Skd3 (human ClpB) is a potent mitochondrial protein disaggregase that is inactivated by 3-methylglutaconic aciduria-linked mutations
Figures
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Skd3 is homologous to Hsp104 and Hsp78 and is conserved across diverse metazoan lineages.
(A) Domain map depicting S. cerevisiae Hsp104, S. cerevisiae Hsp78, and H. sapiens Skd3. Hsp104 is composed of a N-terminal domain (NTD), nucleotide-binding domain 1 (NBD1), middle domain (MD), nucleotide-binding domain 2 (NBD2), and C-terminal domain (CTD). Hsp78 is composed of a mitochondrial-targeting signal (MTS), NBD1, MD, NBD2, and CTD. Skd3 is composed of a MTS, a short hydrophobic stretch of unknown function, an ankyrin-repeat domain (ANK) containing four ankyrin repeats, an NBD that is homologous to Hsp104 and Hsp78 NBD2, and a CTD. (B) Phylogenetic tree depicting a Clustal Omega alignment of Skd3 sequences from divergent metazoan lineages and the protozoan Monosiga brevicollis. The alignment shows conservation of Skd3 across diverse species and shows high similarity between mammalian Skd3 proteins.
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Skd3 NBD alignment to other AAA+ proteins reveals high similarity to Hsp104 and Hsp78.
Alignment of the NBD from H. sapiens Skd3 and NBD2 from S. cerevisiae Hsp104, S. cerevisiae Hsp78, E. coli ClpB, E. coli ClpA, and S. aureus ClpC. Alignments were constructed using Clustal Omega. Bottom row shows consensus sequence of alignment. Highlighted in red are the Walker A and Walker B motifs. Highlighted in green are the Pore-Loop motifs. Highlighted in blue are the Sensor I, Sensor II, and Arginine-Finger motifs.
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Skd3 is a protein disaggregase.
(A) Domain map depicting the Mitochondrial-processing peptidase (MPP) cleavage site and mature-length Skd3 (MPPSkd3). The MTS was predicted using MitoProt in agreement with previous work, as outlined in the Materials and methods. The positions of the Walker A mutation (K387A) predicted to disrupt ATP binding and hydrolysis, pore-loop tyrosine mutation (Y430A) predicted to disrupt substrate binding, and Walker B mutation (E455Q) predicted to disrupt ATP hydrolysis are shown. (B) MPPSkd3 is an ATPase. ATPase assay comparing MPPSkd3 and Hsp104. MPPSkd3 and Hsp104 ATPase were compared to buffer using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 4, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001). (C) Luciferase disaggregation/reactivation assay showing that MPPSkd3 has disaggregase activity in the presence but not absence of ATP. Luciferase activity was buffer subtracted and normalized to Hsp104 + Hsp70/Hsp40. Luciferase activity was compared to buffer using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 4, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001). (D) ATPase assay comparing MPPSkd3, MPPSkd3K387A (Walker A mutant), MPPSkd3E455Q (Walker B mutant), and MPPSkd3Y430A (Pore-Loop mutant), showing that both Walker A and Walker B mutations abolish Skd3 ATPase activity, whereas the Pore Loop mutation reduces ATPase activity. ATPase activity was compared to buffer using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 4, individual data points shown as dots, bars show mean ± SEM, *p<0.05, ****p<0.0001). (E) Luciferase disaggregation/reactivation assay comparing MPPSkd3 to Walker A, Walker B, and Pore-Loop variants demonstrating that ATP binding, ATP hydrolysis, and pore-loop contacts are essential for Skd3 disaggregase activity. Luciferase activity was buffer subtracted and normalized to Hsp104 + Hsp70/Hsp40. Luciferase activity was compared to buffer using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 4, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001).
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Recombinant Skd3 is highly pure and immunoreactive with several commercially available antibodies.
(A) Representative gel of MPPSkd3 showing high purity via Coomassie Brilliant Blue stain. (B) Western blot with Skd3 antibody (Abcam ab76179) showing immunoreactivity of a singular band of purified MPPSkd3. (C) Western blot with Skd3 antibody (Abcam ab87253) showing immunoreactivity of a singular band of purified MPPSkd3. (D) Western blot with Skd3 antibody (Proteintech #15743–1-AP) showing immunoreactivity of a singular band of purified MPPSkd3.
