Research ArticleCELL BIOLOGY

Integrated proteogenetic analysis reveals the landscape of a mitochondrial-autophagosome synapse during PARK2-dependent mitophagy

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Science Advances  06 Nov 2019:
Vol. 5, no. 11, eaay4624
DOI: 10.1126/sciadv.aay4624
  • Fig. 1 Visualization and proteomic characterization of a depolarized mitochondria-autophagosome synapse using APEX2F-OPTN.

    (A) Scheme depicting spatial analysis of APEX2F-OPTN on the MOM. OPTN is depicted in an extended dimeric coiled-coil form with each UBAN domain in the dimer associated with two Ub molecules in a chain attached to a PARK2 substrate on the MOM. The proximity biotinylation zone is indicated by the gray sphere. The position of E50K and D474N mutations in OPTN is shown in yellow and orange, respectively. (B) HFT-PARK2WT;TKO;mt-Keima cells were reconstituted with WT, E50K, or D474N APEX2F-OPTN, and these cells or the corresponding HFT-PARKWT cells were left untreated or depolarized for 1 hour. Cell lysates were subjected to immunoblotting with the indicated antibodies. (C and D) EM analysis of depolarized (1 hour) APEX2-OPTNWT–expressing cell thin sections after staining with DAB/H2O2 and osmium, which deposits an electron-rich signal where APEX2F-OPTN is concentrated (38). Scale bars are indicated. (E) Scheme for 8-plex TMT analysis of proteins in proximity to APEX2F-OPTN in response to mitochondrial depolarization. Duplicate cultures of HFT-PARK2WT;OPTN−/− cells expressing APEX2F-OPTNWT were left untreated, incubated with BP, or incubated with BP and AO (1 or 3 hours) before treatment with H2O2 (1 min). Cells were quenched and lysed in denaturing buffer, and biotinylated proteins were purified using streptavidin beads before on-bead trypsinization and TMT labeling. Mixed peptide samples were subjected to SPS-MS3–based proteomics, and the intensity of TMT reporter ions for individual peptides was determined. (F) A heat map depicting four major clusters of proteins identified in the experiment outlined in (E) with log2 FC > 1.3 and −log (P value) > 2.0 is shown. Untreated samples are omitted from the heat map for simplicity. (G) Venn diagram of overlapping biotinylated proteins in proximity to either APEX2F-OPTNWT or APEX2F-OPTND474N. (H) Venn diagram of proximity biotinylated proteins identified at 1 hour after depolarization in 9-plex (fig. S2, B to D) and 8-plex APEX2F-OPTNWT experiments and in the APEX2F-TAX1BP1 experiment (fig. S2, G and H). Tier 1 proteins are found in two or more multiplexed experiments, while tier 2 proteins were found in a single experiment.

  • Fig. 2 A proximity map of autophagy receptors on damaged mitochondria.

    (A) Overview of tier 1 and tier 2 biotinylated proteins identified with APEX2F-OPTN and APEX2F-TAX1BP1. Tier 1 proteins are identified in at least two experiments, while tier 1 proteins were identified in only a single experiment. GTPase, guanosine triphosphatase. (B) Tier 1 protein fold enrichment (FE) classified according to roles for proteins in PARK2-dependent mitophagy. (C) HFT-PARK2WT;OPTN−/− cells expressing APEX2F-OPTNWT were left untreated, incubated with BP, or incubated with BP and AO (1 hour) before treatment with H2O2 (1 min). Cell extracts were either immunoblotted directly or used for streptavidin pull-downs, followed by immunoblotting with the indicated antibodies. (D) Rank of individual biotinylated proteins identified with OPTN or TAX1BP1 according to the relative abundance of 700 mitochondrial proteins measured by label-free mass spectrometry. (E) HFT_PARK2WT;WIPI2−/− cells stably expressing APEX2-WIPI2 were depolarized for 1 hour, and processes for DAB/osmium staining were followed by EM of thin sections. (F) HFT_PARK2WT;RAB7A−/− cells stably expressing APEX2-RAB7A were depolarized for 1 hour, and processes for DAB/osmium staining were followed by EM of thin sections.

  • Fig. 3 A screen for mitophagic flux regulators identifies the genetic landscape for mitophagy.

