Functional and biochemical analysis of oligomeric intermediates of yeast prions formed in vivo

For over a century there has been a near universal acceptance of Mendel's Laws of Inheritance to explain the faithful transmission of genetic information from one generation to the next. A nucleic acid-based hereditary unit, the gene, carries this information and the characteristics of an organism are defined by its set of genes. Over the last century a number of exceptions to Mendel's Laws have been identified, but a new challenge to the dogma that all inheritance is nucleic acid based has recently arisen from the discovery of prions. Although prions have been intensively studied as the disease-causing agents associated with 'Mad Cow Disease' and the human equivalent, Creutzfeldt-Jakob Disease (CJD), there is now a considerable body of scientific evidence that prions might actually represent an entirely new form of heredity, based on a protein molecule rather than a nucleic acid. This evidence comes from the analysis of novel prion-based 'genetic' determinants in Baker's Yeast (Saccharomyces cerevisiae) one of which (the so-called [PSI+] element) is being studied in this project. Our aim is to fully understand how such a protein-based determinant is generated de novo and then replicated aand passed on to other yeast cells when they grow and divide. We know that this requires cells to produce prion seeds which can be passed on to other cells and it is our objective to define the molecular composition of these seeds. Uisng advanced genetic and biochemical methods we will study different yeast prions and look in particular at the type of physical changes they go through in taking up their prion form. One outcome of this research might be identifying steps in the prion cycle that can be targetted by new drugs to block the formation of prion seeds.

Saccharomyces cerevisiae has three established prions: Sup35p/[PSI+], Ure2p/[URE3+] and Rnq1p/[PIN+]. Two models have been used to explain the self-propagation of prions in both yeast and mammals, the template-directed refolding model and the 'seeded polymerisation'. The latter model has gained general acceptance and invokes the formation of oligomeric seed that drives the polymerisation of soluble molecules of the prion protein thus forming the characteristic high molecular weight amyloid fibrils. Stable propagation of a yeast prion also requires the generation of such new prion seeds (which we refer to as 'propagons') which must be efficiently distributed during cell division and meiosis. However, what constitutes a yeast propagon at the molecular level and how its structure relates to the high molecular weight prion aggregates found in [PSI+] cells remains to be established. In vitro prion conversion assays have identified several different oligomeric forms of Sup35p and we have obtained preliminary data for the existence of several different prion oligomers in the cell. The overall aim of this project is to identify and characterise biochemically the prion protein oligomer(s) that are important for the de novo formation and propagation of the [PSI+] and [PIN+] prions of yeast. We will develop methods for the fractionation and analysis of oligomeric intermediates of both the Sup35p and Rnq1p and then use both an in vivo and an in vitro assay to identify those oligomers that are important for seeding polymerisation of the soluble forms of the protein i.e. define the propagons. Once we have identified these oligomer(s) they will be fully characterised both in terms of their morphology and biochemical composition. Finally, we will establish the nature of the functional interplay between Sup35p, Rnq1p and Hsp104, a molecular chaperone that is essential for prion propagation, during de novo conversion to [PSI+].