Complex Adaptations Can Drive the Evolution of the Capacitor [PSI+], Even with Realistic Rates of Yeast Sex

The [PSI⁺] prion may enhance evolvability by revealing previously cryptic genetic variation, but it is unclear whether such evolvability properties could be favored by natural selection. Sex inhibits the evolution of other putative evolvability mechanisms, such as mutator alleles. This paper explores whether sex also prevents natural selection from favoring modifier alleles that facilitate [PSI⁺] formation. Sex may permit the spread of “cheater” alleles that acquire the benefits of [PSI⁺] through mating without incurring the cost of producing [PSI⁺] at times when it is not adaptive. Using recent quantitative estimates of the frequency of sex in Saccharomyces paradoxus, we calculate that natural selection for evolvability can drive the evolution of the [PSI⁺] system, so long as yeast populations occasionally require complex adaptations involving synergistic epistasis between two loci. If adaptations are always simple and require substitution at only a single locus, then the [PSI⁺] system is not favored by natural selection. Obligate sex might inhibit the evolution of [PSI⁺]-like systems in other species.     

Complex Adaptations Can Drive the Evolution of the Capacitor [PSI +], Even with Realistic Rates of Yeast Sex

The [PSI⁺] prion may enhance evolvability by revealing previously cryptic genetic variation, but it is unclear whether such evolvability properties could be favored by natural selection. Sex inhibits the evolution of other putative evolvability mechanisms, such as mutator alleles. This paper explores whether sex also prevents natural selection from favoring modifier alleles that facilitate [PSI⁺] formation. Sex may permit the spread of “cheater” alleles that acquire the benefits of [PSI⁺] through mating without incurring the cost of producing [PSI⁺] at times when it is not adaptive. Using recent quantitative estimates of the frequency of sex in Saccharomyces paradoxus, we calculate that natural selection for evolvability can drive the evolution of the [PSI⁺] system, so long as yeast populations occasionally require complex adaptations involving synergistic epistasis between two loci. If adaptations are always simple and require substitution at only a single locus, then the [PSI⁺] system is not favored by natural selection. Obligate sex might inhibit the evolution of [PSI⁺]-like systems in other species.     

The Strength of Selection Against the Yeast Prion [PSI+]

The [PSI⁺] prion causes widespread readthrough translation and is rare in natural populations of Saccharomyces, despite the fact that sex is expected to cause it to spread. Using the recently estimated rate of Saccharomyces outcrossing, we calculate the strength of selection necessary to maintain [PSI⁺] at levels low enough to be compatible with data. Using the best available parameter estimates, we find selection against [PSI⁺] to be significant. Inference regarding selection on modifiers of [PSI⁺] appearance depends on obtaining more precise and accurate estimates of the product of yeast effective population size N_e and the spontaneous rate of [PSI⁺] appearance m. The ability to form [PSI⁺] has persisted in yeast over a long period of evolutionary time, despite a diversity of modifiers that could abolish it. If mN_e < 1, this may be explained by insufficiently strong selection. If mN_e > 1, then selection should favor the spread of [PSI⁺] resistance modifiers. In this case, rare conditions where [PSI⁺] is adaptive may permit its persistence in the face of negative selection.     

The [URE3] phenotype: evidence for a soluble prion in yeast

The aggregation of the two yeast proteins Sup35p and Ure2p is widely accepted as a model for explaining the prion propagation of the phenotypes [PSI⁺] and [URE3], respectively. Here, we demonstrate that the propagation of [URE3] cannot simply be the consequence of generating large aggregates of Ure2p, because such aggregation can be found in some conditions that are not related to the prion state of Ure2p. A comparison of [PSI⁺] and [URE3] aggregation demonstrates differences between these two prion mechanisms. Our findings lead us to propose a new unifying model for yeast prion propagation.     

The Spontaneous Appearance Rate of the Yeast Prion [PSI+] and Its Implications for the Evolution of the Evolvability Properties of the [PSI+] System

