CLPB disaggregase dysfunction impacts the functional integrity of the proteolytic SPY complex

Abstract

CLPB is a mitochondrial intermembrane space AAA+ domain-containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease. Utilizing proteomic and biochemical approaches, we show a stress-specific aggregation phenotype in a CLPB-null environment and define the CLPB substrate profile. We illustrate an interplay between intermembrane space proteins including CLPB, HAX1, HTRA2, and the inner membrane quality control proteins (STOML2, PARL, YME1L1; SPY complex), with CLPB deficiency impeding SPY complex function by virtue of protein aggregation in the intermembrane space. We conclude that there is an interdependency of mitochondrial QC components at the intermembrane space/inner membrane interface, and perturbations to this network may underscore CLPB disease pathology.

Figure S1.

Mutation of Walker A or B motifs may impact CLPB localization. (A) Schematic representation of indels present in the CLPB^KO HEK FlpIn-TREx cell line (Clone #4), introduced using CRISPR-Cas9 genome editing, according to CLPB transcript variant 1 (NM_030813.6). Exon 1 is shared by all CLPB variants. (B) Schematic representation of CLPB domain architecture, with relative positions of Walker A (K387A) and Walker B (E455Q) mutant loci indicated within the NBD. MTS, mitochondrial targeting signal; TMD, transmembrane domain. (C–E) Mitochondria were isolated from (C) CLPB^WT-FLAG, (D) CLPB^WA-FLAG, and (E) CLPB^WB-FLAG stable cell lines and treated for sub-fractionation of the mitochondrial compartments (left), and carbonate extraction (right). Samples were run on SDS-PAGE and analyzed via immunoblotting (IB). * = protease protected protein. S = supernatant, P = pellet. (F) Schematic depicting processing of L-OPA1 splice variants 1 (b) and 7 (a) (Sp1/7). OMA1 cleavage of L-OPA1 a or b at S1 yields S-OPA1 c and e splice variants, respectively. Subsequent processing by YME1L1 at S2 of c-form S-OPA1 yields d-form S-OPA1. MTS = mitochondrial targeting sequence. TM = transmembrane domain. GED = GTPase effector domain. Figure adapted from Baker et al. (2014). Source data are available for this figure: SourceData FS1.

Figure 1.

Loss of CLPB influences mitochondrial health. (A) Mitochondria were isolated from control and CLPB^KO cells and subjected to LFQ MS. Data are presented as a volcano plot depicting the level of proteins in CLPB^KO relative to control (n = 3). Proteins above the horizontal cutoff (P value <0.05, Student's t test) and outside of either vertical line (1.5-fold absolute change) are regarded as significantly altered in abundance. Functional annotations correspond to MitoCarta 3.0 categorization. (B) Mitochondria were isolated from control and CLPB^KO cells and prepared for BN-PAGE (left) and SDS-PAGE (right). For BN-PAGE analysis, mitochondria were solubilized in digitonin-containing buffer and separated on a 4–16% acrylamide BN-PAGE gel, followed by immunoblotting (IB) with the indicated antibodies. For SDS-PAGE, mitochondrial pellets were resuspended in SDS-containing loading buffer and run on a 10–16% acrylamide tris-tricine gel followed by immunoblotting with the indicated antibodies. (C) OCRs were quantified in control and CLPB^KO cells using a Seahorse analyzer. Left: Oligomycin, FCCP, and antimycin A/rotenone were added at the indicated time points to measure basal, maximal, and non-mitochondrial respiration rates. Right: Calculated basal and maximal OCR rates. Data averaged over three independent experiments. Error bars represent mean ± SD (n = 3), two-sample t test; *P < 0.05, **P < 0.01. (D) ECAR (mpH/min)/OCR (pmol/min) ratio calculations as obtained in C. Error bars represent mean ± SD (n = 3), two-sample t test; **P < 0.01. (E) Measurement of H₂O₂ ROS in control and CLPB^KO cell lines after a 2 h vehicle (DMSO) or 10 µM menadione pre-treatment. Error bars represent mean ± SD (n = 3 for both vehicle and menadione treated). Two-sample t test; ***P < 0.001. ****P < 0.0001. (F) Mitochondrial sub-fractionation (lanes 1–6) and carbonate extraction (lanes 7–10) on mitochondria isolated from HEK wild-type cells to examine endogenous CLPB localization. Samples were run on a 10–16% acrylamide tris-tricine gel and immunoblotted with the indicated antibodies. S = supernatant, P = pellet. TOMM20 = OM localized control, TIMM29 = IM localized control, TIMM44 = matrix localized control, * = protease protected protein. (G) OPA1 processing in control, CLPB^KO, and induced CLPB^WT-FLAG, CLPB^WA-FLAG, and CLPB^WB-FLAG cell lines. Isolated mitochondria were run on a 8–10% acrylamide tris-tricine gel and and immunoblotted with antibodies as indicated. Stable cell lines were induced with 0.1 µg/ml tetracycline for 14 h prior to mitochondrial isolation. Source data are available for this figure: SourceData F1.