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Skd3 is a protein disaggregase.
(A) ATPase assay time course showing that MPPSkd3 ATPase activity is approximately linear over the first five minutes of the assay. (N = 4, bars show mean ± SEM). (B) Luciferase disaggregation/reactivation activity time course showing that MPPSkd3 disaggregates more luciferase over time. Luciferase activity was buffer subtracted and normalized to Hsp104 plus Hsp70 and Hsp40 (N = 4, bars show mean ± SEM). (C) Luciferase disaggregation/reactivation assay showing dose-response relationship between MPPSkd3 concentration and luciferase reactivation. Luciferase activity was buffer subtracted and normalized to Hsp104 plus Hsp70 and Hsp40. (N = 4, dots show mean ± SEM, EC50 = 0.394 μM). (D) Luciferase disaggregation/reactivation assay showing MPPSkd3 disaggregase activity in the presence of different nucleotides. Results show that MPPSkd3 can disaggregate luciferase in the presence of ATP, but not in the absence of ATP, in the presence of ADP, or in the presence of ATP analogues ATPγS (slowly hydrolyzable) or AMP-PNP (non-hydrolyzable). Luciferase assay incubated for 30 min and no ATP regeneration system was used. Luciferase activity was buffer subtracted and normalized to Hsp104 plus Hsp70 and Hsp40 (N = 4, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001).
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PARL cleavage enhances Skd3 disaggregase activity.
(A) Domain map depicting MPPSkd3 and the PARL cleavage site and corresponding PARL-cleaved Skd3 (PARLSkd3). The positions of the Walker A mutation (K387A) predicted to disrupt ATP binding and hydrolysis, pore-loop tyrosine mutation (Y430A) predicted to disrupt substrate binding, and Walker B mutation (E455Q) predicted to disrupt ATP hydrolysis are shown. (B) ATPase assay comparing MPPSkd3 and PARLSkd3. PARLSkd3 is catalytically active, but is slightly less active than MPPSkd3. PARLSkd3 and Hsp104 ATPase were compared to MPPSkd3 ATPase using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 6, individual data points shown as dots, bars show mean ± SEM, *p<0.05, ****p<0.0001). (C) Luciferase disaggregation/reactivation assay comparing MPPSkd3 disaggregase activity to PARLSkd3. PARLSkd3 was over 10-fold more active than MPPSkd3. Luciferase activity was buffer subtracted and normalized to Hsp104 + Hsp70/Hsp40. Luciferase activity was compared to MPPSkd3 using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 6, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001). (D) ATPase assay comparing PARLSkd3, PARLSkd3K387A (Walker A), PARLSkd3E455Q (Walker B), and PARLSkd3Y430A (Pore Loop), showing that both Walker A and Walker B mutations abolish Skd3 ATPase activity, whereas the Pore-Loop mutation reduces ATPase activity. ATPase activity was compared to buffer using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 4, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001). (E) Luciferase disaggregation/reactivation assay comparing PARLSkd3 to Walker A, Walker B, and Pore-Loop variants showing that ATP binding, ATP hydrolysis, and pore-loop contacts are essential for PARLSkd3 disaggregase activity. Luciferase activity was buffer subtracted and normalized to Hsp104 + Hsp70/Hsp40. Luciferase activity was compared to buffer using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 4, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001).
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The auto-inhibitory peptide of Skd3 is hydrophobic.
(A) Kyte-Doolittle hydrophobicity score was calculated for Skd3 using the ExPASy web server (Kyte and Doolittle, 1982; Wilkins et al., 1999). A positive hydrophobicity score indicates highly hydrophobic regions. Analysis shows a spike in hydrophobicity corresponding to the C-terminal portion of the Skd3 auto-inhibitory peptide (92-126), which is removed by PARL cleavage.
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PARL cleavage of Skd3 enhances Skd3 disaggregase activity.
