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Compositionally biased regions (CBRs), ie, tracts that are dominated by a subset of residue types, are common features of eukaryotic proteins. These are often found bounded within or almost coterminous with intrinsically disordered or 'natively unfolded' parts. Here, it is investigated how the function of such intrinsically disordered compositionally biased regions (ID-CBRs) is directly linked to their compositional traits, focusing on the well-characterized yeast (Saccharomyces cerevisiae) proteome as a test case. The ID-CBRs that are clustered together using compositional distance are discovered to have clear functional linkages at various levels of diversity. The specific case of the Sup35p and Rnq1p proteins that underlie causally linked prion phenomena ([PSI+] and [RNQ+]) is highlighted. Their prion-forming ID-CBRs are typically clustered very close together indicating some compositional engendering for [RNQ+] seeding of [PSI+] prions. Delving further, ID-CBRs with distinct types of residue patterning such as 'blocking' or relative segregation of residues into homopeptides are found to have significant functional trends. Specific examples of such ID-CBR functional linkages that are discussed are: Q/N-rich ID-CBRs linked to transcriptional coactivation, S-rich to transcription-factor binding, R-rich to DNA-binding, S/E-rich to protein localization, and D-rich linked to chromatin remodelling. These data may be useful in informing experimental hypotheses for proteins containing such regions. [ABSTRACT FROM AUTHOR]

Chaperone networks are required for the shearing and generation of transmissible propagons from pre‐existing prion aggregates. However, other cellular networks needed for maintaining yeast prions are largely uncharacterized. Here, we establish a novel role for actin networks in prion maintenance. The [PIN+] prion, also known as [RNQ+], exists as stable variants dependent upon the chaperone machinery for the transmission of propagons to daughter cells during cell division and cytoplasmic transfer. Loss of the Hsp104 molecular chaperone leads to the growth of prion particles until they are too large to be transmitted. Here, we isolated a unique [PIN+] variant, which is unstable in actin mutants. This prion loss is observed over many generations, and coincides with the detection of both high molecular weight species of Rnq1 and large visible aggregates that are asymmetrically retained during cell division. Our data suggest that the irregular actin networks found in these mutants may influence propagon number by slowly permitting aggregate growth over time, resulting in the generation of nontransmissible large aggregates. Thus, we show the potential contribution of cytoskeletal networks in the transmission of prion propagons, which parallels models that have been proposed for cell‐to‐cell transmission of small amyloids in neurodegenerative protein aggregation diseases. [ABSTRACT FROM AUTHOR]

Amyloid formation is associated with many incurable diseases. For some of these, sporadic cases are much more common than familial ones. Some reports point to the role of somatic cell mosaicism in these cases via origination of amyloids in a limited number of cells, which can then spread through tissues. However, specific types of sporadic mutations responsible for such effects are unknown. In order to identify mutations capable of increasing the de novo appearance of amyloids, we searched for such mutants in the yeast prionogenic protein Sup35. We introduced to yeast cells an additional copy of the SUP35 gene with mutated amyloidogenic domain and observed that some nonsense mutations increased the incidence of prions by several orders of magnitude. This effect was related to exposure at the C-terminus of an internal amyloidogenic region of Sup35. We also discovered that SUP35 mRNA could undergo splicing, although inefficiently, causing appearance of a shortened Sup35 isoform lacking its functional domain, which was also highly prionogenic. Our data suggest that truncated forms of amyloidogenic proteins, resulting from nonsense mutations or alternative splicing in rare somatic cells, might initiate spontaneous localized formation of amyloids, which can then spread, resulting in sporadic amyloid disease.

The yeast [PSI+] prion, formed by the Sup35 (eRF3) protein, has multiple structural variants differing in the strength of nonsense suppressor phenotype. Structure of [PSI+] and its variation are characterized poorly. Here, we mapped Sup35 amyloid cores of 26 [PSI+] ex vivo prions of different origin using proteinase K digestion and mass spectrometric identification of resistant peptides. In all [PSI+] variants 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. Two distinct digestion patterns were observed for region 2–72, which always correlated with the “strong” and “weak” [PSI+] nonsense suppressor phenotypes. Also, all [PSI+] with a weak pattern were eliminated by multicopy HSP104 gene and were not toxic when combined with multicopy SUP35. [PSI+] with a strong pattern showed opposite properties, being resistant to multicopy HSP104 and lethal with multicopy SUP35. Thus, Sup35 prion cores can be composed of up to four elements. [PSI+] variants can be divided into two classes reliably distinguishable basing on structure of the first element and the described assays.

