Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy
- Montreal Neurological Institute, McGill University, Canada
- McGill University, Canada
- Institute of Neurogenetics, University of Lübeck, Germany
Figures
Figure 1 with 1 supplement
Ultrastructural analysis of ER-mitochondria contact during mitophagy in U2OS cells and dopaminergic neurons.
(A) Representative TEM images of mitochondria (‘M’) in contact with ER (pseudocoloured blue) in untreated and CCCP-treated U2OS:GFP-parkin cells. Scale bars, 500 nm. (B–E) Quantification of TEM from …
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Figure 1—source data 1
Numerical source data for Figure 1B–1D, E, I, J and N and Figure 1—figure supplement 1B to D.
- https://doi.org/10.7554/eLife.32866.004
Figure 1—figure supplement 1
Mitophagy reduces ER-OMM contacts of all intermembrane distances.
(A) Representative TEM image of an untreated U2OS:GFP-parkin WT cell highlighting ER-OMM distances of less than 100, 50 and 25 nm. Intermembrane distances (d) are indicated. Scale bars, 500 and 100 …
Figure 2 with 1 supplement
Mfn2 is rapidly phosphoubiquitinated upon induction of mitophagy.
(A) Immunoblot analysis of protein turnover in glucose-maintained U2OS:GFP-parkin WT and A320R cells treated with 20 μM CCCP for the indicated time. (B) Higher exposures of Mfn2 and TOM20 …
Figure 2—figure supplement 1
LC/MS of immunoprecipitated Mfn2.
(A) Base peak chromatograms indicating equal loading of both DMSO- and CCCP-treated samples from Figure 2F and G. (B) Extracted ion chromatograms of the indicated Ub and Mfn2 peptides from both …
Figure 3 with 5 supplements
Mfn2 antagonizes mitophagy.
(A) Immunoblot analysis of whole-cell lysates from cells cultured in glucose or galactose transfected with control siRNA or siRNA targeting Mfn1 (‘siMfn1’) or Mfn2 (‘siMfn2’). (B) Mitochondrial …
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Figure 3—source data 1
Numerical source data for Figure 3D, E and G, Figure 3—figure supplement 1B–D, Figures 2B, 3C, D, H, I, 4F, H and 5C.
- https://doi.org/10.7554/eLife.32866.013
Figure 3—figure supplement 1
Mfn2 is a mitochondrion-ER tether.
(A) Representative TEM images of U2OS:GFP-parkin cells transfected with the indicated siRNA. ER tubules are pseudocoloured blue. Scale bar, 500 nm. (B–D) Quantification of mitochondrial length (B), …
Figure 3—figure supplement 2
Mitochondrial respiration impedes mitophagy.
(A) Representative confocal images of U2OS:GFP-parkin (green) cells, grown on either glucose or galactose, treated with 20 μM CCCP for the indicated times. Cells were then fixed and stained for …
Figure 3—figure supplement 3
Parkin recruitment kinetics in cells lacking both Mfns and other mitochondria-ER tethering factors.
(A) Immunoblot analysis of whole-cell lysates from cells cultured in glucose or galactose transfected with control siRNA or siRNA targeting Mfn1 (‘siMfn1’), Mfn2 (‘siMfn2’) or both mitofusins …
Figure 3—figure supplement 4
Analysis of mitophagy in Mfn2 KO U2OS cells.
(A) Genomic sequence of human Mfn2 (exon 3) that was mutated in U2OS cells using CRISPR/Cas9. The arrow indicates the codon corresponding to methionine-1; leucine-29 (‘L29’), lysine-30 (‘K30’) and …
Figure 3—figure supplement 5
Parkin recruitment in Mfn2-depleted cells requires PINK1 and phosphoubiquitin binding.
(A) Immunoblot analysis of PINK1 depletion in WT and Mfn2 KO (clone A4) U2OS cells treated with 20 μM CCCP for four hours. The arrowhead indicates the PINK1 band, while the asterisk indicates a …
Figure 4 with 1 supplement
Parkin ubiquitinates Mfn2 in the HR1 domain to derepress mitophagy.
(A) Mfn2 KO:YFP-parkin WT cells were transfected with the indicated plasmid and CFP in a 3:1 ratio, then fixed and immunostained for TOM20 (red) and counterstained with Hoechst 33342 (blue). Scale …
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Figure 4—source data 1
Numerical source data for Figure 4C, F, G and K.
- https://doi.org/10.7554/eLife.32866.016
Figure 4—figure supplement 1
Location and conservation of ubiquitination and phosphorylation sites in Mfn2.
(A) Sequence alignment of sites of Mfn2 modification across species. Ubiquitinated lysines and phosphorylated serines and threonines are indicated by arrowheads. Residue numbering is according to …
Figure 5
p97 governs ER-OMM contact via the extraction of Mfn2 complexes.
