Using a genetic screen we discovered that (named gene of unknown function suppresses the toxicity of an α-synuclein (α-syn) mutant (A30P) that is associated with early onset Parkinson’s disease. dynamics (and also participates in pheromone-triggered receptor-mediated endocytosis. Our data reveal that mediates the trafficking of A30P to the vacuole via the endocytic pathway. Introduction Parkinson’s disease is usually characterized by the SAHA selective degeneration of dopamine-producing neurons that comprise the and the presence of proteinaceous inclusion body (Lewy body) in the affected neurons (Dawson and Dawson 2003 The principal component of Lewy body is usually α-synuclein (α-syn) which is an intrinsically unfolded protein of unknown function (Weinreb et al. 1996 Uversky et al. 2000 SAHA Wild-type (WT) α-syn and two mutants A30P and A53T (Polymeropoulos et al. 1997 Kruger et al. 1998 associated with early onset PD have been linked to a plethora of defects including proteasomal and mitochondrial dysfunction (Tanaka et al. 2001 Martin et al. 2006 the accumulation of reactive oxygen species (ROS) (Xu et al. 2002 blockage of ER to Golgi traffic (Cooper et al. 2006 and histone acetylation inhibition (Kontopoulos et al. 2006 Using a genetic screen that exploits the super sensitivity of α-syn-expressing yeast cells to killing by H2O2 (Blossom et al. 2005 we discovered that orthologues understanding the function of this gene may shed light on how human neurons safeguard themselves from α-syn. Given that codes for an α-synuclein protective protein was named interacts genetically with (Davierwala et al. 2005 Other studies have indicated that functions in MAPK pathways (Roberts et al. 2000 Hazbun et al. 2003 Mnaimneh et al. 2004 Ypp1p-GFP SAHA localizes in a punctate pattern round the plasma membrane (Huh et al. 2003 although there is no indication from its sequence that Ypp1p is usually a membrane protein. Although information exists about its synthetic genetic interactions and localization Ypp1p’s function has remained obscure. Here we show that Ypp1p binds A30P (but not WT or A53T) and mediates a sequence of events in which A30P is usually encapsulated into vesicles at the plasma membrane and the vesicles then transit to and merge with the vacuole where the A30P protein is usually proteolytically degraded. Results A genetic screen identifies as a suppressor of A30P toxicity A high copy yeast genomic library was used to identify suppressors of the super sensitivity of A30P expressing cells to killing by hydrogen peroxide (Fig. 1 A). Herein we describe the characterization of one suppressor 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 … Table I. Yeast strains and plasmids codes for any protein with a theoretical molecular mass of 95.4 kD. Cells expressing α-syn and transformed with a plasmid harboring a myc-tagged fusion indeed expressed myc-Ypp1p as judged by Western blot analysis and staining with an anti-myc antibody (Fig. 1 B). Specifically myc-Ypp1p was detected in cells that coexpressed WT α-syn or A30P (lanes SAHA 2 and 4); however in each case a ladder of bands ranging from 95 to >200 kD was observed. We attribute the band at 95 kD to myc-Ypp1p and suggest SAHA that the bands at higher molecular mass are posttranslationally altered forms of Ypp1p. The four major bands of myc-tagged Ypp1p were absent in lysates of cells that did not harbor the plasmid (lanes 1 and 3). The effect of Ypp1p overexpression around the viability of cells expressing the various α-syns was also evaluated. A viability assay Mouse monoclonal to NFKB1 (Fannjiang et al. 2004 was conducted using the dye FUN1 on cells induced for 12 h. Dead cells stained green; metabolically active and hence viable cells stained reddish; and a small percentage of cells (～10%) failed to stain. The percentage of crimson cells indicated viability. For cells expressing A30P with Ypp1p overexpression 90 from the cells had been viable whereas just 10% of cells had been practical when A30P was portrayed without Ypp1p overexpression SAHA (Fig. 1 D) and C. On the other hand for cells expressing WT α-syn or A53T with or without Ypp1p overexpression just ～10% from the cells had been practical (Fig. 1 D). This viability assay showed that in high duplicate particularly enhances the viability of A30P expressing cells however not of cells expressing the various other two α-syns (WT or A53T). suppresses ROS deposition in A30P expressing cells A personal feature of α-syn is normally it induces oxidative tension.