The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis.

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For materials and further information, please contact the lead contact, Judith Frydman ( jfrydman@stanford.edu ). DETAILED EXPERIMENTAL PROCEDURES EXPERIMENTAL MODEL AND SUBJECT DETAILS Yeast strains and growth conditions CRISPR strains: Primers were designed for guide sequence (see Table S4 ) and used to amplify the pCAS9 vector( Ryan et al., 2016 ). The yeast Api4 EEE plasmid (see Cloning) was used for amplifying homology sequence containing the EEE mutation for recombination, guide recognition sequence was mutated using overlapping PCR. The EEE CRISPR was carried out on WT BY4742 yeast and Δpfd5 strain by transforming pCAS9-Api4guide and PCR product into strains. Transformants were plated onto G418 and allowed to grow for 48 hrs at 37 °C. Individual colonies were selected and grown overnight in YPD. Overnight cultures were spread on YPD plates, and mutations in individual colonies were confirmed by PCR and sequencing. Other strains: PFD5 add back strains were generated by transforming clone YGPM10b11 from the yeast genomic tiling collection from Dharmacon into indicated strains and selecting on –Leu plates. GFP/CBP CCT yeast strains were generated by taking haploid strains containing the pCuCCTx(Ura3) vector ( Tam et al., 2006 ) and deleted for the corresponding cctx gene and transforming with the pAB-CCTx (Leu) vector containing the desired insertion (for instance CCT1–GFP). Counter selection against the WT (Ura3) plasmid was performed using (5’–FOA) plates. Obtained colonies were tested for growth on –Leu plates to verify presence of pAB vector, and – Ura plates to verify loss of pCu vector. Growth conditions: For experiments involving the EEE strain, all strains in experiment were grown at 37 °C overnight, and then diluted to an OD600 under 0.1 and grown to an OD600 of 0.3 to 0.9 at reported temperatures (minimum of 3 hrs of growth). For experiments involving the PFD5 vector, cells were grown in –Leu media. For experiments not involving the EEE strain, strains were grown at 30 C, and treated as above. For drop tests involving the PFD5 vector, cells were plated on –LEU plates. For all other drop tests cells were plated on YPD. Plates were incubated at indicated temperatures for drop tests.

METHOD DETAILS Cloning

CCT1–8 apical domains: Each apical domain was cloned into the super folder GFP between Beta sheets 10 and 11 of the beta-barrel structure with a glycine serine linker added to provide some flexibility. Constructs were cloned into pST39( Tan, 2001 ) using XbaI and BamHI restriction sites for Api domains 1,2,3,4,5,7 and 8, and XbaI and HindIII for Api domain 6. For the yeast apical domains, pRS vectors containing genomic CCTX were used as template. Mutations were made by overlapping PCR with primers containing the desired mutations. Fastbac Dual hTRiC construct cloning: Individual TRiC subunits were cloned into the pFastbac dual vector (ThermoFisher Scientific) using SalI/NotI for CCT1,2,6 and 7 and XhoI/KpnI for CCT3,4,5 and 8. The dual vectors consisted of CCT1,8, CCT2,4, CCT3,6 and CCT5,7 with CCT2, CCT5, CCT6 and CCT8 being cloned initially followed by CCT4, CCT7, CCT3 and CCT1 respectively. For GFP tagged vectors, BspEI sites were cloned into flexible loops using overlapping PCR. GFP was inserted into BspEI sites using standard techniques. Fastbac Dual hPFD construct cloning: Individual Pfd subunits were cloned into the pFastbac dual vector (ThermoFisher Scientific) using SalI/NotI for Pfd1,4 and 5, and XhoI/KpnI for Pfd2,3, and 6. The dual vectors consisted of Pfd1,2, Pfd5,3 and Pfd4,6 with Pfd1, Pfd4, and Pfd5 being cloned initially followed by Pfd2, Pfd3 and Pfd6 respectively. Yeast GFP and CBP vectors: BspEI sites were cloned into flexible loops of CCT subunits using site directed mutagenesis in pAB vectors. Either GFP or CBP was cloned into BspEI sites using standard cloning techniques. Protein Expression and Purification hTRiC: We used the Bac to Bac baculovirus expression system for recombinant protein production (Invitrogren). P3 virus for human TRiC subunits was prepared as described in the protocol. 1 l of High-Five insect cells (Invitrogen) at a concentration of ~3X10 6 cells/ml were co- infected with 5 mls of each of the viruses. and cells were harvested 2 to 3 days after infection by centrifugation. Cells were resuspended in 100 mls lysis buffer (100 mM NaCl, 100 mM HEPES pH 7.4, complete protease inhibitors EDTA free (Roche), 1 mM DTT, PMSF, Benzonase 5 U/ml (Sigma-Aldrich)) and lysed using an emusiflex (Avestin). Lysate was clarified by ultracentrifugation (50,000XG, 1 h). Cleared lysate was passed over a FFQ column equilibrated with MQA buffer (50 mM NaCl, 20 mM HEPES pH 7.4, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 10% glycerol), and then eluted with 40% MQB buffer (1 M NaCl, 20 mM HEPES pH 7.4, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 10% glycerol). TRiC eluted from FFQ column was passed over a Heparin column equilibrated with 20% MQB buffer and washed until UV returned to baseline. TRiC was eluted with a 200 ml gradient from 20% MQB to 100% MQB, and fractions containing TRiC identified by SDS-PAGE. TRiC containing fractions were concentrated down using an Amicon Ultra 100 kDa MWCO centrifugal filter (Millipore) to a volume of ~ 2 mls. The concentrated TRiC was further purified by size exclusion chromatography over a Superdex200 26/60 (GE Healthcare Life Sciences) equilibrated with column buffer (100 mM NaCl, 50 mM HEPES pH 7.4). Eluted TRiC was identified by SDS- PAGE and purified by Talon resin (Clontech). TRiC eluted from the talon resin was purified by anion exchange chromatography using a MonoQ 10/100 column equilibrated with MQA and eluted by a 150 ml gradient to 100% MQB. Fractions containing TRiC were identified by SDS- PAGE, concentrated to ~ 1 ml, aliquoted and snap frozen. Typical yields were greater than 10 mg/L and TRiC assayed by mass spec is ~ 95% pure. yTRiC: Approximately 6 L of yeast at an OD ~2 expressing CCT2 with a CBP tag was harvested by centrifugation. Cells were resuspended in equal volume lysis buffer (200 mM NaCl, 100 mM HEPES pH 7.4, 5 mM CaCl2, 20% glycerol, complete protease inhibitors (Roche), benzonase 5U/ml, PMSF). Cells were lysed using a MM301 mixer mill grinder MM301 (Retsch). Lysate was cleared by centrifugation 20,000Xg for 30 min. Approximately 9 mls of calmodulin resin was equilibrated in wash buffer A (200 mM NaCl, 50 mM HEPES pH 7.4, 5 mM CaCl2, 20% glycerol) and added to the cleared lysate. Binding was carried out at 4 C on a mutator for 2.5 h. Resin/lysate was loaded onto a gravity column and washed with 20 mls buffer A, 40 mls buffer B (500 mM NaCl, 50 mM HEPES pH 7.4, 5 mM CaCl2, 20% glycerol), and 20 mls buffer A. TRiC was eluted from the calmodulin resin with elution buffer (100 mM NaCl, 50 mM HEPES pH 7.4, 10 mM EDTA, 20% glycerol). Eluted protein was further purified by binding to two 5 ml HiTrap Heparin columns (GE Healthcare) equilibrated with MQA buffer. Heparin columns were washed with 20% MQB, and then eluted with a 60 ml gradient to 100% MQB. ATP to 1 mM was added to eluted TRiC, and TRiC was concentrated down to ~ 600 μl using an Amicon Ultra concentrator 10,000 MWCO. Concentrated protein was run over a Superose 6 gel filtration column equilibrated with MQA. Fractions containing TRiC were concentrated and snap frozen in liquid nitrogen. hPFD: 1 L of High five insect cells (Invitrogen) at a concentration of ~3X10^6 cells/ml are infected with 8 mls of each of the viruses. and cells are harvested 2 to 3 days after infection by centrifugation. Cells are resuspended in 100 mls lysis buffer (100 mM NaCl, 100 mM HEPES pH 7.4, complete protease inhibitors EDTA free (Roche), PMSF, Benzonase 5 U/ml (Sigma- Aldrich), ~ 10 mM imidazole), and lysed using an emusiflex (Avestin). Lysate is clarified by ultracentrifugation (50,000*G, 1 hr). Lysate is cleared at 50,000 G for 30 min. Cleared lysate was run over a gravity column containing 10 mls of Nickel resin equilibrated with buffer A (50 mM HEPES pH 7.4, 100 mM NaCl). Resin was washed with buffer A, buffer A with an additional 400 mM NaCl, and buffer A again. PFD was eluted with buffer A containing 300 mM imidazole. Eluted protein was further purified on an MonoQ 10/100 anion exchange equilibrated with buffer A + 1 mM DTT. PFD was eluted from the MonoQ by a gradient to 100% buffer B (1 M NaCl, 50 mM HEPES pH 7.4, 1 mM DTT). Eluted PFD was concentrated down to ~ 1 ml using Amicon Ultra 30 KD MWCO and run over an SDX200 26/600 gel filtration column (GE Healthcare) equilibrated with buffer A + 1 mM DTT. Fractions containing PFD were concentrated using an Amicon Ultra 30 KD MWCO and 50% glycerol was added to a final concentration of 10%. Alexa647-hPFD: Alexa647 maleimide (Life Technologies) was used to label purified hPFD. 117 μM hPFD was mixed with freshly dissolved Alexa647 dye at 1.17 mM (10 molar excess), dye was reacted for 1 hr at room temperature, and quenched by addition of DTT. Labeled complex was purified by size exclusion chromotagraphy using an SDX200 10/300 (GE life sciences). Final concentrations were determined using extinction coeffiicient, labeling efficiency was 0.59 dyes/complex. yPFD: A polycistronic vector for co-expression of PFD subunits was transformed into BL21 Rosetta2 pLysS cells and grown to ~ 0.3 OD and induced O/N at 23 °C. Cells were pelleted, washed with PBS containing PMSF, and resuspended in lysis buffer (100 mM NaCl, 100 mM HEPES pH 7.4, complete protease inhibitors EDTA free (Roche), PMSF, Benzonase 5 U/ml (Sigma-Aldrich), ~ 10 mM imidazole). Cells were lysed using an emulsiflex and lysate cleared at 20,000 G for 30 min. ScPFD was purified from lysate as described for HsPFD. Apical domains: Apical domains were purified as described in ( Joachimiak et al., 2014 ). Briefly, each Apical domain construct was transformed into BL21 Rosetta2 pLysS cells grown to ~ 0.3 OD and induced O/N at 23 °C. Cells were pelleted, washed with PBS containing PMSF, and resuspended in lysis buffer. Cells were lysed using an emusilflex, and lysate cleared at 20,000XG for 30 min. Cleared lysate was passed over Talon affinity resin (Clonetech), washed with column buffer + Triton X 100, column buffer + 500 mM NaCl, and column buffer + 1 mM ATP. Protein was eluted with column buffer + 300 mM imidazole and concentrated down to ~ 2 mls. Concentrated protein was run over an SDX200 26/60 size exclusion chromatography column equilibrated with column buffer + 1 mM DTT, the peak fraction was collected, concentrated and 50% glycerol was added to 10%. Protein was than aliquoted and snap frozen for future use.

