Hsp104 is a protein remodeling factor that is crucially important for induced thermotolerance and prion propagation in yeast. Recent work demonstrates that Hsp104 is able to directly recognize and interact with synthetic polypeptide substrates, and that this interaction is dependent on the amino acid composition or sequence (Lum et al., 2008). Here this concept is applied to the in vivo substrate Sup35. Sup35, a translation termination factor, also forms the yeast prion [PSI+]. The maintenance of the prion is critically dependent on the expression levels of Hsp104. Over-expression of Hsp104 leads to the loss of prions, as does inhibition of this protein remodeling factor. As part of this thesis, an in vitro assay was established in which spontaneous nucleation, the event preceding of fiber formation, was suppressed. Fibrilization itself then becomes strictly dependent on the chaperones Hsp104, huHsp70p and Ydj1. In line with in vivo observations, Hsp104 mutants that fail to propagate [PSI+] also fail to overcome nucleation inhibition in this assay. Following this, the next part of this work established that the middle (M) domain of Sup35 inhibited this process, while not affecting spontaneous fibrilization under non-inhibitory conditions. This finding was reproduced in vivo, as middle domain over-expression also led to curing of weak [PSI+]. This suggested that the M-domain contains an Hsp104 binding site. This hypothesis is supported by data presented in this thesis which show that a small segment 129-148 within the Middle domain has enhanced Hsp104 binding properties. Deletion of this 20-mer peptide also reduced the Hsp104 ability to interact with this prion substrate; it also results in the destabilization of the prion and enhanced curing by the prion curing agent guandidinium hydrochloride. This represents the first ever Hsp104 binding site identified within a natural substrate.

Hsp104 is a protein remodeling factor that is crucially important for induced thermotolerance and prion propagation in yeast. Recent work demonstrates that Hsp104 is able to directly recognize and interact with synthetic polypeptide substrates, and that this interaction is dependent on the amino acid composition or sequence (Lum et al., 2008). Here this concept is applied to the in vivo substrate Sup35. Sup35, a translation termination factor, also forms the yeast prion [PSI+]. The maintenance of the prion is critically dependent on the expression levels of Hsp104. Over-expression of Hsp104 leads to the loss of prions, as does inhibition of this protein remodeling factor. As part of this thesis, an in vitro assay was established in which spontaneous nucleation, the event preceding of fiber formation, was suppressed. Fibrilization itself then becomes strictly dependent on the chaperones Hsp104, huHsp70p and Ydj1. In line with in vivo observations, Hsp104 mutants that fail to propagate [PSI+] also fail to overcome nucleation inhibition in this assay. Following this, the next part of this work established that the middle (M) domain of Sup35 inhibited this process, while not affecting spontaneous fibrilization under non-inhibitory conditions. This finding was reproduced in vivo, as middle domain over-expression also led to curing of weak [PSI+]. This suggested that the M-domain contains an Hsp104 binding site. This hypothesis is supported by data presented in this thesis which show that a small segment 129-148 within the Middle domain has enhanced Hsp104 binding properties. Deletion of this 20-mer peptide also reduced the Hsp104 ability to interact with this prion substrate; it also results in the destabilization of the prion and enhanced curing by the prion curing agent guandidinium hydrochloride. This represents the first ever Hsp104 binding site identified within a natural substrate.

S. cerevisiae Sup35p inhabits two metastable states: functional translation termination factor; and prion-like aggregate [PSI+], which propagates by converting soluble Sup35p to its own misfolded form. Once initiated, Sup35p polymerization in [PSI+] cells is spontaneous, but [PSI+] prion inheritance depends on the Hsp104p disaggregase. To identify Hsp104-interacting sequences, Sup35p was subjected to a systematic deletion screen. [PSI+] maintenance by mutant Sup35p was assessed in both presence and absence of plasmid-encoded WT Sup35p in haploid sup35 cells. Large deletions abolished [PSI+], implying perturbations of prion structure, while others imparted [PSI+]-dependent toxicity. Removal of a single 25aa segment destabilised [PSI+] inheritance, resulting in enhanced rates of prion loss. This is consistent with the expected prion propagation defect in response to reduced Hsp104p interaction. However, several mutants containing this 25aa segment share the destabilised prion phenotype, suggesting chaperone/prion interactions are strongly context-dependent, and no one Sup35p region is solely responsible for Hsp104p recognition.

S. cerevisiae Sup35p inhabits two metastable states: functional translation termination factor; and prion-like aggregate [PSI+], which propagates by converting soluble Sup35p to its own misfolded form. Once initiated, Sup35p polymerization in [PSI+] cells is spontaneous, but [PSI+] prion inheritance depends on the Hsp104p disaggregase. To identify Hsp104-interacting sequences, Sup35p was subjected to a systematic deletion screen. [PSI+] maintenance by mutant Sup35p was assessed in both presence and absence of plasmid-encoded WT Sup35p in haploid sup35 cells. Large deletions abolished [PSI+], implying perturbations of prion structure, while others imparted [PSI+]-dependent toxicity. Removal of a single 25aa segment destabilised [PSI+] inheritance, resulting in enhanced rates of prion loss. This is consistent with the expected prion propagation defect in response to reduced Hsp104p interaction. However, several mutants containing this 25aa segment share the destabilised prion phenotype, suggesting chaperone/prion interactions are strongly context-dependent, and no one Sup35p region is solely responsible for Hsp104p recognition.

