A Size Threshold Limits Prion Transmission and Establishes Phenotypic Diversity

Abstract

According to the prion hypothesis, atypical phenotypes arise when a prion protein adopts an alternative conformation and persist when that form assembles into self-replicating aggregates. Amyloid formation in vitro provides a model for this protein-misfolding pathway, but the mechanism by which this process interacts with the cellular environment to produce transmissible phenotypes is poorly understood. Using the yeast prion Sup35/[PSI + ], we found that protein conformation determined the size distribution of aggregates through its interactions with a molecular chaperone. Shifts in this range created variations in aggregate abundance among cells due to a size threshold for transmission, and this heterogeneity, along with aggregate growth and fragmentation, induced age-dependent fluctuations in phenotype. Thus, prion conformations may specify phenotypes as population averages in a dynamic system. Prion proteins adopt a spectrum of conformations or strains, which create phenotypes of distinct severity and stability in vivo (1-3). These phenotypes are linked to the assembly of the protein into aggregates that, at unique rates, template the conversion of newly-made prion protein to a similar state and are fragmented (4). But, how do these biochemical events translate into distinct phenotypes? One possibility is an "abundance-based" model, in which phenotypes are linked to an equilibrium between aggregated and soluble prion protein that determines protein activity and the number of heritable prions (propagons) (5,6). However, the conversion and fragmentation reactions also create heterogeneity in aggregate size, raising the possibility of a second, "size-based" model in which a subpopulation of aggregates establishes and propagates phenotypes (7).To distinguish between these models, we focused on the [PSI + ] Weak and [PSI + ] Strong conformations of the yeast prion protein Sup35, which create phenotypes of different stabilities in vivo (8). To sustain these phenotypes in a dividing culture, Sup35 protein in the prion conformation must be inherited (7). To test whether conformational differences impact phenotypic stability by altering protein transmissibility, we monitored Sup35-GFP transfer to daughter cells. Using fluorescence loss in photobleaching (FLIP), a [PSI + ] Weak strain transferred half as much Sup35-GFP (~15% vs. ~30%; Fig. 1A) and contained ~50% fewer ** This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the

Prions in Yeast

“…Yeast cultures bearing a weak [PSI + ] variant exhibit asymmetric accumulation of larger prion polymers in aged cells (Derdowski et al 2010). It was proposed that larger polymers are less likely to be transmitted to a daughter cell (bud) during mitosis.…”

Distinct Amino Acid Compositional Requirements for Formation and Maintenance of the [PSI+] Prion in Yeast

“…Removal of all or part of the ORD (14,22,23,25) or replacement of the ORD with a random sequence (28) destabilizes [PSI ϩ ]. Such mutations appear to reduce prion aggregate fragmentation, resulting in larger aggregates that are frequently lost as a result of imperfect segregation of aggregates into daughter cells (29). The chaperone protein Hsp104 is essential for [PSI ϩ ] maintenance (30); Hsp104 cleaves prion fibers into smaller fragments better suited to segregate into daughter cells (21,31,32).…”

Insights into prion biology

“…138,139 Collectively, our studies on the yeast prion [PSI + ] have begun to bridge this gap by demonstrating that the cellular environment dramatically influences the physiological consequences of Sup35 misfolding. Notably, conformation-based phenotypes are created through the interplay among Sup35 folding, protein quality control and other aspects of yeast cell biology, 24 and imbalances in these forces lead to the dominant inhibition of prion propagation in vivo. 134 Given the many parallels between prion propagation in yeast and in mammals, the insight gained through these studies may provide a new framework for connecting protein misfolding pathways in vitro to disease mechanisms in vivo.…”

“…77 Moreover, mammalian prion biology is altered by changes in expression of the prion protein, as is the case for yeast prion biology. 24,[63][64][65][66][67] For example, the incubation time for clinical disease, but not for the generation of prion infectivity, is highly dependent on PrP expression level. 78,79 While other mechanisms are possible, 78,80,81 an intriguing model to explain these observations is that infectivity increases with aggregate abundance until reaching a plateau, which represents a limitation on the system.…”