Alpha-synuclein (alfaSyn) is a vertebrate-specific protein of 140 amino acids, expressed mainly in neuronal tissue and localized in presynaptic termini. Accumulation of aggregated forms of alfaSyn into Lewy bodies is a known hallmark associated with neuronal cell death in Parkinsons disease and other neurodegenerative disorders, collectively called the synucleinopathies. When expressed in the yeast Saccharomyces cerevisiae, alfaSyn interacts with the plasma membrane, forms inclusions and causes a concentration-dependent growth defect. We have used yeast mutants, that are particularly sensitive to moderate alfaSyn expression, for the screening of a mouse brain-specific cDNA library in order to identify mammalian proteins that counteract alfaSyn toxicity. A first screening, with cells expressing alfaSyn from a multicopy plasmid, did not result in confirmable suppressors of alfaSyn toxicity. We discovered that the colonies were growing because the yeast cells had recombined a cDNA-encoding plasmid with the alfaSyn-encoding plasmid, enabling them to grow on minimal medium without alfaSyn expression. When two galactose-inducible copies of alfaSyn were integrated into the genome of alfaSyn-sensitive strain cog6Δ, a strong growth defect was observed. Using this strain (cog6Δ2alfaSyn) for the screening of the brain cDNA-library resulted in the discovery of two suppressors of the alfaSyn-induced growth defect, the proteasome subunit alpha type 2 (PSMA2) and the ribosomal protein S3A (RPS3A), and one enhancer, the C-terminal part of actin organizer protein Spir1 (Spir1CT).The fact that RPS3A also suppresses the alfaSyn-induced growth defect in all other alfaSyn-sensitive deletion strains tested indicates that it acts directly on alfaSyn toxicity, rather than on the sensitivity of the strains. RPS3A has two homologues in the yeast genome, RPS1A and RPS1B. Because overexpression of these yeast genes has no effect on alfaSyn toxicity, we conclude that the alfaSyn-modifying activity is specific for the mammalian gene. In SDD-AGE experiments, we observed that the alfaSyn in extracts from cells co-expressing RPS3A behaves differently than the alfaSyn in the other extracts, which indicates that RPS3A induces a conformational change or makes some other type of interaction. We were, however; unable to demonstrate co-immunoprecipitation of the two proteins. Fluorescence microscopy studies of cog6Δ cells expressing alfaSyn-GFP and RPS3A-mRFP showed that RPS3A-mRFP strongly reduces the amount of cells with alfaSyn-GFP inclusions, but no co-localization of RPS3A-mRFP and alfaSyn-GFP was observed. We did find that the cells with lower RPS3A-mRFP expression have a higher likelihood to contain alfaSyn-GFP inclusions.We have established a novel method for identifying mammalian effectors of alfaSyn, by directly screening a mammalian brain cDNA library in yeast cells. This method allows to identify proteins which have no homologues in yeast, or proteins of which the yeasthomologue has no alfaSyn-modifying activity. The screening has resulted in the identification of mouse RPS3A as a suppressor of alfaSyn toxicity in yeast, independent of the genetic background. Our results suggest that the recently identified chaperonin function of RPS3A may also apply to alfaSyn, possibly by stabilizing its recently identified helically folded tetrameric form.