The first 38 aa of α-synuclein act as a localization determinant to peripheral vesicular structures containing endosomal markers. (A) Schematic diagrams of the GMAP_N-GFP and α-synuclein chimera–GFP probes and their localization in yeast cells. (B) Localization of α-synuclein chimera–GFP (α-syn chim) with FM4-64 (top) and Rtn1p-mRFP (bottom) and of α-synuclein chimera–mCherry with Lucifer yellow (LY; middle). (A and B) Bars, 2 µm. (C) Quantifications of the experiments shown in B. (D) Strain IPY6 α-synuclein chimera–GFP was grown overnight under repressing conditions and then shifted (open symbols) or not shifted (closed symbols) to induction medium for the indicated times, and absorbance (Abs) of the cultures at OD₆₀₀ was monitored. (E) BY4742 cells expressing the indicated constructs were grown overnight under inducing conditions and treated with FM4-64, and the level of fluorescent signal in vacuolar structures was determined as described in Materials and methods. (F and G) Representative EM images of BY4742 cells expressing α-synuclein chimera–GFP induced for 16 h. Arrows show vesicular structures. N, nucleus; V, vacuole; CW, cell wall. Means and SDs of at least three independent experiments are shown.