(A) Immunoblot analysis of NP-40-solubilized mitochondria, isolated from U2OS:GFP-parkin WT cells treated with 20 μM CCCP for the indicated time, separated by blue native- (BN-) and SDS-PAGE. (B, C) …
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Figure 5—source data 1
Numerical source data for Figure 5G and H.
- https://doi.org/10.7554/eLife.32866.018
Figure 6 with 1 supplement
Degradation of ubiquitinated Mfn2 involves p97 recruitment and activity.
(A) Representative confocal images of p97 recruitment to mitochondria in cells treated with 20 μM CCCP and/or 25 μM NMS-873 for the indicated time. Blue asterisks denote cells with mitochondrial …
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Figure 6—source data 1
Numerical source data for Figure 6I and K and Figure 6—figure supplement 1C.
- https://doi.org/10.7554/eLife.32866.021
Figure 6—figure supplement 1
Analysis of pUb interactors from mouse brain.
(A) Workflow of protein purification, phosphorylation, pull-down and LC/MS. GST-4xUb G76V, which cannot be cleaved by the cellular Ub processing machinery, was phosphorylated on S65 by Tribolium …
Figure 7
p97 and Mfn2 effect mitophagy through parkin substrate availability.
(A) U2OS:mtKeima cells were transfected with the indicated siRNA and GFP-parkin WT, and were treated with 20 μM CCCP (or DMSO) for five hours in the presence (dark grey box) or absence (light grey …
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Figure 7—source data 1
Numerical source data for Figure 7B, D–F and H.
- https://doi.org/10.7554/eLife.32866.023
Figure 8 with 1 supplement
In organello ubiquitination of Mfn2 and VDAC1.
(A) Workflow for the in organello ubiquitination assay, where HeLa cells are depolarized with 20 μM CCCP for four hours and mitochondria are isolated (‘mitoCCCP’, with control ‘mitoDMSO’). These are …
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Figure 8—source data 1
Numerical source data for Figure 8K.
- https://doi.org/10.7554/eLife.32866.026
Figure 8—figure supplement 1
Effect of NMS-873 on cytosol-free ubiquitination.
In organello ubiquitination reactions were performed in the presence or absence of 25 μM NMS-873. Reactions were incubated on ice for 30 min prior to a 60 min 37°C incubation. In the immunoblot …
Figure 9
Dismantling of Mfn2 interorganellar bridges by PINK1, parkin and p97 during mitophagy.
(A) PINK1-phosphorylated Ub on Mfn2 initially recruits parkin to Mfn2 complexes, where it is phosphorylated and activated by PINK1. (B) Parkin and PINK1 cooperate to catalyze a pUb burst on Mfn2. (C)…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| cell line (Homo sapiens) | U2OS | PMID 24446486 | ||
| cell line (Hs) | U2OS:GFP | PMID 24446486 | ||
| cell line (Hs) | U2OS:GFP-parkin WT | PMID 24446486 | ||
| cell line (Hs) | U2OS:GFP-parkin A320R | PMID 28276439 | ||
| cell line (Hs) | Mfn2 KO | this paper | see Plasmids and transfection | |
| cell line (Hs) | Mfn2 KO:YFP-parkin WT | this paper | see Plasmids and transfection | |
| cell line (Hs) | Mfn2 KO:YFP-parkin C431S | this paper | see Plasmids and transfection | |
| cell line (Hs) | HeLa | PMID 24446486 | ||
| cell line (Hs) | control-1 | NIH | NCRM-1 | |
| cell line (Hs) | control-2 | PMID 27641647 | ||
| cell line (Hs) | PRKN(del) | PMID 20885945 | ||
| transfected construct (Hs) | HA-Ub | PMID 25216678 | ||
| transfected construct (Hs) | DsRed2-LC3 | PMID 18596167 | ||
| transfected construct (Hs) | Mfn1-HA | PMID 15878861 | ||
| transfected construct (Hs) | Mfn2 WT | PMID 15878861 | ||
| transfected construct (Hs) | Mfn2 K406R | this paper | see Plasmids and transfection | |
| transfected construct (Hs) | Mfn2 K416R | this paper | see Plasmids and transfection | |
| transfected construct (Hs) | Mfn2 K420R | this paper | see Plasmids and transfection | |
| transfected construct (Hs) | Mfn2 HR1 | this paper | see Plasmids and transfection | |
| transfected construct (Hs) | Mfn2 HR2 | this paper | see Plasmids and transfection | |
| transfected construct (Hs) | Mfn2 TS/AA | this paper | see Plasmids and transfection | |
