Toxicity and infectivity: insights from de novo prion formation

Abstract

Prions are infectious misfolded proteins that assemble into oligomers and large aggregates, and are associated with neurodegeneration. It is believed that the oligomers contribute to cytotoxicity, although genetic and environmental factors have also been shown to have additional roles. The study of the yeast prion [PSI ⁺] has provided valuable insights into how prions form and why they are toxic. Our recent work suggests that SDS-resistant oligomers arise and remodel early during the prion formation process, and lysates containing these newly formed oligomers are infectious. Previous work shows that toxicity is associated with prion formation and this toxicity is exacerbated by deletion of the VPS5 gene. Here, we show that newly made oligomer formation and infectivity of vps5∆ lysates are similar to wild-type strains. However using green fluorescent protein fusions, we observe that the assembly of fluorescent cytoplasmic aggregates during prion formation is different in vps5∆ strains. Instead of large immobile aggregates, vps5∆ strains have an additional population of small mobile foci. We speculate that changes in the cellular milieu in vps5∆ strains may reduce the cell's ability to efficiently recruit and sequester newly formed prion particles into central deposition sites, resulting in toxicity.

Keywords: Infectivity; Oligomer; Prion; Sup35; Vps5; Yeast.

Figure 1

vps5Δ strains have reduced aggregate formation frequency, yet show no change in SDS-resistant oligomers. A. The VPS5 open reading frame (YOR069w) and VAM10 open reading frame (YOR068c) are located on chromosome 15 in the yeast genome. Site directed mutagenesis was performed to generate plasmids that contain a mutation in the initiator methionine of either VPS5 or VAM10. Two nucleotide substitutions replaced the initiation methionine with an arginine in the VPS5 open reading frame, while leaving the VAM10 open reading frame untouched. In a second plasmid, a single nucleotide substitution at the beginning of the VAM10 open reading frame leads to a mutation that changes methionine for isoleucine, while maintaining the same wildtype amino acid (serine) in the VPS5 sequence encoded by the opposite strand. All plasmids were sequenced in both directions to confirm the engineered mutation and the opposite open reading frame sequence. B. Plasmids containing wildtype versions of both genes (rescue), or mutated versions that maintain wildtype versions of only one gene (VAM10 or VPS5) were transformed into vps5Δ [PIN⁺] 74D-694 strains (Manogaran et al., 2011) along with a plasmid containing a copper inducible Sup35PrD-GFP allele. Sup35PrD-GFP was overexpressed for 24 hours in wildtype, vps5Δ, or vps5Δ strains with the indicated plasmid. The number of cells containing ring, line, or dot-like aggregates was counted from a population of at least 300 cells from three independent transformants. Standard deviation is shown. Statistically significant differences from wildtype or vps5Δ strains were determined by unpaired two-tailed t-test * p<0.005. C. Sup35PrD-GFP was overexpressed in wildtype and vps5Δ strains for 24 hours. Cultures were lysed and immediately subjected to SDD-AGE immunoblots using anti-GFP antibody (left) to detect Sup35PrD-GFP and anti-Sup35C antibody to detect full length Sup35p (BE4; right) according to Sharma et al., 2017. [PSI⁺] lysates are run for the detection of established [PSI⁺] oligomers.

Figure 2

Sup35PrD-GFP forms additional small anomalous aggregates during prion induction. A. Sup35PrD-GFP was overexpressed in vps5Δ cells for 18 hours, and then imaged using 8-well glass slides for an additional 6-12 hours by 4D microscopy. Because of the reduced aggregate formation in vps5Δ, we selectively captured fields of cells in which in the initial stages of early foci formation could be captured. Of the 149 cells imaged, we were able to view aggregate formation in 33 cells (17 cells in G1, and 16 cells in G2/M phase). We followed the formation of early foci into larger aggregates and categorized them into four distinct pathways previously characterized for wildtype cells by Sharma et al., 2017. Statistical analysis using Chi-square goodness of fit tests indicate that while wildtype cells have an equal probability for each pathway, vps5Δ cells do not (p < 0.05). B. Diagrammatic representation of the four pathways in vps5Δ strains. While the pathways were similar between wildtype and vps5Δ strains, we noticed small anomalous aggregates, many of which were mobile, associated with pathways I, II, and IV in vpsΔ. C. Representative images of wildtype (WT) and vps5Δ strains are shown. Arrows indicate small anomalous aggregates. All images were subjected to 3D deconvolution using Autoquant deconvolution algorithms (Media Cybernetics) and are shown as maximum projection images.