Heritable yeast prions have a highly organized three-dimensional architecture with interfiber structures

Abstract

Yeast prions constitute a "protein-only" mechanism of inheritance that is widely deployed by wild yeast to create diverse phenotypes. One of the best-characterized prions, [PSI + ], is governed by a conformational change in the prion domain of Sup35, a translation-termination factor. When this domain switches from its normal soluble form to an insoluble amyloid, the ensuing change in protein synthesis creates new traits. Two factors make these traits heritable: (i) the amyloid conformation is self-templating; and (ii) the protein-remodeling factor heat-shock protein (Hsp)104 (acting together with Hsp70 chaperones) partitions the template to daughter cells with high fidelity. Prions formed by several other yeast proteins create their own phenotypes but share the same mechanistic basis of inheritance. Except for the amyloid fibril itself, the cellular architecture underlying these protein-based elements of inheritance is unknown. To study the 3D arrangement of prion assemblies in their cellular context, we examined yeast [PSI + ] prions in the native, hydrated state in situ, taking advantage of recently developed methods for cryosectioning of vitrified cells. Cryo-electron tomography of the vitrified sections revealed the prion assemblies as aligned bundles of regularly spaced fibrils in the cytoplasm with no bounding structures. Although the fibers were widely spaced, other cellular complexes, such as ribosomes, were excluded from the fibril arrays. Subtomogram image averaging, made possible by the organized nature of the assemblies, uncovered the presence of an additional array of densities between the fibers. We suggest these structures constitute a self-organizing mechanism that coordinates fiber deposition and the regulation of prion inheritance.

Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process

“…4). The increased expression of Hsp31 at the diauxic shift and in stationary phase could possibly be due to the stress of decreased nutrients or accumulation of metabolites associated with these growth phases and is part of a global transcriptional response program (70). An increase in Hsp31 expression in response to ROS has been observed previously (71), and we confirmed that our tagged strain behaved in a similar manner by demonstrating an increase in expression upon exposure to H 2 O 2 (Fig.…”

Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits

“…Examples are the fibrils formed from sup35 or pml17 protein. Sup35-derived fibrils play a role in phenotypic inheritance in yeast cells (33) and were found to adopt ring-like assemblies underneath the plasma membrane or dot-like cytoplasmic inclusions with a regular mosaic structure (33). Pml17 fibrils are important for melanosome formation, and tomographic studies revealed intracellular sheet likeassemblies that consist of parallel fibrils of pml17 protein (34).…”

Specific Chaperones and Regulatory Domains in Control of Amyloid Formation

“…The HET-s prion, implicated in non-genetic inheritance of yeast, forms a ␤-solenoid with its ␤-strands arranged in a triangular shape (4). Electron microscopy additionally suggests that yeast prion fibers are capable of forming higher-order structures linked by organizational elements (5). Spider silk, which shares key characteristics with amyloid fibers (6,7), is composed of ␤-crystalline blocks connected by unstructured segments, an architecture that gives rise to the exceptional properties of silk (8 -10).…”