(A) Growth of yeast cells harbouring 1, 2 and 3 yeast integrative plasmids that encode HA-tagged α-synuclein gene under the control of the MET25 promoter, one plate shows strains harbouring 1 copy, 2 and 3 copies of α-synuclein-HA, grown on SD agar plates containing 670 µM methionine. The other plate shows the same strains on SD agar plates that do not contain any methionine. Plates were incubated at 30°C for 96 h. (B) Growth of BC300 derived yeast strains expressing 1, 2, or 3 copies of α-synuclein-HA, from the MET25p, in a minimal liquid medium (SD) where glucose is the carbon source along with controls. Strains expressing 1–3 copies of α-synuclein-HA did not grow throughout 48 h. There was a significant difference between the three yeast strains with the respective controls (P<0.05). Multiple comparisons also indicated that there was significant difference in growth between 1 and 2, and 1 and 3 copies of α-syn (P<0.05), but no significant difference between 2 and 3 copies (P>0.05). (C) Percentage of cells undergoing death upon expression of 1 copy, 2 and 3 copies of α-synuclein under the control of MET25 promoter via Phloxine B staining. The 1-copy strain shows the least cell death. ANOVA test showed a statistically significant difference in cell death (P<0.05) between strains expressing increasing copies of α-synuclein when considering three independent experiments for Phloxine B staining. (D) Comparison of the amounts of NDF, as observed using the TUNEL assay, in yeast strains that express 1 copy, 2 and 3 copies of α-synuclein under the control of MET25 promoter along with controls. ANOVA test showed a statistically significant difference in the levels of NDF (P<0.05) in the three strains expressing α-synuclein. (E) Comparison of the MMP of yeast strains expressing 1 copy, 2 and 3 copies of α-synuclein-HA from the MET25p, along with controls. The 1-copy strain has the highest MMP. In between sample ANOVA test was performed which revealed that differences in MMP between cells containing different copy numbers of α-synuclein and their respective controls were significant (P<0.05); there was significant membrane potential decrease with an increase in copy number, from 1 copy to 2 copies, and from 2 copies to 3 copies (P<0.05). (F) Growth of BC300 yeast cells harbouring the 1 copy of the untagged α-synuclein gene (1) and 1 copy control (2), 2 copies of the untagged α-synuclein gene (3) and 2 copies control (4) and 3 copies of the untagged α-synuclein gene (5) and 3 copies control (6). The yeast transformants were grown on minimal medium solid agar plates that contained either glucose (SD) or galactose (SG). Plates were incubated at 30°C for 96 h. (G) Growth of cells harbouring (a) 1, 2, and 3 copies of untagged α-synuclein (b) 1, 2. and 3 copies of the empty plasmid in minimal medium with galactose as the sole carbon source; galactose induces GAL1 promoter. There was a significant difference in growth between the three α-synuclein expressing yeast strains and the corresponding controls (P<0.05), ANOVA test revealed that there was no significant difference in growth between the three α-synuclein expressing strains (P>0.05). (H) Comparison of the MMP of yeast strains expressing 1, 2 or 3 copies of α-synuclein-No HA from GAL1p, along with controls. There was a significant difference in the MMP of the three yeast strains expressing α-synuclein with their respective controls (P<0.05). ANOVA test revealed that there was also a significant difference between 1-copy and 2 or 3-copy α-synuclein strains (P>0.05).