Mutational analysis of the [Het-s] prion analog of Podospora anserina. A short N-terminal peptide allows prion propagation

The het-s locus is one of nine known het (heterokaryon incompatibility) loci of the fungus Podospora anserina. This locus exists as two wild-type alleles, het-s and het-S, which encode 289 amino acid proteins differing at 13 amino acid positions. The het-s and het-S alleles are incompatible as their coexpression in the same cytoplasm causes a characteristic cell death reaction. We have proposed that the HET-s protein is a prion analog. Strains of the het-s genotype exist in two phenotypic states, the neutral [Het-s*] and the active [Het-s] phenotype. The [Het-s] phenotype is infectious and is transmitted to [Het-s*] strains through cytoplasmic contact. het-s and het-S were associated in a single haploid nucleus to generate a self-incompatible strain that displays a restricted and abnormal growth. In the present article we report the molecular characterization of a collection of mutants that restore the ability of this self-incompatible strain to grow. We also describe the functional analysis of a series of deletion constructs and site-directed mutants. Together, these analyses define positions critical for reactivity and allele specificity. We show that a 112-amino-acid-long N-terminal peptide of HET-s retains [Het-s] activity. Moreover, expression of a mutant het-s allele truncated at position 26 is sufficient to allow propagation of the [Het-s] prion analog.     

In vivo aggregation of the HET-s prion protein of the fungus Podospora anserina

We have proposed that the [Het-s] infectious cytoplasmic element of the filamentous fungus Podospora anserina is the prion form of the HET-s protein. The HET-s protein is involved in a cellular recognition phenomenon characteristic of filamentous fungi and known as heterokaryon incompatibility. Under the prion form, the HET-s protein causes a cell death reaction when co-expressed with the HET-S protein, from which it differs by only 13 amino acid residues. We show here that the HET-s protein can exist as two alternative states, a soluble and an aggregated form in vivo. As shown for the yeast prions, transition to the infectious prion form leads to aggregation of a HET-s--green fluorescent protein (GFP) fusion protein. The HET-s protein is aggregated in vivo when highly expressed. However, we could not demonstrate HET-s aggregation at wild-type expression levels, which could indicate that only a small fraction of the HET-s protein is in its aggregated form in vivo in wild-type [Het-s] strains. The antagonistic HET-S form is soluble even at high expression level. A double amino acid substitution in HET-s (D23A P33H), which abolishes prion infectivity, suppresses in vivo aggregation of the GFP fusion. Together, these results further support the model that the [Het-s] element corresponds to an abnormal self-perpetuating aggregated form of the HET-s protein.     

The [Het-s] prion of Podospora anserina and its role in heterokaryon incompatibility

[Het-s] is a prion from the filamentous fungus Podospora anserina and corresponds to a self-perpetuating amyloid aggregate of the HET-s protein. This prion protein is involved in a fungal self/non-self discrimination process termed heterokaryon incompatibility corresponding to a cell death reaction occurring upon fusion of genetically unlike strains. Two antagonistic allelic variants of this protein exist: HET-s, the prion form of which corresponds to [Het-s] and HET-S, incapable of prion formation. Fusion of a [Het-s] and HET-S strain triggers the incompatibility reaction, so that interaction of HET-S with the [Het-s] prion leads to cell death. HET-s and HET-S are highly homologous two domain proteins with a N-terminal globular domain termed HeLo and a C-terminal unstructured prion forming domain (PFD). The structure of the prion form of the HET-s PFD has been solved by solid state NMR and corresponds to a very well ordered β-solenoid fold with a triangular hydrophobic core. The ability to form this β-solenoid fold is retained in a distant homolog of HET-s from another fungal species. A model for the mechanism of [Het-s]/HET-S incompatibility has been proposed. It is believe that when interacting with the [Het-s] prion seed, the HET-S C-terminal region adopts the β-solenoid fold. This would act as a conformational switch to induce refolding and activation of the HeLo domain which then would exert its toxicity by a yet unknown mechanism.     