Crosslinking-Mass Spectrometry

For hPFD with hTRiC samples, TRiC was brought to 1 μM, and Pfd to 2 μM, human PFD alone was prepared at 10 μM. For yPFD with yTRiC samples, TRiC was brought to 1 μM, and Pfd to 10 μM, and yPFD alone was prepared at 10 μM. All samples were prepared in 50 mM HEPES pH 7.4, 50 mM NaCl and 1 mM DTT. Fresh DSS crosslinker dissolved in DMSO was added to a final concentration of 1 mM, and samples were crosslinked for 2 h at room temperature for the human complexes and 37 °C for 30 min for the yeast complexes. Crosslinking was quenched by incubation for 30 minutes after addition of 1 M Tris pH 7.4 to a final concentration of 100 mM for human complexes, and addition of 1 M Ammonium Bicarbonate to 50 mM for yeast complexes. Crosslinked samples were processed as described previously ( Leitner et al., 2014 ). In brief, processing steps included reduction and alkylation of cysteine residues with tris(2- carboxyethyl)phosphine and iodoacetamide, respectively, sequential digestion with endoprotease Lys-C (Wako) and trypsin (Promega), clean-up using solid-phase extraction (Waters Sep-Pak tC18 cartridges) and fractionation of the purified digests by size exclusion chromatography (SEC; GE Superdex Peptide PC 3.2/300). SEC fractions were analyzed by liquid chromatography-tandem mass spectrometry on a Thermo Easy-nLC 1000 system coupled to a Thermo Orbitrap Elite system as described in detail elsewhere ( Greber et al., 2014 ). MS data analysis was performed using xQuest/xProphet ( Walzthoeni et al., 2012 ) using databases consisting of the TRiC and PFD sequences (including tags) of the respective complexes and contaminant proteins, if applicable. Decoy databases were generated as described in ( Walzthoeni et al., 2012 ), and the results were filtered to a false discovery rate of 5%. PFD–TRiC affinity Human PFD (8 μM) and Human TRiC (0.5 μM) in ATPase buffer (30 mM Tris pH 7.4, 120 mM KCl, 5 mM MgCl2 and 1 mM dithiothreitol added fresh) were serial diluted and mixed 1:1 with TRiC yielding a final concentration of 0.25 μM TRiC and 0.063 to 4 μM PFD. Binding was allowed to equilibrate for approximately 30 min at room temperature and samples were run out on clear Native PAGE (either 4–12% or 4–16%). Protein was transferred to nitrocellulose and probed with Rabbit serum SU-230 (1:2000) developed against recombinant human PFD (Cocalico Biologicals). For the lower concentrations of PFD, the signal for free and TRiC-bound PFD was summed and the signal/uM calculated for each blot. This value was then used to calculate the amount of PFD bound to TRiC at each concentration. The amount of free PFD was calculated by subtracting the amount bound from the starting concentration. Binding was done in triplicate, and error bars represent SEM. PFD–TRiC anisotropy TRiC (400 nM) was prepared in 1XATPase buffer leaving room (30% volume) for nucleotide addition (closed)/H 2 O (open) 1/10 volume 10 mM ATP, 10 mM AlNO 3 , and 60 mM NaF were each added in quick succession to close TRiC for 30 min at 37 °C.

Human Alexa647 labeled

PFD was prepared at 50 nM in 1XATPase buffer, and mixed 1:1 with open TRiC, closed TRiC, and 1XATPase for final concentrations of 200 nM TRiC and 25 nM PFD, and allowed to equilibrate at room temperature. Anisotropy was measured using a CLARIOstar plate reader from BMG Tech with the PFD alone sample normalized to 100 prior to readings.

Actin folding assays

Endpoint assays: TRiC variants (bovine TRiC, WT hTRiC, C1-GFP hTRiC, and C6-GFP hTRiC) were diluted to 0.25 μM in ATPase buffer. [ 35 S]actin, prepared as described,in ( Meyer et al., 2003 ; Thulasiraman et al., 2000 ) in 6 M Guanidnium-HCl, 100 mM HEPES pH 7.4 was rapidly diluted 1:100 with TRiC variants or with ATPase buffer alone. Actin was allowed to bind for 30 min on ice. To remove aggregated Actin, samples were spun 10 min at 19000xg. Supernatants were moved and layered onto either water or ATP to a final concentration of 1 mM. Samples were incubated 1 h at 37 °C. DNAseI was added to each sample (5 μg) to bind and compact folded Actin bands for native gel analyses. Samples were incubated 10 min on ice to allow binding, then large aggregates removed by centrifugation for 10 min at 19,000XG. Samples were run out on clear native gels 4–16%, gels were coomassie stained, dried and exposed to a phosphorscreen. Phosphorscreens were imaged using a Typhoon scanner. Kinetic analyses: 0.13 μM [ 35 S]actin in 6 M Guanidnium-HCl, 100 mM HEPES pH 7.4 was rapidly diluted 1:100 with TRiC at a concentration of 0.25 μM with or without PFD at 2.5 μM in ATPase buffer with fresh 1 mM DTT. Samples were incubated 30 min on ice to allow [ 35 S]actin binding. To remove aggregated actin, samples were spun 10 min at 19000 G. Supernatents were moved into fresh tubes, and the TRiC alone sample split into (2) aliquots. When indicated, the TRiC-[ 35 S]actin was further purified by ion-exchange chromatography on a MonoQ column as described ( Frydman and Hartl, 1996 ; Reissmann et al., 2007 ). PFD was added to one aliquot of TRiC alone to a final concentration of 2.5 μM (equal volumes of ATPase buffer was added to the other samples). ATP was added to 1 mM and the folding reactions initiated by moving samples into a 37 °C water bath. Samples were taken at 0,2,4,8,16,32,48,64 and 128 min and the reactions stopped by addition of 0.2 Units of apyrase and moving samples to ice (the apyrase should deplete all ATP is less than 1 minute, and TRiC ATPase activity is negligible at 4 °C). DNAseI was added to each time point (5 μg) to bind folded Actin and compact bands in native gels. Samples were incubated 10 min on ice to allow binding, then large aggregates removed by centrifugation for 10 min at 19000xg. Timepoints were run out on clear native gels 4–16%, gels were coomassie stained, dried and exposed to a phosphorscreen. Phosphorscreens were imaged using a Typhoon scanner. The amount of Actin folding was quantified using image quant, and each experiment was normalized to the final three timepoints (48, 64, and 128 min) of TRiC alone folding which was set as 1 arbritrary unit (AU). Experiments were done in triplicate and error bars represent SEM.