The interaction of [ PSI ] and [ PIN ] factors in yeast Saccharomyces cerevisiae is known as the first evidence of prions networks. In [ PIN ] cells, Rnq1p prion aggregates work as a template for Sup35p aggregation, which is essential for [ PSI ] induction. No additional factors are required for subsequent Sup35p aggregation. Nevertheless, several recent reports provide data that indicate a more complex interplay between these prions. Our results show that the presence of Rnq1p in the cell significantly decreases the loss of [ PSI ] prion, which is caused by a double mutation in SUP35 (Q61K, Q62K substitutions in the Sup35 protein). These observations support the existence of interaction networks that converge on a strong linkage of prionogenic and prion-like proteins, and the participation of Rnq1 protein in the maintenance of prion [ PSI ]. [ABSTRACT FROM AUTHOR]

Mutations in adenine biosynthesis pathway genes ADE1 and ADE2 have been conventionally used to score for prion [ PSI+] in yeast. If ade1-14 mutant allele is present, which contains a premature stop codon, [ psi−] yeast appear red on YPD medium owing to accumulation of a red intermediate compound in vacuoles. In [ PSI+] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing to its amyloid aggregation allows for read-through of the ade1-14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red colour development in ade1 and ade2 mutant yeast requires reduced-glutathione, which helps in transport of the intermediate metabolite P-ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid-induced oxidative stress would deplete reduced-glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid-forming human proteins TDP-43, A β-42 and Poly-Gln-103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour eventually reverted back to red upon turning off the amyloid protein's expression. Also, the aggregate-bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2-1, ade2Δ, and ade1Δ mutant yeast and also in an ade1-14 mutant with erg6 gene deletion that increases cell-wall permeability. This model would be useful tool for drug-screening against general amyloid-induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The baker’s yeast Saccharomyces cerevisiae harbors multiple prions that allow for the creation of heterogeneity within otherwise clonal cell populations. However, in many cases, the consequences of prion infection are entirely unclear. Predictions of prion-induced changes in cell physiology are complicated by pleotropic effects, and detection is often limited to relatively insensitive cell growth assays that may obscure many physiological changes. We previously showed that silica gel high performance thin-layer chromatography-densitometry (HPTLC) can be used to empirically determine prion-induced changes in lipid content in yeast. Here, we conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether-diethyl ether-glacial acetic acid (80:20:1, v/v/v) mobile phase and phosphomolybdic acid (PMA) detection reagent]; steryl esters, methyl esters, and squalene [hexane-petroleum ether-diethyl ether-glacial acetic acid (50:20:5:1, v/v/v/v) and PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform-diethyl ether-acetic acid (65:25:4.5, v/v/v) and cupric sulfate-phosphoric acid) in otherwise clonal prion-infected ([RNQ+]) and prion-free ([rnq−]) cells in both stationary- and logarithmic-growth phases. We detected multiple statistically significant differences between prion-infected and prion-free cells that varied by growth phase, confirming our pr evious observations that prions exert distinct influences on cell physiology between stationary- and log-phase growth. We also found significant differences between cells expressing or lacking the Rnq1 protein which forms the [RNQ+] prion, providing new clues to the as yet unresolved normal biological function of this prion-forming protein. This investigation further emphasizes the utility of HPTLC-densitometry to empirically determine the effects of prions and other presumed innocuous gene deletions on lipid content in yeast, and we expect that additional analyses will continue to resolve the physiological effects of prion infection.