Domain organization and structure-function relationship of the HET-s prion protein of Podospora anserina

The [Het-s] infectious element of the fungus Podospora anserina is a prion protein involved in a genetically controlled cell death reaction termed heterokaryon incompatibility. Previous analyses indicate that [Het-s] propagates as a self-perpetuating amyloid aggregate. The HET-s protein is 289 amino acids in length. Herein, we identify the region of the HET-s protein that is responsible for amyloid formation and prion propagation. The region of HET-s spanning residues 218-289 forms amyloid fibers in vitro and allows prion propagation in vivo. Conversely, a C-terminal deletion in HET-s prevents amyloid aggregation in vitro and prion propagation in vivo, and abolishes the incompatibility function. In the soluble form of HET-s, the region from residue 1 to 227 forms a well-folded domain while the C-terminal region is highly flexible. Together, our data establish a domain structure-function relationship for HET-s amyloid formation, prion propagation and incompatibility activity.     

The HET-s prion protein of the filamentous fungus Podospora anserina aggregates in vitro into amyloid-like fibrils.

The HET-s protein of Podospora anserina is a fungal prion. This protein behaves as an infectious cytoplasmic element that is transmitted horizontally from one strain to another. Under the prion form, the HET-s protein forms aggregates in vivo. The specificity of this prion model compared with the yeast prions resides in the fact that under the prion form HET-s causes a growth inhibition and cell death reaction when co-expressed with the HET-S protein from which it differs by 13 residues. Herein we describe the purification and initial characterization of recombinant HET-s protein expressed in Escherichia coli. The HET-s protein self-associates over time into high molecular weight aggregates. These aggregates greatly accelerate precipitation of the soluble form. HET-s aggregates appear as amyloid-like fibrils using electron microscopy. They bind Congo Red and show birefringence under polarized light. In the aggregated form, a HET-s fragment of approximately 7 kDa is resistant to proteinase K digestion. CD and FTIR analyses indicate that upon transition to the aggregated state, the HET-s protein undergoes a structural rearrangement characterized by an increase in antiparallel beta-sheet structure content. These results suggest that the [Het-s] prion element propagates in vivo as an infectious amyloid.     

The Mechanism of Toxicity in HET-S/HET-s Prion Incompatibility

The HET-s protein from the filamentous fungus Podospora anserina is a prion involved in a cell death reaction termed heterokaryon incompatibility. This reaction is observed at the point of contact between two genetically distinct strains when one harbors a HET-s prion (in the form of amyloid aggregates) and the other expresses a soluble HET-S protein (96% identical to HET-s). How the HET-s prion interaction with HET-S brings about cell death remains unknown; however, it was recently shown that this interaction leads to a relocalization of HET-S from the cytoplasm to the cell periphery and that this change is associated with cell death. Here, we present detailed insights into this mechanism in which a non-toxic HET-s prion converts a soluble HET-S protein into an integral membrane protein that destabilizes membranes. We observed liposomal membrane defects of approximately 10 up to 60 nm in size in transmission electron microscopy images of freeze-fractured proteoliposomes that were formed in mixtures of HET-S and HET-s amyloids. In liposome leakage assays, HET-S has an innate ability to associate with and disrupt lipid membranes and that this activity is greatly enhanced when HET-S is exposed to HET-s amyloids. Solid-state nuclear magnetic resonance (NMR) analyses revealed that HET-s induces the prion-forming domain of HET-S to adopt the β-solenoid fold (previously observed in HET-s) and this change disrupts the globular HeLo domain. These data indicate that upon interaction with a HET-s prion, the HET-S HeLo domain partially unfolds, thereby exposing a previously buried ∼34-residue N-terminal transmembrane segment. The liberation of this segment targets HET-S to the membrane where it further oligomerizes, leading to a loss of membrane integrity. HET-S thus appears to display features that are reminiscent of pore-forming toxins.     

Het up mould unleashes a sporekiller prion

Conclusion het-s/het-S incompatibility is one of only a handful of fungal heterokaryon incompatibility systems that have begun to be molecularly analysed (for a review see Saupe 2000). The spore killer phenotype also is one of only a few segregation ratio distortions that has been studied in any detail. Finally the [HET-s] prion is one of only three examples known in non-mammalian systems (for a review see Wickneret al 1999). Clearly thehet-s/het-S story intersects with several exciting areas of research that offer many opportunities for young scientists.     