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Proteinaceous infectious particles, termed 'prions' are self-perpetuating protein isoforms that transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Each conformational variant of a prion protein is faithfully propagated to a homologous protein in the same cell environment. However, a reduction in the efficiency of prion transmission between different species is often observed and is termed "species barrier". Prion transmission to a heterologous protein may, in some cases, permanently change the structure of the prion variant, and divergent proteins may interfere with prion propagation in a species-specific manner. To identify the importance of both protein sequence and the cell environment on prion interference and cross-species transmission, we employed heterologous Sup35 proteins from three Saccharomyces sensu stricto species: Saccharomyces cerevisiae (Sc), Saccharomyces paradoxus (Sp), and Saccharomyces bayanus (Sb). We performed our experiments in two different cell environments (Sc and Sp). Our data show that Sup35 from one species can form a prion in another, and we employed a transfection procedure to perform cross-species transfer of the prion. Using a shuffle procedure, we demonstrate that the specificity of prion transmission is determined by the protein itself rather than the cell environment. Interestingly, we noted that variant-specific prion patterns can be altered irreversibly during cross-species transmission through S. bayanus module II. We further show that prion interference does not always correlate with cross-species prion transmission, and the identity of particular regions or even a specific amino acid, rather than the overall level of PrD homology is crucial for determining cross-species transmission and interference. Lastly we provide evidence to suggest that prion interference is specific to the cell environment.

Polyglutamine-expanded fragments, derived from the human huntingtin protein, are aggregation-prone and toxic in yeast cells, bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline-rich region targets polyglutamine aggregates to the large perinuclear deposit (aggresome). Aggresome targeting ameliorates polyglutamine cytotoxicity in the presence of the prion form of Rnq1 protein, however, aggresome-forming construct remains toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). Disomy by chromosome II partly ameliorates polyglutamine toxicity in the strains containing Sup35 prion. The chromosome II gene, coding for another release factor, and interaction partner of Sup35, named Sup45 (eRF1), is responsible for amelioration of toxicity. Plasmid-mediated overproduction of Sup45, or expression of the Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into prion aggregates, also ameliorate polyglutamine toxicity. Protein analysis indicates that polyglutamines alter aggregation patterns of the Sup35 prion and promote aggregation of Sup45, while excess Sup45 counteracts these effects. In the absence of Sup35 prion, disomy by chromosome II is still able to decrease polyglutamine toxicity. However, SUP45 is no longer the gene responsible for such an effect. Taken together with the finding that the presence of both the Rnq1 prion and the Sup35 prion has an additive effect on polyQ toxicity, one gene or few genes on chromosome II are able to ameliorate polyQ toxicity through a SUP45-independent pathway. The identification of such a gene is currently ongoing. Monosomy by chromosome VIII in diploid heterozygous by AQT (Anti-polyQ Toxicity mutants that are disomic by chromosome II) counteracted the effect of AQT. Similarly, deletion of the arg4 gene in chromosome VIII in AQT haploid was able to eliminate the AQT effect. Moreover, analysis of genes involved in the arginine and polyamine synthesis indicated that loss of genes in later stages of arginine biosynthesis causes increase of polyglutamine toxicity. Deletion of genes arg1, arg4, arg8 (arginine pathway) and spe1 (polyamine pathway) all suppressed the Sup35 prion phenotype expression in the nonsense suppression system. Further analysis regarding the mechanisms behind those effects is needed. Our data uncover the mechanisms by which genetic and epigenetic factors may influence polyglutamine toxicity, and demonstrate that one and the same type of polyglutamine deposits could be cytoprotective or cytotoxic, depending on the prion composition of a eukaryotic cell.

The word "Prion" refers to self-perpetuating protein aggregates that cause neurodegenerative diseases in mammals. It is a protein isoform that has undergone a conformational change which converts the normal form of the protein into the infectious form with the same amino acid sequence. Yeast [PSI+] prion is the prion isoform of Sup35 protein, a translation termination factor eRF3. It has been suggested that prion [PSI+] is controlled by the ensemble of chaperones with Hsp104 playing the major role. The previous work performed in the Chernoffs lab showed that the defective GET pathway caused by get led to the defect in [PSI+] curing by excess Hsp104. The GET pathway is a system responsible for transporting newly synthesized TA-protein to the ER membrane, and the components which have been proven to be involved in this pathway include: Get1, Get2, Get3, Get4, Get5 and Sgt2. In this study we describe the mechanism underlying the effect of the defective GET pathway on [PSI+]. We demonstrate that Sgt2, one of the components of GET pathway, interacts with Sup35 in both [PSI+] and [psi-] strains through its prion domain. Overproduction of Sgt2 and Hsp70-Ssa is triggered by the defective GET pathway and leads to the protection of [PSI+] aggregates from curing by excess Hsp104. We show that the direct interaction between Sgt2 and Hsp70-Ssa is not required for this protective effect.

9 ABSTRACT For proper function proteins should have a native conformation. If their conformation is impaired due to environmental stress or genetic mutation, proteins become prone to aggregation. There exist various types of protein aggregates. Stable non-membraneous inclusions can form which can serve for clearance of aberrant proteins from place where they can interfere with essential cellular processes. Another type of aggregates can serve as transient deposits of proteins thus protecting them from stress conditions. Stress granules (SG) are a such example of transient granules. Their formation is induced by heat shock for example. SGs contain mRNA, components of translation machinery, and other proteins. One of these proteins is Mmi1, small highly conserved protein with unknown function. Association of Mmi1 with stress granules and partial co-localization with chaperon Cdc48 and proteasom indicates Mmi1 can mediate heat stress damaged protein degradation. We have uncovered that yeast prion protein Sup35 is a component of stress granules as well. With regard to its aggregation capability there existed an assumption that prion domain of Sup35 could serve as scaffold for SG assembly. However as we show deletion of prion domain of Sup35 protein does not affect stress granules formation dynamics. Yeast...