Growth and heat treatment

Yeast strains were cultured at 30 °C, unless stated otherwise. Heat treatments were conducted in a pre-warmed 37 °C or 39 °C incubator, whereas treatment at 50 °C was performed in a 50 °C shaking heat block. Cultures were grown in synthetic complete media lacking amino acids necessary for plasmid selection and supplemented with either 2% glucose or 2% raffinose and 2% galactose. Experimental comparisons were made between identical growth conditions unless indicated otherwise. Cultures were maintained for >18 h with dilution and harvested during log phase.

HEK293T and DnaJB6 knockout cells ⁶² (Supplementary Table ⁶ ) were cultured in DMEM supplemented with 10% Fetal Bovine Serum, Penicillin and Streptomycin in a 37 °C humidified incubator at 5% CO ₂ . Passaging was performed twice weekly. Transient transfections were performed using 1 µg of DNA and PEI transfection reagent (1:6 ratio). Heat shock experiments were performed for 1 h at 43 °C in a water bath. Samples were collected after ~24 h recovery at 37 ^o C, unless otherwise indicated.

Preparation of cell extracts

Yeast pellets were resuspended in 500 μl lysis buffer (25 mM Tris pH 7.5, 50 mM KCl, 10 mM MgCl ₂ , 1 mM EDTA, 5% glycerol, 0.5% Triton X-100) supplemented with protease inhibitors (cOmplete mini, EDTA-free Protease Inhibitor Cocktail, Roche). Cells were lysed by bead-milling with acid washed glass beads in a MP Beadbeater24 (2 × 20 s at 6.0 m/s with intermittent cooling on ice). Extracts were cleared from cell debris by centrifugation at 500 × g for 5 min at 4 °C. Protein levels were normalized using the Bio-Rad Protein Assay (Bio-Rad). Samples containing polyQ expanded Htt were first precipitated with 25% trichloroacetic acid (TCA) before dissolution with formic acid at 37 °C and preparation for SDS-PAGE ¹⁰⁹ .

Mammalian cell pellets were resuspended in RIPA buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1 mM MgCl ₂ , 1% NP40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Roche) and DENARASE (c-LEcta, 50 U/ml) and vortexed on ice for 1 h. Protein levels were normalized using the DC Protein Assay (Bio-Rad).

Cell fractionation

Total yeast cell lysates were separated into soluble and pellet fractions by centrifugation at 15,000 × g for 15 min at 4 °C. After collection of the soluble supernatant fraction, pellets were washed with lysis buffer (2 × 15,000 × g for 5 min at 4 °C).

SDS-PAGE

Protein samples were suspended in sample buffer (50 mM Tris pH 6.8, 8% glycerol, 2% SDS, 20% β-mercaptoethanol, containing bromophenol blue), heated to 95 °C for 3 min and separated by electrophoresis on 10 or 12% self-made Tris-glycine poly-acrylamide (Serva) or TGX FastCast acrylamide solution (Bio-Rad) gels using Laemmli (25 mM Tris, 192 mM glycine, 0.1% SDS) running buffer at 30 mA/gel.

Filter retardation assay

Filter retardation assays for the detection of total and SDS resistant polyQ aggregates were performed as previously described ^{26 , 37 , 45} . Cell lysates were incubated with 125 U benzonase (Novagen) or smDNase (Max Planck Institute for Biochemistry core facility) with rotation for 1 h at 4 °C. Samples were then incubated with or without SDS buffer (2% SDS, 50 mM DTT) before being loaded onto a pre-wetted (0.1% SDS) 0.2 μm pore size cellulose acetate membrane in a Hoefer slot-blot apparatus. Membranes were subsequently washed four times with 0.1% SDS before immunoblotting.

