The Mechanism of Toxicity in HET-S/HET-s Prion Incompatibility

Abstract

A nontoxic functional prion activates toxicity in the HET-S/HET-s fungal heterokaryon incompatibility system by converting HET-S into a cytotoxic membrane protein.

Abstract

The HET-s protein from the filamentous fungus Podospora anserina is a prion involved in a cell death reaction termed heterokaryon incompatibility. This reaction is observed at the point of contact between two genetically distinct strains when one harbors a HET-s prion (in the form of amyloid aggregates) and the other expresses a soluble HET-S protein (96% identical to HET-s). How the HET-s prion interaction with HET-S brings about cell death remains unknown; however, it was recently shown that this interaction leads to a relocalization of HET-S from the cytoplasm to the cell periphery and that this change is associated with cell death. Here, we present detailed insights into this mechanism in which a non-toxic HET-s prion converts a soluble HET-S protein into an integral membrane protein that destabilizes membranes. We observed liposomal membrane defects of approximately 10 up to 60 nm in size in transmission electron microscopy images of freeze-fractured proteoliposomes that were formed in mixtures of HET-S and HET-s amyloids. In liposome leakage assays, HET-S has an innate ability to associate with and disrupt lipid membranes and that this activity is greatly enhanced when HET-S is exposed to HET-s amyloids. Solid-state nuclear magnetic resonance (NMR) analyses revealed that HET-s induces the prion-forming domain of HET-S to adopt the β-solenoid fold (previously observed in HET-s) and this change disrupts the globular HeLo domain. These data indicate that upon interaction with a HET-s prion, the HET-S HeLo domain partially unfolds, thereby exposing a previously buried ∼34-residue N-terminal transmembrane segment. The liberation of this segment targets HET-S to the membrane where it further oligomerizes, leading to a loss of membrane integrity. HET-S thus appears to display features that are reminiscent of pore-forming toxins.

Author Summary

Filamentous fungi have the potential for genetically distinct individuals to fuse, resulting in a cell with multiple nuclei known as a heterokaryon. This fusion event is controlled by genetic variants that determine the compatibility of the individuals, such that the fusion of incompatible genotypes triggers a cell death reaction in the heterokaryon. We have investigated the molecular mechanism of toxicity in the HET-S/HET-s incompatibility system in the fungus P. anserina. HET-s is an infectious yet non-toxic protein (prion) whose interaction with the almost identical protein HET-S has been shown to re-localize HET-S to the cell periphery, an event that is associated with the death of heterokaryons that simultaneously contain both proteins. We find that the HET-s prion converts soluble HET-S into a protein that binds to and destabilizes lipid membranes. Furthermore, we identify a potential transmembrane helix that is normally buried within the soluble fold of HET-S and show that its presence is associated with toxicity. We conclude that upon interaction with a HET-s prion, the HET-S globular domain partially unfolds, exposing a previously buried transmembrane segment that targets HET-S to the membrane. Once there, it further oligomerizes into a structure that causes a loss of membrane integrity, reminiscent of the mode of action of pore-forming toxins.