Illustration of YPP1-mediated protection against A30P. Sla1p, Sla2p, and End3p form complexes at the actin cortical patches. Ypp1p binds to A30P and mediates the encapsulation of A30P into vesicles, which then bud off the membrane and transit to the vacuole. Class E Vps proteins are required for protein sorting into lumenal vesicles. The Mon1p–Ccz1p complex facilitates vesicle–vacuole fusion. MVB, multivesicular body.

Ypp1p alters GFP-α-syn localization. The effect of Ypp1p overexpression on the localization of (A) GFP-A30P, (B) GFP-WT α-syn, (C) GFP-A53T, and (D) GFP at 3 and 12 h of induction. In each group of four panels, the top and bottom rows are of cells expressing GFP-α-syn with and without Ypp1p overexpressed, respectively. S288c cells were transformed with pTF305 (WT α-syn), pTF306 (A30P), pTF307 (A53T), or pTF308 (GFP) and pTF602 (YPP1) or pTF604 (empty vector). Bar, 5 μm.

Effects of gene deletions on YPP1-mediated A30P trafficking. The S288c strain and the two deletion strains transformed with pTF306 (GFP-A30P) and pTF602 (YPP1) were pregrown in noninducing media to mid-log phase, transferred to inducing media, induced for 3 or 12 h, and then visualized by fluorescence microscopy. The SEC1 and SEC12 Hughes strains transformed with pTF306 (GFP-A30P) and pTF602 (YPP1) were pregrown in noninducing media with doxycycline (20 μg/ml) for 7 h, transferred to inducing media with doxycycline, and then visualized at 3 and 12 h by fluorescence microscopy. (A) S288c; (B) Δmon1; (C) Δmon1 and pTF700 (MON1); (D) Δsla1; (E) Δsla1 and pTF701 (SLA1); (F) tet-SEC12 strain + doxycycline; and (G) tet-SEC1 strain + doxycycline. (A–E): Images labeled “MERGE” had brightness and contrast adjustments of −12 and +20, respectively. (F and G): images labeled MERGE had brightness and contrast adjustments of +40 and +20, respectively. Bar, 5 μm.

Transmission electron microscopy images of cells expressing Ypp1p and A30P. (A) S288c cells transformed with pTF302 (A30P) or pTF300 (empty vector) and pTF602 (YPP1) or pTF604 (empty vector) were pregrown in noninducing media to mid-log phase, transferred to inducing media and induced for 3 or 12 h. Top: −A30P/−Ypp1 (cells contained two plasmids with no inserts). Middle: +A30P/−Ypp1 (cells contained two plasmids, one with no insert). Bottom: +A30P/+Ypp1 (cells contained two plasmids). Bars, 1 μm. (B) Magnification of +A30P/+Ypp1 cell at 3 h shows vesicle budding from the plasma membrane. (C and D) Immunogold labeling of cells expressing A30P and overexpressing Ypp1p at 12 h. A secondary antibody conjugated with 50-nm gold particles was used to detect A30P. Several clusters of gold particles occurred in the vicinity of the plasma membrane (C) and also in the interior of cells (D).

Screening for genes that interact withYPP1. The indicated deletion strains transformed with pTF302 (A30P) and pTF602 (YPP1) were pregrown in noninducing media to mid-log phase, transferred to inducing media, and induced for 3 h. Matched controls consisted of identically treated S288c cells transformed with the same two plasmids. The dye DHR 123 was added at 2 h after induction. After several washing steps, cells were aliquoted into 96-well plates and the DHR 123 fluorescent product was detected using a plate reader. Cells incubated in dilute acetic acid exhibit ROS (Ludovico et al., 2002), and this served as a positive control. Latrunculin A was used at 25 μM. Experiments were performed in two to five independent experiments, each in triplicate. Bars indicate mean ROS signal ± SD. Comparing each deletion strain to matched controls (S288c +A30P/+Ypp1) yielded P-values: red, P < 0.001; orange, P < 0.05; and blue, P > 0.05. Exact P values are given in Table S1 (available at http://www.jcb.org/cgi/content/full/jcb.200610071/DC1).

Pheromone protects cells from A30P toxicity. (A) Effect of α-factor on cells expressing Ypp1p-GFP. Top (−α-factor): no change in the localization of Ypp1p-GFP occurred over 20 min. Bottom (+α-factor): 10 μM α-factor was added to cells and then images were acquired from 2 to 20 min. Strain: YPP1-GFP cultured in YPD. Identical instrumental conditions were used for all images. Bar, 5 μm. The ROS assay using the DHR 123 dye is shown in (B) WT α-syn, (C) A30P, and (D) vector control. The FY23 strain was transformed with pTF201 (WT α-syn), pTF202 (A30P), pTF203 (A53T), or pTF200 (empty vector) and pregrown in noninducing media to mid-log phase. 10 μM pheromone was added after shifting to inducing media; after 2 h in inducing media the DHR 123 dye was added to 5 μg/ml; and, before acquiring images at 3 h, cells were washed and resuspended in inducing media. The two images labeled “MERGE” (in A) had brightness and contrast adjustments of +10 and +40, respectively. (E) Plot of mean ROS (%) (± SD). Cells (n = 292–696) were counted in two independent experiments. *, P = 1.1 × 10⁻⁷. DIC, differential interference contrast; DHR, rhodamine 123.

