Extrachromosomal regulators of phenotypic heterogeneity in Saccharomyces species

Phenotypic heterogeneity in the yeast Saccharomyces cerevisiae has input from both genetic and epigenetic determinants. In addition to changes in DNA sequence induced by mutagens and error-prone mechanisms, there are a variety of 'non-mutagenic' chemicals that can trigger inherited changes in phenotype. One such agent, guanidine hydrochloride (GdnHCl), can both generate mitochondrial petite mutants and also induce the loss of various prions from this yeast species. Prions are novel protein-based epigenetic determinants that undergo self-perpetuating,heritable changes in their structure, resulting in the aggregation of an alternative conformational form of a protein. To date, prions have been almost exclusively studied in laboratory-bred strains of S. cerevisiae, and the work in this thesis broadens the study to wild strains i.e. non-laboratory strains of S. cerevisiae and three related Saccharomyces species: S. bayanus, S. mikatae and S. kudriavzevii. The studies outlined here sought to answer two questions; whether the mechanism of petite induction by GdnHCl is the same as leads to prion loss i.e. by inhibition ofthe molecular chaperone Hsp104; and whether we can identify prion-associated traits in other Saccharomyces species by GdnHCl-mediated curing. To answer the first question, the use of S. cerevisiae mutants lacking either Hsp104 or the related mitochondrial chaperone Hsp78 ruled out inhibition of both of these proteins as the mechanism of petite induction by GdnHCl, as both mutants maintained mitochondrial function. Furthermore, GdnHCl also generates respiratory deficient petite mutants in the three Saccharomyces species under test, and a detailedcomparative analysis of the impact of GdnHCl on the ultrastructure and respiratory functions of mitochondria in these species is reported. The search for prion-related phenotypes in these Saccharomyces species uncovered the possible existence of one or more endogenous prion which may control phenotypic traits that impact on a cell's chance of survival in fluctuating environments. Finally, the ability of these genetically-related species to facilitate and perpetuate the formation of other amyloid-forming proteins; Alzheimer's disease associated protein Aβ42 and Huntingtin (Htt)-associated polyglutamine (polyQ) was analysed. The results obtained indicate that each of these species can propagate the amyloid forms of Aβ42 and polyQ, but these amyloid states are differentially impacted upon by the endogenous prion state of the host yeast species. The expansion of studies into alternate species of Saccharomyces has uncovered the possible existence of prions and prion-related phenotypes in these species; and provides considerable potential to develop more disease-relevant models in the future.