa) amyloid aggregates resolved in agarose gels. The technique is suitable for the analysis of any large proteins or SDS‐stable high molecular weight complexes.

Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. eRF1 recognizes nonsense codons UAA, UAG, and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRF1-dependent manner. In the yeast Saccharomyces cerevisiae, eRF1 and eRF3 are encoded by the SUP45 and SUP35 genes, respectively. Here we show that in yeast shortage of any one of the release factors was accompanied by a reduction in the levels of the other release factor and resulted in a substantial increase of nonsense codon readthrough. Besides, repression of the genes encoding these factors caused different effects on cell morphology. Repression of the SUP35 gene caused accumulation of cells of increased size with large buds. This was accompanied by the disappearance of actin cytoskeletal structures, impairment of the mitotic spindle structure, and defects in nuclei division and segregation in mitosis. The evolutionary conserved C-terminal domain of eRF3 similar to the elongation factor EF-1alpha was responsible for these effects. Repression of the SUP45 gene caused accumulation of unbudded cells with 2C and higher DNA content, indicating that DNA replication is uncoupled from budding. The data obtained suggest that eRF1 and eRF3 play additional, nontranslational roles in the yeast cell.

Yeast SUP7 or SUP11 nonsense suppressors have no phenotypic expression in strains deficient in the isopentenylation of A37 in tRNA. Here we show that such strains spontaneously produce cells with a nonsense suppressor phenotype which is related to the cytoplasmically inherited determinant and manifests all the key features of the [PSI +] prion. A screen of a multicopy yeast genomic library for genes that inactivate the [PSI +]-related suppressor phenotype resulted in the isolation of the SSB1 gene. Moreover, we demonstrate that multicopy plasmid encoding the Ssb1 chaperone cures cells of the [PSI +] prion.

The SUP35 gene of Saccharomyces cerevisiae encodes the polypeptide chain release factor eRF3. This protein (also called Sup35p) is thought to be able to undergo a heritable conformational switch, similarly to mammalian prions, giving rise to the cytoplasmically inherited Psi+ determinant. A dominant mutation (PNM2 allele) in the SUP35 gene causing a Gly58-->Asp change in the Sup35p N-terminal domain eliminates Psi+. Here we observed that the mutant Sup35p can be converted to the prion-like form in vitro, but such conversion proceeds slower than that of wild-type Sup35p. The overexpression of mutant Sup35p induced the de novo appearance of Psi+ cells containing the prion-like form of mutant Sup35p, which was able to transmit its properties to wild-type Sup35p both in vitro and in vivo. Our data indicate that this Psi+-eliminating mutation does not alter the initial binding of Sup35p molecules to the Sup35p Psi+-specific aggregates, but rather inhibits its subsequent prion-like rearrangement and/or binding of the next Sup35p molecule to the growing prion-like Sup35p aggregate.

