Plasmid construction and protein purification
For expression and purification of tagged Sup35NM proteins in Escherichia coli, we followed the previous protocols 31. Briefly, Sup35NM with a 7xHis or 7xHis-Cys tag were overexpressed in E. coli Rosetta (DE3) cells (Nonagen) in LB media and purified by Ni-NTA affinity chromatography (GE healthcare) and anion-exchange chromatography under the denatured condition 31,34,44. The buffer exchange to acetonitrile/water was performed using HPLC (Hitachi). The sample was lyophilized and stored at -20oC until use.
cDNAs of Hsp104, Sis1 and their mutants were cloned into pET28a vector including N-terminally His-MBP-tag 45. A plasmid of Hsp104-SNAP was generated by addition of a SNAP-tag to the C-terminus in the expression vector. cDNA of Sis1 with a C-terminal cysteine was introduced into the pET28a plasmid. Hsp104 and Sis1 were overexpressed in E. coli Rosetta (DE3) in LB media and purified by Ni-NTA affinity chromatography (GE Healthcare), as previously described with modifications 46. Briefly, the eluent from Ni-NTA resin was purified by amylose-resin affinity chromatography (BioLabs). After digestion of a His-MBP-tag by the TEV enzyme, Hsp104 and Sis1 was purified by anion-exchange chromatography using HiTrap Q 6 ml column (GE Healthcare) with the gradient system. To remove any remaining His-tagged proteins, the eluent was subjected to the 2nd Ni-NTA gravity flow purification and the flow-through was obtained. Only pure fractions (>95%) were pooled, and concentration and buffer exchange were performed. Expression and purification of Ssa1 with a with a C-terminal 7xHis tag was followed by the previous report 47,48. Briefly, Ssa1 was purified by Ni-NTA affinity (GE Healthcare) and HiTrap Q (GE Healthcare) columns with the gradient system. Only pure fractions (>95%) were pooled, and concentration and buffer exchange were performed. Purity of all the proteins were rigorously checked throughput the purification procedures by Coomassie Brilliant Blue staining and the absorbance at 280 nm. Quality control of Hsp104 hexamer was performed by AUC as described later. All of the proteins were aliquoted into a small volume, flash frozen by liquid nitrogen and stored at -80°C. Typically, we used up the proteins within 1-2 months to prevent a decrease of their activities.
As a plasmid for expressing TEV, pRK793 was purchased from Addgene (RRID:Addgene_8827) 49. A TEV protease with a 7xHis tag was overexpressed in E. coli Rosetta (DE3) in LB media and was purified by Ni-NTA, as previously reported 45. The pure fractions of the eluent from Ni-NTA affinity chromatography was used for digestion of the tagged proteins. A plasmid for expressing ClpP was kindly provided by Dr. Axel Mogk (Universität Heidelberg). ClpP was overexpressed as a C-terminally 6xHis tagged protein in E. coli Rosetta (DE3) in LB media and purified by Ni-NTA affinity chromatography (GE Healthcare), as previously described 50.
Fluorescence and chemical labeling of proteins
For fluorescence labeling of Sup35NM, Sup35NM with a 7xHis-tag-Cys tag was mixed with a 10-fold excess of STELLA650-maleimide (GORYO Chemical) or Alexa-488-maleimide (ThermoFisher Scientific). An excess amount of fluorescence dye was removed by passing the protein through a Bio-Gel P-6 Gel (Biorad #154130). Labeling efficiencies were spectrophotometrically determined (Hitachi U-3900), using the absorbances at 646 nm (extinction coefficient: 110,000 M-1 cm-1) for STELLA-650-maleimide, at 493 nm (extinction coefficient: 72,000 M-1 cm-1) for Alexa-488-maleimide, combined with the absorbance at 280 nm (extinction coefficient: 29,800 M-1 cm-1) for Sup35NM protein. For biotin labeling of Sup35NM, Sup35NM including an Avi-tag was overexpressed in E. coli Rosetta (DE3) that contains a pBirA plasmid in LB media. After optical density of the culture reaches ~0.6, d-biotin (Nacalai, the final concentration of 50 mM) and IPTG (the final concentration of 0.6 mM) were added for biotin labeling. Biotinylated Sup35NM was purified as described above, and the incorporation of biotin and elimination of non-biotinylated Sup35NM were confirmed by anti-Biotin antibody (Jackson Immuno Research Laboratories, Inc.).
