Analysis of the Swi1 prion domain via mutagenesis

Prions are proteins capable of adopting altered, transmissible conformations implicated in numerous diseases. The namesake prion protein typically adopts a normal fold, PrPC but may also adopt a toxic alternative conformation PrPSc that leads to neurodegenerative diseases termed transmissible spongiform encephalopathies. Other illnesses including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis have proteins implicated to act as prions. Prions have been identified in mammals as well as plants, Drosophila, Aplysia, and fungi such as the budding yeast, Saccharomyces cerevisiae. In fact, S. cerevisiae has at least nine identified yeast prions – endogenous proteins that can adopt altered conformations, aggregate, and act as epigenetic elements. Many yeast prion proteins (and some human prion-like proteins) are highly enriched in glutamine (Q) and/or asparagine (N) residues; however, the effects of various amino-acids on prion characteristics are only partially understood. Our lab discovered the yeast prion [SWI+] whose protein determinant, Swi1 consists of three domains – an N-terminal N-rich domain, a Q-rich domain, and a functional C-terminal domain. We previously demonstrated that the first 38 amino acids of Swi1 are necessary and sufficient for prionization and maintenance and propagation of [SWI+]. This prion domain (PrD) of Swi1 remains the smallest PrD described and uniquely contains a high number of N residues without any Q residues. Within this PrD, we have conducted site-directed mutagenesis to elucidate how the various amino-acid residues contribute towards the prion properties of the Swi1 PrD and characteristics of [SWI+]. Using the robust yeast system, we have analyzed the ability for these mutants to aggregate with wild-type Swi1 as well as maintain [SWI+] in the absence of full-length Swi1. Initial results suggest differing contributions to aggregation of the two N-terminal phenylalanine residues as well as lack of importance for the lone C-terminal threonine residues. Additional analysis via a saturated random mutagenesis approach will provide further unbiased insight. The combined efforts of these experiments shall increase understanding of what makes a protein capable of forming, maintaining, and propagating a prion.