The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L

Abstract

The SPFH (stomatin, prohibitin, flotillin, HflC/K) superfamily is composed of scaffold proteins that form ring-like structures and locally specify the protein-lipid composition in a variety of cellular membranes. Stomatin-like protein 2 (SLP2) is a member of this superfamily that localizes to the mitochondrial inner membrane (IM) where it acts as a membrane organizer. Here, we report that SLP2 anchors a large protease complex composed of the rhomboid protease PARL and the i-AAA protease YME1L, which we term the SPY complex (for SLP2-PARL-YME1L). Association with SLP2 in the SPY complex regulates PARL-mediated processing of PTEN-induced kinase PINK1 and the phosphatase PGAM5 in mitochondria. Moreover, SLP2 inhibits the stress-activated peptidase OMA1, which can bind to SLP2 and cleaves PGAM5 in depolarized mitochondria. SLP2 restricts OMA1-mediated processing of the dynamin-like GTPase OPA1 allowing stress-induced mitochondrial hyperfusion under starvation conditions. Together, our results reveal an important role of SLP2 membrane scaffolds for the spatial organization of IM proteases regulating mitochondrial dynamics, quality control, and cell survival.

Keywords: OMA1; SLP2; YME1L; membrane scaffold; mitochondria; rhomboid.

Figure EV1. Proteomic identification of the SPY complex

1. A, B

   Mitochondria of PARL ^−/− FITR293T cells expressing wild‐type PARL‐FLAG were subjected to immunoprecipitation using FLAG‐specific antibodies. (A) Eluate fractions were separated by SDS–PAGE and (B) by immunoblotting using the indicated antibodies. When indicated (+CCCP; 20 μM, 1 h), mitochondria were depolarized before solubilization. Input (4%); IP, immune precipitate (100%). *Specific bands in PARL‐FLAG IP.

2. C

   BN‐PAGE analysis of mitochondria isolated from wild‐type (WT) and Slp2 ^−/− MEFs. Immunoblotting was performed using AFG3L2‐, SLP2‐, and PARL‐specific antibodies.

Figure 1. PARL is part of a large assembly in the IM

1. Volcano plot representation of PARL interaction partners. Mitochondria isolated from PARL ^−/− FITR293T cells expressing PARL‐FLAG were subjected to co‐immunoprecipitation (IP). Co‐purifying proteins were separated by SDS–PAGE and identified by quantitative MS (n = 3) (Dataset EV1).

2. Heat map of log2‐transformed LFQ intensities for three independent experiments. All significant (FDR < 0.05) proteins showing a positive ratio between PARL IP and control in (A) are shown. Gray color shows missing quantitative information. Clustering was performed using the complete method with Euclidean distance.

3. Complexome analysis of MEF mitochondria (n = 3). Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARL immunoprecipitates at a FDR level of 0.05 (A) and identified by mass spectrometry after BN‐PAGE analysis are shown after hierarchical clustering.

4. BN‐PAGE analysis of mitochondria lacking SLP2, YME1L, or PARL. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3–13% BN‐PAGE and analyzed using SLP2‐, YME1L‐, and PARL‐specific antibodies.

5. High‐resolution complexome analysis of SPY complex members SLP2, YME1L, and PARL using MEF mitochondria (n = 3). Heat maps and migration profiles are shown after separation using a 3–9% BN‐PAGE. SLP2, YME1L, and PARL migrate in a high molecular weight complex (˜2 MDa), whereas SLP2 is additionally present in complexes of ˜1.6 MDa).

Figure 2. SLP2 forms a proteolytic hub in the IM

1. A

   Co‐immunoprecipitation of endogenous SLP2, PARL, and YME1L in mitochondria isolated from human FITR293T cells using either SLP2‐ or PARL‐specific antibodies. IgG was used as a negative control. In, input (10%).

2. B, C

   Co‐immunoprecipitation of endogenous SLP2 and YME1L with PARL‐specific antibodies in mitochondria isolated from wild‐type MEFs (WT) and MEFs lacking YME1L, SLP2, or PARL. In, input (10%).

3. D

   Assembly of SLP2, PARL, and YME1L into a high molecular weight complex. Mitochondria isolated from FITR293T cells (Con) and cells inducibly expressing SLP2‐FLAG (FLAG) were solubilized in digitonin and subjected to immunoprecipitation using FLAG‐specific antibodies. Native eluates of the precipitate were analyzed by BN‐PAGE and immunoblotting using SLP2‐, PARL‐, YME1L‐, and PHB2‐specific antibodies. In, input (8%); E, eluate (100%).