For fluorescence labeling of a SNAP tag, Hsp104 with a SNAP-tag was mixed with a 2-fold excess of SNAP-Surface549 (Biolabs) together with 1 mM DTT for 1 hour in dark. An excess dye was removed by passing of the protein through a P6 column as above. Labeling efficiencies were spectrophotometrically determined using the absorbance at 558 nm (extinction coefficient: 140,300 M-1 cm-1), combined with the absorbance at 280 nm (extinction coefficient: 49,070 M-1 cm-1) for Hsp104-SNAP protein. Ssa1 was labeled on the cysteine residue by a 5-fold excess of Alexa-488-maleimide (ThermoFisher Scientific) with 1 mM TCEP. The labeling efficiency of Ssa1 was determined by the spectrophotometer using the absorbance at 493 nm (extinction coefficient: 72,000 M-1 cm-1) for Alexa488, combined with the absorbance at 280 nm (extinction coefficient: 18,490 M-1 cm-1) for Ssa1. Sis1 was labeled on the cysteine residue with a 5-fold excess of Cy3-maleimide (GE healthcare) with 1 mM TCEP for 1 hour in dark. The labeling efficiency of Sis1 was spectrophotometrically determined using the absorbance at 550 nm (extinction coefficient: 150,000 M-1 cm-1) for Cy3, combined with the absorbance at 280 nm (extinction coefficient: 22,330 M-1 cm-1) for Sis1. The efficiency of fluorescent labeling of chaperones were typically in the range of 90–98%.
Analytical Ultracentrifugation (AUC)
The sedimentation velocity experiments of purified Hsp104, Ssa1 and Sis1 was performed using a proteome Lab XL-A analytical ultracentrifuge system with an AN60Ti rotor (Beckman Counter). All chaperone concentrations were approximately 30 mM in AUC Buffer (20 mM Tris (pH 7.5), 75 mM NaCl, 75 mM KCl, 20 mM MgCl2, 2 mM TCEP). All protein concentrations refer to the concentration of monomeric protein. Ultracentrifugation analysis was performed as follows; Hsp104 (Velocity: 40,000 rpm, Number of scans: 280, Scan interval: 2 min, Temperature: 20oC), Ssa1 or Sis1 (Velocity: 40,000 rpm, Number of scans: 140, Scan interval: 6 min, Temperature: 20oC). Acquired data were analyzed with the SEDFIT software (NIH) using the c(S) distribution method 51. and the parameters of the density 1.0018 g cm-3 and viscosity 1.023 cP were determined using the SEDNTERP software (http://www.jphilo.mailway.com/download.htm).
ATPase activity assay
An ATPase activity of 0.25 mM Hsp104 and 1 mM Ssa1 was measured using a commercially available kit (Innova) in the ATPase buffer (40 mM Hepes-KOH pH 7.4, 150 mM KCl, 20 mM MgCl2, 2 mM DTT) including 1 mM ATP. The assay was followed by manufacturer’s protocol and the previous report 52.
Disaggregation assay
The Sc4 or Sc37 amyloid was freshly prepared by mixing 5uM Sup35NM with 5% (mol/mol) of corresponding Sc4 or Sc37 seeds, respectively, as previously reported 16,32. The disaggregation assay of Sc4 or Sc37 amyloid was typically performed in the presence of 1 mM Hsp104, 2 μM Ssa1, 2 μM Sis1 with ATP or an ATP regeneration system (10 mM creatine phosphate and 0.1 mg/ml creatine kinase) in the disaggregation buffer (25 mM Hepes-KOH (pH 7.5), 150 mM potassium acetate, 10 mM magnesium acetate, 2 mM DTT) at 30oC. Hsp104KT (K218T, K620T), an ATPase-deficient mutant of Hsp104, was used as a negative control. The extent of amyloid disaggregation was measured by thioflavin T fluorescence, sedimentation assay, atomic force microscopy (AFM). The thioflavin T (ThT) assay was followed by the previous report 31. ThT fluorescence was measured by SpectraMax M2 (Molecular devices) without shaking and data were collected by the SoftMaxPro software (Molecular devices). For the spin down assay, soluble fraction of Sup35NM (S) were separated by ultracentrifugation (200,000 g, 30 min at 4oC) from the total fraction. The supernatant (S) and total fraction (T) were subjected to the western blotting with a polyclonal anti-Sup35NM antibody 31. Corresponding horseradish peroxidase-conjugated secondary antibodies were used (GE Healthcare), and images were visualized by ImageQuant LAS 4000 mini (GE Healthcare). For morphological imaging of amyloid fibrils by AFM, the samples were applied to mica (Agar Scientific) for 30 s and observed by MultiMode8 Scan Asyst for NanoScope V (Bruker). The AFM images were analyzed by Nanoscope Analysis software (Bruker).
