Research ArticleCELL BIOLOGY

RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway

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Science Advances  21 Nov 2018:
Vol. 4, no. 11, eaav0443
DOI: 10.1126/sciadv.aav0443
  • Fig. 1 TBK1 is dynamically activated in response to mitochondrial depolarization and promotes phosphorylation of RAB7A on S72 within switch II.

    (A and B) HFT PARKINWT cells treated with doxycycline (DOX) were depolarized with AO for the indicated periods of time before analysis of whole-cell extracts (WCE) by SDS–polyacrylamide gel electrophoresis (PAGE) using the indicated antibodies. (C) Schematic for the creation of HFT-PARKINWT cells (with or without previous deletion of PINK1) expressing GFP-TBK1 using CRISPR-Cas9. (D) HFT-PARKINWT cells with or without either PINK1 or GFP-TBK1 were depolarized with AO for 1 hour before immunoblotting (IB) of whole-cell extracts with the indicated antibodies. (E and F) Quantitative analysis of pS172 in TBK1 in the presence and absence of AO (1 hour) was performed using PRM. Error bars represent SEM from biological triplicate measurements. UT, untreated. (G) Schematic workflow for TBK1-dependent phosphoproteome discovery. Whole-cell extracts from the indicated cells (duplicates for untreated and 1-hour treatments and a single replicate for 1.5 hours of depolarization) were cleaved with Lys-C and trypsin, phosphopeptides were enriched using TiO2, and samples were labeled for 10-plex TMT before analysis using SPS-MS3 (26). m/z, mass/charge ratio. (H) Peptide and protein quantification from TMT proteomics of experiment outlined in (G). (I) Dynamics of pS65-Ub in response to mitochondrial depolarization from the experiment outlined in (G). (J) Dynamics of phosphorylation sites in RAB7A, RMD3, VIM, and SQSTM1 from the experiment outlined in (G). (K) Structure of RAB7A (Protein Data Bank: 1T91) showing the location of switch I in blue, switch II in magenta, S72 in green, and GTP in red.

  • Fig. 2 Dynamic phosphorylation of a pool of RAB7A on S72 in vivo in response to mitochondrial depolarization requires PARKIN and PINK1.

    (A) Schematic for experiments examining the phosphorylation of S72 in RAB7A by TBK1. The indicated cells were subjected to depolarization, and cell extracts were subjected to α-FLAG immunoprecipitation (IP) before either Phostag-PAGE or PRM proteomics. (B) Immunoblotting of whole-cell extracts from the indicated cells using α-RAB7A or α-actin as a loading control. (C) α-FLAG immunoprecipitates from the indicated cells were separated by Phostag-PAGE followed by immunoblotting with the indicated antibodies. (D and E) Quantitative analysis of pS72 in RAB7A in the presence and absence of AO (1 hour) was performed using PRM. Error bars represent SEM from triplicate measurements. (F) TBK1 requirement for RAB7AS72 phosphorylation in response to AO (1 hour). The indicated cells were subjected to depolarization, and cell extracts were subjected to α-FLAG immunoprecipitation before Phostag-PAGE and immunoblotting with α-RAB7A antibodies. (G) HFT-PARKINWT or S65A cells with or without PINK1 were depolarized for the indicated times and purified mitochondria enriched for phosphopeptides using immobilized metal anion chromatography (IMAC) before TMT-based proteomics. RP, reversed-phase. (H) Volcano plot of −log(P value) versus the log2FC (fold change) for quantified phosphopeptides for PARKINWT cells with or without AO treatment for 1 hour. Phosphorylation site and number of peptides quantified are shown in parenthesis. (I) Relative abundance of pS72 RAB7A normalized to RAB7A abundance also measured by TMT. Error bars represent SEM from biological triplicate measurements. n.s., not significant.

  • Fig. 3 TBK1 phosphorylates RAB7AS72 but not the equivalent residue in other RABs in vitro.