Apical domain assays

PFD binding: Apical domains and yPFD/hPFD were each diluted to 2 μM in ATPase buffer. Samples were mixed 1:1 and allowed to bind at room temperature for ~10 min. Samples were run out on a 15% SDS PAGE for a loading control, and on a clear Native PAGE 4–16%. for mobility shift analysis. Protein from native page gels was transferred to nitrocellulose membranes and antibodies raised against recombinant yeast PFD/human PFD at 1:2000 were used to detect proteins Competitive inhibition of PFD binding TRiC: hPFD/yPFD at 2 μM was mixed 1:1 with Apical domain 4 serially diluted from 40 μM to 1.25 μM and allowed to equilibrate ~ 10 min at room temperature. These samples were then mixed 1:1 with 0.5 μM TRiC in ATPase buffer and allowed to equilibrate for 10 min at room temperature. Samples were run out on a 15% SDS PAGE for a loading control, and on a clear Native PAGE 4–16%. for mobility shift analysis. Protein from native page gels was transferred to nitrocellulose membranes and antibodies raised against recombinant yeast PFD/human PFD at 1:2000 were used to detect PFDs.

ATPase measurements

ATPase rates were determined as described in. ( Reissmann et al., 2007 ). Briefly, ATP hydrolysis by 0.25 μM hTRiC or 0.25 μM hTRiC + 2.5 μM hPFD was measured at 37 °C in ATPase buffer, in the presence of 188 to 1500 M [α− 32 P]ATP. After 5 min of preincubation, the reaction was started by mixing 6 l [α− 32 P]ATP solution with 34 l 1.176-fold concentrated reaction mix. At the indicated time points, 2- l samples were taken and transferred onto PEI-cellulose F thin-layer chromatography plastic sheets (EMD Chemicals). The plates were developed in a solvent system containing 1 M LiCl and 0.5 M formic acid in H 2 O, air-dried and exposed to a phosphorimager (Kodak). After the screen was scanned in a Typhoon 9410 imager, the amount of [α− 32 P]ATP was quantified using ImageQuant 5.2, amount of [α− 32 P]/ATP was determined from a standard curve from a dilution set on the thin- layer chromatography sheet. TRiC–PFD CryoEM specimen preparation Purified 20 μM hPFD was diluted 10-fold into MQ buffer containing 1 μM purified hTRiC, resulting in a 2:1 molar ratio of hPFD to hTRiC. The complex mixture was incubated for 30 min in room temperature and Octyl glucoside (OG) was added to a concentration of 0.1 % before grid placement to increase the yield of side views in electron micrographs. The resulting samples were applied to 200-mesh R1.2/1.3 holey-carbon grids (Quantifoil) and vitrified using a Gatan CP3 (Gatan). We loaded grids into a JEM3200FSC (300 KeV) electron microscope with an in- column omega filter (25 eV energy slit). We recorded images at 1.5–3 m underfocus on a K2 Summit direct electron detector (Gatan) in super-resolution mode at nominal 20K magnification, corresponding to a sampling of 1.7 Å/pixel (super-resolution sampling: 0.85 Å/pixel).

Image processing

We initially binned each movie stack by two and then corrected for drift and radiation damage using DE_process_frames.py (Direct Election Ltd). The first three frames were ignored during movie processing. We used EMAN2 to automatically select particle images ( Bell et al., 2016 ). Contrast transfer-function (CTF) parameters were estimated internally based on the boxed particles (e2ctf.py). We then performed two-dimensional reference-free averaging with ~2,000 particle images using the default parameters in EMAN2 and generated an initial three- dimensional model based on the two-dimensional class averages, with no symmetry imposed. We converted a set of CTF-corrected particles to a RELION-compatible format using E2refinetorelion3d.py. We performed all further refinements using RELION 1.4 ( Scheres, 2012 ), starting with maps that were low-pass filtered to 50 Å from the initial model generated by EMAN2. At first, we perform three-dimensional classification (K=3) with C1 symmetry to exclude non-TRiC-like particles. Then, we subsequently refined the remaining particles using the “auto_refine” command, which resulted in a 6.3 Å map based on a gold-standard Fourier shell correlation (FSC) at 0.143. In this step we were identified noisy density around TRiC’s apical domains, which stems from conformational heterogeneity of PFD. Focused classification and localized reconstruction of individual subunits The overall workflow using RELION 1.4 for the focused classification ( Bai et al., 2015 ) and localized reconstruction ( Ilca et al., 2015 ; Roh et al., 2017 ) of TRiC–PFD is described in Figure S4 . We split the 6.5 Å map into two TRiC ring maps by masking out one ring at a time ( Figure S4A , a – b ). We then applied a soft mask around each map of TRiC ring and used the resulting masked map for subtraction of the signal from each raw particle image. This process generated two sub-particles from each raw particle image, which corresponds to either cis or trans ring of TRiC ( Figure S4A , c ). Using these sub-particles, we performed three-dimensional classification (K=5) with C1 symmetry and excluded 15% of particles with sub-optimally resolved features ( Figure S4A , d ). Then, we subjected the remaining 85% sub-particles for auto-refinement with a mask for single TRiC ring to determine the best orientation for each single TRiC ring ( Figure S4A , e ). This resulted in a 6.4 Å map containing only 8 subunits of TRiC. We next performed a focused 3D classification (K=8) on the putative PFD binding area with a spherical mask without orientation search ( Figure S4A , f ). This operation resulted in three groups of unbound TRiC and five groups with extra density around apical domains of TRiC ( Figure S4A , g ). Three unbound groups were combined as an unbound class. Therefore, one unbound class (Class1) and each of five bound classes (Class2–6) was respectively reconstructed with corresponding original two ring particles, while keeping the particle orientation from the single ring refinement ( Figure S4A , h ). In final, each map was sharpened using auto B-factor in RELION post processing step. Model building We first fit the crystal structure of yeast TRiC (4V94) ( Leitner et al., 2012 ) using Chimera ( Pettersen et al., 2004 ) in closed conformation to the CryoEM map of the open conformation; the model matches the map only in the equatorial domain. Then, molecular dynamics flexible fitting (MDFF) ( Trabuco et al., 2008 ) was used on the fitted model and map. This method applies forces at atom position in the direction of the density gradient (density gradient weight of 0.3), while performing a molecular dynamics (MD) simulation at a given temperature (300K). After ~10 5 MD steps, the model matched the map much better, especially in the apical domains. This procedure thus opened the closed conformation of yeast TRiC to the open conformation of TRiC seen in the 6.3Å cryoEM map. We then threaded the sequence of each subunit in the Human TRiC through the open state of the yeast TRiC model based on sequence similarity. We performed a final round of model optimization with Phenix.real_space_refine for global minimization ( Afonine et al., 2012 ). Since at this resolution the density does not constrain the structure tightly, we used probabilistic modeling to estimate the uncertainty at atomic positions using ProMod ( Pintilie et al., 2016 ). Uncertainty values are stored in the Bfactor column of the PDB/model. For Human PFD model, two copies of Archeal PFD crystal structures (2ZDI) ( Sahlan et al., 2010 ) fitted into the extra density in Class 6, considering the right arrangement from XL/MS results ( Figure 5 ). The PFD structure fitted well around the helical tips of TRiC, and contacts are clearly seen. We then threaded the sequence of each subunit of the Human PFD through the Archeal PFD model based on sequence similarity. We rigid-body fitted this human PFD model into each extra density in Class 2–6 and then combined with TRiC model, which generated previously. The tips of CCT were manually adjusted to the density to avoid overlapping with PFD. Each combined model was flexible fitted again to respective map by MDFF ( Trabuco et al., 2008 ). Each resulting model was optimized into each map using Phenix.real_space_refine for global minimization ( Afonine et al., 2012 ), and again probabilistic modeling was used to measure uncertainties at atomic positions ( Afonine et al., 2012 ). yTRiC pulldowns with CNBr-yPFD One liter of WT or EEE yeast strains were grown to ~ 0.5 OD600 at 37 °C (permissive temperature for EEE mutant) and harvested by pelleting at 4000XG for 10 min. Pellets were resuspended in PBS with PMSF, pelleted for 5 min at 4000xg, and then resuspended in equal volume lysis buffer (100 mM HEPES-KOH pH 7.4, 50 mM KCl, 5 mM MgCl 2 , 2 mM DTT, 20% glycerol, 0.25% NP40 and complete protease inhibitors Roche). Pellet was then frozen by slowly dripping into liquid nitrogen for lysis. Cells were lysed using a cryogrinder for 1 minute at 20 Hertz. Lysate was cleared by centrifugation at 19000*G for 10 min at 4 °C, and supernatent was isolated. Protein concentration for each cleared lysate was determined using a bradford assay, and all cultures were diluted to 2 mg/ml using lysis buffer. 1 ml of cleared lysate was mixed with 200 μl of CNBr-yPFD resin and incubated for 2 hr at 4 °C. Lysate/CNBr-yPFD mixtures were split into three tubes of 300 μl. Resin was pelleted by centrifugation 22*G for 1 minute and unbound protein/supernatent was removed. Resin was washed 3 times by pelleting and resuspension with 500 μl of column buffer (100 mM HEPES-KOH pH 7.4, 50 mM KCl, 5 mM MgCl2, 2 mM DTT, 20% glycerol, 0.25% NP40) supplemented with an additional 50 mM, 150 mM or 350 mM KCl for final concentrations of 100, 200, and 400 mM KCl respectively. Resin was moved to a fresh tube after final wash, and bound protein was eluted with 1X Lamealli buffer.