For disaggregation of cell lysates containing in vivo Sc4 prions, [PSI+] Sc4 (74D694, PIN+, MAT a) was cultured until the OD reached ~0.8. Yeast cells were collected by centrifugation (1000 g, 5 min) and lysed in SDD-AGE lysis buffer (30 mM Tris (pH 7.5), 150 mM NaCl, 1% TritonX, 1 mM DTT, 2 mM PMSF, inhibitor cocktail) 53. Cell debris were removed by brief centrifugation (1000 g, 5 min, 4oC). The cell lysate (5 mg/ml) was incubated with 2 μM Hsp104, 4 μM Ssa1, 4 μM Sis1 with ATP and an ATP regeneration system in the disaggregation buffer at 30oC for 2 hours. Sup35 prions and monomers in the lysate were separated by Semi-Denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE), as previously described 35,54, and Sup35 was detected by a rabbit polyclonal anti-Sup35NM antibody 31.
Acceleration assay of amyloid formation
For acceleration of amyloid formation, 5 μM Sup35NM monomer was incubated with 5 μM Hsp104, 5 μM Ssa1 and 5 μM Sis1 with ATP and an ATP regeneration system in the fiber acceleration buffer (20 mM Tris-Cl pH 7.4, 75 mM KCl, 75 mM NaCl, 20 mM MgCl2, 2 mM DTT) at 15oC. The extent of amyloid formation was measured by ThT fluorescence, as described above. After the formation of Sup35NM amyloid, aggregate structures were observed by AFM as described above.
Fluorescence-detected sedimentation velocity AUC
To detect only Sup35NM in the mixture with chaperones by AUC, Sup35NM with a C-terminal cysteine was labeled by Alexa488-maleimide. 20 mM Sup35NM including 20 nM Alexa488-labeled Sup35NM was incubated at 20oC with 20 mM Hsp104, 20 mM Ssa1 and 20 mM Sis in the presence of 5 mM ATPgS or ADP. AUC was performed as follows; velocity: 42000 rpm, number of scans: 600, scan interval: 90 sec, temperature: 20oC 30. The acquired data were analyzed using the SEDFIT software, and parameters of the density of 1.0068 g cm-3 and the viscosity of 1.023 cP were determined using the Sednterp software.
Microscale thermophoresis (MST)
5 mM Sc4 amyloid was sonicated (20%, 14 sec) and diluted to 200 nM by the MST buffer (25 mM Hepes-KOH pH 7.5, 150 mM potassium acetate, 10 mM magnesium acetate, 10 mM DTT, 0.05% tween20). The diluted amyloid solution was mixed with 20 nM (final) fluorescence dye using a Monolith His-Tag labeling Kit RED-tris-NTA 2nd Generation (Nano Temper Technologies) and incubated at room temperature for 30 min. Before the measurements, the efficiency of the His-tag labeling was confirmed by a pre-test. For the binding affinity assay, a serial dilution of Hsp104 trap, an ATPase-deficient mutant of Hsp104, in the MST buffer including 5 mM ATP and the regeneration buffer was prepared in 16 tubes and the same amount of labeled Sc4 amyloid from the above solution (100 nM protein, 10 nM dye) was added to each vial. For evaluation of cooperative binding of Hsp104 with Ssa1 and Sis1 to Sc4 amyloid, 2 mM Ssa1 and Sis1 were included in the labeled Sc4 amyloid solution, and were mixed with a serial dilution of Hsp104. Binding affinity assays were performed using Monolith NT.115 Pico Microscale Thermophoresis (Nano Temper Technologies) and data were collected by the MO. Control software (Nano Temper Technologies). The MST power was set to high and the excitation power used was 20% in each analysis. To determine Kd, the curve fitting of normalized intensity was performed using the MO. Affinity Analysis software (Nano Temper Technologies).
Fluorescence anisotropy
0.5 mM Sup35NM-Alexa488 monomer was incubated with 5 mM Hsp104 WT or Hsp104 trap at 30oC for 5 min in disaggregation buffer. After 5 min incubation, 0.5 mM ATPgS was added to the mixture, following addition of 20 mM ADP. Fluorescence anisotropy was measured using a black 96-well plate (ThermoFisher Scientific) on a SpectraMax iD5 (Molecular Devices, San Jose, CA, USA) and data were collected by the SoftMaxPro software (Molecular devices). The measurement volume was 120 μl. Excitation/emission wavelengths for anisotropy measurements of Sup35NM-Alexa488 was 490/530 nm. Three independent experiments were performed (n = 3).