Figure 2.

CLPB deficiency triggers excessive protein aggregation. (A) Schematic representation of the aggregation assay pipeline utilized in this study. (B and C) Protein insolubility in CLPB^KO without (B) and with (C) heat shock (+HS). Isolated mitochondria from control and CLPB^KO cells (untreated or following a 2 h HS at 42°C) were prepared for LFQ MS as outlined in A. Data is presented as a volcano plot depicting the level of proteins in the CLPB^KO pellets relative to control (n = 3). Proteins above the horizontal cutoff (P value <0.05, Student's t test) and outside of either vertical line (1.5-fold absolute change) are regarded as significantly altered in abundance. To correct for anticipated differences in protein abundance within CLPB^KO, data were normalized against CLPB^KO proteomics (Fig. 1 A and Table S3). Proteins labeled in red had a fold difference >1.5 following normalization and are regarded as most strongly insoluble. Proteins labeled in dark red in C are also identified as insoluble in B. (D) Fold change comparison between proteins identified in the pellet fraction of: CLPB^KO (−HS) (B), CLPB^WT-FLAG (−HS), CLPB^KO (+HS) (C), and CLPB^WT-FLAG (+HS) following normalization to CLPB^KO proteomics (Fig. 1 A and Table S3). The CLPB^WT-FLAG stable cell line was induced with 0.1 µg/ml tetracycline for 12 h prior to immediate HS, or 14 h without HS. Values in bold are “strongly insoluble,” with fold difference >1.5 following normalization to CLPB^KO proteomics. ND = not detected. (E) Immunofluorescence imaging reveals discrete HTRA2 foci in HeLa CLPB^KO cells. HeLa control, CLPB^KO, and CLPB^KO cells stably expressing CLPB^WT-FLAG or CLPB^WB-FLAG were stained with MitoTracker Deep Red (magenta) and subjected to immunofluorescence analysis with antibodies directed against HTRA2 (green) and FLAG (cyan). Scale bar is equivalent to 20 or 5 µM (magnified) as indicated. (F) Monitoring HTRA2 foci clearance following CLPB^WT-FLAG re-expression in HeLa CLPB^KO background. CLPB^WT-FLAG was transiently expressed in control and CLPB^KO HeLa for indicated time points. Cells were stained with MitoTracker Deep Red (magenta) and subjected to immunofluorescence analysis with antibodies directed against HTRA2 (green) and FLAG (cyan). Scale bar is equivalent to 5 µM.

Figure S2.

Aggregation-prone proteins are lost from corresponding CLPB^KO soluble fractions. (A) Mitochondria (50 µg pellets) isolated from control and CLPB^KO were solubilized in Triton X-100 containing buffer (in duplicate) and incubated prior to fractionation. Samples were run on a 10–16% tris-tricine gel followed by immunoblotting (IB) with indicated antibodies. S = supernatant, P = pellet. (B) Quantification of protein abundance in the respective soluble fraction as in A. Calculated as mean percentage ± SD of total for each protein, normalized to the level of succinate dehydrogenase (complex II) subunit A (SDHA). Significance determined by Student’s two-sample t test (n = 3). ****P < 0.0001, NS = not significant, P > 0.05. (C and D) Supernatant fractions corresponding to (C) basal and (D) + HS aggregation assay pellet data from Fig. 2, B and C, respectively. Data are presented as a volcano plot depicting the level of proteins in the CLPB^KO supernatants relative to control (n = 3). Proteins above the horizontal cutoff (P value <0.05, Student's t test) and outside of either vertical line (1.5-fold absolute change) are regarded as significantly altered in abundance. To correct for anticipated differences in protein abundance within the CLPB^KO cell line, data were normalized against CLPB^KO proteomics (Fig. 1 A and Table S3). Proteins labeled in red had a fold difference >1.5 following normalization and are regarded as most depleted from the soluble fraction. Source data are available for this figure: SourceData FS2.

Figure 3.