TRiC closure assay

Yeast lysates were prepared as in the yTRiC pulldowns. Cleared lysates were diluted to 2 mg/ml to a final concentration of 30 mM HEPES-KOH pH 7.4, 100 mM KCl, 5 mM MgCl 2 , 20% glycerol and 1 mM DTT. Samples were prepared leaving 30% volume for addition of ATP, AlNO 3 , and NaF. 35 μl of each sample was brought to 30 °C, and either 15 μl of H 2 O (Open) or 5 μl of 10 mM ATP, 5 μl of 10 mM AlNO 3 , and 5 μl of 60 mM NaF in quick succession (closed) was added. Samples were incubated for 15 min to allow TRiC to fully close. TRiC was separated using 4% Native-PAGE. Protein from native page gels was transferred to nitrocellulose membranes and an antibody raised against yTRiC apical domains (Bio277) was used to detect TRiC.

Drop tests

Yeast strains were grown to log phase (~ 0.6 OD600) at 37 °C. Cells were diluted back to an OD600 of 0.08, and then serially diluted 1:10 three times. Cells were plated onto YPD and incubated at 37 °C and 30 °C for 48 h, and 23 °C for 72 h and imaged. Proteostat/aggregate staining Yeast strains were grown to approximately 0.2 OD600 at 37 C and then shifted to either 37 °C or 30 °C. Cells were allowed at least (2) doubling times (3 h) at the respective temperatures. Cells were fixed by addition of paraformaldehyde to a final concentration of 4% (1.5 mls culture, 0.5 mls 16% paraformaldehyde) for 15 min. Cells were pelleted and washed 2 times with PBS to remove paraformaldehyde. Cells were resuspended in proteostat buffer (PBS with 5% BSA and 0.1% Tween20) for 30 min. Proteostat was prepared by 1:2000 dilution in the buffer, and each pellet was resuspended with 250 μl and incubated for 30 min at 4 °C. Cells were washed 3X using proteostat buffer, and 2X with PBS. Cells were adhered to concanavalin A coverslips using Prolong antifade with DAPI and imaged with a Ziess LSM700 with the 100X oil immersion lens. Aggregate purifications Yeast strains were grown overnight at 30 °C and diluted in the morning into 100 mls of YPD grown to ~ 0.6 OD after at least two doublings and harvested by pelleting cultures at 3000 G. Pellets were resuspended in 1 ml PBS with PMSF, pelleted and snap frozen in liquid N 2 and stored at – 80 C until further processing. Cell lysates were prepared by resuspending each pellet in 1 ml of lysis buffer (50 mM Na-phosphate, pH 6.8, 50 mM Potassium Acetate, 10 mM DTT, 1 mM EDTA, 1 mM PMSF, complete protease inhibitor cocktail (Roche), 3 mg/ml zymolyase and 1.25 U/ml benzonase) and incubation at room temperature for 20 min. Chilled samples were treated with tip sonication (3 s on, 7 s off, 25% power) for 1 min using a Qsonica Q500 sonicator. Lysates were cleared at 200*G for 20 min, and concentrations determined by bradford assay. Each lysate was adjusted to 1 mg/ml with lysis buffer. Aggregates were pelleted at 16000*G for 20 min, resuspended in 1 ml of wash buffer (50 mM Na-phosphate, pH 6.8, 50 mM Potassium Acetate, 10 mM DTT, 1 mM EDTA, 1 mM PMSF, 2% NP40) and tip sonicated (3 s on, 7 s off, 25% power), and pelleted again at 16000*G for 20 min. Wash step was repeated, and pellets were resuspended in SDS sample buffer and analyzed by SDS-PAGE followed by coomassie staining/western blot analysis.

QUANTIFICATION AND STATISTICAL ANALYSIS

Quantification for bands in Figure 1J , S1F , 2HI , S2E , 3C – F , and S3B , C were all performed using imagequant software. Data in figures for 2HI , S2E , 3C – F , and S3B , C represent the mean ± standard error of the mean (SEM) of three independent experiments (n = 3). For Figure S7D , the enriched proteins from each sample were subjected to pathway analysis to search for enriched GO categories using the DAVID database. Shown are categories using p < 0.05 using Fisher’s exact test followed by Benjamini-Hochberg multiple testing correction.

DETAILED EXPERIMENTAL PROCEDURES EXPERIMENTAL MODEL AND SUBJECT DETAILS Yeast strains and growth conditions CRISPR strains: Primers were designed for guide sequence (see Table S4 ) and used to amplify the pCAS9 vector( Ryan et al., 2016 ). The yeast Api4 EEE plasmid (see Cloning) was used for amplifying homology sequence containing the EEE mutation for recombination, guide recognition sequence was mutated using overlapping PCR. The EEE CRISPR was carried out on WT BY4742 yeast and Δpfd5 strain by transforming pCAS9-Api4guide and PCR product into strains. Transformants were plated onto G418 and allowed to grow for 48 hrs at 37 °C. Individual colonies were selected and grown overnight in YPD. Overnight cultures were spread on YPD plates, and mutations in individual colonies were confirmed by PCR and sequencing. Other strains: PFD5 add back strains were generated by transforming clone YGPM10b11 from the yeast genomic tiling collection from Dharmacon into indicated strains and selecting on –Leu plates. GFP/CBP CCT yeast strains were generated by taking haploid strains containing the pCuCCTx(Ura3) vector ( Tam et al., 2006 ) and deleted for the corresponding cctx gene and transforming with the pAB-CCTx (Leu) vector containing the desired insertion (for instance CCT1–GFP). Counter selection against the WT (Ura3) plasmid was performed using (5’–FOA) plates. Obtained colonies were tested for growth on –Leu plates to verify presence of pAB vector, and – Ura plates to verify loss of pCu vector. Growth conditions: For experiments involving the EEE strain, all strains in experiment were grown at 37 °C overnight, and then diluted to an OD600 under 0.1 and grown to an OD600 of 0.3 to 0.9 at reported temperatures (minimum of 3 hrs of growth). For experiments involving the PFD5 vector, cells were grown in –Leu media. For experiments not involving the EEE strain, strains were grown at 30 C, and treated as above. For drop tests involving the PFD5 vector, cells were plated on –LEU plates. For all other drop tests cells were plated on YPD. Plates were incubated at indicated temperatures for drop tests.

EXPERIMENTAL MODEL AND SUBJECT DETAILS Yeast strains and growth conditions

CRISPR strains: Primers were designed for guide sequence (see Table S4 ) and used to amplify the pCAS9 vector( Ryan et al., 2016 ). The yeast Api4 EEE plasmid (see Cloning) was used for amplifying homology sequence containing the EEE mutation for recombination, guide recognition sequence was mutated using overlapping PCR. The EEE CRISPR was carried out on WT BY4742 yeast and Δpfd5 strain by transforming pCAS9-Api4guide and PCR product into strains. Transformants were plated onto G418 and allowed to grow for 48 hrs at 37 °C. Individual colonies were selected and grown overnight in YPD. Overnight cultures were spread on YPD plates, and mutations in individual colonies were confirmed by PCR and sequencing. Other strains: PFD5 add back strains were generated by transforming clone YGPM10b11 from the yeast genomic tiling collection from Dharmacon into indicated strains and selecting on –Leu plates. GFP/CBP CCT yeast strains were generated by taking haploid strains containing the pCuCCTx(Ura3) vector ( Tam et al., 2006 ) and deleted for the corresponding cctx gene and transforming with the pAB-CCTx (Leu) vector containing the desired insertion (for instance CCT1–GFP). Counter selection against the WT (Ura3) plasmid was performed using (5’–FOA) plates. Obtained colonies were tested for growth on –Leu plates to verify presence of pAB vector, and – Ura plates to verify loss of pCu vector. Growth conditions: For experiments involving the EEE strain, all strains in experiment were grown at 37 °C overnight, and then diluted to an OD600 under 0.1 and grown to an OD600 of 0.3 to 0.9 at reported temperatures (minimum of 3 hrs of growth). For experiments involving the PFD5 vector, cells were grown in –Leu media. For experiments not involving the EEE strain, strains were grown at 30 C, and treated as above. For drop tests involving the PFD5 vector, cells were plated on –LEU plates. For all other drop tests cells were plated on YPD. Plates were incubated at indicated temperatures for drop tests.

