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. 2008 Dec 1;17(23):3784-95.
doi: 10.1093/hmg/ddn276. Epub 2008 Sep 4.

Novel suppressors of alpha-synuclein toxicity identified using yeast

Jun Liang  1 , Cheryl Clark-Dixon, Shaoxiao Wang, Todd R Flower, Tara Williams-Hart, Richard Zweig, Lucy C Robinson, Kelly Tatchell, Stephan N Witt
Affiliations

Novel suppressors of alpha-synuclein toxicity identified using yeast

Jun Liang et al. Hum Mol Genet. .

Abstract

The mechanism by which the Parkinson's disease-related protein alpha-synuclein (alpha-syn) causes neurodegeneration has not been elucidated. To determine the genes that protect cells from alpha-syn, we used a genetic screen to identify suppressors of the super sensitivity of the yeast Saccharomyces cerevisiae expressing alpha-syn to killing by hydrogen peroxide. Forty genes in ubiquitin-dependent protein catabolism, protein biosynthesis, vesicle trafficking and the response to stress were identified. Five of the forty genes--ENT3, IDP3, JEM1, ARG2 and HSP82--ranked highest in their ability to block alpha-syn-induced reactive oxygen species accumulation, and these five genes were characterized in more detail. The deletion of any of these five genes enhanced the toxicity of alpha-syn as judged by growth defects compared with wild-type cells expressing alpha-syn, which indicates that these genes protect cells from alpha-syn. Strikingly, four of the five genes are specific for alpha-syn in that they fail to protect cells from the toxicity of the two inherited mutants A30P or A53T. This finding suggests that alpha-syn causes toxicity to cells through a different pathway than these two inherited mutants. Lastly, overexpression of Ent3p, which is a clathrin adapter protein involved in protein transport between the Golgi and the vacuole, causes alpha-syn to redistribute from the plasma membrane into cytoplasmic vesicular structures. Our interpretation is that Ent3p mediates the transport of alpha-syn to the vacuole for proteolytic degradation. A similar clathrin adaptor protein, epsinR, exists in humans.

