Relationships between the sequence of alpha-synuclein and its membrane affinity, fibrillization propensity, and yeast toxicity

J Mol Biol. 2007 Mar 9;366(5):1510-22. doi: 10.1016/j.jmb.2006.12.044. Epub 2006 Dec 21.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Cell Membrane / metabolism*
  • Electroporation
  • Escherichia coli / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Parkinson Disease / etiology
  • Parkinson Disease / genetics
  • Parkinson Disease / physiopathology
  • Point Mutation
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / physiology*
  • Sequence Homology, Amino Acid
  • Transformation, Genetic
  • Two-Hybrid System Techniques
  • alpha-Synuclein / chemistry*
  • alpha-Synuclein / genetics
  • alpha-Synuclein / isolation & purification
  • alpha-Synuclein / metabolism
  • alpha-Synuclein / toxicity*


  • Nerve Tissue Proteins
  • Recombinant Fusion Proteins
  • alpha-Synuclein
  • Green Fluorescent Proteins