    (A) Schematic outline of the mitophagic flux CRISPR-Cas9 screen. Duplicate cultures of HFT-PARK2 cells expressing mt-Keima were infected with one of three pathway-focused gRNA libraries (trafficking and autophagy, mitochondrial proteome, or Ub pathway). PARK2 expression was induced, and cells were depolarized for 16 hours before sorting on the basis of mean emission intensity ratio at 620 nm (excitation 561 nm/excitation 488 nm). PCRed barcodes from sorted cells were subjected to sequencing and MAGeCK analysis. (B to D) Plots of MAGeCK scores [−log (FDR) versus log2 (FE)] for genes identified in trafficking and autophagy (B), mitochondrial proteome (C), and Ub pathway (D) screens. Red dots indicate candidate genes with log2 FE > 0.5 and FDR < 0.05. (E) Modules and functional classes of candidate genes identified in CRISPR-Cas9 screens for mitophagic flux. Genes in yellow are identified in the screen. Genes indicated with a single or double asterisk were enriched with a log2 FC of >0.28 or had a –log (FDR) of 0.05, respectively. (F) Heat map for candidate genes identified in each of the three CRISPR-Cas9 libraries used. Genes not detected (N.D.) with log2 FE > 0.5 are shown in gray. (G) Validation screen for candidate identified from primary screens. (H) A screen for identification of genes whose depletion increases mitophagic flux. As in (B) but with sorting at 4 hours after depolarization.

  • Fig. 4 Proteogenetic landscape of PARK2-dependent mitophagy.

    A summary of functional modules involved in autophagy and trafficking of damaged mitochondria for mitophagy is shown with individual components identified in one of the three CRISPR-Cas9 screens or by proximity biotinylation with either APEX2F-OPTN or APEX2F-TAX1BP1 indicated as shown in the legend.

  • Fig. 5 HK2, but not HK1, promotes assembly of PINK1 with the mitochondrial translocon and its activation upon mitochondrial depolarization.

    (A) HFT_PARK2WT;HK2−/− cells stably reconstituted with HK2-APEX2 were depolarized with AO (1 hour) and subjected to staining with DAB/H2O2 and osmium, which deposits an electron-rich signal where HK2-APEX2 is concentrated. Sections were then analyzed by EM. Scale bars are indicated. (B) The indicated cells (HFT-PARK2WT, HFT-PARK2WT;HK2−/−, and HFT-PARK2WT;HK2−/− expressing HK2-APEX2F) were subjected to depolarization for 32 hours, and mitochondrial DNA was stained with α-DNA antibody in biological triplicate. More than 100 cells were classified and quantified (mean ± SEM) as having unaggregated mitochondria, aggregated mitochondria, or mitochondria cleared by mitophagy. Dox, doxycycline. (C and D) The indicated HFT-PARK2WT, HFT-PARK2WT;HK2−/−, and HFT-PARK2WT;HK2−/− expressing HK2-APEX2F were either left untreated or treated with AO (1 hour). Whole-cell lysates (C) or purified mitochondria (D) were immunoblotted using the indicated antibodies. (E) The indicated HFT-PARK2WT, HFT-PARK2WT;HK1−/−, and HFT-PARK2WT;HK2−/− cell lines were either left untreated or treated with AO (1 hour) before immunoblotting as in (C). (F) Mitochondria isolated from cells in (E) were subjected to immunoblotting with indicated antibodies. (G) The indicated HFT-PARK2WT, HFT-PARK2WT;HK1−/−, HFT-PARK2WT;HK2−/−, and HFT-PARK2WT;HK2−/− cells reconstituted with either HK2WT, HK2T473A, or HK2KD-APEX2F were either left untreated or treated with AO (1 hour) and crude mitochondria immunoblotted with indicated antibodies. KD, kinase dead mutant. (H) The indicated HFT-PARK2WT;HK2−/− cells reconstituted with either HK2WT or HK23KR (K176R, K323R, K337R)-APEX2F were either left untreated or treated with AO (1 hour), and purified mitochondria were immunoblotted with the indicated antibodies. (I) Mitochondria isolated from the indicated HFT-PARK2WT, HFT-PARK2WT;HK2−/−, and HFT-PARK2WT;HK2−/− expressing HK2-APEX2F cell lines were solubilized and subjected to BN-PAGE followed by immunoblotting with indicated antibodies. (J) Quantification of high–molecular weight PINK1 complex as in (H) by densitometry analysis (ImageJ) from biological triplicate experiments (mean ± SEM).