(A) Sequence logo depicting the conservation of the auto-inhibitory peptide (orange) and PARL-cleavage motif (green) of Skd3. Arrows indicate MPP and PARL cleavage sites. Logo shows a high level of homology suggesting conserved importance. Sequence Logo was built with WebLogo using Skd3 protein sequence from 42 different mammalian species. (B) ATPase assay time course showing that PARLSkd3 ATPase activity is approximately linear over the first five minutes of the assay (N = 4, bars show mean ± SEM). (C) Luciferase disaggregation/reactivation activity time course showing that PARLSkd3 disaggregates more luciferase over time. Luciferase activity was buffer subtracted and normalized to Hsp104 plus Hsp70 and Hsp40 (N = 4, bars show mean ± SEM). (D) Luciferase disaggregation/reactivation assay showing dose-response relationship between PARLSkd3 concentration and luciferase reactivation. Luciferase activity was buffer subtracted and normalized to Hsp104 plus Hsp70 and Hsp40 (N = 4, dots show mean ± SEM, EC50 = 0.836 μM). (E) Luciferase disaggregation/reactivation assay showing PARLSkd3 disaggregase activity in the presence of different nucleotides. Results show that PARLSkd3 can disaggregate luciferase in the presence of ATP, but not in the absence of ATP, in the presence of ADP, or in the presence of ATP analogues ATPγS (slowly hydrolyzable) or AMP-PNP (non-hydrolyzable). Luciferase assay incubated for 30 min and no ATP regeneration system was used. Luciferase activity was buffer subtracted and normalized to Hsp104 plus Hsp70 and Hsp40 (N = 4, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001).
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PARLSkd3, but not MPPSkd3, ATPase activity is stimulated by a model substrate.
(A) ATPase assay showing that PARLSkd3 but not MPPSkd3 is stimulated by the model substrate β-casein. ATPase activity with substrate was compared to controls without substrate using a two-tailed, unpaired t-test. (N = 4, individual data points shown as dots, bars show mean ± SEM, *p<0.05).
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Skd3 disaggregates α-synuclein fibrils.
(A) Representative dot blot of α-synuclein disaggregation assay. Blot shows solubilization of α-synuclein fibrils by PARLSkd3 in the presence of an ATP regeneration system (ARS), but not in the presence of PARLSkd3 or ARS alone. (N = 3). (B) Quantification of α-synuclein disaggregation assay showing that PARLSkd3 in the presence of an ARS disaggregates α-synuclein fibrils. Results are normalized as fraction in the supernatant relative to the fraction in the supernatant and the pellet. The fraction of α-synuclein in the supernatant was compared to buffer using a repeated measure one-way ANOVA and a Dunnett’s multiple comparisons test (N = 3, individual data points shown as dots, bars show mean ± SEM, *p<0.05).
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The ankyrin-repeat domain and NBD are required for Skd3 disaggregase activity.
(A) Domain maps showing the Skd3ANK and Skd3NBD constructs. (B) ATPase assay comparing Skd3ANK and Skd3NBD ATPase activity. Results show that Skd3ANK, Skd3NBD, and Skd3ANK + Skd3NBD do not have ATPase activity. Data are from the same experiments as Figure 3B. ATPase activity was compared to buffer using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 4, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001). (C) Luciferase disaggregation/reactivation assay comparing Skd3ANK, Skd3NBD, and Skd3ANK + Skd3NBD d activity. Results show that Skd3ANK, Skd3NBD, or Skd3ANK + Skd3NBD are inactive disaggregases. Data are from same experiments as Figure 3C. Luciferase activity was buffer subtracted and normalized to Hsp104 plus Hsp70 and Hsp40. Luciferase disaggregase activity was compared to buffer using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 4, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001).
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Skd3 does not collaborate with Hsp70 and Hsp40 in protein disaggregation.
(A) Luciferase disaggregation/reactivation comparing MPPSkd3 disaggregase activity in the presence or absence of Hsp70 (Hsc70) and Hsp40 (Hdj1). Luciferase activity was buffer subtracted and normalized to Hsp104 plus Hsp70 and Hsp40. Results show a stimulation of Hsp104 disaggregase activity by Hsp70 and Hsp40, but no stimulation of disaggregase activity for MPPSkd3. MPPSkd3 plus Hsp70 and Hsp40 was compared to MPPSkd3 using a two-tailed, unpaired t-test. Test found no significant difference in disaggregation activity. (N = 4, individual data points shown as dots, bars show mean ± SEM). (B) Luciferase disaggregation/reactivation comparing PARLSkd3 disaggregase activity in the presence or absence of Hsp70 and Hsp40. Luciferase activity was buffer subtracted and normalized to Hsp104 plus Hsp70 and Hsp40. Results show no stimulation of disaggregase activity for PARLSkd3 by Hsp70 and Hsp40. PARLSkd3 plus Hsp70 and Hsp40 was compared to PARLSkd3 using a two-tailed, unpaired t-test. Test found no significant difference in disaggregation activity. (N = 4, individual data points shown as dots, bars show mean ± SEM).