Epigenetically inherited aggregates of the yeast prion [PSI+] cause genomewide readthrough translation that sometimes increases evolvability in certain harsh environments. The effects of natural selection on modifiers of [PSI+] appearance have been the subject of much debate. It seems likely that [PSI+] would be at least mildly deleterious in most environments, but this may be counteracted by its evolvability properties on rare occasions. Indirect selection on modifiers of [PSI+] is predicted to depend primarily on the spontaneous [PSI+] appearance rate, but this critical parameter has not previously been adequately measured. Here we measure this epimutation rate accurately and precisely as 5.8 × 10⁻⁷ per generation, using a fluctuation test. We also determine that genetic “mimics” of [PSI+] account for up to 80% of all phenotypes involving general nonsense suppression. Using previously developed mathematical models, we can now infer that even in the absence of opportunities for adaptation, modifiers of [PSI+] are only weakly deleterious relative to genetic drift. If we assume that the spontaneous [PSI+] appearance rate is at its evolutionary optimum, then opportunities for adaptation are inferred to be rare, such that the [PSI+] system is favored only very weakly overall. But when we account for the observed increase in the [PSI+] appearance rate in response to stress, we infer much higher overall selection in favor of [PSI+] modifiers, suggesting that [PSI+]-forming ability may be a consequence of selection for evolvability.THE yeast phenotype [PSI+] is characterized by prion aggregates of the protein Sup35. Cells are in either a [psi−] (normal) or [PSI+] state, depending on the absence or presence of the prion aggregates (Figure 1, a and b). Sup35 prion aggregates replicate in a similar fashion to mammalian prions but are cytoplasmic and, as such, the prion state is cytoplasmically inherited (Wickner et al. 1995).Open in a separate windowFigure 1.—Comparison between the three possible modes ([PSI+], genetic mimic, point mutation revertant) of the expression of 3′-UTR sequences in yeast. (a) The normal [psi−] phenotypic state; (b) the [PSI+] prion causes readthrough and low-level expression of 3′-UTRs across multiple genes, appearing at rate m_PSI; (c) a genetic mimic of [PSI+] such as the sal3-4 mutant of Sup35 (Eaglestone et al. 1999) appearing at rate m_mimic not reversible by the application of guanidine hydrochloride; (d) a point mutation in a single stop codon at rate μ_point, leading to incorporation of formerly 3′-UTR into a single coding sequence. (e) [PSI+] can act as a “stop-gap” mechanism, buying a lineage more time to acquire one or more adaptive stop codon readthrough point mutations. When this genetic assimilation is complete, [PSI+] can revert to [psi−] (Masel and Bergman 2003; Griswold and Masel 2009).When not part of an aggregate, Sup35 helps mediate translation termination in yeast (Stansfield et al. 1995b; Zhouravleva et al. 1995). Sup35 molecules that are incorporated into nonfunctional prion aggregates are presumably not available for translation termination, which can lead to the translation of stop codons by near-cognate tRNAs (Figure 1b) (Tuite and Mclaughlin 1982; Pure et al. 1985; Lin et al. 1986). This partial loss of Sup35 function leads to an increased frequency of readthrough translation of 3′-untranslated regions (3′-UTR) across all genes (Figure 1b). This increase is modest in wild-type yeast, from an average readthrough rate of 0.3% in [psi−] cells up to 1% in [PSI+] cells (Firoozan et al. 1991). Some [PSI+] yeast strains grow faster than [psi−] controls in certain harsh environments, suggesting that readthrough translation of some 3′-UTRs may be adaptive in certain conditions (True and Lindquist 2000; Joseph and Kirkpatrick 2008). This directly shows that [PSI+]-mediated capacitance may increase evolvability in the laboratory. [PSI+]-mediated phenotypes have a complex genetic basis, involving multiple loci (True et al. 2004).As an epigenetically inherited protein aggregate, [PSI+] can easily be lost after some generations (Cox et al. 1980). This returns the lineage to its normal [psi−] state and restores translation fidelity. If a subset of revealed phenotypic variation is adaptive, it may have lost its dependence on [PSI+] by this time (True et al. 2004). This process of genetic assimilation may, for example, involve one or more point mutations in stop codons, increasing readthrough up to 100% (Figure 1e) (Griswold and Masel 2009). This leaves the yeast with a new adaptive trait and with no permanent load of other, deleterious variation.In general, stop codons can be lost either directly through point mutations or indirectly through upstream indels. This leads to novel coding sequence coming from in-frame and out-of-frame 3′-UTRs, respectively. [PSI+] is expected to facilitate only the former, while mutation bias favors the latter. Yeasts show a much higher ratio of in-frame to out-of-frame 3′-UTR incorporation events than mammals do (Giacomelli et al. 2007), confirming a role for [PSI+] in capacitance-mediated evolvability in natural populations.The adaptive evolution both of evolvability in general (Sniegowski and Murphy 2006; Lynch 2007; Pigliucci 2008) and of capacitance in particular (Dickinson and Seger 1999; Wagner et al. 1999; Partridge and Barton 2000; Brookfield 2001; Pal 2001; Meiklejohn and Hartl 2002; Ruden et al. 2003) is highly controversial. In general, any costs of evolvability are borne in the present, while the benefits lie in the future, making it difficult for natural selection to favor an evolvability allele. For example, mutation rates seem to be set according to a trade-off between metabolic cost (favoring higher mutation rates) and the avoidance of deleterious effects (favoring lower mutation rates) (Sniegowski et al. 2000). The fact that mutation creates variation, the ultimate source of evolvability, is merely a fortuitous consequence of the metabolic cost of fidelity.Previous theoretical population genetic studies have, however, suggested that modifier alleles promoting the formation of [PSI+] might, unlike mutator alleles, be favored for their evolvability properties (King and Masel 2007; Masel et al. 2007; Griswold and Masel 2009; Masel and Griswold 2009). These models depend, however, on a number of parameter estimates. In particular, a number of predictions depend on the spontaneous rate of [PSI+] formation (Masel and Griswold 2009).

