Screening for Toxic Amyloid in Yeast Exemplifies the Role of Alternative Pathway Responsible for Cytotoxicity

Abstract

The relationship between amyloid and toxic species is a central problem since the discovery of amyloid structures in different diseases. Despite intensive efforts in the field, the deleterious species remains unknown at the molecular level. This may reflect the lack of any structure-toxicity study based on a genetic approach. Here we show that a structure-toxicity study without any biochemical prerequisite can be successfully achieved in yeast. A PCR mutagenesis of the amyloid domain of HET-s leads to the identification of a mutant that might impair cellular viability. Cellular and biochemical analyses demonstrate that this toxic mutant forms GFP-amyloid aggregates that differ from the wild-type aggregates in their shape, size and molecular organization. The chaperone Hsp104 that helps to disassemble protein aggregates is strictly required for the cellular toxicity. Our structure-toxicity study suggests that the smallest aggregates are the most toxic, and opens a new way to analyze the relationship between structure and toxicity of amyloid species.

The toxicity of an “artificial” amyloid is related to how it interacts with membranes

“…This suggestion is, however, unlikely, as M8 does not appear to be specifically localized to the vacuole. 11 Moreover, the identification of YPT7 (a Rab-GTPase involved in binding the Vps-C HOPS complex to the vacuole membrane 37 ) as a modulator of M8 toxicity argues for a role of the Vps-C complex that is independent to the presence of the vacuole. The vacuole is highly fragmented in the ER membrane.…”

“…We used multicopy (2 μ) yeast expression plasmids with the URA3 selectable marker in this study: they include pYeYGFP2U (GFP), pYecHetsYGFP2U (WT), pYecHetsm8YG-FP2U (M8). 11 The fusion of the HET-s(PrD) and yeast optimized GFP 42 is expressed under control of a GAL10 promoter in a pYeHFN2U.…”

In Vivo and In Vitro Analyses of Toxic Mutants of HET-s: FTIR Antiparallel Signature Correlates with Amyloid Toxicity

“…as were yeast culture conditions and spotting assays (serial 10-fold dilutions). 38 Yeast cells expressing Cterminal GFP fusions were observed under a DMRB microscope (Leica, Wetzlar, Germany) equipped with a PL APO 63× objective. GFP expression in the yeast spotting assay was evaluated using an Invitrogen Safe Imager™ (Fisher Scientific, Illkirch, France) blue light transilluminator coupled with the VersaDoc Imaging system and Quantity One software (Bio-Rad, Hercules, CA, USA).…”

“…Mutant 8 (M8) is the most toxic mutant, presenting 10 mutations all along its amino acid sequence. 38 We established that the toxic M8 amyloid displayed different biochemical and structural features with a totally different organization. 41 M8 is assembled in mainly FTIR antiparallel amyloid nanofibers (thicker in diameter; ≈10 nm), whereas WT long fibers are organized into parallel β-sheets (about 5 nm in diameter).…”

“…Our amyloid model is [wild type (WT)] and is not toxic to yeast. 38 HET-s is a prion protein (289 amino acids in length) from the filamentous fungi Podospora anserina and is responsible for a process termed "heterogeneous incompatibility". 39 The prion-forming domain HET-s (218-289) forms amyloid fibers, the structure of which has been determined by solidstate NMR.…”