Characterisation of the function, sequence polymorphism and toxicity of the yeast Rnq1p prion protein

The Saccharomyces cerevisiae Rnq 1 p prion protein plays an integral role in the dynamics of other aggregation-prone proteins, be they other prion proteins or glutamine-rich proteins. The conformational conversion of a prion protein to its prion state is often associated with changes to cellular physiology and two interesting questions arise from this. One, what impact do these physiological changes have on our ability to interpret experimental findings in this model organism? Two, are these changes non-random, representing a novel means of altering cellular physiology? An understanding of the cellular function of Rnq 1 P is important in addressing these questions. Further, the role of Rnq 1 P in its [PIN+] prion state as a universal catalyst for amyloid-formation provides a useful model for dissecting mechanisms of amyloid toxicity, and once again, cellular function is one parameter of multiple that determine the [mal toxicity profile of a protein. To identify a cellular function for Rnqlp both phenotypic assays and a mass spectrometry-based label-free quantitative proteomics analysis were performed. A role for Rnq 1 P as a negative regulator of translation termination was characterised

and eo-localisation of Rnq 1 p with P-bodies, tightly packed clusters of untranslating rnRNA, was observed indicating that Rnqlp is intimately associated with mRNA dynamics within the cell. Additionally, evidence for a role in the maintenance of mitochondrial respiratory capacity pertaining to ATP-generation is presented, along with indications that this latter role may be through transcriptional regulation. To further understand the mechanisms of toxic protein aggregation, two analyses were performed. One, a study on the impact of natural Rnqlp polymorphisms identified fifty-three novel RNQI alleles and sequence features affecting Rnq 1 P toxicity. Two, a screen for genetic modifiers of both Rnqlp and mutant huntingtin demonstrated the role of P-bodies and mRNA degradation pathways in modulating amyloid or glutamine-based toxicity, along with a possible role for energy. homeostasis. The results presented in this thesis provided new insight into the functional roles of Rnq 1 P within the cell, and consequently the possible impact on cellular physiology associated with Rnqlp's [pin-] to [PIN+] conversion, and also identified novel modulators of toxic aggregation events in the cell.