[PSI+] appearance rates and the fluctuation test:

The most widely cited spontaneous appearance rate for [PSI+] is m_PSI ∼ 10⁻⁷–10⁻⁵, on the basis of experiments by Lund and Cox (1981). This estimate was calculated as the proportion of colonies scored as [PSI+] after growth over multiple generations from a single founding [psi−] clone. If [PSI+] happens to appear in the first generation of growth, this leads to a “jackpot” event with only one switching event, but many [PSI+] colonies. The proportion of colonies scored as [PSI+] therefore yields a systematic overestimation of the [PSI+] appearance rate.Various implementations of the fluctuation test (Luria and Delbrück 1943) can address such effects. The mutation rate experiment is replicated many times using independent populations, and a Luria–Delbrück distribution is fitted to the results across all replicates. In a simulation study, Stewart (1994) examined a number of estimators of the underlying Luria–Delbrück distribution and found that the maximum-likelihood estimator performed the best.Originally developed to study mutation rates, the fluctuation test can also be used for estimating epimutation rates. Fluctuation tests have been used to estimate the rate of gene silencing in Chinese hamster ovary cells (Holliday and Ho 1998) and in the yeast Schizosaccharomyces pombe (Singh and Klar 2002). However, fluctuation tests do not appear to be used routinely for epimutation rate estimates. For example, although the rates of spontaneous appearance and disappearance of [ISP+], a prion-like element in yeast, have been measured using the fluctuation test (Volkov et al. 2002), to the best of our knowledge there are no published estimates of the spontaneous rate of [PSI+] appearance as measured using a fluctuation test. Although results from the fluctuation test can be confounded by reverse epimutation, or back-switching, this is an issue only if the rate of back-switching is very high, e.g., 10⁻¹–10⁻² per generation (Saunders et al. 2003). This is not the case for [PSI+], for which the reverse epimutation rate (loss of [PSI+]) is <2 × 10⁻⁴ (Tank et al. 2007).

Other [PSI+]-like phenotypes, including genetic mimics:

[PSI+] causes partial loss of Sup35 function, leading to elevated rates of translational readthrough at all stop codons (Figure 1b). There are many other spontaneous changes, presumably mutations, that also lead to elevated translational readthrough (Lund and Cox 1981). Mutations that affect readthrough at all stop codons (Figure 1c) (sometimes called “[PSI+]-like”) can be considered as genetic “mimics” because they produce the same phenotype as the Sup35 aggregate, but are generally not epigenetically inherited. A specific example of such a genetic mimic was characterized by Eaglestone et al. (1999), who identified the sal3-4 point mutation in the SUP35 gene. This leads to a defect in the Sup35 protein structure rendering the termination process less efficient (Eaglestone et al. 1999). The sal3-4 mutant can therefore be considered a partial loss-of-function genetic mimic of [PSI+], since it generates the same readthrough phenotype. Translation termination could also potentially be impaired through other point mutations or deletions, for example, in either the SUP35 or the SUP45 gene (Stansfield et al. 1995a) or in a tRNA that mutates to recognize stop codons at a higher rate. The presence of genetic mimics, whose effects are less reversible than those of [PSI+], can affect the evolution of the evolvability properties of the [PSI+] system such as its epimutation rate (Lancaster and Masel 2009). Note that genetic mimics are quite different from much rarer point mutations that convert stop codons into coding sequence (Figure 1d), resulting in readthrough at a single gene rather than multiple genes.Here we performed experiments to obtain accurate and precise estimates of the baseline appearance rates of both [PSI+] and [PSI+]-like phenotypes in permissive laboratory conditions, excluding stop codon point mutations that affect only a single gene. Our estimates are superior to previous estimates, since we use the fluctuation test. We consider the consequences of these estimates for the evolution of the [PSI+] system.     

Antagonistic Interactions between Yeast Chaperones Hsp104 and Hsp70 in Prion Curing

The maintenance of [PSI], a prion-like form of the yeast release factor Sup35, requires a specific concentration of the chaperone protein Hsp104: either deletion or overexpression of Hsp104 will cure cells of [PSI]. A major puzzle of these studies was that overexpression of Hsp104 alone, from a heterologous promoter, cures cells of [PSI] very efficiently, yet the natural induction of Hsp104 with heat shock, stationary-phase growth, or sporulation does not. These observations pointed to a mechanism for protecting the genetic information carried by the [PSI] element from vicissitudes of the environment. Here, we show that simultaneous overexpression of Ssa1, a protein of the Hsp70 family, protects [PSI] from curing by overexpression of Hsp104. Ssa1 protein belongs to the Ssa subfamily, members of which are normally induced with Hsp104 during heat shock, stationary-phase growth, and sporulation. At the molecular level, excess Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble (cellular) state in the presence of excess Hsp104. Overexpression of Ssa1 also increases nonsense suppression by [PSI] when Hsp104 is expressed at its normal level. In contrast, hsp104 deletion strains lose [PSI] even in the presence of overproduced Ssa1. Overproduction of the unrelated chaperone protein Hsp82 (Hsp90) neither cured [PSI] nor antagonized the [PSI]-curing effect of overproduced Hsp104. Our results suggest it is the interplay between Hsp104 and Hsp70 that allows the maintenance of [PSI] under natural growth conditions.     