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Skd3 maintains the solubility of key mitochondrial proteins in human cells.
(A) Schematic showing sedimentation assay design. HAP1 cells were lysed and the mitochondrial fraction was separated from the cytosolic fraction. The mitochondrial fraction was then lysed, and the soluble fraction was separated from the insoluble fraction via sedimentation. The samples were then either analyzed via mass-spectrometry or western blotting. (B) Volcano plot showing the log2 fold change of protein in the Skd3 (CLPB) knockout pellet compared to the wild-type (WT) pellet. The 99 proteins that were enriched in the Skd3 pellet are highlighted in red. The 53 proteins that were enriched in the wild-type (WT) pellet are highlighted in green. Significance cutoffs were set as fold change >2.0 and p<0.05, indicated with blue dashed lines (N = 3, p<0.05). (C) Select statistically significant terms for GO biological processes from the enriched proteins in the Skd3 knockout pellet. Dashed line shows p=0.05 (p<0.05). For full list see Figure 7—figure supplement 2b. (D) Representative western blot of sedimentation assay showing relative solubility of HAX1 protein in wild-type (WT) and Skd3 (CLPB) knockout cells. Results show a marked decrease in HAX1 solubility when Skd3 is knocked out. (N = 3). (E) Quantification of HAX1 sedimentation assay shows an overall increase in the insoluble HAX1 relative to the total protein in the Skd3 (CLPB) knockout cell line. Quantification is normalized as signal in the pellet divided by the sum of the signal in the pellet and supernatant. The fraction in the pellet for the Skd3 knockout was compared to the wild-type cells using a two-way, unpaired, t-test. (N = 3, individual data points shown as dots, bars show mean ± SEM, *p<0.05).
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Verification of Skd3 knockout and mitochondria isolation in HAP1 cells.
(A) Representative western blot of HAP1 cells showing knockout of Skd3. First and second lane show 100 ng load of recombinant MPPSkd3 and PARLSkd3. Anti-Skd3 (Abcam CAT# ab235349) and anti-COXIV (Abcam CAT# ab14744) antibodies were used. (B) Representative western blot of HAP1 whole cell lysate and mitochondria extract showing enrichment of the mitochondrial protein COXIV and depletion of the cytoplasmic protein GAPDH from whole cell lysates to mitochondrial extract (N = 3). (C) Quantification of the western blots from (B) showing the relative enrichment of COXIV:GAPDH in the purified mitochondria.
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Skd3 deletion increases insolubility of mitochondrial inner membrane and intermembrane space proteins.
(A) Terms for GO cellular component associated with the enriched proteins in the Skd3 knockout pellet. Dashed line shows p=0.05 (p<0.05). (B) Full list of terms for GO biological processes associated with the enriched proteins in the Skd3 knockout pellet. Dashed line shows p=0.05 (p<0.05).
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Skd3 maintains the solubility of MICU2 in human cells.
(A) Representative western blot of sedimentation assay showing relative solubility of MICU2 protein in wild-type and Skd3 (CLPB) knockout cells. Results show a decrease in MICU2 solubility when Skd3 is knocked out. (N = 3). (B) Quantification of MICU2 sedimentation assay shows an overall increase in the insoluble MICU2 relative to the total protein in the Skd3 (CLPB) knockout cell line. Quantification is normalized as signal in the pellet divided by the sum of the signal in the pellet and supernatant. The fraction in the pellet for the Skd3 knockout was compared to the wild-type cells using a two-way, unpaired, t-test. (N = 3, individual data points shown as dots, bars show mean ± SEM, *p<0.05).
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HAX1 is a highly disordered protein.
(A) Domain map of HAX1 with IUPRED disorder prediction score plotted underneath (Mészáros et al., 2018; Mészáros et al., 2009). IUPRED scores higher than 0.5 predict disorder. Analysis suggests that HAX1 is a highly disordered protein. Acidic domain labeled in green, BH1 and BH2 domains labeled in purple, HD1 and HD2 domains labeled in blue, PEST domain labeled in orange, and transmembrane domain (TMD) labeled in tan.