    (A) Coomassie blue PAGE analysis of purified GST-RAB7AWT and RAB7AS72A after purification from E. coli. (B) The indicated GST-RAB7A proteins were incubated with recombinant TcPINK1 (E. coli) (27) or GST-TBK1 (insect cells) (1 hour at 30°C, with or without ATP), and reaction products were separated by Phostag-PAGE before immunoblotting with α-RAB7A antibody. (C) Alignment of the amino acid sequence around S72 in RAB7A (red asterisk) and selected RABs. (D) Coomassie blue SDS-PAGE analysis of purified GST-RAB1A, GST-RAB1B, and GST-RAB7L (with or without mutation of the S72 equivalent to Ala) after purification from E. coli. (E) The indicated GST-RABs were incubated with or without GST-TBK1 (1 hour at 30°C, with ATP), and reaction products were separated by Phostag-PAGE and immunoblotted with α-GST antibody. (F) Coomassie blue SDS-PAGE analysis of purified GST-RAB8AWT, RAB8AS72A, and RAB8AS111A after purification from E. coli. (G) The indicated GST-RAB8A proteins were incubated with or without GST-TBK1 and analyzed as in (E).

  • Fig. 4 Quantitative proteomic analysis of nonphosphorylatable and phosphomimetic RAB7AS72 reveals a phosphorylation-dependent interaction with FNIP1/FLCN.

    (A) Triton X-100 (1%) lysates from HFT PARKINWT;RAB7A−/− cells reconstituted with FLAG-HA-RAB7AWT or the S72A or S72E mutants (see Fig. 2B) were subjected to α-FLAG immunoprecipitation-MS in triplicate, and interacting proteins were quantified by TMT-based proteomics. Volcano plots of −qlog(P value) versus the log2FC of the indicated pairs of proteins for triplicate measurements are shown. (B) As in (A) but depolarized for 1 hour with AO. (C) Histogram of the relative abundance of selected proteins found in association with RAB7AWT and either S72A or S72E mutants. Error bars represent SEM from biological triplicate measurements. (D) The indicated cell lines were left untreated or depolarized for 1 hour with AO, and α-FLAG immunoprecipitates were subjected to immunoblotting with the indicated antibodies. (E) GST-RAB7A and pS72-GST-RAB7A were made in E. coli. For pS72-GST-RAB7A, protein was made using the amber codon suppressor system in the presence of a tRNA system for activation of phospho-Ser (35). Both proteins were purified using glutathione (GSH)–Sepharose and separated by SDS-PAGE before Coomassie staining. (F) The stoichiometry of GST-RAB7A phosphorylation on S72 was measured using PRM proteomics and found to be ~70%. (G) Schematic for use of immobilized pS72-GST-RAB7A for binding to FLCN in cell extracts. (H) Immobilized GST-RAB7A, pS72-GST-RAB7A, or GSH-resin was incubated with cell extracts (4 hours at 4°C), and washed beads were subjected to SDS-PAGE and immunoblotting with either α-FLCN or α-RAB7A antibodies. Extract (2% of input) was run as a loading control. (I) HA-FLCN/FLAG-FNIP1 complexes (6 μg, purified from mammalian cells and immobilized on α-FLAG resin) were incubated with 20 μg of GST-RAB7AWT or S72A, S72E, or T22N mutants (3 hours at 4°C). Washed resin was subjected to SDS-PAGE and immunoblotting with the indicated antibodies. Input proteins were similarly analyzed in parallel.

  • Fig. 5 RAB7AS72A fails to support recruitment of FNIP1/FLCN to depolarized mitochondria.

    (A) The indicated HFT-PARKINWT cell lines were treated with DOX (16 hours) to induce PARKIN and depolarized with AO (1 hour), and mitochondrial extracts were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (B) Quantification of biological triplicate experiments as described in (A). Error bars represent SEM. (C) Schematic describing how S72 in RAB7A was replaced by Ala using CRISPR-Cas9. The replacement template used was single-stranded DNA containing S72A. (D and E) HFT-PARKINWT cell lines were treated with DOX (16 hours) to induce PARKIN and depolarized with AO for 1 hour, and either whole-cell lysates (D) or mitochondrial extracts (E) were subjected to SDS-PAGE and immunoblotting using the indicated antibodies. (F) Quantification of biological triplicate experiments as described in (B). Error bars represent SEM.

  • Fig. 6 Cells harboring nonphosphorylatable RAB7A are defective in mitophagic flux and fail to efficiently recruit ATG9A to damaged mitochondria.