Defining the CLPB interactome and substrate profile. (A–C) Mitochondria were isolated from control, (A) CLPB^WT-FLAG, (B) CLPB^WA-FLAG, or (C) CLPB^WB-FLAG stable cell lines following 14 h induction with 0.1 µg/ml tetracycline. Samples were processed for FLAG IP and eluates were prepared for LFQ MS. Data are presented as a volcano plot depicting the level of enrichment relative to control (n = 3). Proteins above the horizontal cutoff (P value <0.05, Student's t test) and to the right of the vertical line (eight-fold absolute change) are regarded as significantly enriched. Functional annotations correspond to MitoCarta 3.0 categorization. (D) Table containing all significantly enriched proteins across CLPB^WT-FLAG (WT), CLPB^WA-FLAG (WA), and CLPB^WB-FLAG (WB) IP datasets with corresponding fold change across each experiment. Mitochondrial compartmentalization is listed according to MitoCarta 3.0 categorization. HAX1, HTRA2, TIMM13, TIMM8A, and TIMM8B were manually assigned to the IMS. (E) Venn diagram of all detected CLPB^WA-FLAG and CLPB^WB-FLAG interacting proteins against all proteins that were predominantly more insoluble in CLPB^KO with or without HS. Proteins common to both groups are defined as CLPB interactors. (F) Visualization of common interacting partners between CLPB^WA-FLAG and CLPB^WB-FLAG IP datasets using the STRING database webtool (Szklarczyk et al., 2019). All active interaction sources were utilized. Functional annotations correspond to MitoCarta 3.0 categorization, and proteins with “import” or “OXPHOS” annotations have been excluded for clarity.

Figure S3.

Neither STOML2^KO or HAX1^KO impede CLPB localization or disaggregase activity. (A) Schematic representation of indels present in the STOML2^KO HEK FlpIn-TREx cell line, introduced using CRISPR-Cas9 genome editing, according to STOML2 transcript variant 1 (NM_013442.3). Exon 3 is shared by all STOML2 variants. (B) Schematic representation of the homozygous indel present in the HAX1^KO HEK FlpIn-TREx cell line, introduced using CRISPR-Cas9 genome editing, according to HAX1 transcript variant 1 (NM_006118.4). This mutation impacts both HAX1 isoforms a and b. (C and D) STOML2^KO and HAX1^KO HEK mitochondrial proteomics. Mitochondria were isolated from control and (C) STOML2^KO or (D) HAX1^KO HEK cell lines and subjected to LFQ MS. Data are presented as a volcano plot depicting the level of proteins in (C) STOML2^KO or (D) HAX1^KO relative to control (n = 3). Proteins above the horizontal cutoff (P value <0.05, Student's t test) and outside of either vertical line (1.5-fold absolute change) are regarded as significantly altered in abundance. Functional annotations correspond to MitoCarta 3.0 categorization. (E and F) Mitochondrial sub-fractionation and carbonate extraction on mitochondria isolated from (E) STOML2^KO cells and (F) HAX1^KO cells to examine endogenous CLPB localization. Samples were run a 10–16% acrylamide tris-tricine gel and analyzed via immunoblotting (IB). * = protease protected protein. S = supernatant, P = pellet. (G and H) STOML2^KO or (H) HAX1^KO cells were exposed to heat shock (HS) for 2 h at 42°C and pellet fractions (insoluble proteins) were prepared for LFQ MS as outlined in Fig. 2 A. Data is presented as a volcano plot depicting the level of proteins in the STOML2^KO pellet relative to control (n = 3). Proteins above the horizontal cutoff (P value <0.05, Student's t test) and outside of either vertical line (1.5-fold absolute change) are regarded as significantly altered in abundance. To correct for anticipated differences in protein abundance within the STOML2^KO or HAX1^KO, data were normalized against STOML2^KO mitochondrial proteomics (Fig. S3 C and Table S3) or HAX1^KO mitochondrial proteomics (Fig. S3 D and Table S3). Proteins labeled in red had a fold difference >1.5 following normalization and are regarded as most strongly insoluble. Source data are available for this figure: SourceData FS3.

Figure S4.

