The [Het-s] infectious element of the fungus Podospora anserina is a prion protein involved in a genetically controlled cell death reaction termed heterokaryon incompatibility. Previous analyses indicate that [Het-s] propagates as a self-perpetuating amyloid aggregate. The HET-s protein is 289 amino acids in length. Herein, we identify the region of the HET-s protein that is responsible for amyloid formation and prion propagation. The region of HET-s spanning residues 218-289 forms amyloid fibers in vitro and allows prion propagation in vivo. Conversely, a C-terminal deletion in HET-s prevents amyloid aggregation in vitro and prion propagation in vivo, and abolishes the incompatibility function. In the soluble form of HET-s, the region from residue 1 to 227 forms a well-folded domain while the C-terminal region is highly flexible. Together, our data establish a domain structure-function relationship for HET-s amyloid formation, prion propagation and incompatibility activity.

Although autophagy is characteristic of type II programmed cell death (PCD), its role in cell death is currently debated. Both cell death-promoting and prosurvival roles of autophagy have been reported depending on the organism and the cell type. In filamentous fungi, a cell death reaction known as an incompatibility reaction occurs when cells of unlike genotype fuse. Cell death by incompatibility is characterized by a dramatic vacuolar enlargement and cell lysis. In Podospora anserina, autophagy is induced early during this cell death reaction. Cell death by incompatibility in Podospora is a model of type II PCD used here to assess the role of autophagy in this type of cell death. We have inactivated PaATG1, the Podospora ortholog of the Saccharomyces cerevisiae ATG1 gene involved in the early steps of autophagy in yeast. The ⌬PaATG1 mutant displays developmental defects characteristic of abrogated autophagy in Podospora. Using the green fluorescent proteinPaATG8 autophagosome marker, we show that autophagy is abolished in this mutant. Neither cell death by incompatibility nor vacuolization are suppressed in ⌬PaATG1 and ⌬PaATG8 autophagy mutants, indicating that a vacuolar cell death reaction without autophagy occurs in Podospora. Our results thus provide a novel example of a type II PCD reaction in which autophagy is not the cause of cell death. In addition, we found that cell death is accelerated in ⌬PaATG null mutants, suggesting that autophagy has a protective role in this type II PCD reaction.Macroautophagy, generally referred to as autophagy, occurs coincidently with cell death in type II programmed cell death (PCD) (17). Autophagy has been first described as a cellular response to nutrient starvation. This intracellular catabolic system allows degradation and recycling of long-lived proteins and organelles in eukaryotic cells (44). During this process, the cytoplasmic material is sequestered in double-membrane vesicles called autophagosomes (44). These vesicles then fuse by their outer membrane to the vacuolar/lysosomal membrane and deliver single-membrane vesicles called autophagic bodies inside the lumen of these organelles for degradation. The autophagic/vacuolar type of cell death (type II) is defined by morphological features that are the presence of autophagosomes or autophagic bodies inside the lysosome/vacuole degradative compartment with or without large vacuoles (8). Thus, type II PCD is defined as autophagic cell death, but this classification does not imply that autophagy directly contributes to cell death. In fact, two opposing views on the role of autophagy in type II PCD have emerged. Autophagy is either viewed as a medium of cellular demise or, on the contrary, as a prosurvival mechanism. Autophagy is also associated with various diseases in humans, including cancer and neurodegenerative disorders (25). Whether autophagy protects from or causes disease is also unclear (45).The molecular dissection of autophagy has been mostly performed in Saccharomyces cerevisiae and led to the identifica...

In Saccharomyces cerevisiae, the actin cytoskeleton is depolarized by NaCl stress. In this study, the response was maximal after 30 min, and then actin patches repolarized. Rvs161p was required for actin repolarization because the rvs161⌬ mutant did not repolarize actin patches after growth in a salt medium. Mutations suppressing the rvs161⌬-related salt sensitivity all occurred in genes required for sphingolipid biosynthesis: FEN1, SUR4, SUR2, SUR1, and IPT1. These suppressors also suppressed act1-1-related salt sensitivity and the defect in actin repolarization of the rvs161⌬ mutant, providing a link between sphingolipids and actin polarization. Indeed, deletion of the suppressor genes suppressed the rvs161⌬ defect in actin repolarization in two ways: either actin was not depolarized at the wild-type level in a set of suppressor mutants, or actin was repolarized in the absence of Rvs161p in the other suppressor mutants. Rvs161p was localized as cortical patches that concentrated at polarization sites, i.e., bud emergence and septa, and was found to be associated with lipid rafts. An important link between sphingolipids and actin polarization is that Rvs161p was required for actin repolarization and was found to be located in lipid rafts.