Increased [PSI+] Appearance by Fusion of Rnq1 with the Prion Domain of Sup35 in Saccharomyces cerevisiae

During propagation, yeast prions show a strict sequence preference that confers the specificity of prion assembly. Although propagations of [PSI⁺] and [RNQ⁺] are independent of each other, the appearance of [PSI⁺] is facilitated by the presence of [RNQ⁺]. To explain the [RNQ⁺] effect on the appearance of [PSI⁺], the cross-seeding model was suggested, in which Rnq1 aggregates act as imperfect templates for Sup35 aggregation. If cross-seeding events take place in the cytoplasm of yeast cells, the collision frequency between Rnq1 aggregates and Sup35 will affect the appearance of [PSI⁺]. In this study, to address whether cross-seeding occurs in vivo, a new [PSI⁺] induction method was developed that exploits a protein fusion between the prion domain of Sup35 (NM) and Rnq1. This fusion protein successfully joins preexisting Rnq1 aggregates, which should result in the localization of NM around the Rnq1 aggregates and hence in an increased collision frequency between NM and Rnq1 aggregates. The appearance of [PSI⁺] could be induced very efficiently, even with a low expression level of the fusion protein. This study supports the occurrence of in vivo cross-seeding between Sup35 and Rnq1 and provides a new tool that can be used to dissect the mechanism of the de novo appearance of prions.Prions were originally defined as proteinaceous infectious particles responsible for transmissible spongiform encephalopathies in mammals (reviewed in reference 23). Since a non-Mendelian genetic element, [URE3], was identified as a yeast prion (37), however, this concept has been expanded to include protein-based genetic elements. In addition to [URE3], there are at least two more proteinaceous genetic elements in Saccharomyces cerevisiae, namely, [PSI⁺] and [RNQ⁺] (20, 22, 28). [Het-s] was also identified as a prion in the filamentous fungus Podospora anserina (2).Despite the absence of any structural and functional homologies between various prion proteins from different organisms, they share a common feature, i.e., prion proteins can adopt two distinct conformational states. One of these, the aggregated prion state, can stimulate the soluble, nonprion conformation to convert into the prion form. Gaining intermolecular β-sheet structures, purified yeast prion proteins aggregate and form amyloid fibers in vitro (8, 12, 28, 32). Protein extract from yeast cells in the prion state can facilitate the in vitro polymerization of soluble prion protein from nonprion cells (21), and amyloid fibers of purified yeast prion proteins can convert the cells into the prion state when introduced into yeast cells, demonstrating the protein-only hypothesis (15, 31). Thus, intracellular prion aggregates are thought to have the same structural basis as amyloid fibers formed in vitro.Yeast prion biology has provided invaluable insights into the prion concept at the molecular level. Because of its experimental convenience, [PSI⁺] has been investigated most intensively among various yeast prions. [PSI⁺] results from the aggregation of Sup35 protein, which is essential for terminating the translation at stop codons. When Sup35 is in the [PSI⁺] aggregated state, ribosomes often fail to release polypeptides at stop codons, causing a non-Mendelian trait which is easily detected by nonsense suppression. ade1 or ade2 nonsense mutants are used as marker genes to determine the [PSI⁺] state. These mutants cannot grow on adenine-deficient medium and form red colonies on medium supplemented with a limiting amount of adenine, such as yeast extract-peptone-dextrose (YPD). ade mutants in the [PSI⁺] state, however, can grow on adenine-deficient medium and form white colonies, as they produce functional Ade1 or Ade2 by virtue of a nonsense mutation readthrough. To sustain propagation, all yeast prions need the disaggregation activity of Hsp104, which can be inhibited by guanidine hydrochloride (GuHCl) (9). Since yeast prions are cured by growth on guanidine-containing medium, prion phenotypes can easily be distinguished from chromosomal suppressor mutants.Sup35 (eRF3) of S. cerevisiae has a prion-determining N-terminal domain (N), a highly charged middle domain (M) that confers solubility on the molecule, and an essential C-terminal domain that binds guanine nucleotides and stimulates the polypeptide release reaction catalyzed by Sup45 (eRF1) (17, 29, 33). The de novo appearance of [PSI⁺] can be induced by overexpression of SUP35 or its prion domain-containing fragments (NM) (6). [PSI⁺] induction, however, can be achieved only in [RNQ⁺] cells that harbor the prion state of the Rnq1 protein (4, 19). Two hypotheses about how [RNQ⁺] can affect the appearance of [PSI⁺] have been suggested. One is an inhibitor titration model that postulates the molecules preventing the aggregation of Sup35 and the recruitment of these inhibitors to Rnq1 aggregates in [RNQ⁺] cells. The other is a cross-seeding model in which Rnq1 aggregates directly catalyze the polymerization of Sup35. In vitro cross-seeding between different amyloidogenic proteins was reported, and Rnq1 amyloid fiber can also act as a seed for Sup35 polymerization in vitro (7, 13). These in vitro data support the possibility of cross-seeding between Rnq1 and Sup35. However, because the milieu of cytoplasm is very different from that of a test tube, whether this cross-seeding really occurs in vivo is still obscure. For this study, we developed a new, robust [PSI⁺] induction method that confirms the cross-seeding events in the cytoplasmic environment.     