SDD-AGE

Semi-denaturing detergent agarose gel electrophoresis was performed as previously described ⁷⁵ with a modified lysis buffer (25 mM Tris pH 7.5, 50 mM KCl, 10 mM MgCl ₂ , 1 mM EDTA, 5% glycerol, 0.5% Triton X-100). Yeast extracts were incubated at 30 °C for 7 min in SDD-AGE sample buffer (50 mM Tris pH 6.8, 8% glycerol, 2% SDS, supplemented with bromophenol blue). Samples were then run on an agarose gel (1.5% agarose, 0.1% SDS, in 25 mM Tris, 192 mM glycine) in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 160 V for 1 h at 4 °C.

Immunoblotting

Proteins were transferred from poly-acrylamide gels to nitrocellulose membranes in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) at 110 V for 1 h using the Mini Trans-Blot Cell (Bio-Rad) or for 10 min using the Trans-Blot Turbo Transfer System (Bio-Rad). Proteins were transferred from agarose gels (SDD-AGE) to nitrocellulose membranes in transfer buffer containing 0.01% SDS at 7 V for 14 h using the Genie Blotter System (Research Products International). Membranes were washed with TBST (10 mM Tris-HCl pH7.5, 150 mM NaCl, 0.1% Tween-20), blocked with 4% powdered milk in TBST, and incubated with primary antibodies overnight at 4 °C. Information regarding the primary antibodies used in this study can be found in Supplementary Table ⁷ . After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h and washed again. Imaging was performed using Luminata Classico substrate and the ImageQuant LAS 4000 mini detector or Bio-Rad ChemiDoc imaging system. Densitometric analyses were performed using LICOR Image Studio Lite or Image Lab (Bio-Rad) software.

Immunoprecipitation after crosslinking

Yeast cells were pelleted and resuspended in crosslinking (XL) buffer (1.2 M sorbitol, 5 mM EDTA, 0.1 M KH ₂ PO ₄ /K ₂ HPO ₄ pH 7.5). DSP solution (50 mM dithiobis(succinimidyl propionate) in DMSO) was added to 1 mM final concentration and samples were incubated at 30 °C for 20 min before quenching by addition of 0.1 volume 1 M Tris pH 7.5 for 15 min. Lysates were prepared as above and soluble fractions isolated by centrifugation at 15,000 × g for 15 min at 4 °C. Thousand microgram of protein (determined by Bradford assay) were diluted in lysis buffer, total fractions removed, and remaining samples incubated with 50 μl Milltenyi μMACS magnetic anti-Myc beads for 1 h at 4 °C. Samples were passed over the magnetic columns and washed (4× lysis buffer with 1% Triton X-100 and 2 × 20 mM Tris-HCl pH 7.5) before elution with hot SDS sample buffer.

Confocal imaging

Mid-to-late log phase yeast cells (0.5–1 OD ₆₀₀ ) were adhered to the chamber of a concanavalin A coated µ-Slide (Ibidi, 80626) for 5 min at room temperature. Cells were washed three times with culture media. Imaging was done with an Olympus (Tokyo, Japan) FV1000 confocal microscope setup equipped with an Olympus PLAPON 60×/NA1.42 oil immersion objective. The GFP fluorophore was detected using an excitation wavelength of 488 nm and emission of 505–540 nm. The mCherry fluorophore was detected using an excitation wavelength of 559 nm and emission of 575–675 nm. Image analysis was carried out in Fiji ¹¹⁰ .

Analysis of aggregate density

Cells were prepared as described above and imaged with an Olympus (Tokyo, Japan) FV1000 confocal microscope setup equipped with an Olympus PLAPON 60×/NA1.42 oil immersion objective using the same nonsaturating acquisition settings for all samples. Image analysis was carried out in Fiji ¹¹⁰ . To analyze the fluorescent density of aggregates, circular regions within the aggregate were selected and the average fluorescence signal was compared between samples. To obtain the total cellular fluorescence signal, the integrated fluorescence density over the whole area of the analyzed cells was calculated.