Methods for the in vivo and in vitro analysis of [Het-s] prion infectivity

Prions have been described in mammals and fungi. The [Het-s] infectious genetic element of the filamentous fungus Podospora anserina is the prion form of the HET-s protein. This protein is involved in the control of a cell death reaction termed heterokaryon incompatibility. The infectious form of HET-s corresponds to a self-perpetuating amyloid. The purpose of the present paper is to describe the techniques that can be used to analyse [Het-s] prion propagation in vivo and HET-s amyloid aggregation in vitro. In addition, we report several methods that can be used to infect Podospora with recombinant HET-s amyloid.     

Localization of HET-S to the cell periphery, not to [Het-s] aggregates, is associated with [Het-s]-HET-S toxicity

Prion diseases are associated with accumulation of the amyloid form of the prion protein, but the mechanisms of toxicity are unknown. Amyloid toxicity is also associated with fungal prions. In Podospora anserina, the simultaneous presence of [Het-s] prion and its allelic protein HET-S causes cell death in a self-/nonself-discrimination process. Here, using the prion form of a fragment of HET-s ([PrD(157)(+)]), we show that [Het-s]-HET-S toxicity can be faithfully recapitulated in yeast. Overexpression of Hsp40 chaperone, Sis1, rescues this toxicity by curing cells of [PrD(157)(+)]. We find no evidence for toxic [PrD(157)(+)] conformers in the presence of HET-S. Instead, [PrD(157)(+)] appears to seed HET-S to accumulate at the cell periphery and to form aggregates distinct from visible [PrD(157)(+)] aggregates. Furthermore, HET-S mutants that cause HET-S to be sequestered into [PrD(157)(+)] prion aggregates are not toxic. The localization of HET-S at the cell periphery and its association with cell death was also observed in the native host Podospora anserina. Thus, upon interaction with [Het-s], HET-S localizes to the cell periphery, and this relocalization, rather than the formation of mixed HET-s/HET-S aggregates, is associated with toxicity.     

Genomic clustering and homology between HET-S and the NWD2 STAND protein in various fungal genomes

Background

Prions are infectious proteins propagating as self-perpetuating amyloid polymers. The [Het-s] prion of Podospora anserina is involved in a cell death process associated with non-self recognition. The prion forming domain (PFD) of HET-s adopts a β-solenoid amyloid structure characterized by the two fold repetition of an elementary triangular motif. [Het-s] induces cell death when interacting with HET-S, an allelic variant of HET-s. When templated by [Het-s], HET-S undergoes a trans-conformation, relocates to the cell membrane and induces toxicity.

Methodology/Principal Findings

Here, comparing HET-s homologs from different species, we devise a consensus for the HET-s elementary triangular motif. We use this motif to screen genomic databases and find a match to the N-terminus of NWD2, a STAND protein, encoded by the gene immediately adjacent to het-S. STAND proteins are signal transducing ATPases which undergo ligand-induced oligomerisation. Homology modelling predicts that the NWD2 N-terminal region adopts a HET-s-like fold. We propose that upon NWD2 oligomerisation, these N-terminal extensions adopt the β-solenoid fold and template HET-S to adopt the amyloid fold and trigger toxicity. We extend this model to a putative prion, the σ infectious element in Nectria haematococca, because the s locus controlling propagation of σ also encodes a STAND protein and displays analogous features. Comparative genomic analyses indicate evolutionary conservation of these STAND/prion-like gene pairs, identify a number of novel prion candidates and define, in addition to the HET-s PFD motif, two distinct, novel putative PFD-like motifs.

Conclusions/Significance

We suggest the existence, in the fungal kingdom, of a widespread and evolutionarily conserved mode of signal transduction based on the transmission of an amyloid-fold from a NOD-like STAND receptor protein to an effector protein.     

