Contribution of Alanine-76 and Serine Phosphorylation in α-Synuclein Membrane Association and Aggregation in Yeasts
Figure 4
Characterization of alanine-76 in TCP1. (a) Live-cell GFP microscopy of TCP1 cells expressing WT, A76E, or A76R at 24 and 48 hours (left). Quantification: ~750 cells of each type were scored for these localization patterns: diffuse, aggregate, and aggregate/diffuse (right). Phenotypes were plotted as a percent of total cells that fluoresced (). (b) TCP1 cells expressing WT, A76E, or A76R were grown in inducing (EMM−T) media for 48 hours. Vector alone and GFP served as controls. Equal number of cells were serially diluted 5-fold and spotted onto repressing (EMM+T) or inducing (EMM−T) plates and grown for two days (right). The A76E and A76R mutants were equally toxic as WT (). (c) Western blot of TCP1 cells expressing WT, A76E, or A76R at 24 and 48 hours. Lysates were probed with anti-V5 (α-synuclein) or anti-β-actin (loading control). Expression is equivalent between WT and the alanine-76 mutants at 24 and 48 hours ().
