Relationships between the Sequence of α-Synuclein and its Membrane Affinity, Fibrillization Propensity, and Yeast Toxicity

To investigate the α-synuclein protein and its role in Parkinson's disease, we screened a library of random point mutants both in vitro and in yeast to find variants in an unbiased way that could help us understand the sequence–phenotype relationship. We developed a rapid purification method that allowed us to screen 59 synuclein mutants in vitro and discovered two double-point mutants that fibrillized slowly relative to wild-type, A30P, and A53T α-synucleins. The yeast toxicity of all of these proteins was measured, and we found no correlation with fibrillization rate, suggesting that fibrillization is not necessary for synuclein-induced yeast toxicity. We found that β-synuclein was of intermediate toxicity to yeast, and γ-synuclein was non-toxic. Co-expression of Parkinson's disease-related genes DJ-1, parkin, Pink1, UCH-L1, or synphilin, with synuclein, did not affect synuclein toxicity. A second screen, of several thousand library clones in yeast, identified 25 non-toxic α-synuclein sequence variants. Most of these contained a mutation to either proline or glutamic acid that caused a defect in membrane binding. We hypothesize that yeast toxicity is caused by synuclein binding directly to membranes at levels sufficient to non-specifically disrupt homeostasis.