Total intensity reflection fluorescence (TIRF) microscopy
All coverslips (18 mm×18 mm and 24 mm×45 mm) were cleaned by sonication in 1M KOH for 30 min, in H2O by 3 times for 5 min each, then in methanol for 10 min as described 55. The coverslips were coated by amino silane using the amino silane buffer (2% of N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane (Tokyo Chemical Industry), 135 mM Acetic acid (Wako Pure Chemical cooperation), 4% H2O in methanol (Wako Pure Chemical Industries, Ltd.)) at room temperature for 3 hours as described 55. The coverslips were layered with 0.1 M NaHCO3 (Wako Pure Chemical Industries, Ltd.) including 200 mg ml-1 mPEG-succinimidyl valerate (Laysan Bio) and 2 mg /ml of Biotin-PEG-succinimidyl valeric acid ester (Laysan Bio) and incubated at room temperature for 3 hours 55. After the PEG coating, all coverslips were washed by H2O and dried by N2 blow and stored in methanol. All PEG-coated coverslips were used up within 1 weeks.
Before TIRF experiments, PEG-coated coverslips were rinsed by H2O and flow-cells (18 mm×18 mm and 24 mm×45 mm) were assembled by attaching them, using double-sided tape. The flow cell was incubated with 0.2 mg/ml neutravidin protein (ThermoFisher Scientific) for 10 min and then incubated with Sc4 amyloid (5 mM final, 10% STELLA650 labeled, 1% biotinylated) or Sc37 amyloid (5 mM final, 10% STELLA650 labeled, 1% biotinylated) for 10 min and equilibrated by the TIRF reaction buffer (25 mM Hepes-KOH pH7.5, 150 mM potassium acetate, 10 mM magnesium acetate, 10 mM DTT, 50 unit ml-1 glucose-oxidase, 50 unit ml-1 catalase, 4.5 mg ml-1 glucose 5 mM ATP, 0.1 mg/ml BSA, 10 mM Creatine Phosphate and 0.05 mg ml-1 Creatine kinase). Chaperone mixtures in the TIRF reaction buffer were incubated at 30oC for 5 min before flowing into the flow-cell.
TIRF imaging analysis was performed using an inverted microscope, eclipse Ti-E with TIRF module (Nikon Corp., Tokyo, Japan), equipped with Continuous Wave (CW) Solid-State Laser, Sapphire (Coherent Inc., CA, USA), a TIRF-objective lens, Apo TIRF 100x NA1.49 (Nikon Corp., Tokyo, Japan) and an EMCCD camera, Ixon3 (Andor Technology Ltd, Belfast UK). Solid-State lasers were used for excitation at wavelength of 488 nm (Sapphire, 20 mW, Coherent Inc.), 561 nm (Sapphire, 20 mW, Coherent Inc.), and 640 nm (CUBE, 40 mW, Coherent Inc.). STELLA650 fluorescence dye was excited by 640 nm. Alexa488 fluorescence dye was excited by 488 nm. Cy3 fluorescence dye was excited by 561 nm. During the measurements, optical focus was maintained by the perfect focus system (Nikon Corp., Tokyo, Japan). Images were acquired every 500 msec for Fig. 2b, 2c, 4a, 5b, 6a and Supplementary Fig. 3, 5, and 7 and every 10 sec for Fig. 2e, 3a, 3e and Supplementary Fig. 4c.
TIRF data analysis
Acquired image sequences were converted to 16-bit TIFF files using NIS Elements (Nikon Instruments Inc.) and all images was analyzed by ImageJ (NIH, Bethesda, MD). All images were automatically aligned using micropattern images by a plugin, Template Matching and Slice Alignment 56. Background fluorescence was subtracted using the background subtraction tool (rolling ball radius: 50 pixel) in ImageJ. We calculated the fragmentation frequency of Sc4 and Sc37 amyloids in Fig. 5c and Supplementary Fig. 7c by manually counting fragmentation events. The number of fragmentation events was divided by the initial length of amyloid.
The number of Hsp104 binding on Sc4 amyloid in Fig. 2d was counted per 600 sec and the number was subtracted by the number of control experiments without Sc4 amyloid. For binding of Ssa1-Alexa488 or Sis1-Cy3, Sc4 amyloid-STELLA650 was traced over time, and the traces were used to determine the corresponding fluorescence profiles in other channels. Mean fluorescence intensities of Ssa1 and Sis1 were normalized to the length of Sc4 amyloid (Fig. 2f and Supplementary Fig. 4d). The same method of the trace analysis was used for our preparation of spatiotemporal profiles by the Plot Profile tool. The binding rates of Ssa1 and Sis1 to Sc4 amyloid in Fig. 3d were determined by fitting the fluorescence data with a single exponential function with x offset constant using IgorPro (Wavemetrics , OR, USA).
For binding of Hsp104-SNAP549 and Ssa1-Alexa488, Sc4 amyloid-STELLA650 was traced over time using the plot profile tool. Then, the trace was used to determine the corresponding fluorescence profiles in other channels. The time of the first appearance of Hsp104-SNAP549 (THsp104 arrival) and Ssa1-Alexa488 (TSsa1 arrival) in Fig. 3g was determined by analysis of the time when fluorescence reached 10% of the maximum intensity in a 3x3 pixel area on amyloid. Distribution of the interval times of Hsp104 and Ssa1 was calculated as THsp104 arrival - TSsa1 arrival.
To determine fragmented and non-fragmented sites on amyloid, the spots in a 3x3 pixel area where amyloid was finally fragmented or not fragmented were traced, and fluorescence intensities were measured. The same pixel regions were also used to determine the corresponding fluorescence profiles in other channels. To subtract background signals, fluorescence intensities at the spots in a 3x3 pixel area that did not contain amyloid were traced. Fragmented sites were defined as the sites showing a more than 70% decrease of the initial fluorescence intensity at 600 sec, while that of non-fragmented (dissolution) sites showed over 30% of the initial fluorescence intensity at 600 sec. The first appearance time of Ssa1 was determined when fluorescence intensity reached 10% of the maximum in a 3x3 pixel area (Fig. 4c, 6c). The time of the first appearance of Hsp104-SNAP549 (THsp104 arrival) and Ssa1-Alexa488 (TSsa1 arrival) was determined when fluorescence intensity reached 10% of the maximum intensity in a 3x3 pixel area on amyloid (Fig. 4d, 6d). Cumulative intensity of Hsp104 and Ssa1 fluorescence was integrated from 0 to 600 sec (Fig. 4e, 4f, 6e, 6f, Supplementary Fig.7f, g). The number of Hsp104 appearance on amyloid in a 3x3 pixel area, which represents the number of Hsp104 binding to the same site, was counted from 0 to 600 sec (Fig. 4g, 6g and Supplementary Fig. 3d, 7h). Dwell time of Hsp104 was determined as the period of time when fluorescence intensity was over 100 a.u..
Negative staining with electron microscopy
Sc4 or Sc37 (5 mM Sup35NM monomer) amyloid was prepared as described above. Sc4 or Sc37 amyloid was centrifuged (10,000 g, 10 min, 4oC) to eliminate small aggregates or monomers. We removed 90% of the supernatant and resuspend the pellet fraction with the same volume of disaggregation buffer. A 3 ml volume of the samples was applied to the carbon-coated holy microgrid (EM Japan). Subsequently, the applied sample was negatively stained with 2% (w/v) uranyl acetate three times. Images were recorded on a Falcon II direct electron detector using a Tecnai TF20 transmission electron microscope (Thermo Fisher Scientific) at the acceleration voltage of 200kV and at the magnification of 62,000.
Confocal microscopy
All coverslips and labeled proteins (Sc4 or Sc37 amyloid-STELLA650 and Ssa1-Alexa488) were prepared as described by TIRF analysis. First, fluorescence images of Sc4 or Sc37 amyloid-STELLA650 immobilized on cover glass was acquired. Next, 2 mM Ssa1-Alexa488 and 0.5 mM Sis1 was flowed into the flow-cell and then fluorescence images at the identical place were acquired. A Leica TCS SP8 confocal microscope equipped with 638 nm diode and 488 nm OPSL laser (Leica Microsystems, Wetzlar, Germany) was used to collect z-stacks of the amyloid images, and these images were processed by deconvolution using the Leica Application Suite X (LAS X) software (Leica Microsystems, Wetzlar, Germany). Each image was compiled by Image J software
Quantification and statistical analysis
Statistical significance was tested using two-tailed, unpaired, Student’s t-test in the GraphPad Prism version 8 (GraphPad Software, La Jolla, CA) and Excel (Microsoft).