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

FIGURE 1.

FIGURE 1.

αSyn is sumoylated in S. cerevisiae and impairment of sumoylation increases αSyn toxicity and foci formation. A, total protein extract of ulp1 temperature-sensitive yeast cells, defective in SUMO deconjugation, co-expressing integrated αSyn (driven by GAL1 promoter at TRP1 locus) and the His6-tagged yeast SUMO protein Smt3 (driven by ADH1 promoter at HIS3 locus). Enriched sumoylated proteins in the ulp1ts strain in comparison with the control W303 were detected by Western blot with Smt3 antibody (α-Smt3). EV, yeast cells, transformed with empty vector. B, nickel pulldown of His6-tagged yeast SUMO protein Smt3 (His6-smt3) in ulp1ts cells co-expressing αSyn. Sumoylated αSyn (αSyn and A30PSyn) was detected in the pulldown fractions with αSyn antibody (upper panel). Unmodified αSyn was detected in the flow-through. Yeast cells transformed with empty vector were used as a control. Western hybridization of the same blot with Smt3 antibody (lower panel) verified the Ni2+ pulldown (lower panel). C, Western hybridization with Smt3 antibody of total protein extract of smt3 temperature-sensitive yeast cells, co-expressing αSyn or empty vector (EV) at permissive (25 °C) or restrictive temperature (30 °C). D, spotting assay of conditional smt3ts mutant strain expressing αSyn-GFP or A30P-GFP at permissive (25 °C; +SUMO; Smt3 functional) or restrictive temperature (30 °C; −SUMO; Smt3 dysfunctional). GAL1-driven αSyn-GFP is expressed from two genomically integrated copies. GAL1-driven A30P-GFP is expressed from a 2-μm plasmid. GFP, expressed from the same promoter, is used as a control. Yeast cells were spotted in 10-fold dilutions on selection plates containing glucose (αSyn 'OFF') or galactose (αSyn 'ON'). E, fluorescence microscopy of smt3ts cells expressing αSyn-GFP or A30P-GFP at permissive (25 °C; +SUMO) or restrictive temperature (30 °C; −SUMO). Scale bar, 1 μm. F, quantification of the percentage of cells displaying αSyn inclusions. W303 cells expressing αSyn-GFP or A30P-GFP at 25 or 30 °C were used in comparison with smt3ts cells expressing αSyn-GFP or A30P-GFP at permissive (25 °C; +SUMO) or restrictive temperature (30 °C; −SUMO). At least 300 cells were counted per strain and experiment. Significance of differences was calculated with t test (***, p < 0.001, n = 3).