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. 2014 Sep 17;289(45):31224–31240. doi: 10.1074/jbc.M114.559237

FIGURE 4.

FIGURE 4.

Increased αSyn Ser-129 phosphorylation level by GRK5/PLK2 expression alleviates the toxicity and reduces foci formation associated with impaired sumoylation in smt3ts cells. A, spotting assay of smt3ts cells co-expressing αSyn-GFP or S129A-GFP with GRK5 or PLK2 either at permissive (25 °C; +SUMO) or restrictive temperature (30 °C; −SUMO). Yeast cells were spotted in 10-fold dilutions on selection plates containing glucose (αSyn 'OFF'; kinases 'OFF') or galactose (αSyn 'ON'; kinases 'ON'). B, spotting assay of W303 yeast cells, carrying two copies of GAL1-driven αSyn-GFP and K96R/K102R-GFP in the presence of GRK5 and PLK2 or empty vector. Yeast cells were spotted in 10-fold dilutions on selection plates containing glucose (αSyn 'OFF'; kinases 'OFF') or galactose (αSyn 'ON'; kinases 'ON'). C, fluorescence microscopy of smt3ts cells expressing αSyn in the presence or absence of GRK5 or PLK2 (left panel). Scale bar, 1 μm. Quantification of percentage of cells displaying αSyn inclusions in the presence or absence of GRK5/PLK2 (upright) and quantification of cells with foci formation expressing S129A-GFP with and without overexpression of GRK5/PLK2. Significance of differences was calculated with t test with respect to αSyn-GFP at the same temperature (**, p < 0.01, n = 3).