SPY complex instability is specific to CLPB^KO. (A) Mitochondria from control, CLPB^KO, HAX1^KO, and STOML2^KO were resuspended in solubilization buffer containing 1% digitonin. Lysates were run on a 4–16% acrylamide BN-PAGE gel and analyzed by immunoblotting (IB) with an anti-STOML2 antibody to assess integrity of the SPY complex. (B) Mitochondria isolated from control and CLPB^KO clones #1, #4, and #6 were analyzed by BN-PAGE (left) and SDS-PAGE (right). Mitochondria were solubilized in 1% digitonin for BN-PAGE prior to electrophoresis or resuspended in SDS-loading dye prior to SDS-PAGE. Gels were analyzed by immunoblotting with antibodies as indicated. (C–E) SPY complex integrity was assessed by BN-PAGE following induction of CLPB^WT-FLAG, CLPB^WA-FLAG, and CLPB^WB-FLAG with 1 µg/ml tetracycline for the indicated duration. Isolated mitochondria were resuspended in solubilization buffer containing 1% digitonin and lysates were run on a continuous 4–10% acrylamide BN-PAGE gel followed by immunoblotting with an anti-STOML2 antibody to assess SPY complex recovery over time. (F) Table of Log₂ fold changes of known YME1L1 substrates from CLPB^KO (Fig. 1 A and Table S1) and STOML2^KO (Fig. S3 C and Table S1) isolated mitochondrial proteomics. YME1L1 substrates listed are as defined in MacVicar et al. (2019). (G and H) Mitochondria were isolated from control and STOML2^WT-FLAG stable cell lines following (G) 4 h (minimal expression) or (H) 16 h (extended expression) induction with 1 µg/ml tetracycline. STOML2^WT-FLAG interactors were captured via IP and eluates were then prepared for LFQ MS. Data is presented as a volcano plot depicting the level enrichment relative to control (n = 3). Proteins above the horizontal cutoff (P value <0.05, Student's t test) and to the right of the vertical line (eight-fold change) are regarded as significantly enriched. Proteins in red are components of the SPY complex. Source data are available for this figure: SourceData FS4.

Figure 4.

CLPB disaggregase is required to maintain SPY integrity. (A) Mitochondria isolated from control, CLPB^KO, CLPB^WT-FLAG, and CLPB^WB-FLAG cells were prepared for BN-PAGE (left) and SDS-PAGE (right) analysis. For BN-PAGE, mitochondrial pellets were solubilized in digitonin-containing buffer and lysates were analyzed on a 4–16% gradient BN-PAGE gel and immunoblotted (IB) with the indicated antibodies. SDS-PAGE samples were solubilized in SDS-loading buffer prior to tris-tricine electrophoresis and immunoblotting with the indicated antibodies. *, stably expressed CLPB band lacking FLAG. (B) Quantification of STOML2 signal on BN-PAGE from A. Calculated as mean percentage ± SD of STOML2 signal, normalized to the level of SDHA (n = 4). (C) Mitochondria from control, CLPB^KO, CLPB^WT-FLAG, or CLPB^WB-FLAG were solubilized in digitonin-containing buffer and separated into insoluble and soluble fractions by centrifugation and analyzed by SDS-PAGE and immunoblotting. (D) Quantification of STOML2 abundance in “Soluble” fraction shown in C. Calculated as mean percentage ± SD of “Total solubilized” for each cell line, normalized to SDHA level (n = 4). (E) Control, CLPB^KO, and STOML2^KO cell pellets were harvested following CHX treatment and solubilized in RIPA lysis buffer prior to SDS-PAGE and immunoblotting with antibodies as indicated. p, precursor form; m, mature form. (F) Quantification of PGAM5 processing as shown in E. Calculated as mean percentage ± SD of total PGAM5 signal (sum of p-PGAM5 and m-PGAM5 signals) across each time point per cell line, normalized to acylglycerol kinase (AGK) level (n = 4). (G) Mitochondria were isolated from control STOML2^WT-FLAG (STOML2^KO + STOML2^WT-FLAG) and STOML2/CLPB^dKO stable cell lines expressing STOML2^WT-FLAG. Interactors of STOML2^WT-FLAG were captured via IP and eluates were analyzed by LFQ MS. Data are presented as a volcano plot depicting the levels of interacting partners in CLPB^KO as compared with control (n = 3). Proteins on the right of the volcano are more enriched in CLPB^KO and proteins on the left of the volcano are more enriched in the control. Proteins above the horizontal cutoff (P value <0.05, Student's t test) and outside of the vertical lines (1.5-fold absolute change) are regarded as significantly altered in abundance. Functional annotations correspond to MitoCarta 3.0 categorization. (H) Mitochondria were isolated from control CLPB^WB-FLAG (CLPB^KO + CLPB^WB-FLAG) and CLPB/STOML2^dKO stable cell line expressing CLPB^WB-FLAG and processed as described in G. Throughout this figure, statistical significance was determined by Student’s two-sample t test = *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS = not significant, P > 0.05. Source data are available for this figure: SourceData F4.