Prion and non-prion amyloids of the HET-s prion forming domain

HET-s is a prion protein of the fungus Podospora anserina. A plausible structural model for the infectious amyloid fold of the HET-s prion-forming domain, HET-s(218-289), makes it an attractive system to study structure-function relationships in amyloid assembly and prion propagation. Here, we report on the diversity of HET-s(218-289) amyloids formed in vitro. We distinguish two types formed at pH 7 from fibrils formed at pH 2, on morphological grounds. Unlike pH 7 fibrils, the pH 2 fibrils show very little if any prion infectivity. They also differ in ThT-binding, resistance to denaturants, assembly kinetics, secondary structure, and intrinsic fluorescence. Both contain 5 nm fibrils, either bundled or disordered (pH 7) or as tightly twisted protofibrils (pH 2). We show that electrostatic interactions are critical for the formation and stability of the infectious prion fold given in the current model. The altered properties of the amyloid assembled at pH 2 may arise from a perturbation in the subunit fold or fibrillar stacking.     

Conformational transition occurring upon amyloid aggregation of the HET-s prion protein of Podospora anserina analyzed by hydrogen/deuterium exchange and mass spectrometry

The [Het-s] infectious element of the filamentous fungus Podospora anserina corresponds to the prion form of the HET-s protein. HET-s (289 amino acids in length) aggregates into amyloid fibers in vitro. Such fibers obtained in vitro are infectious, indicating that the [Het-s] prion can propagate as a self-perpetuating amyloid aggregate of the HET-s protein. Previous analyses have suggested that only a limited region of the HET-s protein is involved in amyloid formation and prion propagation. To document the conformational transition occurring upon amyloid aggregation of HET-s, we have developed a method involving hydrogen/deuterium exchange monitored by MALDI-MS. In a first step, a peptide mass fingerprint of the protein was obtained, leading to 87% coverage of the HET-s primary structure. Amyloid aggregates of HET-s were obtained, and H/D exchange was monitored on the soluble and on the amyloid form of HET-s. This study revealed that in the soluble form of HET-s, the C-terminal region (spanning from residues 240-289) displays a high solvent accessibility. In sharp contrast, solvent accessibility is drastically reduced in that region in the amyloid form. H/D exchange rates and levels in the N-terminal part of the protein (residues 1-220) are comparable in the soluble and the aggregated state. These results indicate that amyloid aggregation of HET-s involves a conformational transition of the C-terminal part of the protein from a mainly disordered to an aggregated state in which this region is highly protected from hydrogen exchange.     

As a toxin dies a prion comes to life: A tentative natural history of the [Het-s] prion

A variety of signaling pathways, in particular with roles in cell fate and host defense, operate by a prion-like mechanism consisting in the formation of open-ended oligomeric signaling complexes termed signalosomes. This mechanism emerges as a novel paradigm in signal transduction. Among the proteins forming such signaling complexes are the Nod-like receptors (NLR), involved in innate immunity. It now appears that the [Het-s] fungal prion derives from such a cell-fate defining signaling system controlled by a fungal NLR. What was once considered as an isolated oddity turns out to be related to a conserved and widespread signaling mechanism. Herein, we recall the relation of the [Het-s] prion to the signal transduction pathway controlled by the NWD2 Nod-like receptor, leading to activation of the HET-S pore-forming cell death execution protein. We explicit an evolutionary scenario in which formation of the [Het-s] prion is the result of an exaptation process or how a loss-of-function mutation in a pore-forming cell death execution protein (HET-S) has given birth to a functional prion ([Het-s]).     

As a toxin dies a prion comes to life: A tentative natural history of the [Het-s] prion

A variety of signaling pathways, in particular with roles in cell fate and host defense, operate by a prion-like mechanism consisting in the formation of open-ended oligomeric signaling complexes termed signalosomes. This mechanism emerges as a novel paradigm in signal transduction. Among the proteins forming such signaling complexes are the Nod-like receptors (NLR), involved in innate immunity. It now appears that the [Het-s] fungal prion derives from such a cell-fate defining signaling system controlled by a fungal NLR. What was once considered as an isolated oddity turns out to be related to a conserved and widespread signaling mechanism. Herein, we recall the relation of the [Het-s] prion to the signal transduction pathway controlled by the NWD2 Nod-like receptor, leading to activation of the HET-S pore-forming cell death execution protein. We explicit an evolutionary scenario in which formation of the [Het-s] prion is the result of an exaptation process or how a loss-of-function mutation in a pore-forming cell death execution protein (HET-S) has given birth to a functional prion ([Het-s]).     