The amino acid sequence of protein Gln3 in yeast Saccharomyces cerevisiae has a region enriched with Gln (Q) and Asn (N) residues. In this study, we analyzed the effects of overexpression of Gln3 and its Q/N-rich fragment fused with yellow fluorescent protein (YFP). Being overexpressed, full-length Gln3-YFP does not form aggregates, inhibits vegetative growth, and demonstrates nuclear localization, while the Q/N-rich fragment (Gln3QN) fused with YFP forms aggregates that do not colocalize with the nucleus and do not affect growth of the cells. Although detergent-resistant aggregates of Gln3QN are formed in the absence of yeast prions, the aggregation of Gln3QN significantly increases in the presence of [ PIN] prion, while in the presence of two prions, [ PSI] and [ PIN], the percentage of cells with Gln3QN aggregates is significantly lower than in the strain bearing only [ PIN]. Data on colocalization demonstrate that this effect is mediated by interaction between Gln3QN aggregates and [ PSI] and [ PIN] prions. [ABSTRACT FROM AUTHOR]

Prions, infectious proteins, can transmit diseases or be the basis of heritable traits (or both), mostly based on amyloid forms of the prion protein. A single protein sequence can be the basis for many prion strains/variants, with different biological properties based on different amyloid conformations, each rather stably propagating. Prions are unique in that evolution and selection work at both the level of the chromosomal gene encoding the protein, and on the prion itself selecting prion variants. Here, we summarize what is known about the evolution of prion proteins, both the genes and the prions themselves. We contrast the one known functional prion, [Het-s] of Podospora anserina, with the known disease prions, the yeast prions [PSI+] and [URE3] and the transmissible spongiform encephalopathies of mammals. [ABSTRACT FROM AUTHOR]

Maintenance of cellular redox homoeostasis forms an important part of the cellular defence mechanism and continued cell viability. Despite extensive studies, the role of the chaperone Hsp104 (heat-shock protein of 102 kDa) in propagation of misfolded protein aggregates in the cell and generation of oxidative stress remains poorly understood. Expression of RNQ1-RFP in Saccharomyces cerevisiae cells led to the generation of the prion form of the protein and increased oxidative stress. In the present study, we show that disruption of Hsp104 in an isogenic yeast strain led to solubilization of RNQ1-RFP. This reduced the oxidative stress generated in the cell. The higher level of oxidative stress in the Hsp104-containing (parental) strain correlated with lower activity of almost all of the intracellular antioxidant enzymes assayed. Surprisingly, this did not correspond with the gene expression analysis data. To compensate for the decrease in protein translation induced by a high level of reactive oxygen species, transcriptional up-regulation takes place. This explains the discrepancy observed between the transcription level and functional enzymatic product. Our results show that in a Hsp104 strain, due to lower oxidative stress, no such mismatch is observed, corresponding with higher cell viability. Thus Hsp104 is indirectly responsible for enhancing the oxidative stress in a prion-rich environment. [ABSTRACT FROM AUTHOR]

Abstract: The formation of amyloid-like fibrils is a hallmark of several neurodegenerative diseases. How the assembly of amyloid-like fibrils contributes to cell death is a major unresolved question in the field. The budding yeast Saccharomyces cerevisiae is a powerful model organism to study basic mechanisms for how cellular pathways regulate amyloid assembly and proteotoxicity. For example, studies of the amyloidogenic yeast prion [RNQ +] have revealed novel roles by which molecular chaperones protect cells from the accumulation of cytotoxic protein species. In budding yeast there are a variety of cellular assays that can be employed to analyze the assembly of amyloid-like aggregates and mechanistically dissect how cellular pathways influence proteotoxicity. In this review, we describe several assays that are routinely used to investigate aggregation and toxicity of the [RNQ +] prion in yeast. [Copyright &y& Elsevier]

Protein conformational diseases are associated with the aberrant accumulation of amyloid protein aggregates, but whether amyloid formation is cytotoxic or protective is unclear. To address this issue, we investigated a normally benign amyloid formed by the yeast prion (RNQ+]. Surprisingly, modest overexpression of Rnql protein was deadly, but only when preexisting Rnql was in the [RNQ+] prion conformation. Molecular chaperones protect against protein aggregation diseases and are generally believed to do so by solubilizing their substrates. The Hsp40 chaperone, Sisi, suppressed Rnq1 proteotoxicity, but instead of blocking Rnq1 protein aggregation, it stimulated conversion of soluble Rnql to [RNQ] amyloid. Furthermore, interference with Sisi-mediated [RNQ+] amyloid formation exacerbated Rnq1 toxicity. These and other data establish that even subtle changes in the folding homeostasis of an amyloidogenic protein can create a severe proteotoxic gain-of-function phenotype and that chaperone-mediated amyloid assembly can be cytoprotective. The possible relevance of these findings to other phenomena, including prion-driven neurodegenerative diseases and heterokaryon incompatibility in fungi, is discussed. [ABSTRACT FROM AUTHOR]

