(a) Expression
(b) Localization & Quantification
(c) Toxicity
Figure 3: Characterization of phosphorylation mutants in BY4741. (a) Western blot of WT, phosphorylation-deficient mutants (S87A, S129A, and S87A/S129A), and phosphorylation-mimic mutants (S87D, S129D). BY4741 lysates were probed with anti-pS129 (top), anti-V5 (α-synuclein; middle), and anti-PGK, which served as a protein-loading control (bottom). WT, S87A, and S87D are phosphorylated at serine-129 ( ). (b) Live-cell GFP microscopy of BY4741 cells expressing GFP, WT, S87A, S129A, S87A/S129A, S87D, or S129D at 24 and 48 hours (top). Quantification: ~750 cells of each type were scored for these localization patterns: cytoplasmically diffuse, and plasma membrane, aggregate, plasma membrane/diffuse, plasma membrane/aggregate (bottom). Phenotypes were plotted as a percent of total cells that fluoresced ( ). (c) Cells expressing WT, S87A, S129A, S87A/S129A, S87D, or S129D 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 (bottom). No phosphorylation-specific toxicity was apparent ( ).