4. E

   High‐resolution BN‐PAGE analysis of mitochondria lacking PARL, YME1L, or SLP2. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3–9% gradient gel containing 0–10% glycerol and analyzed using SLP2‐specific antibodies.

5. F, G

   Submitochondrial localization of SLP2. (F) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated. (G) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet (P) and supernatant (S) fractions by centrifugation. Fractions were analyzed by SDS–PAGE and immunoblotting. MFN2 served as OM marker, TIMM23, SMAC/DIABLO, and YME1L as IMS markers, and AFG3L1 and AFG3L2 as matrix marker proteins.

6. H

   Topology of the SLP2–PARL–YME1L (SPY) complex in the IM. IM, inner membrane; IMS, intermembrane space.

Source data are available online for this figure.

Figure EV2. Submitochondrial localization of PARL and YME1L

Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet (P) and supernatant (S) fraction by centrifugation. Fractions were analyzed by SDS–PAGE and immunoblotting using the indicated antibodies.

Figure 3. SLP2 modulates PGAM5 processing by PARL in the SPY complex

1. Steady state levels of proteolytic substrates of YME1L. Whole‐cell extracts of Yme1l ^−/−, Parl ^−/−, and Slp2 ^−/− MEFs were analyzed by SDS–PAGE and immunoblotting using the indicated antibodies. a–b, L‐OPA1 forms. c–e, S‐OPA1 forms.

2. PGAM5 processing depends on proteolytically active PARL. PARL and catalytic inactive PARL^S277A harboring C‐terminal FLAG epitopes were expressed under the control of a tetracycline (tet)‐inducible promoter in wild‐type (WT) and PARL ^−/− FITR293T cells as indicated. Processing of L‐PGAM5 to S‐PGAM5 was monitored by immunoblotting.

3. Accelerated processing of L‐PGAM5 in Slp2 ^−/− cells. Processing of L‐PGAM5 was analyzed by immunoblotting in wild‐type (WT), Yme1l ^−/−, Parl ^−/−, and Slp2 ^−/− MEFs after inhibition of cytosolic protein synthesis by cycloheximide (CHX). A quantification of L‐PGAM5 levels at different time points is shown in the lower panel. Two‐way ANOVA analysis (n = 3; ****P < 0.0001). Error bars indicate SEM.

4. The accelerated processing of L‐PGAM5 in Slp2 ^−/− cells is mediated by PARL. PARL was depleted from Slp2 ^−/− cells by RNAi prior to CHX treatment. A quantification of L‐PGAM5 levels at different time points is shown. Two‐way ANOVA analysis (n = 3; **P < 0.01. ****P < 0.0001). Error bars indicate SEM.

5. PGAM5 associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL ^−/− FITR293T cells expressing PARL‐FLAG (PARL‐WT) or PARL^S277A‐FLAG (PARL‐S277A) were solubilized in digitonin and were analyzed by BN‐PAGE and immunoblotting using FLAG‐ and PGAM5‐specific antibodies.

Figure EV3. Stability of YME1L substrate proteins in cells expressing YME1L^E381Q

1. After tetracycline‐induced expression of wild‐type YME1L (YME1L^WT) or a mutant variant thereof harboring a mutation in the Walker B motif (YME1L^WB) in FITR293T cells and inhibition of cytosolic protein synthesis with cycloheximide (CHX), cells were further incubated to monitor the stability of YME1L and its substrates PRELID1 and TIMM23. Samples were analyzed by SDS–PAGE and immunoblotting. *Unspecific cross‐reaction.

2. Quantification of S‐OPA1 form d and PRELID1 in experiments shown in Fig 3A. Parametric t‐tests (unpaired, two‐tailed) were performed. P‐values were categorized as follows: *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001 (n = 3). Error bars represent SD.

Figure EV4. Submitochondrial localization of PGAM5

PGAM5 is localized in the IM. Subfractionation of mitochondria by osmotic swelling and membrane solubilization with Triton X‐100 (TX‐100) combined with proteinase K protection assays identify both forms of PGAM5, L‐PGAM5, and S‐PGAM5, in the IM fraction. The following marker proteins were used: TOMM20 (OM), YME1L (IMS), PARL (IM), and HSP70 (matrix).

Figure 4. The SPY complex facilitates PINK1 processing by PARL

1. PARL‐dependent cleavage of PINK1‐HA is reduced in Slp2 ^−/− and Yme1l ^−/− MEFs. Whole‐cell extracts of Slp2 ^−/−, Parl ^−/−, and Yme1l ^−/− MEFs expressing PINK1‐HA were analyzed by SDS–PAGE and immunoblotting using the indicated antibodies. Cells were treated with 20 μM MG132 or 20 μM CCCP for 4 h.

