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

Abstract

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 μm 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.

Mass Analysis by Scanning Transmission Electron Microscopy and Electron Diffraction Validate Predictions of Stacked β-Solenoid Model of HET-s Prion Fibrils

“…Plasmids-Construction of Escherichia coli expression plasmids for C-terminally histidine-tagged versions of both polypeptides has been described (6,15). In brief, the reading frame of HET-s-(218 -289) and HET-s-(157-289) was amplified by PCR and cloned into pET-21a using NdeI and HindIII restriction sites.…”

“…When expressed in P. anserina, the HET-s prion domain alone can propagate the prion, but is not functional in heterokaryon incompatibility. To restore this function, at least parts of its N-terminal domain are needed (15).…”

“…Axial packing density may be determined from mass-per-unit-length measurements made by dark-field scanning transmission electron microscopy (STEM) 3 (25,26). In this study, we have applied these techniques to fibrils of the prion domain HET-s-(218 -289), and of HET-s-(157-289), a construct containing an additional segment that, when appended to the prion domain, restores competence in heterokaryon incompatibility (15). Our observations lend strong support to the model.…”

Correlation of structural elements and infectivity of the HET-s prion

“…Red lines encircle cross-peaks that show a virtually complete loss of intensity after t = 6 weeks. c,d, Homonuclear 13 ex C-13 C DREAM correlation spectra of 13 C, 15 N-labeled, HETs(218-289) fibrils. The carbonyl region of the DREAM spectra (c) was recorded at 40 kHz MAS.…”

Probing the Structure of the Infectious Amyloid Form of the Prion-forming Domain of HET-s Using High Resolution Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

“…Are Infectious-The proteinase K-resistant amyloid core of HET-s amyloids corresponds to the region spanning residue 218 -289 (22,38). We have previously shown that HET-s fibrils submitted to proteinase K digestion retain infectivity (21).…”