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Skd3 disaggregase activity predicts the clinical severity of MGCA7-associated mutations.
(A) Domain map depicting all published mutations in Skd3 that have been associated with MGCA7. Mutants in red are studied further here. (B) ATPase assay showing the effect of four homozygous MGCA7 mutations on Skd3 activity. PARLSkd3T268M has increased ATPase activity, PARLSkd3R475Q and PARLSkd3A591V have decreased ATPase activity, and PARLSkd3R650P has unchanged ATPase activity compared to wild type. PARLSkd3 MGCA7 mutants ATPase activities were compared to PARLSkd3 wild-type using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 3, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001). (C) Luciferase disaggregation/reactivation assay showing the effect of the same four homozygous MGCA7 mutations on Skd3 activity. PARLSkd3T268M had reduced disaggregase activity, whereas PARLSkd3R475Q, PARLSkd3A591V, and PARLSkd3R650Pwere almost completely inactive compared to wild type. Luciferase activity was buffer subtracted and normalized to Hsp104 plus Hsp70 and Hsp40. Luciferase disaggregase activity was compared to PARLSkd3 wild type using one-way ANOVA and a Dunnett’s multiple comparisons test (N = 3, individual data points shown as dots, bars show mean ± SEM, ****p<0.0001). (D) Table summarizing the clinical severity of each MGCA7 mutation as well as the ATPase activity and luciferase disaggregase activity. The table shows a relationship between luciferase disaggregase activity and clinical severity, but no relationship between either the ATPase activity and clinical severity or ATPase and luciferase disaggregase activity. Values represent ATPase activity and luciferase disaggregase activity normalized to wild-type PARLSkd3 activity. Values show mean ± SEM (N = 3).
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Skd3 is a protein disaggregase that is activated by PARL and inactivated by MGCA7-linked mutations.
(A) Schematic illustrating (i) that Skd3 is a protein disaggregase that is activated by PARL cleavage of a hydrophobic auto-inhibitory peptide, (ii) that Skd3 works to solubilize key substrates in the mitochondrial intermembrane space and inner membrane that are involved in apoptosis, protein import, calcium handling, and respiration, and (iii) that mutations in Skd3 associated with MGCA7 result in defective Skd3 disaggregase activity in a manner that predicts the clinical severity of disease.
Tables
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Mouse monoclonal α-α-synuclein (SYN211) | ThermoFisher Scientific | AHB0261; RRID:AB_10978319 | (1:500 dilution) |
Antibody | Rabbit polyclonal α-CLPB (Skd3) | abcam | ab235349; RRID:AB_2847899 | (1:1000 dilution) |
Antibody | Rabbit polyclonal α-CLPB (Skd3) | abcam | ab76179; RRID:AB_1310087 | (1:1000 dilution) |
Antibody | Rabbit polyclonal α-CLPB (Skd3) | abcam | ab87253; RRID:AB_1952530 | (1:1000 dilution) |
Antibody | Rabbit polyclonal α-CLPB (Skd3) | Proteintech | 15743–1-AP; RRID:AB_2847900 | (1:1000 dilution) |
Antibody | Mouse monoclonal α-COXIV | abcam | ab14744; RRID:AB_301443 | (1:1000 dilution) |
Antibody | Mouse monoclonal α-GAPDH | abcam | ab8245; RRID:AB_2107448 | (1:1000 dilution) |
Antibody | Rabbit polyclonal α-HAX1 | abcam | ab137613; RRID:AB_2847902 | (1:500 dilution) |
Antibody | Rabbit polyclonal α-MICU2 | abcam | ab101465; RRID:AB_10711219 | (1:1000 dilution) |
Antibody | IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody | Li-Cor | 926–68071; RRID:AB_10956166 | (1:2500 dilution) |
Antibody | IRDye 800CW Goat anti-Mouse IgG Secondary Antibody | Li-Cor | 926–32210; RRID:AB_621842 | (1:2500 dilution) |
Cell line (Escherichia coli) | BL21-CodonPlus (DE3) -RIL competent cells | Agilent | 230245 | Chemically competent cells |
Cell line (Homo sapiens) | Human HAP1 Knockout Cell Lines - WT | Horizon Discovery | HZGHC81570;; RRID:CVCL_Y019 | Human control cell line. The HAP1 cell line was bought directly from Horizon Discovery and has been control quality checked by the vendor. |
Cell line (Homo sapiens) | Human HAP1 Knockout Cell Lines - CLPB | Horizon Discovery | HZGHC007326c001 | Human knockout cell line. The HAP1 ClpB knockout cell line was bought directly from Horizon Discovery and has been control quality checked by the vendor. |
Commercial assay or kit | ATPase Activity Kit (Colorimetric) | Innova Biosciencens | 601–0120 | |
Commercial assay or kit | Luciferase Assay Reagent | Promega | E1483 | |
Chemical compound, drug | Creatine phosphate | Roche | 10621722001 | |
Chemical compound, drug | ADP | MP Biomedicals | 150260 | |
Chemical compound, drug | AMPPNP | Roche | 10102547001 | |
Chemical compound, drug | ATP | Sigma-Aldrich | A3377 | |
Chemical compound, drug | ATPγS | Roche | 10102342001 | |
Chemical compound, drug | cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail | Sigma-Aldrich | 4693159001 | |
Chemical compound, drug | Pepstatin A, microbial,≥90% (HPLC) | Sigma-Aldrich | P5318 | |
Chemical compound, drug | Protease Inhibitor Cocktail (mammalian) | Sigma-Aldrich | P8340 | |
Gene (Homo sapiens) | CLPB | NA | HGNC:30664 | |