By analogy with the yeast model, the fragmentation of prion and amyloid polymers performed by Hsp104p homologs or other factors should influence their propagation and accumulation. The fragmentation might accelerate accumulation of prion and amyloid aggregates, but it might also serve to dissolve these aggregates if the fragmenting activity is high enough. The level of chaperone activity could control the appearance of amyloids and explain their age-dependent onset. The fragmentation of prions would be essential for their transmissibility, but would not be so critical for amyloid polymers, which are not transmissible and can appear by nucleation.The different frequency of fragmentation could explain the different properties of prions in two human diseases. The absence of observable fibrils in some cases of human CJD probably reflects a high level of PrPSc fragmentation. Another human prion disease, Gerstmann-Straussler-Sheinker syndrome, is characterized by filamentous, amyloid-like PrP deposits and a slower disease progression than CJD. These phenotypes could be related: more frequent fragmentation of CJD fibrils could mean higher overall polymerization speed and faster disease progression.In vitro, yeast Hsp104p interacted specifically with Sup35p, PrP and β-amyloid 1–42 peptide (Schirmer and Lindquist 1997xSchirmer, E.C and Lindquist, S. Proc. Natl. Acad. Sci. USA. 1997; 94: 13932–13937Crossref | PubMed | Scopus (75)See all ReferencesSchirmer and Lindquist 1997), and moreover, promoted the prion conversion of PrP (DebBurman et al. 1997xDebBurman, S.K, Raymond, G.J, Caughey, B, and Lindquist, S. Proc. Natl. Acad. Sci. USA. 1997; 94: 13938–13943Crossref | PubMed | Scopus (202)See all ReferencesDebBurman et al. 1997). These results suggest that Hsp104p analogs in mammals could also interact with such proteins and participate in prion and amyloid formation. Hsp104p is likely to occur in animals, since it shows similarity to the Clp/Hsp100 family of bacterial and eukaryotic chaperones and is functionally conserved between yeast and plants. However, in contrast to yeast prions, which propagate inside cells, mammalian prions and most amyloids occur extracellularly. Hsp104p is not secreted in yeast and it is likely to be so in mammals as well. At least one might expect different levels of chaperone fragmenting activity and probably even different factors causing fragmentation in intra- and extracellular compartments.Thus, in terms of the type and levels of fragmenting activity the best analogy with yeast prions may be found in intracellular amyloid diseases, related to the expansion of polyglutamine tracts in some human proteins. These diseases are accompanied by neuronal nuclear inclusions, formed by the respective polyglutamine proteins. The expanded polyglutamine domain of one such protein, huntingtin, was shown to form amyloid fibrils in vitro (Scherzinger et al. 1997xScherzinger, E, Lurz, R, Turmaine, M, Mangiarini, L, Hollenbach, B, Hasenbank, R, Bates, G.P, Davies, S.W, Lehrach, H, and Wanker, E.E. Cell. 1997; 90: 549–558Abstract | Full Text | Full Text PDF | PubMed | Scopus (833)See all ReferencesScherzinger et al. 1997). The abundance of glutamine and the related amino acid asparagine is also a characteristic feature of prion domains of Sup35p and Ure2p of S. cerevisiae. This feature is common for Sup35p from different yeast species and fungi, although not from higher eukaryotes. The importance of these residues was further demonstrated by the finding that the mutations which interfere with the prion properties of Sup35p fall within a short amino-terminal fragment, which is rich in Gln and Asn (DePace et al. 1998xDePace, A.H, Santoso, A, Hillner, P, and Weissman, J.S. Cell. 1998; 93: 1241–1252Abstract | Full Text | Full Text PDF | PubMed | Scopus (276)See all ReferencesDePace et al. 1998). The yeast prions and polyglutamine proteins might be considered as a single structural class, in contrast to other amyloidogenic proteins, which are not abundant in Gln and Asn. These similarities suggest that chaperones like Hsp104p might play an important role in diseases associated with expanded polyglutamine tracts.

The yeast cytoplasmically inherited genetic determinant [ PSI + ] is presumed to be a manifestation of the prion-like properties of the Sup35 protein (Sup35p). Here, cell-free conversion of Sup35p from [ psi − ] cells (Sup35p psi − ) to the prion-like [ PSI + ]-specific form (Sup35p PSI + ) was observed. The conversion reaction could be repeated for several consecutive cycles, thus modeling in vitro continuous [ PSI + ] propagation. Size fractionation of lysates of [ PSI + ] cells demonstrated that the converting activity was associated solely with Sup35p PSI + aggregates, which agrees with the nucleation model for [ PSI + ] propagation. Sup35p PSI + was purified and showed high conversion activity, thus confirming the prion hypothesis for Sup35p.

