Quantitative analysis of amyloid clearance in the Saccharomyces cerevisiae model system [PSI+]

Abstract

Protein misfolding is a hallmark of several neurodegenerative diseases, leading to the formation of non‐native amyloid deposits that correlate with disease progression. Alzheimer's disease, Parkinson's disease, Huntington's disease, and Creutzfeldt‐Jakob disease are examples of neurodegenerative diseases that share the neuropathological feature of amyloid deposits. Molecular chaperones are a class of proteins that catalyze the folding or activation of nascent proteins, the refolding and reactivation of misfolded proteins, and the disaggregation of protein aggregates. Several proteins, including the ATP‐dependent chaperone Hsp104, have been shown to disassemble amyloid fibers in vitro and in vivo. In the Saccharomyces cerevisiae model for prion propagation and inheritance, [PSI+], reduced amyloid burden correlates with the development of pigmentation attributable to an impaired adenine biosynthesis pathway. This colorimetric conversion assay has been extensively utilized to qualitatively compare the ability of chaperones to cure the cells of amyloid. We developed a quantitative assay to compare the pigmentation in yeast cell lysates, which allows for screening of mutants partially defective for amyloid curing. Using this assay, we compared the extent of amyloid clearance resulting from expression of several chaperones, including Hsp104 wild type and mutant proteins. These studies provide a platform to compare the effectiveness of different chaperone systems and partial loss of function mutants for reducing amyloid burden.