Figure S5.

STOML2/CLPB^dKO has a cumulative impact on OXPHOS integrity. (A) Quantification of HAX1 turnover (left) and residual HAX1 remaining in isolated mitochondria following 20 h CHX treatment (right). HAX1 processing was calculated as mean percentage ± SD of HAX1 signal across each time point per cell line, relative to control signal and normalized to AGK level. Residual HAX1 was calculated as mean percentage ± SD of HAX1 signal remaining following CHX treatment. Significance determined by Student’s t test (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. (B) Mitochondria were isolated from control and uninduced STOML2/CLPB^dKO HEK cells and subjected to LFQ MS. Volcano plot depicts the level of proteins in STOML2/CLPB^dKO relative to control (n = 3). Proteins above the horizontal cutoff (P value <0.05, Student's t test) and outside of either vertical line (1.5-fold absolute change) are regarded as significantly altered in abundance. Functional annotations correspond to MitoCarta 3.0 categorization. (C) Topographical heatmap showing Log₂ fold difference values of CI subunits mapped against CI and supercomplex (SC) PDB structures from STOML2/CLPB^dKO mitochondria, as determined in B (Table S1). N = NADH oxidation module (N-module). Q = ubiquinone reduction module (Q-module). CI: PDB 5LDW, CIII: PDB 5XTE, CIV: PDB 5Z62. (D) Left: Mitochondria isolated from control, CLPB^KO, STOML2^KO, and STOML2/CLPB^dKO (no induction) were resuspended in solubilization buffer containing 1% digitonin. Lysates were run on a 4–16% acrylamide BN-PAGE gel, followed by immunoblotting (IB) with anti-NDUFA9 and anti-COXIV antibodies to assess OXPHOS supercomplex integrity. Right: Isolated mitochondria were resuspended in SDS-containing buffer and run on a 10–16% tris tricine gel followed by immunoblotting with indicated antibodies. Source data are available for this figure: SourceData FS5.

Figure 5.

HAX1 aggregates colocalize with SPY complex in CLPB^KO. (A and B) Control and CLPB^KO HEK cells were transiently transfected with HAX1^WT-FLAG and treated for immunofluorescence with anti-FLAG (magenta) and (A) anti-STOML2 or (B) anti-TIM44 (green) antibodies. Arrowheads denote areas of increased signal intensity consistent with the formation of HAX1-containing punctae. Scale bar is equivalent to 10 or 2 µM (zoom) as indicated. Dashed box indicates the zoom region shown on the far right. (C) Pearson’s correlation coefficient (r) analysis of 1 μm linescans of HAX1^WT-FLAG and endogenous STOML2 or TIMM44 signal in HEK control or CLPB^KO cell lines. n = ≥160 measurements per condition from three independent experiments, ≥28 images/condition. ****P <0.0001, two tailed Mann-Whitney test. (D) Mitochondria were isolated from control CLPB^WB-FLAG (CLPB^KO + CLPB^WB-FLAG) and CLPB/HAX1^dKO stable cell line expressing CLPB^WB-FLAG. Interactors were captured via IP and eluates were then prepared for LFQ MS. Data are presented as a volcano plot depicting the levels of CLPB^WB-FLAG interacting partners in HAX1^KO as compared with control (n = 3). Proteins on the right of the volcano are more enriched in the absence of HAX1. Proteins above the horizontal cutoff (P value <0.05, Student's t test) and outside of the vertical lines (1.5-fold absolute change) are regarded as significantly altered in abundance. Functional annotations correspond to MitoCarta 3.0 categorization. (E) Schematic depicting the relationship between IMS and IM protein QC mechanisms. CLPB maintains HAX1 solubility thus enabling HAX1-HTRA2 interactivity and promoting their association with SPY (left). In the absence of CLPB, HAX1 is vulnerable to aggregation, colocalizing with SPY in discrete punctae (right). Functional (soluble) interactions between HAX1-HTRA2 and SPY are lost in the absence of CLPB. Excessive aggregation in a CLPB-null environment likely drives overloading at IMS QC machinery, leading to dysregulated proteostasis. The broad phenotypic outcomes of CLPB deficiency, such as reduced OXPHOS capacity, overabundance of apoptotic factors, and extreme ROS accumulation, may be due to an inefficiency of supporting QC factors in the face of acute proteotoxic stress.