Peptide, recombinant protein | α-synuclein fibrils | Luk et al., 2012 | Preformed α-synuclein fibrils were a gift from Kelvin Luk. | |
Peptide, recombinant protein | β-casein | Sigma-Aldrich | C6905 | |
Peptide, recombinant protein | Creatine kinase | Roche | 10127566001 | |
Peptide, recombinant protein | Firefly luciferase | Sigma-Aldrich | L9506 | |
Peptide, recombinant protein | Hdj1 | Michalska et al., 2019 | ||
Peptide, recombinant protein | Hsc70 | Michalska et al., 2019 | ||
Peptide, recombinant protein | Hsp104 | DeSantis et al., 2012 and Jackrel et al., 2014 | ||
Peptide, recombinant protein | Lysozyme | Sigma-Aldrich | L6876 | |
Peptide, recombinant protein | MPPSkd3 | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | MPPSkd3K387A | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | MPPSkd3E455Q | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | MPPSkd3Y430A | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | PARLSkd3 | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | PARLSkd3K387A | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | PARLSkd3E455Q | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | PARLSkd3Y430A | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | PARLSkd3T268M | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | PARLSkd3R475Q | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | PARLSkd3A591V | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | PARLSkd3R650P | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | Skd3ANK | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Peptide, recombinant protein | Skd3NBD | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | Hdj1 in pE-SUMO | Michalska et al., 2019 | ||
Recombinant DNA reagent | Hsc70 in pE-SUMO | Michalska et al., 2019 | ||
Recombinant DNA reagent | Hsp104 in pNOTAG | DeSantis et al., 2012 and Jackrel et al., 2014 | ||
Recombinant DNA reagent | MPPSkd3 in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | MPPSkd3K387A in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | MPPSkd3E455Q in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | MPPSkd3Y430A in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | PARLSkd3 in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | PARLSkd3K387A in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | PARLSkd3E455Q in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | PARLSkd3Y430A in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | PARLSkd3T268M in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | PARLSkd3R475Q in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | PARLSkd3A591V in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | PARLSkd3R650P in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | Skd3ANK in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Recombinant DNA reagent | Skd3NBD in pMAL C2 with TEV site | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | ClpA (Escherichia coli) | This study | UniProtKB:P0ABH9 | Full description can be found in Materials and methods: Purification of Skd3 |
Sequence-based reagent | ClpB (Escherichia coli) | This study | UniProtKB:P63284 | Full description can be found in Materials and methods: Purification of Skd3 |
Sequence-based reagent | ClpC (Staphylococcus aureus) | This study | UniProtKB:Q2G0P5 | Full description can be found in Materials and methods: Purification of Skd3 |
Sequence-based reagent | Hsp104 (Saccharomyces cerevisiae) | This study | UniProtKB:P31539 | Full description can be found in Materials and methods: Purification of Skd3 |
Sequence-based reagent | Hsp78 (Saccharomyces cerevisiae) | This study | UniProtKB:P33416 | Full description can be found in Materials and methods: Purification of Skd3 |
Sequence-based reagent | HAX1 (Homo sapiens) | This study | UniProtKB:O00165 | Full description can be found in Materials and methods: Purification of Skd3 |
Sequence-based reagent | Skd3 (Ailuropoda melanoleuca) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Anolis carolinensis) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Bos taurus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Callithrix jacchus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Callorhinus ursinus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Canis lupus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Capra hircus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Carlito syrichta) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Cebus capucinus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Ceratotherium simum) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Cercocebus atys) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Chlorocebus sabaeus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Colobus angolensis) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Danio rerio) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Equus asinus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Equus caballus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Equus przewalskii) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Felis catus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Geospiza fortis) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Gorilla gorilla) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Gulo gulo) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Grammomys surdaster) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Homo sapiens) | This study | UniProtKB:Q9H078 | Full description can be found in Materials and methods: Purification of Skd3 |
