Synphilin-1 Enhances α-Synuclein Aggregation in Yeast and Contributes to Cellular Stress and Cell Death in a Sir2-Dependent Manner
Figure 2
Synphilin-1 induces inclusion formation of α-Syn in yeast.
A: Fluorescence microscopic visualization and intracellular localization of the α-Syn–EGFP (upper panels), dsRed-SYWT (middle panels) or dsRed-SYR621C (lower panels) fusion proteins expressed separately in the BY4741 wild-type yeast strain. The panels display cells without (left) or with (right) aggregates. The percentages refer to the number of cells with or without inclusions in an exponential culture. Cells expressing native EGFP or dsRed served as controls and showed a dispersed cytoplasmic localization. B: Fluorescence microscopic visualization and intracellular localization of α-Syn–EGFP and dsRed-SYWT upon combined expression in the BY4741 wild-type yeast strain. The upper panels display cells where both fusion proteins co-localize, the middle panels cells with intracellular inclusions of synphilin-1 and plasma membrane localized α-Syn, and the lower panels cells with peripheral inclusions of α-Syn and a dispersed cytoplasmic distribution of synphilin-1. C: Western blot analysis of strains transformed with an empty plasmid (Ctrl.) or a construct allowing the expression of dsRed-SYWT or dsRed-SYR621C. Immunodetection was performed using primary antibodies directed against synphilin-1 or Adh2 as indicated on the left. Molecular weight markers are indicated on the right. D and E: The percentage of cells containing inclusions of wild-type or mutant synphilin-1 (D) or α-Syn (E) in exponential cultures. Data represent the combined results of at least three independent experiments. Error bars represent the variation between different counts. Significance was assayed on the total amount of cells counted using a t-test (*** = p<0.001).