Figure 1.

Identification of YPP1 as a suppressor of A30P α-syn toxicity. Suppression of A30P toxicity by a high-copy yeast genomic library. FY23 cells transformed with pTF202 (A30P) and plated on SGal–Trp (left plate). FY23 cells transformed with pTF202 (A30P) and a 2-μm yeast genomic library and plated on SGal–Trp–Leu (right plate). 2-d incubation at 30°C. The disk in the center of each plate contained 10 μl of ∼8% hydrogen peroxide. (B) Western blot analysis of cells expressing α-syn with or without myc-Ypp1p overexpression. FY23 cells transformed with pTF201 (WT α-syn), pTF202 (A30P), or pTF203 (A53T) and pTF504 (myc-Ypp1) or pTF503 (empty vector) were pregrown in noninducing media to mod-log phase, shifted to inducing media, and incubated for 3 h at 30°C. Cell extracts were prepared and subjected to SDS-PAGE and immunoblotting. The proteins were visualized using monoclonal antibodies (anti-α-syn; anti-myc). Lane 1, WT α-syn; lane 2, myc-Ypp1p and WT α-syn; lane 3, A30P; lane 4, myc-Ypp1p and A30P. (C) Effect of Ypp1p overexpression on cell viability after 12 h of induction. The FUN1 dye stains metabolically active cells red and dead cells green. The top panel shows that cells expressing A30P (plus plasmid with no insert) are dead (green). The bottom panel shows that cells expressing A30P with Ypp1p overexpression are viable (red). The FY23 strain, harboring pTF302 (A30P) and pTF602 (YPP1) or pTF604 (empty vector), was used in these experiments. (D) Plot of percent viability (mean ± SD). The number of cells counted for each group was n = 400 to 1,400. *, P = 0.000013.