YPP1 suppresses ROS accumulation in A30P expressing cells. ROS accumulation in cells expressing A30P (pTF302) (A), WT α-syn (pTF301) (B), or vector control cells (pTF300) (C). The “Hughes” YPP1 strain that contains a titratable promoter was used in these experiments. This strain transformed with pTF602 (YPP1) or pTF604 (empty vector) was used for these experiments. For shutting off YPP1 (+doxy), cells were pregrown in noninducing media with 20 μM doxycycline for 7 h, and then shifted into inducing media with doxycycline. For experiments with Ypp1p overexpression, cells were pregrown in noninducing media to mid-log, and then shifted into inducing media. The images shown in all panels were obtained at 3 h in inducing media. The dye DHR123 was added after 2 h in inducing media to give 5 μg/ml, and the cells were then washed before image acquisition. DHR, rhodamine 123. DIC, differential interference contrast. (D) Plot of the percentage of cells that exhibited red fluorescence (mean ± SD). The number of cells counted for each group was n = 640 to 1,200. The symbols "−", “e”, and “+” indicate decreased (with doxycycline), endogenous, and increased levels of Ypp1p, respectively. (E) Coimmunoprecipitation shows selective binding of myc-Ypp1p to A30P. Cells expressing the various α-syns and overexpressing myc-tagged Ypp1p were lysed, the extract was incubated with an anti-myc antibody-protein A resin, and the resin was boiled and reduced and subjected to SDS-PAGE and immunoblotting. The top panels show the ∼95-kD band due to myc-Ypp1p; the bottom panels show α-syn. Lane 1, WT α-syn lysate; lane 2, myc-Ypp1p fails to pull down WT α-syn; lane 3, A30P lysate; lane 4, myc-Ypp1p pulls down A30P; lane 5, A53T lysate; lane 6, myc-Ypp1p fails to pull down A53T. The FY23 strain was transformed with pTF201 (WT α-syn), pTF202 (A30P), or pTF203 (A53T) and with pTF504 (myc-YPP1) or pTF503 (empty vector).

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.

Ypp1p drives A30P to the vacuole. (A) Two-color fluorescence microscopy experiments of cells expressing GFP-α-syn with Ypp1p overexpression. S288c cells transformed with pTF305 (WT α-syn), pTF306 (A30P), pTF307 (A53T), or pTF308 (empty vector) and pTF602 (YPP1) or pTF604 (empty vector) were pregrown in noninducing media to mid-log phase, transferred to inducing media, and induced for 3 or 12 h. Before the analysis cells were stained with the dye FM4-64. Aliquots were removed, washed, resuspended in YPD, and incubated with 40 μM FM4-64 for 10 min at 30°C. Cells were then washed twice, resuspended in YPD, incubated an additional 30 min at 30°C, and then visualized by fluorescence microscopy. The white arrows (top right-hand panel, GFP-A30P) indicate areas of overlap between the GFP inclusions with the red structures. Bar, 5 μm. (B) Ypp1p promotes degradation of A30P. Western blot analysis was conducted to monitor the level of A30P in cells. Blots (i and ii): Ypp1p overexpression. 10 μM cycloheximide was used to halt protein synthesis in cells with (i) or without (ii) Ypp1p overexpression. S288c cells transformed with pTF202 (A30P) and pTF602 (YPP1) or pTF604 (empty vector) were pregrown in noninducing media to mid-log phase, transferred to inducing media, inhibited with cycloheximide, and induced for 12 h. Indicated times are after addition of cycloheximide. Blot (iii): Deletion of PEP4. A Δpep4 deletion strain transformed with pTF202 (A30P) and pTF602 (YPP1) was pregrown in noninducing media to mid-log phase, transferred to inducing media, inhibited with 10 μM cycloheximide, and induced for 12 h. Indicated times are after addition of cycloheximide. Blot (iv): Proteasome inhibition. S288c cells transformed with pTF202 (A30P) and pTF602 (YPP1) were pregrown in noninducing media to mid-log phase, transferred to inducing media, inhibited with 10 μM cycloheximide and 50 μM of MG132, and induced for 12 h. Indicated times are after addition of inhibitors. For each experiment, cell extracts were prepared, subjected to SDS-PAGE, and immunoblotted using a monoclonal antibody specific for α-syn. In each blot, 20 μg of protein was loaded per well. Experiments were conducted two or three times. (C) Degradation of A30P. Band intensities in the blots were determined using a scanner with image quantization software and plotted against time. (i) Blue, A30P + Ypp1p overexpression; (ii) red, A30P + endogenous level of Ypp1p; (iii) green, A30P + Ypp1p overexpression in Δpep4 strain; (iv) yellow, A30P + Ypp1 overexpression + 50 μM MG132.