A mutation within the C-terminal domain of Sup35p that affects [PSI+] prion propagation

The epigenetic factor [PSI+] in the yeast Saccharomyces cerevisiae is due to the prion form of Sup35p. The N-terminal domain of Sup35p (N), alone or together with the middle-domain (NM), assembles in vitro into fibrils that induce [PSI+] when introduced into yeast cells. The Sup35p C-terminal domain (C), involved in translation termination, is essential for growth. The involvement of Sup35p C-terminal domain into [PSI+] propagation is subject to debate. We previously showed that mutation of threonine 341 within Sup35p C-domain affects translation termination efficiency. Here, we demonstrate that mutating threonine 341 to aspartate or alanine results in synthetic lethality with [PSI+] and weakening of [PSI+] respectively. The corresponding Sup35D and Sup35A proteins assemble into wild-type like fibrils in vitro, but with a slower elongation rate. Moreover, cross-seeding between Sup35p and Sup35A is inefficient both in vivo and in vitro, suggesting that the point mutation alters the structural properties of Sup35p within the fibrils. Thus, Sup35p C-terminal domain modulates [PSI+] prion propagation, possibly through a functional interaction with the N and/or M domains of the protein. Our results clearly demonstrate that Sup35p C-terminal domain plays a critical role in prion propagation and provide new insights into the mechanism of prion conversion.     

酿酒酵母Sup35朊蛋白结构域的自组装机理及其应用的研究进展

Sup35是酿酒酵母的翻译终止因子,其朊蛋白结构域在体内外都能形成淀粉样蛋白纤维。由于其高度有序的交叉β-片层构象与其他物种中的淀粉样蛋白纤维相似,因此,Sup35的分子自组装机理的研究可以作为蛋白质错误折叠性疾病及朊病毒生物学等相关研究的理想模型。而Sup35朊蛋白结构域自组装成纳米线的能力在生物技术和纳米材料等方面已得到广泛的应用。     

Prion properties of the Sup35 protein of yeast Pichia methanolica

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

Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber

The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.     

PSI+] Prion transmission barriers protect Saccharomyces cerevisiae from infection: intraspecies 'species barriers'

[PSI+] is a prion of Sup35p, an essential translation termination and mRNA turnover factor. The existence of lethal [PSI+] variants, the absence of [PSI+] in wild strains, the mRNA turnover function of the Sup35p prion domain, and the stress reaction to prion infection suggest that [PSI+] is a disease. Nonetheless, others have proposed that [PSI+] and other yeast prions benefit their hosts. We find that wild Saccharomyces cerevisiae strains are polymorphic for the sequence of the prion domain and particularly in the adjacent M domain. Here we establish that these variations within the species produce barriers to prion transmission. The barriers are partially asymmetric in some cases, and evidence for variant specificity in barriers is presented. We propose that, as the PrP 129M/V polymorphism protects people from Creutzfeldt-Jakob disease, the Sup35p polymorphisms were selected to protect yeast cells from prion infection. In one prion incompatibility group, the barrier is due to N109S in the Sup35 prion domain and several changes in the middle (M) domain, with either the single N109S mutation or the group of M changes (without the N109S) producing a barrier. In another, the barrier is due to a large deletion in the repeat domain. All are outside the region previously believed to determine transmission compatibility. [SWI+], a prion of the chromatin remodeling factor Swi1p, was also proposed to benefit its host. We find that none of 70 wild strains carry this prion, suggesting that it is not beneficial.     

Hsp110 chaperones regulate prion formation and propagation in S. cerevisiae by two discrete activities

The cytosolic chaperone network of Saccharomyces cerevisiae is intimately associated with the emergence and maintenance of prion traits. Recently, the Hsp110 protein, Sse1, has been identified as a nucleotide exchange factor (NEF) for both cytosolic Hsp70 chaperone family members, Ssa1 and Ssb1. We have investigated the role of Sse1 in the de novo formation and propagation of [PSI(+)], the prion form of the translation termination factor, Sup35. As observed by others, we find that Sse1 is essential for efficient prion propagation. Our results suggest that the NEF activity is required for maintaining sufficient levels of substrate-free Ssa1. However, Sse1 exhibits an additional NEF-independent activity; it stimulates in vitro nucleation of Sup35NM, the prion domain of Sup35. We also observe that high levels of Sse1, but not of an unrelated NEF, very potently inhibit Hsp104-mediated curing of [PSI(+)]. Taken together, these results suggest a chaperone-like activity of Sse1 that assists in stabilization of early folding intermediates of the Sup35 prion conformation. This activity is not essential for prion formation under conditions of Sup35 overproduction, however, it may be relevant for spontaneous [PSI(+)] formation as well as for protection of the prion trait upon physiological Hsp104 induction.     

