Propagating Prions in Fungi and Mammals Mick F Tuite, Nadejda Koloteva-Levin  Molecular Cell  Volume 14, Issue 5, Pages 541-552 (June 2004) DOI: 10.1016/j.molcel.2004.05.012

Figure 1 An Overview of Prionogenesis: De Novo Conversion versus Seeded Propagation The normal form of the prion protein P (Psol) exists in equilibrium with a structurally distinct but unstable form (P*). This can then be converted to the stable prion form ([P]) by one of two routes. Propagation: through an interaction with a pre-existing prion “seed” (Pseed) the P* form can be stabilized and take up a stable prion form ([P]), a process that does not require the involvement of any cellular factors. Over time, through an interaction with other [P] forms, a high molecular weight polymer ([P]n: amyloid in nature) forms. The [P]n and possibly the [P] form define the protease resistant state of this protein. New prion seeds are generated by the disaggregation of the [P]n form with the help of one or more cellular factors (Y). De novo conversion: either through an interaction with a cellular factor (X) or through a rare misfolding event a stable [P] form is spontaneously generated leading to propagation as described above. Molecular Cell 2004 14, 541-552DOI: (10.1016/j.molcel.2004.05.012)

Figure 2 Assaying Prion Conversion and Propagation in Yeast Cells (A) Expression of a Sup35p-GFP fusion protein in a prion-free [psi−] cell results in a uniform fluorescence in the cell's cytoplasm whereas expression in a prion-containing [PSI+] cell leads to the appearance of defined aggregates (or foci) of the fusion protein. (B) Cells which carry the ade1-14 nonsense suppressible (UGA) allele give rise to red colonies on rich medium but if these cells carry the [PSI+] prion, the colonies appear pink/white due to suppression of the ade1-14 allele. A failure to efficiently propagate the [PSI+] prion will often lead to the appearance of red [psi−] sectors. Molecular Cell 2004 14, 541-552DOI: (10.1016/j.molcel.2004.05.012)

Figure 3 The In Vitro Formation of Amyloid Fibrils of the Yeast Sup35p Prion Protein (A) Conversion of the soluble form of the prion-forming domain of Sup35p (Sup35-PrD) to amyloid fibrils monitored by the binding of Congo Red. Amyloid fibrils begin to appear after 2 hr in the unseeded reaction. However, addition of pre-formed fibrils of the same protein significantly reduces the 2 hr lag phase with effectively no lag seen when 3% (wt/wt) pre-formed fibrils are added. (B) Amyloid fibrils of the Sup35p-PrD protein formed in vitro as visualized by atomic force microscopy. The scale bars are 1 μm. Figure is taken from DePace et al. (1998) with permission from the publishers. Molecular Cell 2004 14, 541-552DOI: (10.1016/j.molcel.2004.05.012)

Figure 4 New Prion Seeds Are Generated by the Shearing of the Growing Amyloid Fibril into Smaller Polymers The amyloid fibril (gray) is sheared either mechanically or by a molecular chaperone-based disaggregase complex. In yeast the latter involves the molecular chaperone Hsp104 and efficient shearing requires a degree of conformational flexibility within the amyloid fibril provided by the prion-forming domain (arrow). The new seeds capture the conformationally distinct but unstable P* form (red) of the prion protein P and stabilize it in the new prion conformer by incorporating it as part of the growing amyloid chain. New amyloid growth can be from either end of the seed although one end is believed to predominate (DePace and Weissman, 2002). Propagation requires on-going synthesis of Psol (blue). Molecular Cell 2004 14, 541-552DOI: (10.1016/j.molcel.2004.05.012)

Figure 5 Functional Dissection of the Prion-Forming Domain of the Yeast Sup35p Protein The Saccharomyces cerevisiae Sup35p-PrD contains two structurally distinct regions, the Asn/Gln-rich region (QNR; residues 1-40) and the oligopeptide repeat containing region (OPR; residues 41-97). The behavior of the indicated regions in three different assays are shown: aggregation, as defined by the formation of fluorescent foci when fused to GFP (see Figure 2); induction, the ability to induce the de novo conversion of wild-type Sup35p to its prion form, when the indicated region is overexpressed; and propagation, the ability to propagate the prion form of the indicated Sup35p protein in the absence of the wild-type Sup35p. The data in (A) and (B) are taken from Osherovich et al. (2004). The oligopeptide repeats in the OPR region are numbered 1 to 5 with the partial 6th repeat indicated by a *. (A) Deletion analysis of the Sup35p-PrD defines the location of the regions important for protein self-aggregation (the QNR plus repeats 1 and 2 of the OPR; residues 1-64), and for propagation of the prion form (repeats 1-5 in the OPR; residues 41-97). The PNM2-1 Sup35p-OPR contains an Gly58Asp substitution in repeat 2 (Doel et al., 1994) that leads to a defect in prion propagation (see text). (B) Substitution of the Sup35p QNR with a polyglutamine tract of 62 Gln residues (poly(Gln)62). To propagate the prion form of this protein (designated [Q+]) requires the continued synthesis of the poly (Gln)62-Sup35p variant in place of the wild-type Sup35p (Osherovich et al., 2004). (C) The sequences of the regions functionally corresponding to the S. cerevisiae Sup35p-QNR region, from a range of yeast species illustrating that there is no primary sequence conservation but all have a high density of Gln and Asn residues (data from Hara et al., 2003). The peptide that may constitute the amyloid core of the S. cerevisiae Sup35p-QNR is boxed. Molecular Cell 2004 14, 541-552DOI: (10.1016/j.molcel.2004.05.012)