| transfected construct (Hs) | GFP-parkin WT | PMID 24446486 | ||
| biological sample (Mus musculus) | parkin WT brain cytosol | this paper | see In organello ubiquitination assays | |
| biological sample (Mm) | parkin KO brain cytosol | this paper | see In organello ubiquitination assays | |
| antibody | anti-actin | Millipore | MAB1501 | |
| antibody | anti-B-III-tubulin | Sigma | T8660 | |
| antibody | anti-MAVS | Enzo | ALX-210–929 C100 | |
| antibody | anti-cytochrome c | BD | 556432 | |
| antibody | anti-GFP | Abcam | ab6673 | IP |
| antibody | anti-GFP | Invitrogen | A6455 | WB |
| antibody | anti-Grp78 | Santa Cruz | sc-376768 | |
| antibody | anti-HA | Abcam | ab9134 | |
| antibody | anti-HK1 | Cell Signaling | 2024S | |
| antibody | anti-Mfn1 | Santa Cruz | sc-50330 | |
| antibody | anti-Mfn2 | Sigma | M6319 | WB in Figure 3—figure supplement 2D |
| antibody | anti-Mfn2 | Cell Signaling | 9482 | all other assays (IF, WB, IP) |
| antibody | anti-CIV-COXI | Abcam | ab14705 | |
| antibody | anti-p62 | Progen | GP62-C | |
| antibody | anti-PDH E1a | Abcam | ab110330 | |
| antibody | anti-PDH E2/E3bp | Abcam | ab110333 | |
| antibody | anti-PDI | Abcam | ab2792 | |
| antibody | anti-PINK1 | Cell Signaling | 6946 | |
| antibody | anti-pS65 Ub | Millipore | ABS1513-I | |
| antibody | anti-Rab11A | Cell Signaling | 2413 | |
| antibody | anti-Miro1 | Sigma | HPA010687 | |
| antibody | anti-CII-SDHA | Abcam | ab14715 | |
| antibody | anti-Stx17 | ProteinTech | 17815–1-AP | |
| antibody | anti-TH | Pel-Freez | P40101-150 | |
| antibody | anti-TIM23 | BD | 611222 | |
| antibody | anti-TOM20 | Santa Cruz | sc-11414 | |
| antibody | anti-TOM70 | Santa Cruz | sc-390545 | |
| antibody | anti-Ub [FK2] | Enzo | BML-PW8810 | IF |
| antibody | anti-Ub [P4D1] | Santa Cruz | sc-8017 | WB |
| antibody | anti-CIII-core2 | Abcam | ab14745 | |
| antibody | anti-CIII-Rieske | Abcam | ab14746 | |
| antibody | anti-p97 | Abcam | ab11433 | |
| antibody | anti-VDAC1 | Abcam | ab14734 | |
| recombinant protein (Rattus norvegicus) | GST-R0RBR WT | PMID 23661642 | ||
| recombinant protein (Rn) | GST-R0RBR A320R | this paper | see Plasmids and transfection | |
| recombinant protein (Rn) | GST-parkin WT | PMID 28276439 | ||
| recombinant protein (Rn) | GST-parkin C431A | PMID 28276439 | ||
| recombinant protein (Hs) | UbcH7 | PMID 28276439 | ||
| recombinant protein (Hs) | UBE1 | BostonBiochem | E-305 | |
| recombinant protein (Hs) | Ubiquitin | BostonBiochem | U-100H | |
| recombinant protein (Hs) | Usp2 catalytic domain | BostonBiochem | E-504 | |
| recombinant protein (Tribolium castaneum) | TcPINK1 | PMID 24784582 | ||
| recombinant protein (Hs) | GST-4xUb G76V | PMID 23670163 | ||
| recombinant protein (Mm) | His-p97 | PMID 19506019 | ||
| commercial assay or kit | QuikChange II site-directed mutagenesis kit | Agilent | 200523 | |
| commercial assay or kit | BCA protein assay | ThermoFisher | 23227 | |
| chemical compound, drug | CCCP | Sigma | C2759 | |
| chemical compound, drug | MG132 | Sigma | M8699 | |
| chemical compound, drug | Hoechst 33342 | ThermoFisher | H3570 | |
| chemical compound, drug | NMS-873 | ApexBio | B2168 | |
| software, algorithm | BioTools | Bruker | ||
| software, algorithm | MASCOT | Matrix Science | ||
| software, algorithm | Data Analysis | Bruker | ||
| software, algorithm | ImagJ | NIH | ||
| software, algorithm | PyMOL | Schrodinger | ||
| software, algorithm | Excel | Microsoft | ||
| software, algorithm | Prism | GraphPad |
Additional files
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Supplementary file 1
MS identification of selective Ub and pUb interactors.
Table depicting GST-4xUb interactors that are selective for S65-phosphorylated (top) or unphosphorylated (bottom) Ub. p97-related data (shaded in yellow) are also depicted in Figure 6—figure supplement 1C.
- https://doi.org/10.7554/eLife.32866.028
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Transparent reporting form
- https://doi.org/10.7554/eLife.32866.029