Alternative assembly pathways of the amyloidogenic yeast prion determinant Sup35-NM

The self-perpetuating conformational change of the translation termination factor Sup35 is associated with a prion phenomenon of Saccharomyces cerevisiae. In vitro, the prion-determining region (NM) of Sup35 assembles into amyloid-like fibres through a mechanism of nucleated conformational conversion. Here, we describe an alternative assembly pathway of NM that produces filaments that are composed of beta-strands and random coiled regions with several-fold smaller diameters than the amyloid fibres. NM filaments are not detectable with either thioflavin T or Congo Red and do not show SDS or protease resistance. As filaments do not self-convert into fibres and do not act as seed, they are not intermediates of amyloid fibre formation. Instead, they represent a stable off-pathway form. Similar to mammalian prion proteins, Sup35 contains oligopeptide repeats located in the NM region. We found that the number of repeats determines the partitioning of the protein between filaments and amyloid-like fibres. Low numbers of repeats favour the formation of the filamentous structure, whereas high numbers of repeats favour the formation of amyloid-like fibres.     

Chimeric yeast prions with unstable inheritance

The Saccharomyces cerevisiae [PSI] factor, a cytoplasmic omnipotent nonsense suppressor, is a conformationally changed (prion) form of translation termination factor eRF3 (Sup35p). Induction and maintenance of the [PSI] factor depend on the prionizing peptide located in the N domain of Sup35p. The N domain of Sup35p was fused with phosphoribosylaminoimidazole carboxylase (Ade2p), a purine biosynthesis enzyme, and the hybrid protein (NM-Sup35p::Ade2p) was tested for induction of the [PSI] factor. Transformation with a centromeric plasmid carrying the gene for NM-Sup35p::Ade2p induced a [PSI]-like factor in yeast cells, which was evident from efficient nonsense suppression. The suppressory effect depended on the presence of the prionizing peptide both in the hybrid protein and in Sup35p synthesized from the chromosomal gene, as well as on the presence of the prion-like [PIN] factor in the cell.     

Yeast Prions Compared to Functional Prions and Amyloids

Saccharomyces cerevisiae is an occasional host to an array of prions, most based on self-propagating, self-templating amyloid filaments of a normally soluble protein. [URE3] is a prion of Ure2p, a regulator of nitrogen catabolism, while [PSI +] is a prion of Sup35p, a subunit of the translation termination factor Sup35p. In contrast to the functional prions, [Het-s] of Podospora anserina and [BETA] of yeast, the amyloid-based yeast prions are rare in wild strains, arise sporadically, have an array of prion variants for a single prion protein sequence, have a folded in-register parallel β-sheet amyloid architecture, are detrimental to their hosts, arouse a stress response in the host, and are subject to curing by various host anti-prion systems. These characteristics allow a logical basis for distinction between functional amyloids/prions and prion diseases. These infectious yeast amyloidoses are outstanding models for the many common human amyloid-based diseases that are increasingly found to have some infectious characteristics.     