METHOD DETAILS Cloning

CCT1–8 apical domains: Each apical domain was cloned into the super folder GFP between Beta sheets 10 and 11 of the beta-barrel structure with a glycine serine linker added to provide some flexibility. Constructs were cloned into pST39( Tan, 2001 ) using XbaI and BamHI restriction sites for Api domains 1,2,3,4,5,7 and 8, and XbaI and HindIII for Api domain 6. For the yeast apical domains, pRS vectors containing genomic CCTX were used as template. Mutations were made by overlapping PCR with primers containing the desired mutations. Fastbac Dual hTRiC construct cloning: Individual TRiC subunits were cloned into the pFastbac dual vector (ThermoFisher Scientific) using SalI/NotI for CCT1,2,6 and 7 and XhoI/KpnI for CCT3,4,5 and 8. The dual vectors consisted of CCT1,8, CCT2,4, CCT3,6 and CCT5,7 with CCT2, CCT5, CCT6 and CCT8 being cloned initially followed by CCT4, CCT7, CCT3 and CCT1 respectively. For GFP tagged vectors, BspEI sites were cloned into flexible loops using overlapping PCR. GFP was inserted into BspEI sites using standard techniques. Fastbac Dual hPFD construct cloning: Individual Pfd subunits were cloned into the pFastbac dual vector (ThermoFisher Scientific) using SalI/NotI for Pfd1,4 and 5, and XhoI/KpnI for Pfd2,3, and 6. The dual vectors consisted of Pfd1,2, Pfd5,3 and Pfd4,6 with Pfd1, Pfd4, and Pfd5 being cloned initially followed by Pfd2, Pfd3 and Pfd6 respectively. Yeast GFP and CBP vectors: BspEI sites were cloned into flexible loops of CCT subunits using site directed mutagenesis in pAB vectors. Either GFP or CBP was cloned into BspEI sites using standard cloning techniques. Protein Expression and Purification hTRiC: We used the Bac to Bac baculovirus expression system for recombinant protein production (Invitrogren). P3 virus for human TRiC subunits was prepared as described in the protocol. 1 l of High-Five insect cells (Invitrogen) at a concentration of ~3X10 6 cells/ml were co- infected with 5 mls of each of the viruses. and cells were harvested 2 to 3 days after infection by centrifugation. Cells were resuspended in 100 mls lysis buffer (100 mM NaCl, 100 mM HEPES pH 7.4, complete protease inhibitors EDTA free (Roche), 1 mM DTT, PMSF, Benzonase 5 U/ml (Sigma-Aldrich)) and lysed using an emusiflex (Avestin). Lysate was clarified by ultracentrifugation (50,000XG, 1 h). Cleared lysate was passed over a FFQ column equilibrated with MQA buffer (50 mM NaCl, 20 mM HEPES pH 7.4, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 10% glycerol), and then eluted with 40% MQB buffer (1 M NaCl, 20 mM HEPES pH 7.4, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 10% glycerol). TRiC eluted from FFQ column was passed over a Heparin column equilibrated with 20% MQB buffer and washed until UV returned to baseline. TRiC was eluted with a 200 ml gradient from 20% MQB to 100% MQB, and fractions containing TRiC identified by SDS-PAGE. TRiC containing fractions were concentrated down using an Amicon Ultra 100 kDa MWCO centrifugal filter (Millipore) to a volume of ~ 2 mls. The concentrated TRiC was further purified by size exclusion chromatography over a Superdex200 26/60 (GE Healthcare Life Sciences) equilibrated with column buffer (100 mM NaCl, 50 mM HEPES pH 7.4). Eluted TRiC was identified by SDS- PAGE and purified by Talon resin (Clontech). TRiC eluted from the talon resin was purified by anion exchange chromatography using a MonoQ 10/100 column equilibrated with MQA and eluted by a 150 ml gradient to 100% MQB. Fractions containing TRiC were identified by SDS- PAGE, concentrated to ~ 1 ml, aliquoted and snap frozen. Typical yields were greater than 10 mg/L and TRiC assayed by mass spec is ~ 95% pure. yTRiC: Approximately 6 L of yeast at an OD ~2 expressing CCT2 with a CBP tag was harvested by centrifugation. Cells were resuspended in equal volume lysis buffer (200 mM NaCl, 100 mM HEPES pH 7.4, 5 mM CaCl2, 20% glycerol, complete protease inhibitors (Roche), benzonase 5U/ml, PMSF). Cells were lysed using a MM301 mixer mill grinder MM301 (Retsch). Lysate was cleared by centrifugation 20,000Xg for 30 min. Approximately 9 mls of calmodulin resin was equilibrated in wash buffer A (200 mM NaCl, 50 mM HEPES pH 7.4, 5 mM CaCl2, 20% glycerol) and added to the cleared lysate. Binding was carried out at 4 C on a mutator for 2.5 h. Resin/lysate was loaded onto a gravity column and washed with 20 mls buffer A, 40 mls buffer B (500 mM NaCl, 50 mM HEPES pH 7.4, 5 mM CaCl2, 20% glycerol), and 20 mls buffer A. TRiC was eluted from the calmodulin resin with elution buffer (100 mM NaCl, 50 mM HEPES pH 7.4, 10 mM EDTA, 20% glycerol). Eluted protein was further purified by binding to two 5 ml HiTrap Heparin columns (GE Healthcare) equilibrated with MQA buffer. Heparin columns were washed with 20% MQB, and then eluted with a 60 ml gradient to 100% MQB. ATP to 1 mM was added to eluted TRiC, and TRiC was concentrated down to ~ 600 μl using an Amicon Ultra concentrator 10,000 MWCO. Concentrated protein was run over a Superose 6 gel filtration column equilibrated with MQA. Fractions containing TRiC were concentrated and snap frozen in liquid nitrogen. hPFD: 1 L of High five insect cells (Invitrogen) at a concentration of ~3X10^6 cells/ml are infected with 8 mls of each of the viruses. and cells are harvested 2 to 3 days after infection by centrifugation. Cells are resuspended in 100 mls lysis buffer (100 mM NaCl, 100 mM HEPES pH 7.4, complete protease inhibitors EDTA free (Roche), PMSF, Benzonase 5 U/ml (Sigma- Aldrich), ~ 10 mM imidazole), and lysed using an emusiflex (Avestin). Lysate is clarified by ultracentrifugation (50,000*G, 1 hr). Lysate is cleared at 50,000 G for 30 min. Cleared lysate was run over a gravity column containing 10 mls of Nickel resin equilibrated with buffer A (50 mM HEPES pH 7.4, 100 mM NaCl). Resin was washed with buffer A, buffer A with an additional 400 mM NaCl, and buffer A again. PFD was eluted with buffer A containing 300 mM imidazole. Eluted protein was further purified on an MonoQ 10/100 anion exchange equilibrated with buffer A + 1 mM DTT. PFD was eluted from the MonoQ by a gradient to 100% buffer B (1 M NaCl, 50 mM HEPES pH 7.4, 1 mM DTT). Eluted PFD was concentrated down to ~ 1 ml using Amicon Ultra 30 KD MWCO and run over an SDX200 26/600 gel filtration column (GE Healthcare) equilibrated with buffer A + 1 mM DTT. Fractions containing PFD were concentrated using an Amicon Ultra 30 KD MWCO and 50% glycerol was added to a final concentration of 10%. Alexa647-hPFD: Alexa647 maleimide (Life Technologies) was used to label purified hPFD. 117 μM hPFD was mixed with freshly dissolved Alexa647 dye at 1.17 mM (10 molar excess), dye was reacted for 1 hr at room temperature, and quenched by addition of DTT. Labeled complex was purified by size exclusion chromotagraphy using an SDX200 10/300 (GE life sciences). Final concentrations were determined using extinction coeffiicient, labeling efficiency was 0.59 dyes/complex. yPFD: A polycistronic vector for co-expression of PFD subunits was transformed into BL21 Rosetta2 pLysS cells and grown to ~ 0.3 OD and induced O/N at 23 °C. Cells were pelleted, washed with PBS containing PMSF, and resuspended in lysis buffer (100 mM NaCl, 100 mM HEPES pH 7.4, complete protease inhibitors EDTA free (Roche), PMSF, Benzonase 5 U/ml (Sigma-Aldrich), ~ 10 mM imidazole). Cells were lysed using an emulsiflex and lysate cleared at 20,000 G for 30 min. ScPFD was purified from lysate as described for HsPFD. Apical domains: Apical domains were purified as described in ( Joachimiak et al., 2014 ). Briefly, each Apical domain construct was transformed into BL21 Rosetta2 pLysS cells grown to ~ 0.3 OD and induced O/N at 23 °C. Cells were pelleted, washed with PBS containing PMSF, and resuspended in lysis buffer. Cells were lysed using an emusilflex, and lysate cleared at 20,000XG for 30 min. Cleared lysate was passed over Talon affinity resin (Clonetech), washed with column buffer + Triton X 100, column buffer + 500 mM NaCl, and column buffer + 1 mM ATP. Protein was eluted with column buffer + 300 mM imidazole and concentrated down to ~ 2 mls. Concentrated protein was run over an SDX200 26/60 size exclusion chromatography column equilibrated with column buffer + 1 mM DTT, the peak fraction was collected, concentrated and 50% glycerol was added to 10%. Protein was than aliquoted and snap frozen for future use.

