Yeast prion physiology

Abstract

Prions, or proteinaceous infections, are caused by proteins that have the unique ability to adopt an alternative, self-replicating structure. These self-replicating structures are the causative agent of a number of mammalian diseases including Bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, and Kuru. More recently, yeast were discovered to carry at least a dozen proteins capable of making this structural conversion. Yeast prions are unique in that their prion-forming domains are intrinsically disordered domains, with unusual compositional biases. This thesis addresses two broad questions about yeast prion physiology. First, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobics are strongly under-represented. The second chapter of this dissertation explores the effects of hydrophobic and aromatic residues on prion formation. Insertion of even a small number of hydrophobic residues is found to strongly increase prion formation. These data, combined with bioinformatics analysis of glutamine/asparagine-rich domains, suggest a limit on the number of strongly prion-promoting residues tolerated in glutamine/asparagine-rich domains. Recent studies have demonstrated that aromatic residues play a key role in the maintenance of yeast prions during cell division. Taken together, these results imply that non-aromatic hydrophobic residues are excluded from prion domains not because they inhibit prion formation, but instead because they too strongly promote aggregation, without promoting prion propagation. Despite more than 20 years of research, we still don’t know why yeast carry so many prion and prion-like domains. It has been proposed that prions may serve some biological function. Chapter Three presents progress on two lines of investigation designed to resolve this issue First, a novel bioinformatics algorithm (GARRF) is used to screen a wide range of proteomes to find examples of Q/N rich domains outside of Saccharomyces cerevisiae. Identifying other species that carry these unusual regions provides insight into their role in cellular biology. We find a wide range species carry prion-like domains at levels comparable to Saccharomyces cerevisiae, and a small number carry up to an order of magnitude more. Second, currently researchers rely primarily on yeast genetic methods to discover and monitor prions. These methods have a number of drawbacks, including a glacially slow readout time. Chapter Three reports on progress towards the development of a novel fluorescence based prion assay. This assay takes advantage of bi-molecular fluorescence complementation, a technique that uses complementary fragments of a fluorescent protein to indicate when two interacting domains are in proximity to one another. When completed, this assay will provide a means to monitor protein aggregations that is both faster and more sensitive than any existing assay.