Heterologous prion-forming proteins interact to cross-seed aggregation in Saccharomyces cerevisiae

Abstract

The early stages of protein misfolding remain incompletely understood, as most mammalian proteinopathies are only detected after irreversible protein aggregates have formed. Cross-seeding, where one aggregated protein templates the misfolding of a heterologous protein, is one mechanism proposed to stimulate protein aggregation and facilitate disease pathogenesis. Here, we demonstrate the existence of cross-seeding as a crucial step in the formation of the yeast prion [PSI ⁺], formed by the translation termination factor Sup35. We provide evidence for the genetic and physical interaction of the prion protein Rnq1 with Sup35 as a predominant mechanism leading to self-propagating Sup35 aggregation. We identify interacting sites within Rnq1 and Sup35 and determine the effects of breaking and restoring a crucial interaction. Altogether, our results demonstrate that single-residue disruption can drastically reduce the effects of cross-seeding, a finding that has important implications for human protein misfolding disorders.

Figure 1

Models for the [RNQ ⁺]-dependent formation of the [PSI ⁺] prion. (A) The inhibitor titration model suggests that an inhibitor molecule (red squares) binds to Sup35 in its soluble state (blue circles). The presence of [RNQ ⁺] (green ring) sequesters the inhibitor away from Sup35, thereby allowing the protein to aggregate and form [PSI ⁺]. (B) The seeding model suggests that there is a physical interaction between Sup35 and aggregated Rnq1 during the formation of [PSI ⁺]. After the interaction, the [RNQ ⁺] and [PSI ⁺] prions are propagated independently.

Figure 2

Rnq1 and Sup35 physically interact. (A) ThT kinetic experiments monitor the polymerization of amyloid via enhanced fluorescent emission. Unseeded Sup35 (blue line) polymerizes and aggregates after approximately 17 hours, as does Sup35 incubated with Rnq1 monomer alone (green line). The addition of Rnq1 fibers (orange line) reduces the lag time for Sup35 polymerization to approximately 12.5 hours. Curves represent data from three experiments. (B) Sup35 was immobilized on resin and utilized as bait for a [RNQ ⁺] trap assay. Cell lysates were incubated with the resin, washed with buffer, and eluted. The presence of trapped, untagged Rnq1 was detected via western blot with anti-Rnq1 antibody. Lanes shown are the unbound (UB) fraction, the final wash fraction and the first two elution fractions. “Wash*” indicates an intermediate wash fraction. Blots are representative images from three independent experiments.

Figure 3

Rnq1-Q298R causes a [PSI ⁺] induction defect. (A) The Rnq1 protein contains an N-terminal domain (NTD) and a glutamine/asparagine-rich prion-forming domain (PFD). The PFD contains four glutamine-dense regions, Q1-4. The Q298R mutation occurs in region Q3 of the PFD. (B) Rnq1 aggregates were treated at a gradient of temperatures in SDS to determine the melting point of the Rnq1-Q298R aggregates versus the WT. There were no significant differences in the thermostable properties of either protein aggregate. Western blot signal from multiple experiments was quantified using ImageJ, normalized by the 95C band, and plotted using Origin 9.0. Error bars represent standard error of the mean (s.e.m). (C) There are no detectable differences in mitotic stability of Rnq1-Q298R aggregates as compared to WT Rnq1 aggregates. [RNQ ⁺] strains containing the [R NQ ⁺] Reporter Protein (RRP), a phenotypic readout for the [RNQ ⁺] prion, were transformed with plasmids harboring either WT RNQ1 or rnq1-Q298R. We assessed the mitotic stability, or spontaneous prion loss, of resulting strains and found that [RNQ ⁺] formed from WT or Rnq1-Q298R was similarly maintained. (D) The rnq1-Q298R mutant shows a strong defect in [PSI ⁺] induction relative to WT cells of the m.d. high variant of [RNQ ⁺]. [PSI ⁺] was induced by overexpression of Sup35 in [psi ⁻][RNQ ⁺] cells of either a RNQ1 or rnq1-Q298R genetic background. Colonies were assessed by color, with white or pink colonies or sectored colonies scored as [PSI ⁺]. Error bars represent mean ± s.e.m. The “_*” symbol represents a significant difference between [PSI ⁺] induction in RNQ1 versus rnq1-Q298R backgrounds, p < 0.001. (E) Results from [PSI ⁺] induction following cytoplasmic transfer of either WT Rnq1 or Rnq1-Q298R protein aggregates into a [rnq ⁻] RNQ1 strain. The propagated [RNQ ⁺] would be templated from the WT or mutant aggregate, but be comprised of only WT protein. Error bars represent mean ± s.e.m.

Figure 4

Sup35 mutations can restore interaction with Rnq1-Q298R. (A) Candidates from a suppressor screen to identify Sup35 mutants that can rescue the [PSI ⁺] induction defect associated with rnq1-Q298R. These mutants enhanced [PSI ⁺] formation in the rnq1-Q298R genetic background relative to WT SP5. Horizontal white bars separate non-adjacent spottings from the same plate. Complete spottings of screen candidates appear in Supplementary Figure S2. (B) The indicated mutations were cloned into SUP35 for further testing.

Figure 5

Sup35-N5Y strongly rescues the [PSI ⁺] induction defect. (A) [PSI ⁺] induction experiments demonstrate that three Sup35 mutants restore [PSI ⁺] formation to nearly WT levels in rnq1-Q298R cells. These mutations do not increase [PSI ⁺] formation in a RNQ1 genetic background. Horizontal axis labels denote SUP35 genetic status. The [rnq ⁻] control cells express RNQ1 and SUP35. More than 13,000 colonies were assessed over five biological replicates. Error bars represent mean ± s.e.m. The “*” symbols represent a significant difference between [PSI ⁺] induction with WT Sup35 in rnq1-Q298R versus induction with the indicated sup35 mutants, p < 0.005. (B) Boiled gel assays allow separate migration of monomeric and aggregated materials, confirming that Sup35 aggregation is reduced in the SUP35 rnq1-Q298R background relative to SUP35 RNQ1 cells. Sup35 aggregation is restored in sup35-N5Y rnq1-Q298R cells. We attribute the slightly aberrant monomer bands as an artifact of the experimental procedure, as samples were unboiled (and likely non-denatured) as per the protocol, and Sup35 may shed unevenly from aggregates in addition to the soluble pool. Western blot is a representative image from three independent experiments.

Figure 6

The N terminal region of Sup35 crosslinks to Rnq1. Site-directed crosslinking of Sup35-G7C to Rnq1 created high-molecular weight aggregates as visualized by SDD-AGE. Rnq1 alone, Sup35-G7C alone, and both proteins together without SMPB did not create large aggregates. The “⁺*” notation in the fifth lane indicates Sup35 monomer following treatment with TCEP. Western blot is a representative image from three independent experiments. In the lower blot, Rnq1 loading was confirmed by SDS-PAGE. Vertical white bars separate non-adjacent lanes of the same blot. The dashed line separates adjacent lanes of the same blot under differing film exposures for image clarity (full blots in Supplementary Figure S4). There is excess Rnq1 in lanes 1 and 3, samples prepared without crosslinker, to adjust for protein that is lost during sample desalting following attachment of the crosslinker (see Methods). Three trials of crosslinking were included in each experiment and the crosslinking experiment was repeated with different batches of purified proteins three times.