Crosslinking-Mass Spectrometry

For hPFD with hTRiC samples, TRiC was brought to 1 μM, and Pfd to 2 μM, human PFD alone was prepared at 10 μM. For yPFD with yTRiC samples, TRiC was brought to 1 μM, and Pfd to 10 μM, and yPFD alone was prepared at 10 μM. All samples were prepared in 50 mM HEPES pH 7.4, 50 mM NaCl and 1 mM DTT. Fresh DSS crosslinker dissolved in DMSO was added to a final concentration of 1 mM, and samples were crosslinked for 2 h at room temperature for the human complexes and 37 °C for 30 min for the yeast complexes. Crosslinking was quenched by incubation for 30 minutes after addition of 1 M Tris pH 7.4 to a final concentration of 100 mM for human complexes, and addition of 1 M Ammonium Bicarbonate to 50 mM for yeast complexes. Crosslinked samples were processed as described previously ( Leitner et al., 2014 ). In brief, processing steps included reduction and alkylation of cysteine residues with tris(2- carboxyethyl)phosphine and iodoacetamide, respectively, sequential digestion with endoprotease Lys-C (Wako) and trypsin (Promega), clean-up using solid-phase extraction (Waters Sep-Pak tC18 cartridges) and fractionation of the purified digests by size exclusion chromatography (SEC; GE Superdex Peptide PC 3.2/300). SEC fractions were analyzed by liquid chromatography-tandem mass spectrometry on a Thermo Easy-nLC 1000 system coupled to a Thermo Orbitrap Elite system as described in detail elsewhere ( Greber et al., 2014 ). MS data analysis was performed using xQuest/xProphet ( Walzthoeni et al., 2012 ) using databases consisting of the TRiC and PFD sequences (including tags) of the respective complexes and contaminant proteins, if applicable. Decoy databases were generated as described in ( Walzthoeni et al., 2012 ), and the results were filtered to a false discovery rate of 5%. PFD–TRiC affinity Human PFD (8 μM) and Human TRiC (0.5 μM) in ATPase buffer (30 mM Tris pH 7.4, 120 mM KCl, 5 mM MgCl2 and 1 mM dithiothreitol added fresh) were serial diluted and mixed 1:1 with TRiC yielding a final concentration of 0.25 μM TRiC and 0.063 to 4 μM PFD. Binding was allowed to equilibrate for approximately 30 min at room temperature and samples were run out on clear Native PAGE (either 4–12% or 4–16%). Protein was transferred to nitrocellulose and probed with Rabbit serum SU-230 (1:2000) developed against recombinant human PFD (Cocalico Biologicals). For the lower concentrations of PFD, the signal for free and TRiC-bound PFD was summed and the signal/uM calculated for each blot. This value was then used to calculate the amount of PFD bound to TRiC at each concentration. The amount of free PFD was calculated by subtracting the amount bound from the starting concentration. Binding was done in triplicate, and error bars represent SEM. PFD–TRiC anisotropy TRiC (400 nM) was prepared in 1XATPase buffer leaving room (30% volume) for nucleotide addition (closed)/H 2 O (open) 1/10 volume 10 mM ATP, 10 mM AlNO 3 , and 60 mM NaF were each added in quick succession to close TRiC for 30 min at 37 °C.

Human Alexa647 labeled

PFD was prepared at 50 nM in 1XATPase buffer, and mixed 1:1 with open TRiC, closed TRiC, and 1XATPase for final concentrations of 200 nM TRiC and 25 nM PFD, and allowed to equilibrate at room temperature. Anisotropy was measured using a CLARIOstar plate reader from BMG Tech with the PFD alone sample normalized to 100 prior to readings.

Actin folding assays

Endpoint assays: TRiC variants (bovine TRiC, WT hTRiC, C1-GFP hTRiC, and C6-GFP hTRiC) were diluted to 0.25 μM in ATPase buffer. [ 35 S]actin, prepared as described,in ( Meyer et al., 2003 ; Thulasiraman et al., 2000 ) in 6 M Guanidnium-HCl, 100 mM HEPES pH 7.4 was rapidly diluted 1:100 with TRiC variants or with ATPase buffer alone. Actin was allowed to bind for 30 min on ice. To remove aggregated Actin, samples were spun 10 min at 19000xg. Supernatants were moved and layered onto either water or ATP to a final concentration of 1 mM. Samples were incubated 1 h at 37 °C. DNAseI was added to each sample (5 μg) to bind and compact folded Actin bands for native gel analyses. Samples were incubated 10 min on ice to allow binding, then large aggregates removed by centrifugation for 10 min at 19,000XG. Samples were run out on clear native gels 4–16%, gels were coomassie stained, dried and exposed to a phosphorscreen. Phosphorscreens were imaged using a Typhoon scanner. Kinetic analyses: 0.13 μM [ 35 S]actin in 6 M Guanidnium-HCl, 100 mM HEPES pH 7.4 was rapidly diluted 1:100 with TRiC at a concentration of 0.25 μM with or without PFD at 2.5 μM in ATPase buffer with fresh 1 mM DTT. Samples were incubated 30 min on ice to allow [ 35 S]actin binding. To remove aggregated actin, samples were spun 10 min at 19000 G. Supernatents were moved into fresh tubes, and the TRiC alone sample split into (2) aliquots. When indicated, the TRiC-[ 35 S]actin was further purified by ion-exchange chromatography on a MonoQ column as described ( Frydman and Hartl, 1996 ; Reissmann et al., 2007 ). PFD was added to one aliquot of TRiC alone to a final concentration of 2.5 μM (equal volumes of ATPase buffer was added to the other samples). ATP was added to 1 mM and the folding reactions initiated by moving samples into a 37 °C water bath. Samples were taken at 0,2,4,8,16,32,48,64 and 128 min and the reactions stopped by addition of 0.2 Units of apyrase and moving samples to ice (the apyrase should deplete all ATP is less than 1 minute, and TRiC ATPase activity is negligible at 4 °C). DNAseI was added to each time point (5 μg) to bind folded Actin and compact bands in native gels. Samples were incubated 10 min on ice to allow binding, then large aggregates removed by centrifugation for 10 min at 19000xg. Timepoints were run out on clear native gels 4–16%, gels were coomassie stained, dried and exposed to a phosphorscreen. Phosphorscreens were imaged using a Typhoon scanner. The amount of Actin folding was quantified using image quant, and each experiment was normalized to the final three timepoints (48, 64, and 128 min) of TRiC alone folding which was set as 1 arbritrary unit (AU). Experiments were done in triplicate and error bars represent SEM.

Apical domain assays

PFD binding: Apical domains and yPFD/hPFD were each diluted to 2 μM in ATPase buffer. Samples were mixed 1:1 and allowed to bind at room temperature for ~10 min. Samples were run out on a 15% SDS PAGE for a loading control, and on a clear Native PAGE 4–16%. for mobility shift analysis. Protein from native page gels was transferred to nitrocellulose membranes and antibodies raised against recombinant yeast PFD/human PFD at 1:2000 were used to detect proteins Competitive inhibition of PFD binding TRiC: hPFD/yPFD at 2 μM was mixed 1:1 with Apical domain 4 serially diluted from 40 μM to 1.25 μM and allowed to equilibrate ~ 10 min at room temperature. These samples were then mixed 1:1 with 0.5 μM TRiC in ATPase buffer and allowed to equilibrate for 10 min at room temperature. Samples were run out on a 15% SDS PAGE for a loading control, and on a clear Native PAGE 4–16%. for mobility shift analysis. Protein from native page gels was transferred to nitrocellulose membranes and antibodies raised against recombinant yeast PFD/human PFD at 1:2000 were used to detect PFDs.

ATPase measurements

ATPase rates were determined as described in. ( Reissmann et al., 2007 ). Briefly, ATP hydrolysis by 0.25 μM hTRiC or 0.25 μM hTRiC + 2.5 μM hPFD was measured at 37 °C in ATPase buffer, in the presence of 188 to 1500 M [α− 32 P]ATP. After 5 min of preincubation, the reaction was started by mixing 6 l [α− 32 P]ATP solution with 34 l 1.176-fold concentrated reaction mix. At the indicated time points, 2- l samples were taken and transferred onto PEI-cellulose F thin-layer chromatography plastic sheets (EMD Chemicals). The plates were developed in a solvent system containing 1 M LiCl and 0.5 M formic acid in H 2 O, air-dried and exposed to a phosphorimager (Kodak). After the screen was scanned in a Typhoon 9410 imager, the amount of [α− 32 P]ATP was quantified using ImageQuant 5.2, amount of [α− 32 P]/ATP was determined from a standard curve from a dilution set on the thin- layer chromatography sheet. TRiC–PFD CryoEM specimen preparation Purified 20 μM hPFD was diluted 10-fold into MQ buffer containing 1 μM purified hTRiC, resulting in a 2:1 molar ratio of hPFD to hTRiC. The complex mixture was incubated for 30 min in room temperature and Octyl glucoside (OG) was added to a concentration of 0.1 % before grid placement to increase the yield of side views in electron micrographs. The resulting samples were applied to 200-mesh R1.2/1.3 holey-carbon grids (Quantifoil) and vitrified using a Gatan CP3 (Gatan). We loaded grids into a JEM3200FSC (300 KeV) electron microscope with an in- column omega filter (25 eV energy slit). We recorded images at 1.5–3 m underfocus on a K2 Summit direct electron detector (Gatan) in super-resolution mode at nominal 20K magnification, corresponding to a sampling of 1.7 Å/pixel (super-resolution sampling: 0.85 Å/pixel).