A Promiscuous Prion: Efficient Induction of [URE3] Prion Formation by Heterologous Prion Domains

The [URE3] and [PSI⁺] prions are the infections amyloid forms of the Saccharomyces cerevisiae proteins Ure2p and Sup35p, respectively. Randomizing the order of the amino acids in the Ure2 and Sup35 prion domains while retaining amino acid composition does not block prion formation, indicating that amino acid composition, not primary sequence, is the predominant feature driving [URE3] and [PSI⁺] formation. Here we show that Ure2p promiscuously interacts with various compositionally similar proteins to influence [URE3] levels. Overexpression of scrambled Ure2p prion domains efficiently increases de novo formation of wild-type [URE3] in vivo. In vitro, amyloid aggregates of the scrambled prion domains efficiently seed wild-type Ure2p amyloid formation, suggesting that the wild-type and scrambled prion domains can directly interact to seed prion formation. To test whether interactions between Ure2p and naturally occurring yeast proteins could similarly affect [URE3] formation, we identified yeast proteins with domains that are compositionally similar to the Ure2p prion domain. Remarkably, all but one of these domains were also able to efficiently increase [URE3] formation. These results suggest that a wide variety of proteins could potentially affect [URE3] formation.AMYLOID fibril formation is associated with numerous human diseases, including Alzheimer''s disease, type II diabetes, and the transmissible spongiform encephalopathies. Yeast prions provide a powerful model system for examining amyloid fibril formation in vivo. [URE3] and [PSI⁺] are the prion forms of the Saccharomyces cerevisiae proteins Ure2p and Sup35p, respectively (Wickner 1994). In both cases, prion formation is thought to result from conversion of the native protein into an inactive amyloid form (Glover et al. 1997; King et al. 1997; Taylor et al. 1999). Both proteins contain an N-terminal glutamine/asparagine (Q/N)-rich prion-forming domain (PFD) and a C-terminal functional domain (Ter-Avanesyan et al. 1993; Ter-Avanesyan et al. 1994; Masison and Wickner 1995; Liebman and Derkatch 1999; Maddelein and Wickner 1999). Sup35p contains an additional highly charged middle domain (M) that is not required either for prion formation or for normal protein function, but stabilizes [PSI⁺] aggregates (Liu et al. 2002).Amyloid fibril formation is thought to occur through a seeded polymerization mechanism. In vitro, amyloid fibril formation from native proteins is generally characterized by a significant lag time, thought to result from the slow rate of formation of amyloid nuclei; addition of a small amount of preformed amyloid aggregates (seeds) eliminates the lag time, resulting in rapid polymerization (Glover et al. 1997; Taylor et al. 1999; Serio et al. 2000).Despite considerable study, the mechanism by which amyloid seeds initially form is unclear. At least some of the amyloid proteins involved in human disease can interact with unrelated amyloidogenic proteins, resulting in cross-seeding and modulation of toxicity. Injecting mice with amyloid-like fibrils formed by a variety of short synthetic peptides promotes amyloid formation by amyloid protein A, a protein whose deposition is found in systemic AA amyloidosis (Johan et al. 1998). In yeast, [PSI⁺] and [PIN⁺], the prion form of the protein Rnq1p (Sondheimer and Lindquist 2000; Derkatch et al. 2001), both promote the aggregation of and increase toxicity of expanded polyglutamine tracts, like those seen in Huntington''s disease (Osherovich and Weissman 2001; Meriin et al. 2002; Derkatch et al. 2004; Gokhale et al. 2005; Duennwald et al. 2006); however, in Drosophila, [PSI⁺] aggregates reduce polyglutamine toxicity (Li et al. 2007). Thus, interactions between heterologous amyloidogenic proteins can influence amyloid formation both positively and negatively in vivo.A variety of interactions have been observed among the yeast prions. Under normal cellular conditions, efficient formation, but not maintenance, of [PSI⁺] requires the presence of [PIN⁺] (Derkatch et al. 2000). Overexpression of various Q/N-rich proteins can effectively substitute for [PIN⁺], allowing [PSI⁺] formation in cells lacking [PIN⁺] (Derkatch et al. 2001; Osherovich and Weissman 2001). In vitro and in vivo evidence suggest that the ability of [PIN⁺] to facilitate [PSI⁺] formation is the result of a direct interaction between Rnq1p aggregates and Sup35p (Derkatch et al. 2004; Bardill and True 2009; Choe et al. 2009). [PIN⁺] also increases the frequency of [URE3] formation, while [PSI⁺] inhibits [URE3] formation (Bradley et al. 2002; Schwimmer and Masison 2002).It is unclear whether the ability of Ure2p, Sup35p, and Rnq1p to cross-react is an intrinsic feature of all similar amyloidogenic proteins, or whether it has specifically evolved to regulate prion formation. There is debate as to whether yeast prion formation is a beneficial phenomenon, allowing for regulation of the activity of the prion protein (True and Lindquist 2000; True et al. 2004), or a deleterious event analogous to human amyloid disease (Nakayashiki et al. 2005). Either way, it is likely that interactions between the yeast prion proteins have specifically evolved, either to minimize the detrimental effects of amyloid formation or to regulate beneficial amyloid formation.For both Ure2p and Sup35p, the amino acid composition of the PFD is the predominant feature that drives prion formation. Scrambled versions of Ure2p and Sup35p (in which the order of the amino acids in the PFD was randomized while maintaining amino acid composition) are able to form prions when expressed in yeast as the sole copy Ure2p or Sup35p (Ross et al. 2004, 2005). To examine whether amino acid composition can similarly drive interactions between heterologous proteins, we tested whether the scrambled PFDs can interact with their wild-type counterparts to stimulate prion formation. When overexpressed, scrambled Ure2 PFDs promoted de novo prion formation by wild-type Ure2p, suggesting that the Ure2p PFD can promiscuously interact with compositionally similar PFDs during prion formation. When we searched the yeast proteome for proteins with regions of high compositional similarity to Ure2p, four of the top five proteins were able to efficiently stimulate [URE3] formation. However, there were limits to this promiscuity; overexpression of wild-type or scrambled Sup35 PFDs did not increase [URE3] levels. We propose that this ability to promiscuously interact may have evolved as a mechanism to regulate Ure2p activity and/or prion formation.     