Prion and Nonprion Amyloids: A Comparison Inspired by the Yeast Sup35 Protein

Yeast prion determinants are related to polymerization of some proteins into amyloid-like fibers. The [PSI⁺] determinant reflects polymerization of the Sup35 protein. Fragmentation of prion polymers by the Hsp104 chaperone represents a key step of the prion replication cycle. The frequency of fragmentation varies depending on the structure of the prion polymers and defines variation in the prion phenotypes, e.g., the suppressor strength of [PSI⁺] and stability of its inheritance. Besides [PSI⁺], overproduction of Sup35 can produce nonheritable phenotypically silent Sup35 amyloid-like polymers. These polymers are fragmented poorly and are present due to efficient seeding with the Rnq1 prion polymers, which occurs by several orders of magnitude more frequently than seeding of [PSI⁺] appearance. Such Sup35 polymers resemble human nonprion amyloids by their nonheritability, mode of appearance and increased size. Thus, a single protein, Sup35, can model both prion and nonprion amyloids. In yeast, these phenomena are distinguished by the frequency of polymer fragmentation. We argue that in mammals the fragmentation frequency also represents a key factor defining differing properties of prion and nonprion amyloids, including infectivity. By analogy with the Rnq1 seeding of nonheritable Sup35 polymers, the “species barrier” in prion transmission may be due to seeding by heterologous prion of nontransmissible type of amyloid, rather than due to the lack of seeding.Key Words: amyloid, prion, Rnq1, Sup35, Ure2, translation termination, yeast     

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.     

A role for the proteasome in the turnover of Sup35p and in [PSI+] prion propagation

Yeast prions are superb models for understanding the mechanisms of self‐perpetuating protein aggregates formation. [PSI⁺] stands among the most documented yeast prions and results from self‐assembly of the translation termination factor Sup35p into protein fibrils. A plethora of cellular factors were shown to affect [PSI⁺] formation and propagation. Clearance of Sup35p prion particles is however poorly understood and documented. Here, we investigated the role of the proteasome in the degradation of Sup35p and in [PSI⁺] prion propagation. We found that cells lacking the RPN4 gene, which have reduced intracellular proteasome pools, accumulated Sup35p and have defects in [PSI⁺] formation and propagation. Sup35p is degraded in vitro by the 26S and 20S proteasomes in a ubiquitin‐independent manner, generating an array of amyloidogenic peptides derived from its prion‐domain. We also demonstrate the formation of a proteasome‐resistant fragment spanning residues 83–685 which is devoid of the prion‐domain that is essential for [PSI⁺] propagation. Most important was the finding that the 26S and 20S proteasomes degrade Sup35p fibrils in vitro and abolish their infectivity. Our results point to an overlooked role of the proteasome in clearing toxic protein aggregates, and have important implications for a better understanding of the life cycle of infectious protein assemblies.     

Evolutionary conservation of prion-forming abilities of the yeast Sup35 protein

Saccharomyces cerevisiae prion [PSI ] is a self-propagating isoform of the eukaryotic release factor eRF3 (Sup35p). Sup35p consists of the evolutionary conserved release factor domain (Sup35C) and two evolutionary variable regions - Sup35N, which serves as a prion-forming domain in S. cerevisiae, and Sup35M. Here, we demonstrate that the prion form of Sup35p is not observed among industrial and natural strains of yeast. Moreover, the prion ([PSI + ]) state of the endogenous S. cerevisiae Sup35p cannot be transmitted to the next generations via heterologous Sup35p or Sup35NM, originating from the distantly related yeast species Pichia methanolica. This suggests the existence of a 'species barrier' in yeast prion conversion. However, the chimeric Sup35p, containing the Sup35NM region of Pichia, can be turned into a prion in S. cerevisiae by overproduction of the identical Pichia Sup35NM. Therefore, the prion-forming potential of Sup35NM is conserved in evolution. In the heterologous system, overproduction of Pichia Sup35p or Sup35NM induced formation of the prion form of S. cerevisiae Sup35p, albeit less efficiently than overproduction of the endogenous Sup35p. This implies that prion induction by protein overproduction does not require strict correspondence of the 'inducer' and 'inducee' sequences, and can overcome the 'species barrier'.     

Growth phase‐dependent changes in the size and infectivity of SDS‐resistant Sup35p assemblies associated with the [PSI+] prion in yeast

The translation termination factor Sup35p can form self‐replicating fibrillar aggregates responsible for the [PSI⁺] prion state. Sup35p aggregation yields detergent‐resistant assemblies detectable on agarose gels under semi‐denaturant conditions and fluorescent puncta within the yeast cytosol when the protein is fused to GFP. It is still unclear whether any of these manifestations of [PSI⁺] truly correspond to the Sup35p assemblies that faithfully transmit the [PSI⁺] prion from mother to daughter cells. The infectious titer of prions in cells can be indirectly assessed by the ability of [PSI⁺] cells lysates to induce the prion state when introduced into naïve cells. Here, we report that the dramatic changes in the size and amounts of SDS‐resistant Sup35p that occur during growth do not correlate with the infectious titer. Our results suggest that fluorescent Sup35‐GFP puncta and detergent‐resistant Sup35p assemblies are good indicators of Sup35p conversion to the prion state but not of infectious particles number.     