Image processing

We initially binned each movie stack by two and then corrected for drift and radiation damage using DE_process_frames.py (Direct Election Ltd). The first three frames were ignored during movie processing. We used EMAN2 to automatically select particle images ( Bell et al., 2016 ). Contrast transfer-function (CTF) parameters were estimated internally based on the boxed particles (e2ctf.py). We then performed two-dimensional reference-free averaging with ~2,000 particle images using the default parameters in EMAN2 and generated an initial three- dimensional model based on the two-dimensional class averages, with no symmetry imposed. We converted a set of CTF-corrected particles to a RELION-compatible format using E2refinetorelion3d.py. We performed all further refinements using RELION 1.4 ( Scheres, 2012 ), starting with maps that were low-pass filtered to 50 Å from the initial model generated by EMAN2. At first, we perform three-dimensional classification (K=3) with C1 symmetry to exclude non-TRiC-like particles. Then, we subsequently refined the remaining particles using the “auto_refine” command, which resulted in a 6.3 Å map based on a gold-standard Fourier shell correlation (FSC) at 0.143. In this step we were identified noisy density around TRiC’s apical domains, which stems from conformational heterogeneity of PFD. Focused classification and localized reconstruction of individual subunits The overall workflow using RELION 1.4 for the focused classification ( Bai et al., 2015 ) and localized reconstruction ( Ilca et al., 2015 ; Roh et al., 2017 ) of TRiC–PFD is described in Figure S4 . We split the 6.5 Å map into two TRiC ring maps by masking out one ring at a time ( Figure S4A , a – b ). We then applied a soft mask around each map of TRiC ring and used the resulting masked map for subtraction of the signal from each raw particle image. This process generated two sub-particles from each raw particle image, which corresponds to either cis or trans ring of TRiC ( Figure S4A , c ). Using these sub-particles, we performed three-dimensional classification (K=5) with C1 symmetry and excluded 15% of particles with sub-optimally resolved features ( Figure S4A , d ). Then, we subjected the remaining 85% sub-particles for auto-refinement with a mask for single TRiC ring to determine the best orientation for each single TRiC ring ( Figure S4A , e ). This resulted in a 6.4 Å map containing only 8 subunits of TRiC. We next performed a focused 3D classification (K=8) on the putative PFD binding area with a spherical mask without orientation search ( Figure S4A , f ). This operation resulted in three groups of unbound TRiC and five groups with extra density around apical domains of TRiC ( Figure S4A , g ). Three unbound groups were combined as an unbound class. Therefore, one unbound class (Class1) and each of five bound classes (Class2–6) was respectively reconstructed with corresponding original two ring particles, while keeping the particle orientation from the single ring refinement ( Figure S4A , h ). In final, each map was sharpened using auto B-factor in RELION post processing step. Model building We first fit the crystal structure of yeast TRiC (4V94) ( Leitner et al., 2012 ) using Chimera ( Pettersen et al., 2004 ) in closed conformation to the CryoEM map of the open conformation; the model matches the map only in the equatorial domain. Then, molecular dynamics flexible fitting (MDFF) ( Trabuco et al., 2008 ) was used on the fitted model and map. This method applies forces at atom position in the direction of the density gradient (density gradient weight of 0.3), while performing a molecular dynamics (MD) simulation at a given temperature (300K). After ~10 5 MD steps, the model matched the map much better, especially in the apical domains. This procedure thus opened the closed conformation of yeast TRiC to the open conformation of TRiC seen in the 6.3Å cryoEM map. We then threaded the sequence of each subunit in the Human TRiC through the open state of the yeast TRiC model based on sequence similarity. We performed a final round of model optimization with Phenix.real_space_refine for global minimization ( Afonine et al., 2012 ). Since at this resolution the density does not constrain the structure tightly, we used probabilistic modeling to estimate the uncertainty at atomic positions using ProMod ( Pintilie et al., 2016 ). Uncertainty values are stored in the Bfactor column of the PDB/model. For Human PFD model, two copies of Archeal PFD crystal structures (2ZDI) ( Sahlan et al., 2010 ) fitted into the extra density in Class 6, considering the right arrangement from XL/MS results ( Figure 5 ). The PFD structure fitted well around the helical tips of TRiC, and contacts are clearly seen. We then threaded the sequence of each subunit of the Human PFD through the Archeal PFD model based on sequence similarity. We rigid-body fitted this human PFD model into each extra density in Class 2–6 and then combined with TRiC model, which generated previously. The tips of CCT were manually adjusted to the density to avoid overlapping with PFD. Each combined model was flexible fitted again to respective map by MDFF ( Trabuco et al., 2008 ). Each resulting model was optimized into each map using Phenix.real_space_refine for global minimization ( Afonine et al., 2012 ), and again probabilistic modeling was used to measure uncertainties at atomic positions ( Afonine et al., 2012 ). yTRiC pulldowns with CNBr-yPFD One liter of WT or EEE yeast strains were grown to ~ 0.5 OD600 at 37 °C (permissive temperature for EEE mutant) and harvested by pelleting at 4000XG for 10 min. Pellets were resuspended in PBS with PMSF, pelleted for 5 min at 4000xg, and then resuspended in equal volume lysis buffer (100 mM HEPES-KOH pH 7.4, 50 mM KCl, 5 mM MgCl 2 , 2 mM DTT, 20% glycerol, 0.25% NP40 and complete protease inhibitors Roche). Pellet was then frozen by slowly dripping into liquid nitrogen for lysis. Cells were lysed using a cryogrinder for 1 minute at 20 Hertz. Lysate was cleared by centrifugation at 19000*G for 10 min at 4 °C, and supernatent was isolated. Protein concentration for each cleared lysate was determined using a bradford assay, and all cultures were diluted to 2 mg/ml using lysis buffer. 1 ml of cleared lysate was mixed with 200 μl of CNBr-yPFD resin and incubated for 2 hr at 4 °C. Lysate/CNBr-yPFD mixtures were split into three tubes of 300 μl. Resin was pelleted by centrifugation 22*G for 1 minute and unbound protein/supernatent was removed. Resin was washed 3 times by pelleting and resuspension with 500 μl of column buffer (100 mM HEPES-KOH pH 7.4, 50 mM KCl, 5 mM MgCl2, 2 mM DTT, 20% glycerol, 0.25% NP40) supplemented with an additional 50 mM, 150 mM or 350 mM KCl for final concentrations of 100, 200, and 400 mM KCl respectively. Resin was moved to a fresh tube after final wash, and bound protein was eluted with 1X Lamealli buffer.

TRiC closure assay

Yeast lysates were prepared as in the yTRiC pulldowns. Cleared lysates were diluted to 2 mg/ml to a final concentration of 30 mM HEPES-KOH pH 7.4, 100 mM KCl, 5 mM MgCl 2 , 20% glycerol and 1 mM DTT. Samples were prepared leaving 30% volume for addition of ATP, AlNO 3 , and NaF. 35 μl of each sample was brought to 30 °C, and either 15 μl of H 2 O (Open) or 5 μl of 10 mM ATP, 5 μl of 10 mM AlNO 3 , and 5 μl of 60 mM NaF in quick succession (closed) was added. Samples were incubated for 15 min to allow TRiC to fully close. TRiC was separated using 4% Native-PAGE. Protein from native page gels was transferred to nitrocellulose membranes and an antibody raised against yTRiC apical domains (Bio277) was used to detect TRiC.