The Schizosaccharomyces pombe Hsp104 Disaggregase Is Unable to Propagate the [PSI +] Prion

The molecular chaperone Hsp104 is a crucial factor in the acquisition of thermotolerance in yeast. Under stress conditions, the disaggregase activity of Hsp104 facilitates the reactivation of misfolded proteins. Hsp104 is also involved in the propagation of fungal prions. For instance, the well-characterized [PSI⁺] prion of Saccharomyces cerevisiae does not propagate in Δhsp104 cells or in cells overexpressing Hsp104. In this study, we characterized the functional homolog of Hsp104 from Schizosaccharomyces pombe (Sp_Hsp104). As its S. cerevisiae counterpart, Sp_hsp104⁺ is heat-inducible and required for thermotolerance in S. pombe. Sp_Hsp104 displays low disaggregase activity and cannot propagate the [PSI⁺] prion in S. cerevisiae. When overexpressed in S. cerevisiae, Sp_Hsp104 confers thermotolerance to Δhsp104 cells and reactivates heat-aggregated proteins. However, overexpression of Sp_Hsp104 does not propagate nor eliminate [PSI⁺]. Strikingly, [PSI⁺] was cured by overexpression of a chimeric chaperone bearing the C-terminal domain (CTD) of the S. cerevisiae Hsp104 protein. Our study demonstrates that the ability to untangle aggregated proteins is conserved between the S. pombe and S. cerevisiae Hsp104 homologs, and points to a role of the CTD in the propagation of the S. cerevisiae [PSI⁺] prion.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells

Anoxia induces a rapid elevation of the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca²⁺ release is important for gene activation and survival in O₂-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca²⁺ to the anoxic [Ca²⁺]_cyt elevation in maize cells. Imaging of intramitochondrial Ca²⁺ levels showed that a majority of mitochondria released their Ca²⁺ in response to anoxia and took up Ca²⁺ upon reoxygenation. We also investigated whether the mitochondrial Ca²⁺ release contributed to the increase in [Ca²⁺]_cyt under anoxia. Analysis of the spatial association between anoxic [Ca²⁺]_cyt changes and the distribution of mitochondrial and other intracellular Ca²⁺ stores revealed that the largest [Ca²⁺]_cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca²⁺]_cyt under aerobic conditions but prevented a [Ca²⁺]_cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca²⁺]_cyt and participate in the signaling of O₂ deprivation.     

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca²⁺]_cyt response. Cyclic-nucleotide-mediated elevations in [Ca²⁺]_cyt involved both internal and external Ca²⁺ stores. Both cAMP- and cGMP-mediated [Ca²⁺]_cyt elevations could be inhibited by the Ca²⁺-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca²⁺]_cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca²⁺]_cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca²⁺ signaling.     

