The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog

Abstract

The het-s locus of Podospora anserina is a heterokaryon incompatibility locus. The coexpression of the antagonistic het-s and het-S alleles triggers a lethal reaction that prevents the formation of viable heterokaryons. Strains that contain the het-s allele can display two different phenotypes, [Het-s] or [Het-s*], according to their reactivity in incompatibility. The detection in these phenotypically distinct strains of a protein expressed from the het-s gene indicates that the difference in reactivity depends on a posttranslational difference between two forms of the polypeptide encoded by the het-s gene. This posttranslational modification does not affect the electrophoretic mobility of the protein in SDS/PAGE. Several results suggest a similarity of behavior between the protein encoded by the het-s gene and prions. The [Het-s] character can propagate in [Het-s*] strains as an infectious agent, producing a [Het-s*] --> [Het-s] transition, independently of protein synthesis. Expression of the [Het-s] character requires a functional het-s gene. The protein present in [Het-s] strains is more resistant to proteinase K than that present in [Het-s*] mycelium. Furthermore, overexpression of the het-s gene increases the frequency of the transition from [Het-s*] to [Het-s]. We propose that this transition is the consequence of a self-propagating conformational modification of the protein mediated by the formation of complexes between the two different forms of the polypeptide.

Figure 1

Immunoblot analysis of proteins isolated from [Het-s] and [Het-s*] strains (A) or from strains that contain the het-s ORF under the control of its promoter or under the control of the promoter of the glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene of A. nidulans (B). Markers used for gel calibration were muscle rabbit phosphorylase B (97.4 kDa), BSA (66 kDa), egg albumin (45 kDa), and carbonic anhydrase from bovine erythrocytes (29 kDa). Arrowheads indicate position of protein encoded by the het-s gene.

Figure 2

Schematic drawing of the experimental device used to analyze the [Het-s*] → [Het-s] transition. The striped square shows the position where a piece of mycelium was picked up 15 hr after the fusion of donor and recipient mycelia to determine the phenotype.

Figure 3

Immunoblot of protein extracts from [Het-s] and [Het-s*] strains after digestion for various times with proteinase K. Numbers on the left show the position of monomers, 1; dimers, 2; trimers, 3; and tetramers, 4.

Figure 4

Model of relations between the proteins encoded at the het-s locus. The het-S allele encodes a protein that can form complexes with the pHET-s polypeptide; coexpression of the two proteins triggers the incompatibility reaction. The het-s allele encodes the pHET-s* polypeptide, which is not reactive in incompatibility, and can be converted into a structurally different form, the prion form pHET-s, which is reactive against pHET-S, and can catalyze the conversion of pHET-s* to the pHET-s conformation.