    (A) Top: Example images of individual HFT-PARKINWT cells 32 hours after depolarization stained with α-DNA antibodies to detect unaggregated, aggregated, or cleared mitochondrial. Bottom: Histogram of the results of PARKIN-dependent mitophagy assays 32 hours after depolarization using the indicated cells and analyzed as in the top panel of (A). Error bars represent SEM from biological triplicate measurements, with >100 cells analyzed per replicate. Cells were treated with DOX for 2.5 hours to induce PARKIN before depolarization. (B) Schematic workflow for analysis of proteins enriched in lysosomes from cells expressing wild-type (WT) or S72A RAB7A. Extracts from the indicated cells were subjected to density centrifugation, and lysosomal fractions were subjected to 10-plex TMT using SPS-MS3 analysis (26). (C) Volcano plot [−log(P value) versus log2FC (RAB7AWT/RAB7AS72A)] of proteins present in the lysosomal fraction 16 hours after depolarization. Proteins in pink or blue represent proteins with P < 0.05 and log2FC of >1.5 or <−1.5, respectively. Mitochondrial proteins (based on MitoCarta2.0) are shown in dark red circles, while the location of RAB7A itself, the HOPS complex, and CCZ1-MON1 is shown in yellow, green, and blue circles, respectively. (D) Histogram of the relative abundance of selected mitophagy-related proteins in the lysosome based on the data in (C). (E) Mitophagy assays as in (A) for cells lacking FLCN with or without rescue with MSCV-FLAG-HA-FLCN. (F) The indicated cells were depolarized with AO for 2 hours, and mitochondria were purified before SDS-PAGE and immunoblotting with the indicated antibodies. SA, S72A. (G) Working model for TBK1-driven phosphorylation of RAB7A and downstream regulation of mitophagy. See text for details. ER, endoplasmic reticulum; HM, heavy mitochondria; LM, light mitochondria; PM, plasma membrane.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/11/eaav0443/DC1

    Fig. S1. Phosphorylation of RAB7A by TBK1.

    Fig. S2. Analysis of RAB7AS72A and RAB7AS72E mutants.

    Fig. S3. Analysis of PARKIN-dependent mitophagy in RAB7A and FLCN mutant cells.

    Data File S1. Phosphoproteomic data for TBK1WT and TBK1−/− cells in response to mitochondrial depolarization (corresponds to experiment in Fig. 1).

    Data File S2. Total proteome quantification for cell extracts used in the phosphoproteomic data in Fig. 1 and data file S1.

    Data File S3. Phosphoproteome data for PARKINWT and PARKINS65A cells, as well as PINK1−/− cells in the presence and absence of depolarization (corresponds to experiment in Fig. 2).

    Data File S4. Protein interaction proteomic data for FLAG-HA-RAB7AWT, S72A, and S72E with and without mitochondrial depolarization.

    Data File S5. Proteomic data for lysosomes purified from RAB7AWT or RAB7AS72A cells in response to depolarization (corresponds to experiment in Fig. 6).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Phosphorylation of RAB7A by TBK1.
    • Fig. S2. Analysis of RAB7AS72A and RAB7AS72E mutants.
    • Fig. S3. Analysis of PARKIN-dependent mitophagy in RAB7A and FLCN mutant cells.

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Phosphoproteomic data for TBK1WT and TBK1−/− cells in response to mitochondrial depolarization (corresponds to experiment in Fig. 1).
    • Data File S2 (Microsoft Excel format). Total proteome quantification for cell extracts used in the phosphoproteomic data in Fig. 1 and data file S1.
    • Data File S3 (Microsoft Excel format). Phosphoproteome data for PARKINWT and PARKINS65A cells, as well as PINK1−/− cells in the presence and absence of depolarization (corresponds to experiment in Fig. 2).
    • Data File S4 (Microsoft Excel format). Protein interaction proteomic data for FLAG-HA-RAB7AWT, S72A, and S72E with and without mitochondrial depolarization.
    • Data File S5 (Microsoft Excel format). Proteomic data for lysosomes purified from RAB7AWT or RAB7AS72A cells in response to depolarization (corresponds to experiment in Fig. 6).

    Files in this Data Supplement:

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