Signal Transduction by a Fungal NOD-Like Receptor Based on Propagation of a Prion Amyloid Fold

In the fungus Podospora anserina, the [Het-s] prion induces programmed cell death by activating the HET-S pore-forming protein. The HET-s β-solenoid prion fold serves as a template for converting the HET-S prion-forming domain into the same fold. This conversion, in turn, activates the HET-S pore-forming domain. The gene immediately adjacent to het-S encodes NWD2, a Nod-like receptor (NLR) with an N-terminal motif similar to the elementary repeat unit of the β-solenoid fold. NLRs are immune receptors controlling cell death and host defense processes in animals, plants and fungi. We have proposed that, analogously to [Het-s], NWD2 can activate the HET-S pore-forming protein by converting its prion-forming region into the β-solenoid fold. Here, we analyze the ability of NWD2 to induce formation of the β-solenoid prion fold. We show that artificial NWD2 variants induce formation of the [Het-s] prion, specifically in presence of their cognate ligands. The N-terminal motif is responsible for this prion induction, and mutations predicted to affect the β-solenoid fold abolish templating activity. In vitro, the N-terminal motif assembles into infectious prion amyloids that display a structure resembling the β-solenoid fold. In vivo, the assembled form of the NWD2 N-terminal region activates the HET-S pore-forming protein. This study documenting the role of the β-solenoid fold in fungal NLR function further highlights the general importance of amyloid and prion-like signaling in immunity-related cell fate pathways.     

A Single Amino Acid Difference Is Sufficient to Elicit Vegetative Incompatibility in the Fungus Podospora Anserina

Vegetative incompatibility is known to limit heterokaryosis in filamentous fungi. It results from genetic differences between incompatible strains at specific loci. The proteins encoded by the two incompatible alleles het-s and het-S of the fungus Podospora anserina differ from each other by 14 amino acids. Two approaches have been used to identify how many and which of these differences are necessary to elicit incompatibility. Twelve alleles of the het-s locus of wild-type isolates of P. anserina and of the related species Podospora comata have been sequenced to determine the extent of the variability of genes controlling s and S specificities. Expression of hybrid het-s/het-S genes and site-specific mutagenesis revealed that the specificities of het-s and het-S are under the control of a limited number of amino acid differences. The results show that vegetative incompatibility between s and S strains can be attributed to a single amino acid difference in the proteins encoded by the het-s locus.     

Mass analysis by scanning transmission electron microscopy and electron diffraction validate predictions of stacked beta-solenoid model of HET-s prion fibrils

Fungal prions are infectious filamentous polymers of proteins that are soluble in uninfected cells. In its prion form, the HET-s protein of Podospora anserina participates in a fungal self/non-self recognition phenomenon called heterokaryon incompatibility. Like other prion proteins, HET-s has a so-called "prion domain" (its C-terminal region, HET-s-(218-289)) that is responsible for induction and propagation of the prion in vivo and for fibril formation in vitro. Prion fibrils are thought to have amyloid backbones of polymerized prion domains. A relatively detailed model has been proposed for prion domain fibrils of HET-s based on a variety of experimental constraints (Ritter, C., Maddelein, M. L., Siemer, A. B., Luhrs, T., Ernst, M., Meier, B. H., Saupe, S. J., and Riek, R. (2005) Nature 435, 844-848). To test specific predictions of this model, which envisages axial stacking of beta-solenoids with two coils per subunit, we examined fibrils by electron microscopy. Electron diffraction gave a prominent meridional reflection at (0.47 nm)(-1), indicative of cross-beta structure, as predicted. STEM (scanning transmission electron microscopy) mass-per-unit-length measurements yielded 1.02 +/- 0.16 subunits per 0.94 nm, in agreement with the model prediction (1 subunit per 0.94 nm). This is half the packing density of approximately 1 subunit per 0.47 nm previously obtained for fibrils of the yeast prion proteins, Ure2p and Sup35p, whence it follows that the respective amyloid architectures are basically different.     