Analysis of Ssa1 enrichment

Cells were prepared as described above and imaged with an Olympus (Tokyo, Japan) FV1000 confocal microscope setup equipped with an Olympus PLAPON 60×/NA1.42 oil immersion objective using the same nonsaturating acquisition settings for all samples. Image analysis was carried out in Fiji ¹¹⁰ . Regions of interest were selected outlining the FLuc aggregate as well as a large area in the cell not containing any aggregate (soluble). The enrichment of Ssa1 in aggregates over soluble signal was calculated as the ratio between the average fluorescence of Ssa1 in these two regions. To obtain the total cellular fluorescence signal, the integrated fluorescence density over the whole area of the analyzed cells was calculated.

FRAP analysis

Samples were prepared as described above. In vivo fluorescence recovery after photobleaching (FRAP) experiments were carried out at MPIB Imaging Facility (Martinsried, Germany) on a ZEISS (Jena, Germany) LSM780 confocal laser scanning microscope equipped with a ZEISS Plan-APO 63×/NA1.46 oil immersion objective. Circular regions of constant size were bleached after 20 frames and monitored for at least 80 s (0.38 s/frame) for fluorescence recovery in a single focal plane. Image analysis was carried out in Fiji ¹¹⁰ . Fluorescence intensity data were corrected for photobleaching and normalized to the average fluorescence intensity before (1) and after (0) bleaching (relative fluorescence), except the experiment including soluble material, which was only normalized pre-bleach due to the rapid recovery (reported as fluorescence intensity).

β-Galactosidase activity measurements

LacZ expression was measured using a standard ß-galactosidase assay ¹⁰³ . Briefly, cell pellets were resuspended in 700 µl of Z buffer (60 mM Na ₂ HPO ₄ , 40 mM NaH ₂ PO ₄ , 10 mM KCl, 1 mM MgSO ₄ , 50 mM ß-mercaptoethanol) supplemented with 50 µl of 0.1% SDS and 50 µl of chloroform, vortexed and incubated for 5 min at 30 °C. Two hundred microliter of substrate (4 mg/ml ONPG (2-Nitrophenyl β- d -galactopyranoside, Sigma-Aldrich) in Z buffer) was added with further incubation (~3–13 min) before quenching with 350 µl 1 M Na ₂ CO ₃ , clearance by centrifugation at 500 × g , and spectrophotometric measurement at A ₄₂₀ . Activity was calculated in standard Miller Units. 1 Miller Unit = 1000 × (A ₄₂₀ / (O.D. ₆₀₀ × ml × t _min )).

mRNA sequencing

Cells were harvested in triplicate and sent to Novogene Co., Ltd. (Hong Kong) for RNA extraction, library preparation, Illumina paired-end sequencing, mapping, and bioinformatics analysis. Briefly, 1 µg RNA per sample was used for library generation using NEB Next Ultra RNA Library Prep Kit for Illumina (NEB). Library preparations were subjected to Illumina paired-end sequencing (150 bp, 10 million reads). Reads were indexed using Bowtie v2.2.3 and aligned to the reference genome using TopHat v2.0.12. Read numbers were counted using HTSeq v0.6.1 and gene expression was calculated using FPKM (Fragments per Kilobase of transcript sequence per Million base pairs sequenced) method. The differentially expressed genes between samples were calculated using the DESeq R package and the resulting p values were adjusted using the Benjamini and Hochberg false discovery rate approach. Differentially expressed genes are defined as having an adjusted p value less than 0.05. Gene ontology (GO) enrichment analysis of the differentially expressed genes was performed using the GOseq R package. Significance was corrected for gene length bias and adjusted p values less than 0.05 indicate a significantly enriched term.

Statistical analysis

Error bars represent standard deviation from at least three experiments. Significance was determined for two-sample comparisons using the unpaired t -test function with a threshold of two-tailed p values less than 0.05. Significance among multiple conditions were determined using ANOVA and Dunnett’s multiple comparisons test with a threshold of p < 0.05. Calculations were performed using Graphpad software.