Date of Award
Spring 2025
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Biological Sciences
First Advisor
Anita Manogaran
Second Advisor
Allison Abbott
Third Advisor
Emily Sontag
Abstract
Prion diseases are infectious neurodegenerative disorders associated with Creutzfeldt-Jakob disease, bovine spongiform encephalopathy (“Mad cow” disease) and kuru. In prion disease, a native protein can misfold and further convert a normal version of itself to the misfolded conformation. These misfolded proteins can form aggregates which are associated with these diseases. While much work has been dedicated to understanding how protein aggregates impact cellular health, less is known about the cellular mechanisms that contribute to the formation of the prion. To dissect these mechanisms, we use the budding yeast, Saccharomyces cerevisiae, which provides a powerful tool for studying prion formation. The yeast translation termination factor, Sup35, is similar to the human prion protein in that it can misfold and aggregate to form [PSI+] prion. Sup35 contains an N-terminal prion-forming domain (PrD), which is rich in glutamine (Q) and Asparagine (N) residues followed by a C-terminus that is important for translation termination. The PrD is required for the formation of the prion. Sup35 as well as the PrD alone, can misfold to form aggregates that are transmitted from mother to daughter cell in a process called propagation. The cellular mechanisms that underlie the formation of [PSI+] are poorly understood. Previous work has shown that deletions of certain endocytic coat protein genes, SLA1 and LAS17, reduce [PSI+] formation. Yeast 2-hybrid (Y2H) studies suggest that Sup35PrD interacts with the C-terminus of Sla1 protein. Similar to Sup35PrD, the C-terminus of Sla1 is Q-rich. I hypothesize that [PSI+] formation is dependent upon early endocytic coat proteins and the Q-rich C-terminus of Sla1. In this study, I elucidated the mechanisms driving de novo prion formation with a focus on the role of endocytic coat proteins. Using in silico methods, I analyzed the amino acid sequences of several early endocytosis coat proteins and showed that Ede1, Ent1, Ent2, Sla1, and Vrp1, all harbor extensive intrinsically disordered regions (IDRs), and some are enriched with glutamine (Q)-rich sequences (Sla1). Focusing on these IDR-containing endocytic coat proteins, I generated several gene deletions in order to test whether loss of these genes would result in changes in prion formation. Using prion induction techniques, my work shows that only loss of SLA1 and LAS17 significantly impairs prion formation, specifically sla1∆ mutants exhibited a 1.6-fold reduction in the percentage of cells with visible Sup35PrD-GFP aggregates, whereas las17∆ mutants showed a 5.1-fold reduction. Similarly, [PSI+] induction frequencies decreased by approximately 4.2-fold in sla1∆ and 4.8-fold in las17∆ mutant strains. To test if the C-terminus of Sla1 influences prion formation, I used a Sla1-mCherry mutant (lacking the C-terminal domain) to test prion induction frequency. These experiments revealed no significant difference in prion induction compared to wildtype, suggesting that the N-terminal region, especially the SH3 domains of Sla1, may play a critical role in prion formation.
