Contribution of Alanine-76 and Serine Phosphorylation in α-Synuclein Membrane Association and Aggregation in Yeasts
Figure 5
Characterization of alanine-76 in BY4741. (a) Live-cell GFP microscopy of BY4741 cells expressing WT, A76E, or A76R at 24 and 48 hours (left). Quantification: ~750 cells of each type were scored for these localization patterns: cytoplasmically diffuse, and plasma membrane, aggregate, plasma membrane/diffuse, plasma membrane/aggregate (right). Phenotypes were plotted as a percent of total cells that fluoresced (). (b) BY4741 cells expressing WT, A76E, or A76R were grown in inducing (galactose) media for 48 hours. Vector alone and GFP served as controls. Equal number of cells were serially diluted 5-fold and spotted onto repressing (glucose) or inducing (galactose) plates and grown for two days (right). No toxicity was apparent in budding yeast (). (c) Western blot of BY4741 cells expressing WT, A76E, or A76R at 24 and 48 hours. Lysates were probed with anti-V5 (α-synuclein) or anti-PGK. Expression is equivalent between WT and the alanine-76 mutants at 24 and 48 hours.