Drop tests

Yeast strains were grown to log phase (~ 0.6 OD600) at 37 °C. Cells were diluted back to an OD600 of 0.08, and then serially diluted 1:10 three times. Cells were plated onto YPD and incubated at 37 °C and 30 °C for 48 h, and 23 °C for 72 h and imaged. Proteostat/aggregate staining Yeast strains were grown to approximately 0.2 OD600 at 37 C and then shifted to either 37 °C or 30 °C. Cells were allowed at least (2) doubling times (3 h) at the respective temperatures. Cells were fixed by addition of paraformaldehyde to a final concentration of 4% (1.5 mls culture, 0.5 mls 16% paraformaldehyde) for 15 min. Cells were pelleted and washed 2 times with PBS to remove paraformaldehyde. Cells were resuspended in proteostat buffer (PBS with 5% BSA and 0.1% Tween20) for 30 min. Proteostat was prepared by 1:2000 dilution in the buffer, and each pellet was resuspended with 250 μl and incubated for 30 min at 4 °C. Cells were washed 3X using proteostat buffer, and 2X with PBS. Cells were adhered to concanavalin A coverslips using Prolong antifade with DAPI and imaged with a Ziess LSM700 with the 100X oil immersion lens. Aggregate purifications Yeast strains were grown overnight at 30 °C and diluted in the morning into 100 mls of YPD grown to ~ 0.6 OD after at least two doublings and harvested by pelleting cultures at 3000 G. Pellets were resuspended in 1 ml PBS with PMSF, pelleted and snap frozen in liquid N 2 and stored at – 80 C until further processing. Cell lysates were prepared by resuspending each pellet in 1 ml of lysis buffer (50 mM Na-phosphate, pH 6.8, 50 mM Potassium Acetate, 10 mM DTT, 1 mM EDTA, 1 mM PMSF, complete protease inhibitor cocktail (Roche), 3 mg/ml zymolyase and 1.25 U/ml benzonase) and incubation at room temperature for 20 min. Chilled samples were treated with tip sonication (3 s on, 7 s off, 25% power) for 1 min using a Qsonica Q500 sonicator. Lysates were cleared at 200*G for 20 min, and concentrations determined by bradford assay. Each lysate was adjusted to 1 mg/ml with lysis buffer. Aggregates were pelleted at 16000*G for 20 min, resuspended in 1 ml of wash buffer (50 mM Na-phosphate, pH 6.8, 50 mM Potassium Acetate, 10 mM DTT, 1 mM EDTA, 1 mM PMSF, 2% NP40) and tip sonicated (3 s on, 7 s off, 25% power), and pelleted again at 16000*G for 20 min. Wash step was repeated, and pellets were resuspended in SDS sample buffer and analyzed by SDS-PAGE followed by coomassie staining/western blot analysis.

Supplementary Material 1 Figure S1. A recombinant system for human TRiC, Related to Figure 1 (A) Location of GFP insertion for CCT1 and CCT6 (B) Cell Growth test for WT vs GFP- insertions in indicated subunits at indicated temperatures. (C) Representative micrograph and Gold Standard FSC at 0.143 for each TRiC-CCT1-GFP and TRiC- CCT6-GFP. Local resolution maps (ResMap) (D) and overall architecture (E) of TRiC-CCT1-GFP and TRiC-CCT6-GFP showing psuedo-two-fold symmetry and the location of a protruding subunit (CCT1 indicated by red arrows). (F) Autoradiogram from native PAGE of denatured [ 35 S]actin folding assay and quantification plot of folded actin vs time , comparing actin folding kinetics of bovine TRiC and hTRiC. 6 Figure S6. TRiC–PFD interaction site mediated by an electrostatic pivot point on CCT4, Related to Figure 6 . (A) Purified yApical domains stabilized in a split-GFP construct were mixed with yPFD and analyzed by Native-PAGE followed by immunoblot for yPFD. Only the apical domain of yCCT4 caused a dramatic shift in PFD mobility. (B) The apical domain of yCCT4 was mixed with yPFD at varying concentrations to look for competitive inhibition of binding to TRiC and analyzed by Native-PAGE followed by immunoblot, binding is inhibited at near equimolar concentrations to PFD, and binding inhibition correlates with a shift in PFD mobility. (C) Electrostatic surface display (unwrapped TRiC and PFD) of TRiC and PFD. Circled numbers indicate the location of respective PFD subunit on TRiC. (D) Cartoon showing that PFD is recruited to TRiC through the electrostatic interaction to align the substrate binding chambers of these two complexes. 7 Figure S7. Loss of TRiC–PFD electrostatic interaction results in proteostasis defects, Related to Figure 7 (A) Outline of aggregation purification protocol. (B) Total (T), soluble (S) and aggregate pellet (P) fractions from indicated yeast strains, separated by SDS-PAGE and stained with coomassie, red dots added to emphasize multiple species present in the TRiC CCT4-EEE strain. (C) Immunoblot of above fractions for TRiC and alpha-Tubulin, note loss of alpha-Tubulin in CCT4- EEE strain. (D) Plot of the functional enrichment of proteins found in CCT4-EEE aggregates separated by GO categories, enriched classes are consistent with general proteostasis disruption. 8 Movie S1. Pivoting trajectory of PFD on TRiC (Density maps) Related to Figure 4 . Conformational trajectory between the Classes (2→3→4→5→6), showing a pivoting mechanism of PFD on TRiC. 9 Movie S2. Pivoting trajectory of PFD on TRiC (Model ribbon diagram) Related to Figure 4 . Conformational trajectory between the Classes (2→3→4→5→6), showing a pivoting mechanism of PFD on TRiC. 10 Table S1. CryoEM Data Collection, 3-D Reconstruction, and Model Refinement, Related to Figures 1 , 4 , 5 , S1 and S4 . Summary table includes parameters for CryoEM map reconstruction and model building. 11 Table S2: Crosslinking/Mass spectrometry of TRiC, PFD, and TRiC–PFD samples, Related to Figures 1 , 2 , 5 , S2 , and S5 . Table includes identified intra and inter crosslinks for hTRiC, hTRiC CCT1-GFP, hTRiC with hPFD, hTRiC CCT1-GFP with hPFD, hPFD alone, yPFD alone, and yPFD with yTRiC. See table legend for description of column headers. 12 Table S3: Mass spectrometry analysis of aggregates from wild type and CCT4 EEE yeast strains, Related to Figures 7 , S7 . Aggregates were run out on a short SDS-PAG, and extracted for analysis. Table includes Log2 MS values for WT and EEE aggregates, and Fisher’s test for functional annotation enrichment of proteins detected in EEE sample, relative to entire proteome. 13 Table S4: Oligonucleotides used in this study Related to STARS methods. 2 Figure S2. PFD XL maps and TRiC–PFD binding, Related to Figure 2 Top and side views of yPFD (A), and hPFD (B) with XLs obtained from DSS crosslinking and MS mapped onto homology models refined using density from hTRiC-hPFD structure. (C) Histograms of Cα-Cα distance of XLs mapped onto the homology model of yPFD for the two subunit arrangements that satisfied the most constraints. (D) SDS-PAGE of hPFD variants with PFD1 or PFD4 tagged with GFP, migration shift allows identification of the respective subunit. (E) Full data points of binding data from hTRiC-hPFD binding experiments show a second TRiC–PFD binding at high concentrations. (F) Raw fluorescence polarization table of Alexa647- hPFD in the absence or presence of TRiC in the open (no nucelotide), and closed (ATP–AlF 4 ) conformation, polarization in the absence of TRiC is normalized to 100. 3 Figure S3. Actin transfer between hPFD and hTRiC ATPase rates, Related to Figure 3 (A) [ 35 S]actin transfer from PFD to TRiC, and from TRiC to PFD in the absence of ATP, and folding after ATP addition allows identification of all Actin bands from autoradiography experiments. (B) Plots of ATP hyrdolyzed/TRiC vs time for TRiC alone, and TRiC+PFD at 0.75 mM [ATP]. (C) ATPase rates for TRiC and TRiC+PFD at [ATP] from 0.188 to 1.5 mM. Measurements done in triplicate, error bars represent SEM, and ATPase rates are determined from linear fits to the data. 4 Figure S4. CryoEM map reconstructions of TRiC–PFD complex, Related to Figure 4 (A) data analysis workflow using local reconstruction and focused classification strategy. (B) Reference-free 2D class averages after 3D classification. Yellow circled area indicates the locations of PFD binding in each class. (C) Euler angle distribution of the particles that were included in the 3D reconstruction indicating overall isotropic coverage of orientations. Inside of sphere, blue map (TRiC/CCT) is shown at default contour (0.1). Green (GFP) and red (PFD) densities are shown as a difference map between the original map and lowpass filtered map (20Å). (D) Gold standard FSC for TRiC–PFD maps of all particle, half-ring and classes. (E) Local resolution map for each class shows structural heterogeneity around apical tips and PFD bound. 5 Figure S5. TRiC and PFD interact in a subunit specific manner. Related to Figure 5 (A) Schematics of the two possible mirror image arrangements of yPfd on yTRiC. Obtained XLs were mapped onto TRiC–PFD models from CryoEM, histograms of Cα-Cα distances establish the correct arrangement of the PFD subunits. (B) TRiC contact points for PFD identified by CryoEM reconstructions of the different classes highlighted in yellow. (C) Surface display and (D) histogram to show Cα RMSD analysis among six models from the class1–6. (E) Overall and zoom-in view to the contact of CCT4 to PFD (4/6) in Class 6 (F) Top and side view of TRiC- PFD complex as a space filling model (Class 6) showing ~40Å fenestration between anchoring subunit (CCT4) and protruding subunit (CCT1).