The [PSI+] prion of Saccharomyces cerevisiae can be propagated by an Hsp104 orthologue from Candida albicans

The molecular chaperone Hsp104 is not only a key component of the cellular machinery induced to disassemble aggregated proteins in stressed cells of Saccharomyces cerevisiae but also plays an essential role in the propagation of the [PSI⁺], [URE3], and [RNQ/PIN⁺] prions in this organism. Here we demonstrate that the fungal pathogen Candida albicans carries an 899-residue stress-inducible orthologue of Hsp104 (CaHsp104) that shows a high degree of amino acid identity to S. cerevisiae Hsp104 (ScHsp104). This identity is significantly lower in the N- and C-terminal regions implicated in substrate recognition and cofactor binding, respectively. CaHsp104 is able to provide all known functions of ScHsp104 in an S. cerevisiae hsp104 null mutant, i.e., tolerance to high-temperature stress, reactivation of heat-denatured proteins, and propagation of the [PSI⁺] prion. As also observed for ScHsp104, overexpression of CaHsp104 leads to a loss of the [PSI⁺] prion. However, unlike that of ScHsp104, CaHsp104 function is resistant to guanidine hydrochloride (GdnHCl), an inhibitor of the ATPase activity of this chaperone. These findings have implications both in terms of the mechanism of inhibition of Hsp104 by GdnHCl and in the evolution of the ability of fungal species to propagate prions.     

Hexamminecobalt(III)-induced condensation of calf thymus DNA: circular dichroism and hydration measurements

The interaction of hexamminecobalt(III), Co(NH₃)₆³⁺, with 160 and 3000–8000 bp length calf thymus DNA has been investigated by circular dichroism, acoustic and densimetric techniques. The acoustic titration curves of 160 bp DNA revealed three stages of interaction: (i) Co(NH₃)₆³⁺ binding up to the molar ratio [Co(NH₃)₆³⁺]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH₃)₆³⁺]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH₃)₆³⁺]/[P] = 0.3. In the case of 3000–8000 bp DNA only two processes were observed: (i) binding up to [Co(NH₃)₆³⁺]/[P] = 0.3; and (ii) precipitation after this point. In agreement with earlier observations, long DNA aggregates without changes in its B-form circular dichroism spectrum, while short DNA demonstrates a positive B→Ψ transition after [Co(NH₃)₆³⁺]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH₃)₆³⁺ binding on volume and compressibility have been obtained. The binding of Co(NH₃)₆³⁺ to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH₃)₆³⁺: ΔV = 9 cm³ mol^–1 and Δκ = 33 × 10^–4 cm³ mol^–1 bar^–1. The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH₃)₆³⁺ and the atomic groups of DNA. This extent of water displacement would be consistent with the formation of two direct, hydrogen bonded contacts between the cation and the phosphates of DNA.     

Prion formation and polyglutamine aggregation are controlled by two classes of genes

Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI ⁺] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN ⁺]. When fused to GFP and overexpressed in [ps⁻] [PIN ⁺] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI ⁺] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI ⁺]. Two classes of gene deletions were identified. Class I deletions (bug1Δ, bem1Δ, arf1Δ, and hog1Δ) reduced the efficiency of [PSI ⁺] induction, but formed rings normally. Class II deletions (las17Δ, vps5Δ, and sac6Δ) inhibited both [PSI⁺] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington''s disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.     

The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

Protein-only (prion) epigenetic elements confer unique phenotypes by adopting alternate conformations that specify new traits. Given the conformational flexibility of prion proteins, protein-only inheritance requires efficient self-replication of the underlying conformation. To explore the cellular regulation of conformational self-replication and its phenotypic effects, we analyzed genetic interactions between [PSI⁺], a prion form of the S. cerevisiae Sup35 protein (Sup35^[PSI+]), and the three N^α-acetyltransferases, NatA, NatB, and NatC, which collectively modify ~50% of yeast proteins. Although prion propagation proceeds normally in the absence of NatB or NatC, the [PSI⁺] phenotype is reversed in strains lacking NatA. Despite this change in phenotype, [PSI⁺] NatA mutants continue to propagate heritable Sup35^[PSI+]. This uncoupling of protein state and phenotype does not arise through a decrease in the number or activity of prion templates (propagons) or through an increase in soluble Sup35. Rather, NatA null strains are specifically impaired in establishing the translation termination defect that normally accompanies Sup35 incorporation into prion complexes. The NatA effect cannot be explained by the modification of known components of the [PSI⁺] prion cycle including Sup35; thus, novel acetylated cellular factors must act to establish and maintain the tight link between Sup35^[PSI+] complexes and their phenotypic effects.     

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca²⁺-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca²⁺]_cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca²⁺]_cyt and inhibition of growth that was similar to the effect of the Ca²⁺ channel blocker La³⁺ and the Ca²⁺ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca²⁺ channel blockers verapamil and nifedipine did not induce a decrease in [Ca²⁺]_cyt in these cells and also failed to inhibit growth. Al and La³⁺, but not verapamil or nifedipine, reduced the rate of Mn²⁺ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca²⁺- and Mn²⁺-permeable channels. These results suggest that Al may act to block Ca²⁺ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.     