2. Quantification of the protein ratio (log2) PINK1‐HA 52 kDa/63 kDa in the presence of MG132 (n = 3; *P < 0.05; one‐way ANOVA). n.s., not significant. Error bars indicate SEM.

3. PINK1‐HA associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL ^−/− FITR293T cells expressing PARL‐FLAG (PARL‐WT) or PARL^S277A‐FLAG (PARL‐S277A) were solubilized in digitonin and analyzed by BN‐PAGE and immunoblotting using FLAG‐, HA‐, and SLP2‐specific antibodies.

4. PINK1 associates with SLP2. Mitochondria isolated from FITR293T cells (WT) or PARL ^−/− FITR293T cells, depleted of SLP2 by siRNA as indicated, were solubilized in digitonin and analyzed by BN‐PAGE and immunoblotting using SLP2‐ and PINK1‐specific antibodies.

Figure 5. OMA1 cleaves PGAM5 under stress and is regulated by SLP2

1. Processing of L‐PGAM5 to S‐PGAM5 in depolarized mitochondria of wild‐type (WT), Slp2 ^−/−, Yme1l ^−/−, and Parl ^−/− MEFs. PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide (CHX). A quantification of L‐PGAM5 levels at different time points is shown. L‐PGAM5 levels at t = 0 were set to 100%. Two‐way ANOVA analysis (n = 3; *P < 0.05, **P < 0.01. ****P < 0.0001). Arrowheads denote intermediate PGAM5 cleavage products. Error bars indicate SEM.

2. OMA1‐mediated processing of PGAM5 in depolarized Parl ^−/− mitochondria. Processing of L‐PGAM5 to S‐PGAM5 was assessed as in (A) in the presence or absence of CCCP (20 μM) in Parl ^−/− mitochondria which were depleted of OMA1 by siRNA as indicated. The arrowhead denotes an intermediate PGAM5 cleavage product. SCR, scrambled.

3. Impaired PGAM5 processing in depolarized Oma1 ^−/− mitochondria lacking PARL. Processing of L‐PGAM5 to S‐PGAM5 was examined as in (A) after 2 h in wild‐type (WT) and Oma1 ^−/− mitochondria in the presence or absence of CCCP (20 μM, 2 h), which were depleted of PARL by siRNA as indicated. *Unspecific cross‐reaction.

4. OMA1 mediates accelerated processing of L‐OPA1 in Slp2 ^−/− MEFs. Processing of OPA1 and PGAM5 was monitored in Slp2 ^−/− , Oma1 ^−/−, and Slp2 ^−/− Oma1 ^−/− cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide (CHX). a–b, L‐OPA1 forms. c–e, S‐OPA1 forms.

5. OMA1 interacts with SLP2. OMA1 ^−/− FITR293T cells ectopically expressing OMA1‐myc were transfected with pcDNA5 (Control) or pcDNA5‐SLP2‐FLAG (FLAG). Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG‐specific antibodies. Native eluates of the precipitate were analyzed by BN‐PAGE and immunoblotting using SLP2‐, YME1L‐, PARL‐, and myc‐specific antibodies. In, input (8%); E, eluate (100%). Samples were analyzed by SDS–PAGE in parallel (lower panel).

6. Quantification of immunoprecipitation efficiencies in (E).

7. Model for the regulation of IM proteases by the membrane scaffold SLP2. SLP2 inhibits processing of L‐OPA1 by OMA1 and of PGAM5 by PARL, while facilitating PINK1 cleavage by PARL, thus modulating mitochondrial morphology, quality control, and cell survival.

Figure EV5. OMA1 cleaves PGAM5 in depolarized mitochondria and is regulated by SLP2

1. Immunoblot analysis of L‐PGAM5 and S‐PGAM5 in wild‐type (WT) and PARL ^−/−, OMA1 ^−/−, and OMA1 ^−/− PARL ^−/− FITR293T cells treated with CCCP for 0, 1, and 4 h.

2. Tubular mitochondria are reduced in Slp2 ^−/− cells and can be rescued by OMA1 depletion. Quantification of mitochondrial morphology (> 100 cells, n = 3; **P < 0.01, ****P < 0.0001). n.s., not significant. Error bars represent SEM. Scale bar, 15 ?m.

3. Stress‐induced mitochondrial hyperfusion (SiMH) induced by CHX (10 μM; 2 h) is impaired in Slp2 ^−/− cells and is rescued by OMA1 depletion. Quantification of mitochondrial morphology (> 100 cells, n = 3; ****P < 0.0001). n.s., not significant. Error bars represent SEM. Scale bar, 15 ?m.