The SUP45 and SUP35 genes of Saccharomyces cerevisiae encode polypeptide chain release factors eRF1 and eRF3, respectively. It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian [PSI+] nonsense suppressor determinant. In a [PSI+] state, Sup35p forms high-molecular-weight aggregates which may inhibit Sup35p activity, leading to the [PSI+] phenotype. Sup35p is composed of the N-terminal domain (N) required for [PSI+] maintenance, the presumably nonfunctional middle region (M), and the C-terminal domain (C) essential for translation termination. In this study, we observed that the N domain, alone or as a part of larger fragments, can form aggregates in [PSI+] cells. Two sites for Sup45p binding were found within Sup35p: one is formed by the N and M domains, and the other is located within the C domain. Similarly to Sup35p, in [PSI+] cells Sup45p was found in aggregates. The aggregation of Sup45p is caused by its binding to Sup35p and was not observed when the aggregated Sup35p fragments did not contain sites for Sup45p binding. The incorporation of Sup45p into the aggregates should inhibit its activity. The N domain of Sup35p, responsible for its aggregation in [PSI+] cells, may thus act as a repressor of another polypeptide chain release factor, Sup45p. This phenomenon represents a novel mechanism of regulation of gene expression at the posttranslational level.

Genetic studies have indicated that the product of the yeast SUP45 gene encodes a component of the translational-termination machinery. In higher eukaryotes, genes similar to SUP45 encode eukaryote release factor 1 (eRF1), which has a stop-codon-dependent peptidyl-release activity. Using a conditional-lethal mutant allele of SUP45 (sup45-2) and a combination of in vivo and in vitro approaches, we demonstrate that the product of the SUP45 gene (Sup45p or eRF1) is a factor required for translation termination in yeast. A homologous in vitro assay based on suppressor-tRNA-mediated readthrough of stop codons is used to show that a translating lysate from a sup45-2 mutant strain exhibits a termination defect when heated for short periods to greater than the non-permissive temperature (37 degrees C). This defect can be complemented with a purified preparation of Sup45p (eRF1) expressed in Eschericha coli. The termination defect in this strain appears to be due to an inability of the Sup45p protein to bind the ribosome, resulting in vivo in a reduced ability of Sup45p to release nascent polypeptides from the ribosome at the non-permissive temperature. Cell-free translation lysates from the sup45-2 strain do not show a defect in sense-codon translation at the non-permissive temperature. These data demonstrate that yeast eRF1 plays a role in translation termination and is functionally equivalent to its higher eukaryotic homologues.

The SUP35 gene of yeast Saccharomyces cerevisiae encodes a 76.5-kD ribosome-associated protein (Sup35p), the C-terminal part of which exhibits a high degree of similarity to EF-1 alpha elongation factor, while its N-terminal region is unique. Mutations in or overexpression of the SUP35 gene can generate an omnipotent suppressor effect. In the present study the SUP35 wild-type gene was replaced with deletion alleles generated in vitro that encode Sup35p lacking all or a part of the unique N-terminal region. These 5'-deletion alleles lead, in a haploid strain, simultaneously to an antisuppressor effect and to loss of the non-Mendelian determinant [psi+]. The antisuppressor effect is dominant while the elimination of the [psi+] determinant is a recessive trait. A set of the plasmid-borne deletion alleles of the SUP35 gene was tested for the ability to maintain [psi+]. It was shown that the first 114 amino acids of Sup35p are sufficient to maintain the [psi+] determinant. We propose that the Sup35p serves as a trans-acting factor required for the maintenance of [psi+].

SUP35 is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C-terminal part similar to the elongation factor EF-1 alpha and a unique N-terminal sequence of 253 amino acids. Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence. Functional studies of these deletion mutants showed that: (i) only the EF-1 alpha-like C-terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N-terminal part of the Sup35 protein or the full-length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of [psi+] strains; (iii) expression of the C-terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N- or C-terminal segments of the Sup35 protein can bind to 80S ribosomes. Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.

The study of suppressor mutations that affect translational fidelity provides an efficient approach for identifying new components of the translational machinery in eukaryotes. Some of the identified genes controlling fidelity in lower eukaryotes presumably code for ribosomal proteins (Dequard-Chablat et al, 1986; All-Robyn et al, 1990; Hinnebusch and Liebman, 1991) and translation factors (Sandbaken and Culbertson, 1988; Kushnirov et al, 1988; Breining and Piepersberg, 1986). However, functions of the majority of these genes have not been established.

Background Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl – dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough.