Early steps of protein disaggregation by Hsp70 chaperone and class B J-domain proteins are shaped by Hsp110
- Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, University of Gdańsk, Poland
Figures
Figure 1 with 3 supplements
Impact of Hsp110 on protein disaggregation by Hsp70 system depends on class of J-domain protein (JDP).
(A) Refolding of aggregated luciferase by Ssa1-Sis1 (1 µM Ssa1, 1 µM Sis1)±µM Sse1 (0.1 µM) (left) or Ssa1-Ydj1 (1 µM Ssa1, 1 µM Ydj1)±Sse1 (0.1 µM) (right). Error bars show SD from three …
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Figure 1—source data 1
Spreadsheet containing data for the graphs shown in Figure 1A.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-data1-v1.xlsx
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Figure 1—source data 2
Spreadsheet containing data for the graph shown in Figure 1B.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-data2-v1.xlsx
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Figure 1—source data 3
Spreadsheet containing data for the graph shown in Figure 1C.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-data3-v1.xlsx
Figure 1—figure supplement 1
J-domain protein-specific impact of Hsp110 on protein disaggregation by Hsp70 system.
(A) Renaturation of heat-aggregated GFP by Ssa1-Sis1±Sse1 or Ssa1-Ydj1±Sse1 (1 µM Ssa1, 1 µM Sis1, 1 µM Ydj1, and 0,1 µM Sse1). (B) Refolding of aggregated luciferase by Ssa1-Sis1±Sse1 (left) or …
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Figure 1—figure supplement 1—source data 1
Spreadsheet containing data for the graphs shown in Figure 1—figure supplement 1A.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp1-data1-v1.xlsx
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Figure 1—figure supplement 1—source data 2
Spreadsheet containing data for the graphs shown in Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp1-data2-v1.xlsx
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Figure 1—figure supplement 1—source data 3
Spreadsheet containing data for the graphs shown in Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp1-data3-v1.xlsx
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Figure 1—figure supplement 1—source data 4
Spreadsheet containing data for the graph shown in Figure 1—figure supplement 1D.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp1-data4-v1.xlsx
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Figure 1—figure supplement 1—source data 5
Spreadsheet containing data for the graph shown in Figure 1—figure supplement 1E and another replicate of the experiment.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp1-data5-v1.xlsx
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Figure 1—figure supplement 1—source data 6
Spreadsheet containing data for the graph shown in Figure 1—figure supplement 1F and another replicate of the experiment.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp1-data6-v1.xlsx
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Figure 1—figure supplement 1—source data 7
Spreadsheet containing data for the graph shown in Figure 1—figure supplement 1G and another replicate of the experiment.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp1-data7-v1.xlsx
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Figure 1—figure supplement 1—source data 8
Spreadsheet containing data for the graphs shown in Figure 1—figure supplement 1H.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp1-data8-v1.xlsx
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Figure 1—figure supplement 1—source data 9
Spreadsheet containing data for the graph shown in Figure 1—figure supplement 1I.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp1-data9-v1.xlsx
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Figure 1—figure supplement 1—source data 10
Spreadsheet containing data for the graph shown in Figure 1—figure supplement 1J and another replicate of the experiment.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp1-data10-v1.xlsx
Figure 1—figure supplement 2
Hsp110 determines level of Hsp70 binding to aggregates.
(A) Upper panel shows the scheme of the biolayer interferometry (BLI) experiment. Sensors covered with luciferase aggregates incubated with Ssa1 labelled with Alexa 488 and Sis1 ±Sse1 or Ydj1±Sse1 …
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Figure 1—figure supplement 2—source data 1
Spreadsheet containing data for the graphs shown in Figure 1—figure supplement 2A.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp2-data1-v1.xlsx
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Figure 1—figure supplement 2—source data 2
Spreadsheet containing data for the graph shown in Figure 1—figure supplement 2B.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp2-data2-v1.xlsx
Figure 1—figure supplement 3
Effects of Fes1 on Hsp70 binding to protein aggregates and disaggregation.
(A) Refolding of aggregated luciferase by Ssa1-Sis1 with 0.1 µM concentration of Sse1 or increasing concentrations of Fes1 measured after 1 hr. (B) Sensor with immobilized luciferase aggregates …
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Figure 1—figure supplement 3—source data 1
Spreadsheet containing data for the graph shown in Figure 1—figure supplement 3A.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp3-data1-v1.xlsx
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Figure 1—figure supplement 3—source data 2
Spreadsheet containing data for the graph shown in Figure 1—figure supplement 3B.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp3-data2-v1.xlsx
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Figure 1—figure supplement 3—source data 3
Spreadsheet containing data for the graphs shown in Figure 1—figure supplement 3C.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig1-figsupp3-data3-v1.xlsx
Figure 2 with 1 supplement
Sse1 promotes modification of aggregates by the Hsp70 system.
(A) Initial incubation of heat-aggregated GFP aggregates with the Hsp70 system, followed by the addition of the Hsp104 D484K F508A variant (0.15 µM). Recovery was initiated by the addition of the …
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Figure 2—source data 1
Spreadsheet containing data for the graph shown in Figure 2A.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig2-data1-v1.xlsx
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Figure 2—source data 2
Spreadsheet containing data for the graph shown in Figure 2B.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig2-data2-v1.xlsx
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Figure 2—source data 3
Uncropped microscopy images presented in Figure 2B and replicates for the calculations in Figure 2—source data 2.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig2-data3-v1.zip
Figure 2—figure supplement 1
Hsp110 impact on aggregate modification by Hsp70 system.
(A) Recovery of FLUC-EGFP aggregates (0.3 µM) by Hsp70 system with indicated combination of Ssa1 (1 µM), Sis1 (1 µM), Ydj1 (1 µM), Hsp104 (1 µM), or Sse1 (0.1 µM). luc-GFP activity was measured …
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Figure 2—figure supplement 1—source data 1
Spreadsheet containing data for the graph shown in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 1—source data 2
Spreadsheet containing data for the graphs shown in Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig2-figsupp1-data2-v1.xlsx
Figure 3 with 1 supplement
Susceptibility of Hsp70 to Hsp110 depends on J-domain protein (JDP) class and phase of disaggregation.
(A) Titration of Sse1 in the refolding of aggregated luciferase by Ssa1-Sis1 (1 µM Ssa1, 1 µM Sis1) (red) or Ssa1-Ydj1 (1 µM Ssa1, 1 µM Ydj1) (blue). Activity of luciferase was measured after 1 hr …
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Figure 3—source data 1
Spreadsheet containing data and model fitting parameters for the graphs shown in Figure 3A.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-data1-v1.xlsx
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Figure 3—source data 2
Spreadsheet containing data for the graphs shown in Figure 3B.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-data2-v1.xlsx
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Figure 3—source data 3
Spreadsheet containing data for the graphs shown in Figure 3C.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-data3-v1.xlsx
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Figure 3—source data 4
Spreadsheet containing data for the graphs shown in the left and middle panels of Figure 3D.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-data4-v1.xlsx
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Figure 3—source data 5
Spreadsheet containing data for the graph shown in the right panel of Figure 3D.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-data5-v1.xlsx
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Figure 3—source data 6
Spreadsheet containing data for the graphs shown in the left and middle panels of Figure 3E.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-data6-v1.xlsx
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Figure 3—source data 7
Spreadsheet containing data and model fitting parameters for the graph shown in the right panel of Figure 3E.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-data7-v1.xlsx
Figure 3—figure supplement 1
Concentration-dependent effects of Hsp110 on aggregate binding and disaggregtion by Hsp70.
(A) Titration of Sse1 in the refolding of aggregated luciferase by Ssa1-Sis1 (red) or Ssa1-Ydj1 (blue) with Hsp104 (1 µM Ssa1, 1 µM Sis1, 1 µM Ydj1, 1 µM Hsp104, and indicated concentrations of …
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Figure 3—figure supplement 1—source data 1
Spreadsheet containing data for the graphs shown in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-figsupp1-data1-v1.xlsx
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Figure 3—figure supplement 1—source data 2
Spreadsheet containing data for the graph shown in Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-figsupp1-data2-v1.xlsx
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Figure 3—figure supplement 1—source data 3
Uncropped microscopy images presented in Figure 3—figure supplement 1B and replicates for the calculations in Figure 3—figure supplement 1—source data 2.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-figsupp1-data3-v1.zip
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Figure 3—figure supplement 1—source data 4
Spreadsheet containing data for the graph shown in Figure 3C.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-figsupp1-data4-v1.xlsx
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Figure 3—figure supplement 1—source data 5
Spreadsheet containing data for the graphs shown in Figure 3D.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-figsupp1-data5-v1.xlsx
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Figure 3—figure supplement 1—source data 6
Spreadsheet containing data for the graph shown in Figure 3E.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-figsupp1-data6-v1.xlsx
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Figure 3—figure supplement 1—source data 7
Spreadsheet containing data for the graph shown in Figure 3F.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig3-figsupp1-data7-v1.xlsx
Figure 4 with 1 supplement
Hsp110 and class B J-domain protein (JDP) show apparent competition for Hsp70.
(A) Upper panel shows the scheme of the biolayer interferometry (BLI) experiment. Binding of Ssa1 (1 µM) in the presence of increasing concentrations of Sse1 to Sis1 immobilised on the BLI sensor …
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Figure 4—source data 1
Spreadsheet containing data for the graph shown in Figure 4A.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig4-data1-v1.xlsx
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Figure 4—source data 2
Spreadsheet containing data for the graphs shown in Figure 4B.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig4-data2-v1.xlsx
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Figure 4—source data 3
Spreadsheet containing data and model fitting parameters for the graphs shown in Figure 4C.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
Competition between Hsp110 and JDP co-chaperones.
(A) Titration of Sse1-2 in the presence of 1 µM Ssa1 in the binding to the immobilised Sis1 on the biolayer interferometry (BLI) sensor. (B) Comparison of the binding signal prior to the …
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Figure 4—figure supplement 1—source data 1
Spreadsheet containing data for the graph shown in Figure 4—figure supplement 1A.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig4-figsupp1-data1-v1.xlsx
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Figure 4—figure supplement 1—source data 2
Spreadsheet containing data for the graph shown in Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig4-figsupp1-data2-v1.xlsx
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Figure 4—figure supplement 1—source data 3
Spreadsheet containing data for the graph shown in Figure 4—figure supplement 1C.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig4-figsupp1-data3-v1.xlsx
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Figure 4—figure supplement 1—source data 4
Spreadsheet containing data for the graphs shown in Figure 4—figure supplement 1D.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig4-figsupp1-data4-v1.xlsx
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Figure 4—figure supplement 1—source data 5
Spreadsheet containing data for the graph and model fitting parameters shown in Figure 4—figure supplement 1E.
- https://cdn.elifesciences.org/articles/94795/elife-94795-fig4-figsupp1-data5-v1.xlsx
Figure 5
Impact of Hsp110 on Hsp70-dependent disaggregation.
Hsp110 stimulates aggregate disassembly by Hsp70 with class B J-domain proteins (JDPs) and Hsp104 (green arrow), contrary to the stage of final folding of the solubilised polypeptide (light purple …
Author response image 1
Chaperone-assisted folding of luciferase after denaturation at 5 M or 6 M GdnHCl.
Luciferase was denatured in 5 M or 6 M GdnHCl according to the protocol in the Materials and Methods section. Luminescence was monitored alone or after incubation with Luminescence was monitored …
Author response image 2
Disaggregaton of heat-aggregated luciferase – impact of sHsps.