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Figures

Figure 1.
Figure 1.
A genome-wide screen identifies suppressors of the super sensitivity of WT α-syn expressing cells to killing by hydrogen peroxide. (A) FY23 cells transformed with pTF201 (WT α-syn) were plated on SGal−Trp. The halo of inhibition is due to the concentrated hydrogen peroxide on the disk in the center of the plate. (B) Suppression of WT α-syn toxicity by a high-copy yeast genomic library. FY23 cells transformed with pTF201 (WT α-syn) and a 2µ yeast genomic library and plated on SGal−Trp−Leu. The disk in the center of each plate contained 10 µl of 8–10% hydrogen peroxide. Plates were incubated for 3 days at 30°C. In general, the relatively low transformation efficiency of the 2µ library resulted in fewer colonies per plate compared with the control plate.
Figure 2.
Figure 2.
ROS fluorescence assay of transformants with 66 yeast genes identified in the hydrogen peroxide screen as potential suppressors of WT α-syn toxicity. The y-axis is the fluorescence signal; the x-axis gives the gene name. The FY23 strain was transformed with pTF201 (WT α-syn) and pBG1805 (Open Biosystems 2µ plasmid with gene of interest) (see Supplementary Material, Table S1). Cells were pre-grown in sucrose drop out media to mid-log phase and then transferred into inducing media and incubated for 3 h at 30°C. The DHR123 dye (5 µg/ml) was added after 2 h in inducing media. The cells were washed several times in phosphate-buffered saline (PBS) and resuspended in PBS before measuring the ROS fluorescence signal. The excitation and emission wavelengths were 485 and 535 nm, respectively. As a control, FY23 was transformed with the pTF201 (WT α-syn), and these cells were treated identically to those described above. The ROS signal from these cells was 152 236 ± 15 177 and 27 382 ± 4710 units of fluorescence in inducing and non-inducing media, respectively. Note that FY23 cells transformed with pTF201 and pJL200 (empty vector) yielded 154 675 ± 2436 and 25 179 ± 895 units of fluorescence in inducing media and non-inducing media, respectively. Other empty vector controls (pTF200 or pTF200 and pJL200) are also shown. Values for the 24 genes with human orthologs are shown in color (magenta and red); values for the 5 genes that were characterized in more detail are shown in red.
Figure 3.
Figure 3.
Growth properties of cells expressing WT α-synuclein. The effect of WT α-synuclein expression on the growth of five deletion strains (ent3Δ, idp3Δ, jem1Δ, arg2Δ and hsp82Δ) and the WT control strain KT2253 was evaluated. These strains were transformed with pJL101 (WT α-syn) or pJL100 (empty vector) and spotted in successive 10-fold serial dilutions on solid sucrose and solid galactose plates and incubated for 3 days at 30°C. Experiments were repeated three times with similar results.
Figure 4.
Figure 4.
ROS accumulation in ent3Δ, idp3Δ, jem1Δ, arg2Δ and hsp82Δ deletion strains expressing WT α-syn. The fluorescence assay using the dye DHR123 was used to measure the ROS accumulation in WT α-synuclein expressing cells after 3 h incubation in inducing media at 30°C. The WT haploid strain KT2253 transformed with pJL101 (WT α-syn) and pJL200 (empty vector) exhibited an ROS signal of 154 675 ± 2436 and 25 179 ± 895 in inducing media (+gal) and non-inducing media (−gal), respectively. KT2253 transformed with two empty vectors (pJL100 and PJL200) (+gal) exhibited an ROS signal of 19 763 ± 349. The arg2Δ strain transformed with pJL101 (WT α-syn) and pJL200 (red bar) or pARG2 (2µ Open Biosystems plasmid containing ARG2) (blue bar) were treated identically to the WT control strain, and ROS was measured at 3 h. The ROS signal from the arg2Δ strain transformed with two empty vectors (pJL100 and pJL200) is represented by the green bar. The other four deletion strains were prepared and analyzed in the same way as arg2Δ.
Figure 5.
Figure 5.
(A) ARG2, ENT3, HSP82, IDP3 and JEM1 in high copy fail to suppress the toxicity of A30P or A53T. The fluorescence assay using DHR123 was used to measure the ROS accumulation in cells expressing various α-synucleins (WT, A30P or A53T). For example, the FY23 strain was transformed with pARG2 (2µ plasmid with ARG2) and with pTF201 (WT α-syn), pTF202 (A30P) or pTF203 (A53T). Matched controls consisted of the FY23 strain transformed with pTF201 (WT α-syn), pTF202 (A30P) or pTF203 (A53T). Cells were pre-grown in non-inducing media, shifted into inducing media and then incubated for 3 h. The DHR123 dye (5 µg/ml) was added after 2 h in inducing media, and the ROS signal was measured 1 h later. Prior to measuring the fluorescence signal, the cells were washed and resuspended in PBS. Each bar in the graph represents the ratio: average ROS signal (multi-copy gene)/average ROS signal (single copy gene). A value of 1 indicates no protection; a value <1 indicates a decrease in ROS; and a value >1 indicates an increase in ROS. The average signals were obtained from triplicate samples recorded on three different days. Error bars were determined by to the method described in Bevington (64). (B) Western blot analysis of α-syn expression in the cultures as shown in (A). Lysates were prepared and then subjected to SDS–PAGE followed by western blot analysis. The cell-signaling monoclonal antibody against α-syn was used to visualize the three α-syns in yeast cells overexpressing the five genes of interest. The loading control was the yeast protein Pgk1p. Identical amounts of protein were loaded per well.
Figure 6.
Figure 6.
ENT3 alters the localization of WT α-syn. The KT2253 strain was transformed with pJL103 (GFP-WT α-syn) and pENT3 or the empty vector pJL200. Cells were pre-grown in non-inducing media until mid-log phase, washed and resuspended in inducing media, and incubated for 12 h at 30°C. Cells were visualized by fluorescence microscopy at 3 and 12 h as discussed in the Materials and Methods section. The top row shows cells expressing GFP-WT α-syn with pJL200 control plasmid; the bottom row shows cells expressing GFP-WT α-syn with ENT3 in high copy (pENT3).
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