Spindle pole body (SPB; the microtubule organizing center in yeast) duplication is essential to form a bipolar spindle. The duplicated SPBs must then separate and migrate to opposite sides of the nucleus. We identified a novel functional relationship in SPB separation between the microtubule stabilizing protein Bik1p/CLIP-170 and the SPB half-bridge protein Sfi1p. A genetic interaction between BIK1 and SFI1 was discovered in a synthetic lethal screen using a strain deficient in the prion protein gene RNQ1. RNQ1 deletion reduced expression from the divergently transcribed BIK1, allowing us to identify genetic interactors with bik1. The sfi1-1 bik1 synthetic lethality was suppressed by over-expression of CIK1, KAR1, and PPH21. Genetic analysis indicated that the sfi1-1 bik1 synthetic lethality was unlikely related to the function of Bik1p in the dynein pathway or to defects in spindle position. Furthermore, a sfi1-1 Δkip2 mutant was viable, suggesting that the Bik1p pool at the cytoplasmic microtubule plus-ends may not be required in sfi1-1. Microscopic examination indicated the sfi1-1 mutant was delayed in SPB duplication, SPB separation, or spindle elongation and the sfi-1 Δbik1 double mutant arrested with duplicated but unseparated SPBs. These results suggest that Bik1p has a previously uncharacterized function in the separation of duplicated SPBs. [ABSTRACT FROM AUTHOR]

Abstract: We report that a null rnq1 mutation in the yeast RNQ1 (YCL028w) prion-like gene of so far unknown function produces the doubling of spores in the asci. This phenotype is possibly due to the lack of inhibition by Rnq1p of an additional mitotic division during ascus formation. This novel phenotype termed “octopus asci” could be similar to prion [PIN+] phenotype. [Copyright &y& Elsevier]

Yeast prions are inherited through proteins that exist in alternate, self-perpetuating conformational states. The mechanisms by which these states arise and are maintained are still poorly defined. Here we demonstrate for the first time that Sis1, a member of the Hsp40 chaperone family, plays a critical role in the maintenance of a prion. The prion [RNQ+] is formed by Rnq1, which is present in the same physical complex as Sis1, but only when Rnq1 is in the prion state. The G/F domain of Sis1 is dispensable for rapid growth on rich medium, but is required for [RNQ+] maintenance, distinguishing essential regions of Sis1 from those needed for prion interaction. A specific Sis1 deletion mutant altered the physical aggregation pattern of Rnq1 without curing the prion. This variant state propagated in a heritable fashion after wild-type Sis1 function was restored, indicating that multiple physical states are compatible with prion maintenance and that changes in chaperone activity can create prion variants. Using a prion chimera we demonstrate that the prion-determinant domain of Rnq1 is genetically sufficient for control by Sis1. [ABSTRACT FROM AUTHOR]

Amyloid formation is associated with many incurable diseases. For some of these, sporadic cases are much more common than familial ones. Some reports point to the role of somatic cell mosaicism in these cases via origination of amyloids in a limited number of cells, which can then spread through tissues. However, specific types of sporadic mutations responsible for such effects are unknown. In order to identify mutations capable of increasing the de novo appearance of amyloids, we searched for such mutants in the yeast prionogenic protein Sup35. We introduced to yeast cells an additional copy of the SUP35 gene with mutated amyloidogenic domain and observed that some nonsense mutations increased the incidence of prions by several orders of magnitude. This effect was related to exposure at the C-terminus of an internal amyloidogenic region of Sup35. We also discovered that SUP35 mRNA could undergo splicing, although inefficiently, causing appearance of a shortened Sup35 isoform lacking its functional domain, which was also highly prionogenic. Our data suggest that truncated forms of amyloidogenic proteins, resulting from nonsense mutations or alternative splicing in rare somatic cells, might initiate spontaneous localized formation of amyloids, which can then spread, resulting in sporadic amyloid disease. [ABSTRACT FROM AUTHOR]

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan's laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.