Luciferase (2 μM) was denatured with (blue) or without (red) Hsp26 (20 μM) at 45 ̊C for 15 min in the buffer A (Materials and Methods). Upon 100-fold dilution with the buffer A, supplemented wih 5 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21(DE3) CodonPlus | Agilent | Cat # 230250; RRID:SCR_013575 | Genotype: E. coli B F- ompT hsdS(rB- mB-) dcm +Tetr gal endA Hte [argU proL Camr] |
| Strain, strain background (E. coli) | RosettaBL21 (DE3) | Novagen | Cat # 70954; RRID:SCR_008441 | Genotype: E. coli F- ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE (CamR) |
| Strain, strain background (Saccharomyces cerevisiae) | W303 | Laboratory collection | Genotype: MATa/MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [phi+] | |
| Peptide, recombinant protein | Ssa1 | DOI:10.1073/pnas.0804187105 | Uniprot ID: P10591 | Expressed from pCA533-His6-SUMO-SSA1 plasmid, KanR, T7 |
| Peptide, recombinant protein | Ssa1 ∆EEVD | DOI:10.1016/j.jmb.2015.02.007 | Expressed from pCA533-His6-SUMO-SSA1 ∆EEVD plasmid, KanR, T7 | |
| Peptide, recombinant protein | Sse1 | DOI:10.1073/pnas.0804187105 | Uniprot ID: P32589 | Expressed from pCA534-SSE1 plasmid KanR, T7 |
| Peptide, recombinant protein | Sse1-2 (N572Y N575A) | This study | Variant generated based on DOI:10.1016/j.jmb.2010.07.004, expressed from pCA534-SSE1-2 plasmid KanR, T7 | |
| Peptide, recombinant protein | Fes1 | DOI:10.1073/pnas.0804187105 | Uniprot ID: P38260 | Expressed from pCA707-FES1 plasmid KanR, T7 |
| Peptide, recombinant protein | Ydj1 | DOI:10.1074/jbc.M112.387589 | Uniprot ID: P25491 | Expressed from pET21a-YDJ1 plasmid AmpR, T7 |
| Peptide, recombinant protein | Sis1 | DOI:11810.1073/pnas.2108163118 | Uniprot ID: P25294 | Expressed from pPROEX-TEV-SIS1 plasmid AmpR, T7 |
| Peptide, recombinant protein | Sis1 E50A | DOI:10.1016/j.jmb.2015.02.007 | Expressed from pPROEX-TEV-SIS1 E50A plasmid AmpR, trc | |
| Peptide, recombinant protein | Hsp104 | DOI:10.1074/jbc.M112.387589 | Uniprot ID: P31539 | Expressed from pET5a-HSP104 plasmid AmpR, T7 |
| Peptide, recombinant protein | Hsp104 D484K F508A | DOI:10.1016/j.jmb.2019.04.014 | Expressed from pET5a-HSP104 D484K F508A plasmid AmpR, T7 | |
| Peptide, recombinant protein | Hsc70 | DOI:10.1038/nature14884 | Uniprot ID: P11142 | Expressed from pPROEX-His-TEV-HSC70 plasmid AmpR, trc |
| Peptide, recombinant protein | DNAJA2 | DOI:10.1038/nature14884 | Uniprot ID: O60884 | Expressed from pPROEX-His-TEV-DNAJA2 plasmid AmpR, trc |
| Peptide, recombinant protein | DNAJB1 | DOI:10.1038/nature14884 | Uniprot ID: P25685 | Expressed from pPROEX-His-TEV-DNAJB1 plasmid AmpR, trc |
| Peptide, recombinant protein | Hsp105 | DOI:10.1038/nature14884 | Uniprot ID: Q92598 | Expressed from pPROEX-His-TEV-HSP105 plasmid AmpR, trc |
| Peptide, recombinant protein | GFP | DOI:10.1074/jbc.M402405200 | Expressed from pGFPuv plasmid (TaKaRa, RRID:SCR_003960) | |
| Peptide, recombinant protein | Fluc-EGFP | This study | EGFP fusion with firefly luciferase, expressed from pET22b-Fluc-GFP plasmid AmpR, T7 | |
| Peptide, recombinant protein | His-tagged EGFP | This study | ||
| Peptide, recombinant protein | OuantiLum Recombinant Luciferase | Promega | Cat # E1701; RRID:SCR_006724 | |
| Peptide, recombinant protein | His-tagged Luciferase | DOI:10.1371/journal.pgen.1008479 | ||
| Peptide, recombinant protein | Creatine Kinase | Roche | Cat # 10127566001; RRID:SCR_001326 | |
| Commercial assay or kit | Luciferase Assay System | Promega | Cat # E151A; RRID:SCR_006724 | |
| Chemical compound, drug | Alexa Fluor 488 C5 Maleimide | Invitrogen | Cat # A10254; RRID:SCR_013378 | |
| Commercial assay or kit | QuikChange Site-Directed Mutagenesis Kit | Agilent | Cat # 200513; RRID:SCR_013575 | |
| Other | Ni-NTA BLI sensors | Sartorius | Cat # 18-5101; RRID:SCR_003935 |