Mutational analyses of dinucleotide and tetranucleotide microsatellites in Escherichia coli: influence of sequence on expansion mutagenesis

Mutagenesis at [GT/CA]₁₀, [TC/AG]₁₁ and [TTCC/AAGG]₉ microsatellite sequences inserted in the herpes simplex virus thymidine kinase (HSV-tk) gene was analyzed in isogenic mutL⁺ and mutL^– Escherichia coli. In both strains, significantly more expansion than deletion mutations were observed at the [TTCC/AAGG]₉ motif relative to either dinucleo­tide motif. As the HSV-tk coding sequence contains an endogenous [G/C]₇ mononucleotide repeat and ~1000 bp of unique sequence, we were able to compare mutagenesis among various sequence motifs. We observed that the relative risk of mutation in E.coli is: [TTCC/AAGG]₉ > [GT/CA]₁₀ ~ [TC/AG]₁₁ > unique ~ [G/C]₇. The mutation frequency varied 1400-fold in mutL⁺ cells between the tetranucleotide motif and the mononucleotide motif, but only 50-fold in mutL^– cells. The [G/C]₇ sequence was destabilized the greatest and the tetranucleotide motif the least by loss of mismatch repair. These results demonstrate that the quantitative risk of mutation at various microsatellites greatly depends on the DNA sequence composition. We suggest alternative models for the production of expansion mutations during lagging strand replication of the [TTCC/AAGG]₉ microsatellite.     

Prion-dependent proteome remodeling in response to environmental stress is modulated by prion variant and genetic background

A number of fungal proteins are capable of adopting multiple alternative, self-perpetuating prion conformations. These prion variants are associated with functional alterations of the prion-forming protein and thus the generation of new, heritable traits that can be detrimental or beneficial. Here we sought to determine the extent to which the previously-reported ZnCl₂-sensitivity trait of yeast harboring the [PSI⁺] prion is modulated by genetic background and prion variant, and whether this trait is accompanied by prion-dependent proteomic changes that could illuminate its physiological basis. We also examined the degree to which prion variant and genetic background influence other prion-dependent phenotypes. We found that ZnCl₂ exposure not only reduces colony growth but also limits chronological lifespan of [PSI⁺] relative to [psi^?] cells. This reduction in viability was observed for multiple prion variants in both the S288C and W303 genetic backgrounds. Quantitative proteomic analysis revealed that under exposure to ZnCl₂ the expression of stress response proteins was elevated and the expression of proteins involved in energy metabolism was reduced in [PSI⁺] relative to [psi^?] cells. These results suggest that cellular stress and slowed growth underlie the phenotypes we observed. More broadly, we found that prion variant and genetic background modulate prion-dependent changes in protein abundance and can profoundly impact viability in diverse environments. Thus, access to a constellation of prion variants combined with the accumulation of genetic variation together have the potential to substantially increase phenotypic diversity within a yeast population, and therefore to enhance its adaptation potential in changing environmental conditions.     

A crystallographic study of the binding of 13 metal ions to two related RNA duplexes

Metal ions, and magnesium in particular, are known to be involved in RNA folding by stabilizing secondary and tertiary structures, and, as cofactors, in RNA enzymatic activity. We have conducted a systematic crystallographic analysis of cation binding to the duplex form of the HIV-1 RNA dimerization initiation site for the subtype-A and -B natural sequences. Eleven ions (K⁺, Pb²⁺, Mn²⁺, Ba²⁺, Ca²⁺, Cd²⁺, Sr²⁺, Zn²⁺, Co²⁺, Au³⁺ and Pt⁴⁺) and two hexammines [Co (NH₃)₆]³⁺ and [Ru (NH₃)₆]³⁺ were found to bind to the DIS duplex structure. Although the two sequences are very similar, strong differences were found in their cation binding properties. Divalent cations bind almost exclusively, as Mg²⁺, at ‘Hoogsteen’ sites of guanine residues, with a cation-dependent affinity for each site. Notably, a given cation can have very different affinities for a priori equivalent sites within the same molecule. Surprisingly, none of the two hexammines used were able to efficiently replace hexahydrated magnesium. Instead, [Co (NH₃)₄]³⁺ was seen bound by inner-sphere coordination to the RNA. This raises some questions about the practical use of [Co (NH₃)₆]³⁺ as a [Mg (H₂O)₆]²⁺ mimetic. Also very unexpected was the binding of the small Au³⁺ cation exactly between the Watson–Crick sites of a G-C base pair after an obligatory deprotonation of N1 of the guanine base. This extensive study of metal ion binding using X-ray crystallography significantly enriches our knowledge on the binding of middleweight or heavy metal ions to RNA, particularly compared with magnesium.