Figure 7

Ca²⁺ rather than Mn²⁺ transport activity of Pmr1p contributes to αSyn toxicity. (a) Spotting assays of WT and Δpmr1 yeast cells expressing either Pmr1p or the point mutants Pmr1p^D53A and Pmr1p^Q783A alone or in combination with αSyn. Cells were grown for 24 h in galactose media and spotted in fivefold serial dilutions onto glucose (Pmr1p and αSyn expression repressed) and galactose (Pmr1p and αSyn expression induced) plates supplemented or not with 1 mM and 4 mM Mn²⁺. (b) Cells described in (a) were subjected to clonogenic survival plating on galactose plates supplemented or not with 1 mM Mn²⁺. Survival has been normalized to WT cells harbouring both empty vectors plated on galactose plates without manganese. Mean±S.E.M., n=12–16. (c) Western blot analysis of Pmr1p, Pmr1p^D53A and Pmr1p^Q783A overexpression as well as of αSyn expression in WT and Δpmr1 yeast cells. Blots were probed with antibodies directed against FLAG-epitope to detect FLAG-tagged Pmr1p variants and αSyn and against glyceraldehyd-3-phosphate dehydrogenase (GAPDH) as loading control