  • Fig. 6 Landscape of the mitochondria-autophagosome synapse.

    (A) A schematic depicting a subset of proteins identified by proximity biotinylation is shown. OPTN, a Ub-binding receptor for mitophagy, associates with Ub via its C-terminal UBAN domain (dark blue) and is thought to interact minimally with a di-Ub moiety. OPTN is a coiled-coil dimeric protein and is expected to be ~20 nm in a fully extended conformation. Recruitment of APEX2-tagged OPTN to ubiquitylated PARK2 targets on the MOM allows the identification of proteins located nearby (within ~10 nm of the APEX2 domain). Biotinylated proteins identified by quantitative mass spectrometry are indicated with a “B.” The autophagosome/phagophore is recruited nearby the MOM (within ~25 to 50 nm). The approximate sizes of the TOMM40 component of the translocon, the VDAC proteins, and dimeric Ub are shown. (B) Schematic showing the potential role of HK2 as a protein that promotes assembly of PINK1 in a ~700-kDa complex with components of the translocon and PARK2. In the absence of HK2, there is reduced accumulation of PINK1 and PARK2 in the ~700-kDa complex and reduced levels of pS65-Ub.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaay4624/DC1

    Fig. S1. Characterization of APEX2F-OPTN recruitment to depolarized mitochondria.

    Fig. S2. Analysis of proximity biotinylation using APEX2F-OPTN and APEX2F-TAX1BP1 in response to mitochondrial depolarization.

    Fig. S3. Benchmarking and validation of an mt-Keima–based screening platform for CRISPR-Cas9 analysis of mitophagic flux.

    Fig. S4. The HK2 requirement for PINK1 activation is not due to ATP depletion and is also observed in H9 hES.

    Dataset S1. Tables containing proteomic identification of proteins in proximity to APEX2-OPTN at 1 and 3 hours after depolarization determined in duplicate.

    Dataset S2. Tables containing proteomic identification of proteins in proximity to APEX2-OPTN at 1 hour after depolarization in triplicate.

    Dataset S3. Tables containing proteomic identification of proteins in proximity to APEX2-OPTND474N at 1 hour after depolarization in triplicate.

    Dataset S4. Tables containing proteomic identification of proteins in proximity to APEX2-TAX1BP1 at 1 hour after depolarization in triplicate.

    Dataset S5. Tables containing target sgRNA sequences used to create custom CRISPR libraries, as well as raw sequence reads and MAGeCK scores from the mitophagic flux screens performed using mt-Keima flux assays.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. Characterization of APEX2F-OPTN recruitment to depolarized mitochondria.
    • Fig. S2. Analysis of proximity biotinylation using APEX2F-OPTN and APEX2F-TAX1BP1 in response to mitochondrial depolarization.
    • Fig. S3. Benchmarking and validation of an mt-Keima–based screening platform for CRISPR-Cas9 analysis of mitophagic flux.
    • Fig. S4. The HK2 requirement for PINK1 activation is not due to ATP depletion and is also observed in H9 hES.

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    Other Supplementary Material for this manuscript includes the following:

    • Dataset S1 (Microsoft Excel format). Tables containing proteomic identification of proteins in proximity to APEX2-OPTN at 1 and 3 hours after depolarization determined in duplicate.
    • Dataset S2 (Microsoft Excel format). Tables containing proteomic identification of proteins in proximity to APEX2-OPTN at 1 hour after depolarization in triplicate.
    • Dataset S3 (Microsoft Excel format). Tables containing proteomic identification of proteins in proximity to APEX2-OPTND474N at 1 hour after depolarization in triplicate.
    • Dataset S4 (Microsoft Excel format). Tables containing proteomic identification of proteins in proximity to APEX2-TAX1BP1 at 1 hour after depolarization in triplicate.
    • Dataset S5 (Microsoft Excel format). Tables containing target sgRNA sequences used to create custom CRISPR libraries, as well as raw sequence reads and MAGeCK scores from the mitophagic flux screens performed using mt-Keima flux assays.

    Files in this Data Supplement:

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