The yeast [PSI+] prion, formed by the Sup35 (eRF3) protein, has multiple structural variants differing in the strength of nonsense suppressor phenotype. Structure of [PSI+] and its variation are characterized poorly. Here, we mapped Sup35 amyloid cores of 26 [PSI+] ex vivo prions of different origin using proteinase K digestion and mass spectrometric identification of resistant peptides. In all [PSI+] variants 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. Two distinct digestion patterns were observed for region 2–72, which always correlated with the "strong" and "weak" [PSI+] nonsense suppressor phenotypes. Also, all [PSI+] with a weak pattern were eliminated by multicopy HSP104 gene and were not toxic when combined with multicopy SUP35. [PSI+] with a strong pattern showed opposite properties, being resistant to multicopy HSP104 and lethal with multicopy SUP35. Thus, Sup35 prion cores can be composed of up to four elements. [PSI+] variants can be divided into two classes reliably distinguishable basing on structure of the first element and the described assays. [ABSTRACT FROM AUTHOR]

The baker's yeast Saccharomyces cerevisiae harbors multiple prions that allow for the creation of heterogeneity within otherwise clonal cell populations. However, in many cases, the consequences of prion infection are entirely unclear. Predictions of prion-induced changes in cell physiology are complicated by pleotropic effects, and detection is often limited to relatively insensitive cell growth assays that may obscure many physiological changes. We previously showed that silica gel high performance thin-layer chromatography-densitometry (HPTLC) can be used to empirically determine prion-induced changes in lipid content in yeast. Here, we conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether-diethyl ether-glacial acetic acid (80:20:1, v / v / v ) mobile phase and phosphomolybdic acid (PMA) detection reagent]; steryl esters, methyl esters, and squalene [hexane-petroleum ether-diethyl ether-glacial acetic acid (50:20:5:1, v / v / v / v ) and PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform-diethyl ether-acetic acid (65:25:4.5, v / v / v ) and cupric sulfate-phosphoric acid) in otherwise clonal prion-infected ([ RNQ + ]) cells in both stationary- and logarithmic-growth phases. We detected multiple statistically significant differences between prion-infected and prion-free cells that varied by growth phase, confirming our pr evious observations that prions exert distinct influences on cell physiology between stationary- and log-phase growth. We also found significant differences between cells expressing or lacking the Rnq1 protein which forms the [ rnq - ]) cells in both stationary- and logarithmic-growth phases. We detected multiple statistically significant differences between prion-infected and prion-free cells that varied by growth phase, confirming our pr evious observations that prions exert distinct influences on cell physiology between stationary- and log-phase growth. We also found significant differences between cells expressing or lacking the Rnq1 protein which forms the [ RNQ + ] prion, providing new clues to the as yet unresolved normal biological function of this prion-forming protein. This investigation further emphasizes the utility of HPTLC-densitometry to empirically determine the effects of prions and other presumed innocuous gene deletions on lipid content in yeast, and we expect that additional analyses will continue to resolve the physiological effects of prion infection., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Yeast prions are heritable protein-based elements, most of which are formed of amyloid aggregates that rely on the action of molecular chaperones for transmission to progeny. Prions can form distinct amyloid structures, known as 'strains' in mammalian systems, that dictate both pathological progression and cross-species infection barriers. In yeast these same amyloid structural polymorphisms, called 'variants', dictate the intensity of prion-associated phenotypes and stability in mitosis. We recently reported that [PSI(+)] prion variants differ in the fundamental domain requirements for one chaperone, the Hsp40/J-protein Sis1, which are mutually exclusive between 2 different yeast prions, demonstrating a functional plurality for Sis1. Here we extend that analysis to incorporate additional data that collectively support the hypothesis that Sis1 has multiple functional roles that can be accomplished by distinct sets of domains. These functions are differentially required by distinct prions and prion variants. We also present new data regarding Hsp104-mediated prion elimination and show that some Sis1 functions, but not all, are conserved in the human homolog Hdj1/DNAJB1. Importantly, of the 10 amyloid-based prions indentified to date in Saccharomyces cerevisiae, the chaperone requirements of only 4 are known, leaving a great diversity of amyloid structures, and likely modes of amyloid-chaperone interaction, largely unexplored.

The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.