Sequence-based reagent | Skd3 (Macaca fascicularis) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Macaca mulatta) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Macaca nemestrina) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Mandrillus leucophaeus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Microcebus murinus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Microtus ochrogaster) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Monosigia brevicollis) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Mus musculus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Mustela putorius) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Nomascus leucogenys) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Nothobranchius rachovii) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Odobenus rosmarus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Orycteropus afer) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Pan paniscus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Pan troglodyte) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Panthera tigris) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Papio anubis) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Piliocolobus tephrosceles) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Pongo abelii) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Propithecus coquereli) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Puma concolor) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Rattus norvegicus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Rhinopithecus bieti) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Rhinopithecus roxellana) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Rousettus aegyptiacus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Sus scrofa) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Theropithecus gelada) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Trachymyrmex septentrionalis) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Trichinella papuae) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Ursus arctos) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Ursus maritimus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Xenopus laevis) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Sequence-based reagent | Skd3 (Zalophus californianus) | This study | Full description can be found in Materials and methods: Purification of Skd3 | |
Software, algorithm | BOXSHADE | Hofmann and Baron, 1996 | ||
Software, algorithm | Clusal Omega | Madeira et al., 2019 | ||
Software, algorithm | EXPASY ProtParam | Kyte and Doolittle, 1982; Wilkins et al., 1999 | ||
Software, algorithm | FigTree | Rambaut, 2012 | ||
Software, algorithm | Gene Ontology (GO) | Ashburner et al., 2000; Mi et al., 2019; The Gene Ontology Consortium, 2019 | ||
Software, algorithm | IUPRED | Mészáros et al., 2018; Mészáros et al., 2009 | ||
Software, algorithm | PhyloPic | http://phylopic.org/ | ||
Software, algorithm | Prism 8 | GraphPad | ||
Software, algorithm | WebLogo | https://weblogo. berkeley.edu/logo.cgi |
Additional files
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Supplementary file 1
Alignment of Skd3 to diverse metazoan lineages shows conservation of key motifs and domains.
Alignment of Skd3 protein from diverse metazoan lineages. Alignment was constructed using Clustal Omega. Alignment shows high level of conservation of Skd3 among species. H. sapiens, G. gorilla, and C. jacchus Skd3 have an additional insertion in the ankyrin-repeat domain that is not conserved in the other species. This alignment was used to generate the phylogenetic tree in Figure 1B. The protozoan M. brevicollis Skd3 sequence was included in the alignment for reference. MTS (mitochondrial-targeting sequence, ANK (ankyrin-repeat domain), NBD (nucleotide-binding domain), and CTD (C-terminal domain).
- https://cdn.elifesciences.org/articles/55279/elife-55279-supp1-v2.docx
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Supplementary file 2
Proteins enriched in HAP1 ΔCLPB pellet.
Proteins from mass spectrometry data in Figure 7b highlighted in red that have >2.0 fold change increase in the ΔCLPB insoluble fraction compared to wild type and a p-value of <0.05.
- https://cdn.elifesciences.org/articles/55279/elife-55279-supp2-v2.xlsx
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Supplementary file 3
Proteins enriched in HAP1 WT pellet.
Proteins from mass spectrometry data in Figure 7b highlighted in green that have >2.0 fold change increase in the wild-type insoluble fraction compared to ΔCLPB and a p-value of <0.05.
- https://cdn.elifesciences.org/articles/55279/elife-55279-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55279/elife-55279-transrepform-v2.docx