Role of Hsp104 in the propagation and inheritance of the [Het-s] prion

The chaperones of the ClpB/HSP100 family play a central role in thermotolerance in bacteria, plants, and fungi by ensuring solubilization of heat-induced protein aggregates. In addition in yeast, Hsp104 was found to be required for prion propagation. Herein, we analyze the role of Podospora anserina Hsp104 (PaHsp104) in the formation and propagation of the [Het-s] prion. We show that DeltaPaHsp104 strains propagate [Het-s], making [Het-s] the first native fungal prion to be propagated in the absence of Hsp104. Nevertheless, we found that [Het-s]-propagon numbers, propagation rate, and spontaneous emergence are reduced in a DeltaPaHsp104 background. In addition, inactivation of PaHsp104 leads to severe meiotic instability of [Het-s] and abolishes its meiotic drive activity. Finally, we show that DeltaPaHSP104 strains are less susceptible than wild type to infection by exogenous recombinant HET-s(218-289) prion amyloids. Like [URE3] and [PIN(+)] in yeast but unlike [PSI(+)], [Het-s] is not cured by constitutive PaHsp104 overexpression. The observed effects of PaHsp104 inactivation are consistent with the described role of Hsp104 in prion aggregate shearing in yeast. However, Hsp104-dependency appears less stringent in P. anserina than in yeast; presumably because in Podospora prion propagation occurs in a syncitium.     

The Molecular Organization of the Fungal Prion HET-s in Its Amyloid Form

The prion hypothesis states that it is solely the three-dimensional structure of the polypeptide chain that distinguishes the prion and nonprion forms of the protein. For HET-s, the atomic-resolution structure of the isolated prion domain HET-s(218-289), consisting of a highly ordered triangular cross-β arrangement, is known. Here we present a solid-state NMR study of fibrils of the full-length HET-s prion in which we compare their spectra with spectra from isolated C-terminal prion domain fibrils and the crystalline N-terminal globular domain HET-s(1-227). The spectra reveal unequivocally that the highly ordered structure of the isolated prion domain HET-s(218-289) is conserved in the context of the full-length fibrils investigated here. However, the globular domain loses much of its tertiary structure while partly retaining its secondary structure, thus exhibiting behavior reminiscent of a molten globule. Flexible residues that may constitute the linker connecting the two domains are detected using INEPT (insensitive nuclei enhanced by polarization transfer) spectroscopy. Based on our data, we propose a structural model that is in line with a general model developed for amyloid fibrils built from a cross-β core decorated with globular domains. The loss of structure in the HET-s globular domain sharply contrasts with the behavior observed for fibrils of Ure2p and suggests that there is considerable structural diversity in the fibrils of globular-domain-containing prions despite their similar appearances at the microscopic level.     

Characterization of the amyloid bacterial inclusion bodies of the HET-s fungal prion

The formation of amyloid aggregates is related to the onset of a number of human diseases. Recent studies provide compelling evidence for the existence of related fibrillar structures in bacterial inclusion bodies (IBs). Bacteria might thus provide a biologically relevant and tuneable system to study amyloid aggregation and how to interfere with it. Particularly suited for such studies are protein models for which structural information is available in both IBs and amyloid states. The only high-resolution structure of an infectious amyloid state reported to date is that of the HET-s prion forming domain (PFD). Importantly, recent solid-state NMR data indicates that the structure of HET-s PFD in IBs closely resembles that of the infectious fibrils. Here we present an exhaustive conformational characterization of HET-s IBs in order to establish the aggregation of this prion in bacteria as a consistent cellular model in which the effect of autologous or heterologous protein quality machineries and/or anti-aggregational and anti-prionic drugs can be further studied.