Prions are infectious, self-propagating amyloid-like protein aggregates of mammals and fungi. We have studied aggregation propensities of a yeast prion domain in cell culture to gain insights into general mechanisms of prion replication in mammalian cells. Here, we report the artificial transmission of a yeast prion across a phylogenetic kingdom. HA epitope-tagged yeast Sup35p prion domain NM was stably expressed in murine neuroblastoma cells. Although cytosolically expressed NM-HA remained soluble, addition of fibrils of bacterially produced Sup35NM to the medium efficiently induced appearance of phenotypically and biochemically distinct NM-HA aggregates that were inherited by daughter cells. Importantly, NM-HA aggregates also were infectious to recipient mammalian cells expressing soluble NM-HA and, to a lesser extent, to yeast. The fact that the yeast Sup35NM domain can propagate as a prion in neuroblastoma cells strongly argues that cellular mechanisms support prion-like inheritance in the mammalian cytosol.PrP ͉ Sup35 P rions are infectious particles composed exclusively or predominantly of proteins. In mammals, prions are the causative agent of transmissible spongiform encephalopathies or prion diseases that are associated with the conversion of the normal host encoded prion protein, PrP C , to its infectious, aggregated prion isoform, PrP Sc (1). Mammalian prion diseases belong to the group of protein misfolding diseases that are associated with the abnormal aggregation of diverse host proteins into highly ordered, ␤-sheet-rich fibrillar aggregates, the so-called amyloids. A hallmark of prions is the existence of different strains that are associated with characteristic disease phenotypes (2). Remarkably, prions have also been identified in fungi, where they represent epigenetic elements of inheritance that replicate via a similar mechanism of selfpropagating protein conformation. Prions of eukaryotic microorganisms have been invaluable in elucidating basic concepts of prion biology. In fact, discoveries in yeast prion biology allowed formal demonstration of the protein-only hypothesis originally proposed for mammalian prions. Unlike mammalian prions, however, yeast prions are generally not lethal. The Saccharomyces cerevisiae epigenetic element [PSI ϩ ] is a prion of the translation termination factor subunit Sup35p (3, 4) that arises from conversion of soluble active monomers to an inactive amyloid (5-9), leading to a change in the yeast metabolic phenotype. Translation termination activity is conferred by the carboxyl-terminal domain C, whereas the amino-terminal Sup35NM domain is necessary and sufficient for prion-based inheritance (10). The N region comprises a prionforming domain (amino acids 1-97), which is defined as the minimal region essential for induction and propagation of the prion state (10). Acting as a linker between the N and C regions, the highly charged M region increases the solubility of the protein (8) and imparts stability of the prion during mitosis and meiosis (11). Yeast prion b...

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In mammalian prion diseases, an abnormally folded, aggregated form of the prion protein (PrP(Sc)) appears to catalyze a conformational switch of its cellular isoform (PrP(C)) to an aggregated state. A similar prion-like phenomenon has been reported for the Saccharomyces cerevisiae translation termination factor Sup35p that can adopt a self-propagating conformation. We have compared aggregation propensities of chimeric proteins derived from the Sup35p prion domain NM and PrP in vitro and in the cytosol of mammalian cells. Sup35p-NM and PrP displayed strikingly different aggregation behaviors when expressed in mammalian cells, with NM remaining soluble and cytosolic PrP spontaneously aggregating due to the globular domain of PrP. When fused to PrP(90-230), Sup35p-M exhibited an inhibitory effect for nucleation but increased aggregate growth, potentially by facilitating recruitment of newly synthesized chimeric proteins into the growing aggregates. This effect, however, could, to some extent, be counteracted by the prion-forming region Sup35p-N, thereby increasing aggregate frequency. Interestingly, a lowered nucleation rate was also observed in the presence of the amino-terminal region of PrP, suggesting that Sup35p-M and PrP(23-90) share some biological function in prion protein assembly. Our results provide new insights into prion protein aggregation behaviors, demonstrating the impact of dynamic interactions between prion domains and suggesting that aggregation of yeast and mammalian prion proteins is strongly influenced by yet unidentified cellular conditions or factors.

Prion diseases are infectious and fatal neurodegenerative disorders of man and animals which are characterized by spongiform degeneration in the central nervous system. In human diseases, the manifestation can be sporadic, familial or acquired by infection. Prion disorders are caused by the accumulation of an aberrantly folded isoform of the cellular prion protein (PrP(c)), commonly named PrP(Sc). Although prion diseases are usually rare, they have the potential to be transferred within and also between species by infection processes, giving then raise even to epidemic scenarios. As pathology is obviously restricted to the central nervous system pre-mortem diagnosis is usually hard to achieve. Promising approaches towards the development of therapeutic and even prophylactic anti-prion regimens were recently made. However, only a profound knowledge of the infectious agent and its replication strategy enables the design of effective anti-prion strategies. Cell culture models were highly instrumental in uncovering fundamental aspects of prion propagation. In this chapter, the cellular and molecular biology of prion proteins in general is discussed and prophylactic and therapeutic concepts derived thereof are introduced. In particular, emphasis is put on strategies targeting PrP(c) which is absolutely needed as substrate for prion conversion, and on intrinsic cellular clearance mechanisms for prions.