Chimeric Yeast Prions with Unstable Inheritance

The Saccharomyces cerevisiae [PSI] factor, a cytoplasmic omnipotent nonsense suppressor, is a conformationally changed (prion) form of translation termination factor eRF3 (Sup35p). Induction and maintenance of the [PSI] factor depend on the prionizing peptide located in the N domain of Sup35p. The N domain of Sup35p was fused with phosphoribosylaminoimidazole carboxylase (Ade2p), a purine biosynthesis enzyme, and the hybrid protein (NM-Sup35p::Ade2p) was tested for induction of the [PSI] factor. Transformation with a centromeric plasmid carrying the gene for NM-Sup35p::Ade2p induced a [PSI]-like factor in yeast cells, which was evident from efficient nonsense suppression. The suppressor effect depended on the presence of the prionizing peptide both in the hybrid protein and in Sup35p synthesized from the chromosomal gene, as well as on the presence of the prion-like [PIN] factor in the cell.     

Effects of randomizing the Sup35NM prion domain sequence on formation of amyloid fibrils in vitro

The mechanism by which proteins aggregate and form amyloid fibrils is still elusive. In order to preclude interference by cellular factors and to clarify the role of the primary sequence of Sup35p prion domain in formation of amyloid fibrils, we generated five Sup35NM variants by randomizing amino acid sequences in PrDs without altering the amino acid composition and analyzed the in vitro process of amyloid fibril formation. The results showed that each of the five Sup35NM variants polymerized into amyloid fibrils in vitro under native conditions. Furthermore, the Sup35NM variants showed differences in their aggregation time courses. These findings indicate that specific amino acid sequence features in PrD can modify the rate of conversion of Sup35p into amyloid fibrils in vitro.     

Molecular population genetics and evolution of a prion-like protein in Saccharomyces cerevisiae

The prion-like behavior of Sup35p, the eRF3 homolog in the yeast Saccharomyces cerevisiae, mediates the activity of the cytoplasmic nonsense suppressor known as [PSI(+)]. Sup35p is divided into three regions of distinct function. The N-terminal and middle (M) regions are required for the induction and propagation of [PSI(+)] but are not necessary for translation termination or cell viability. The C-terminal region encompasses the termination function. The existence of the N-terminal region in SUP35 homologs of other fungi has led some to suggest that this region has an adaptive function separate from translation termination. To examine this hypothesis, we sequenced portions of SUP35 in 21 strains of S. cerevisiae, including 13 clinical isolates. We analyzed nucleotide polymorphism within this species and compared it to sequence divergence from a sister species, S. paradoxus. The N domain of Sup35p is highly conserved in amino acid sequence and is highly biased in codon usage toward preferred codons. Amino acid changes are under weak purifying selection based on a quantitative analysis of polymorphism and divergence. We also conclude that the clinical strains of S. cerevisiae are not recently derived and that outcrossing between strains in S. cerevisiae may be relatively rare in nature.     

The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae.

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

Glucose availability dictates the export of the soluble and prion forms of Sup35p via periplasmic or extracellular vesicles

The yeast [PSI⁺] prion originates from the self-perpetuating transmissible aggregates of the translation termination factor Sup35p. We previously showed that infectious Sup35p particles are exported outside the cells via extracellular vesicles (EV). This finding suggested a function for EV in the vertical and horizontal transmission of yeast prions. Here we report a significant export of Sup35p within periplasmic vesicles (PV) upon glucose starvation. We show that PV are up to three orders of magnitude more abundant than EV. However, PV and EV are different in terms of size and protein content, and their export is oppositely regulated by glucose availability in the growth medium. Overall, our work suggests that the export of prion particles to both the periplasm and the extracellular space needs to be considered to address the physiological consequences of vesicle-mediated yeast prions trafficking.     

Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for prion-dependent regulation.

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