Relationships between the sequence of alpha-synuclein and its membrane affinity, fibrillization propensity, and yeast toxicity.
To investigate the alpha-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 alpha-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 beta-synuclein was of intermediate toxicity to yeast, and gamma-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 alpha-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.
Volles
Michael J
MJ
Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA 02139, USA.
Lansbury
Peter T
PT
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010300:Parkinson Disease / Q000209:etiology / Q000235:genetics / Q000503:physiopathology; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity*
D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D051844:alpha-Synuclein; D049452:Green Fluorescent Proteins