The sequences appended to the amyloid core region of the HET-s prion protein determine higher-order aggregate organization in vivo.

The [Het-s] prion of the fungus Podospora anserina propagates as a self-perpetuating amyloid form of the HET-s protein. This protein triggers a cell death reaction termed heterokaryon incompatibility when interacting with the HET-S protein, an allelic variant of HET-s. HET-s displays two distinct domains, a N-terminal globular domain and a C-terminal unstructured prion-forming domain (residues 218-289). Here, we describe the characterization of HET-s(157-289), a truncated form of HET-s bearing an extensive deletion in the globular domain but retaining full activity in incompatibility and prion propagation. In vitro, HET-s(157-289) polymerizes into amyloid fibers displaying the same core region as full-length HET-s fibers. We have shown previously that fusions of green fluorescent protein (GFP) with HET-s or HET-s(218-289) form dot-like aggregates in vivo upon transition to the prion state. By contrast, a HET-s(157-289)/GFP fusion protein forms elongated fibrillar aggregates in vivo. Such elongated aggregates can reach up to 150 microm in length. The in vivo dynamics of these organized structures is analysed by time lapse microscopy. We find that the large elongate structures grow by lateral association of shorter fibrillar aggregates. When co-expressed with HET-s(157-289), full-length HET-s and HET-s(218-289) can be incorporated into such elongated aggregates. Together, our data indicate that HET-s(157-289) aggregates can adopt an organized higher-order structure in vivo and that the ability to adopt this supramolecular organization is conferred by the sequences appended to the amyloid core region.