Essential genetic interactors of SIR2 required for spatial sequestration and asymmetrical inheritance of protein aggregates.

Yeast strains and growth conditions

Yeast strains used in this study are listed in Supplemental Table S1 . The Yeast conditional temperature-sensitive (ts) collection of essential genes for the SGA analysis was a gift from Prof. Charles Boone. Yeast cells were grown in YPD or synthetic drop-out media with antibiotics added as indicated. SIR2 Synthetic Genetic Array (SGA) analysis using the conditional temperature-sensitive (ts) collection of essential genes The SIR2 (ts) SGA analysis was performed in duplicates as described [28] . The screen was run in the 1536-spot format using a SINGER ROTOR HDA Robot (Singer Instrument Co. Ltd.). Hits with the highest statistical probability to be true interactions were confirmed by microcultivation experiments in triplicate at 30, 34, and 38°C using the Bioscreen C system (Labsystems Oy, Helsinki, Finland). The optical density was measured every 30 minutes for 72 hours. The LSC (Logarithmic Strain Coefficient) values of growth rates were calculated and scored as described [28] . The heat map was made using TreeView [58] and Ospery 1.2.0 [59] was used for SIR2 essential gene network analysis. The physical interactions between SGA hits were obtained based on the BioGRID interaction database [60] . Fluorescence microscopy A Zeiss Axiovert 200 M fluorescence microscope was used to obtain images using GFP, Cy3 and DAPI channels. The ImageJ plugin “Iterative deconvolve 3-D” was used for all deconvolution images.

HttQ103-GFP expression and aggregation retention efficiency assay

Cells containing the pYES2-HttQ103-GFP plasmid were grown at 30°C to exponential phase (OD 600 about 0.5) in YNB-URA 2% raffinose. Htt103Q-GFP expression was induced by adding galactose to a final concentration of 2%. After 4 hours at 30°C cells were washed and resuspended in 1.5 ml Buffer P (10 mM NaH 2 PO 4 , 150 mM NaCl, 2% galactose; pH 7.2). All cells present during the expression of Htt103Q-GFP were marked by staining the cell wall components α-mannopyranosyl and α-glucopyranosyl with 0.2 mg/ml concanavalin A Alexa Fluor 647 (Invitrogen) for 30 minutes at room temperature. The cells were then washed in Buffer P, resuspended in YNB-URA with 2% glucose, which will switch off the Htt103Q-GFP expression, and grown at 30°C for one budding event. This makes it possible to distinguish between concanavalin A stained daughter cells present during Htt103Q-GFP expression and daughter cells produced subsequent to Htt103Q-GFP expression. Cells were fixed in 3.7% formaldehyde and segregation of aggregates was analyzed using fluorescence microscopy. The segregation assay of ts-alleles were performed in the same way but without concanvalin A staining. Cells were grown at 22°C to exponential phase, followed by induction and budding at different temperatures (26°C for sec18-1 and sec53-6 , 28°C for cmd1-1 and 32°C for myo2-14 ). Removal and retention assay of heat induced aggregate using concanavalin A staining The retention efficiency assay was performed as described [14] with concanavalin A staining (as described above) before the heat shock treatment. Aggregate retention and removal was distuinguished upon image analysis. Retention is determined as the percentage of aggregate-containing buds of the total number of buds generated from an aggregate-containing mother cell after the heat shock treatment (buds free of conA); Removal efficiency is determined as the perecentage of aggregate-free buds of the total number of buds already existing before heatshock; i.e. stained with conA. More than 300 budding events were quantified for each strains. Statistical analysis of aggregate segregation as a function of the generation time All statistical calculations were done in R-3.0.0 ( www.r-project.org ). Regression analysis was performed and showed that there is no statistically significant difference at p