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Endosomal Rab cycles regulate Parkin-mediated mitophagy

  1. Koji Yamano  Is a corresponding author
  2. Chunxin Wang
  3. Shireen A Sarraf
  4. Christian Münch
  5. Reika Kikuchi
  6. Nobuo N Noda
  7. Yohei Hizukuri
  8. Masato T Kanemaki
  9. Wade Harper
  10. Keiji Tanaka
  11. Noriyuki Matsuda  Is a corresponding author
  12. Richard J Youle  Is a corresponding author
  1. Tokyo Metropolitan Institute of Medical Science, Japan
  2. National Institutes of Health, United States
  3. Harvard Medical School, United States
  4. School of Medicine, Goethe University, Germany
  5. Institute of Microbial Chemistry, Japan
  6. Kyoto University, Japan
  7. National Institute of Genetics, Research Organization of Information and Systems, Japan
  8. SOKENDAI, Japan
  9. National Institute of Genetics, ROIS, Japan
Research Article
Cite this article as: eLife 2018;7:e31326 doi: 10.7554/eLife.31326
11 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
RAB7A is recruited to damaged mitochondria during mitophagy.

The indicated HCT116 cells stably expressing mCherry-Parkin and YFP-RAB7A (A and B), mCherry-Parkin alone (C), 2HA-RAB7A alone (D), or mCherry-Parkin and indicated RAB7A mutant (E and F) were treated with DMSO or valinomycin for 3 hr followed by immunostaining. Magnified images are also shown for A-C, and E. Bars, 10 μm. (G) Quantification of RAB7A recruitment to damaged mitochondria. Overlapped RAB7A signals with TOMM20 per total RAB7A signals were measured. Total RAB7A signal in each cell set to 100%. Error bars represent mean ± SE of at least two independent experiments. Statistical differences were determined by one-way ANOVA with Dunnett’s multiple comparisons test. ***p<0.001.

https://doi.org/10.7554/eLife.31326.002
Figure 1—source data 1

This excel file contains quantification of recruitment of RAB7 WT and mutants to damaged mitochondria.

https://doi.org/10.7554/eLife.31326.005
Figure 1—figure supplement 1
RAB7A localization under basal conditions.

(A and B) The indicated HCT116 cells stably expressing mCherry-Parkin and YFP-RAB7A were subjected to immunostaining. Bars, 10 μm.

https://doi.org/10.7554/eLife.31326.003
Figure 1—figure supplement 2
Mitochondrial recruitment of endogenous RAB7A during mitophagy.

(A) HCT116 cells were subjected to immunostaining. Bars, 20 μm. (B) HCT116 cells were treated with control or RAB7A siRNAs followed by immunostaining. Bars, 20 μm. (C) The indicated HCT116 cells stably expressing mCherry-Parkin were treated with DMSO or valinomycin for 3 hr followed by immunostaining. The magnified pictures were shown in the right. Bars, 10 μm.

https://doi.org/10.7554/eLife.31326.004
RAB7A directly associates to the outer membrane of damaged mitochondria.

(A and B) The indicated HCT116 cells stably expressing YFP-LC3B, mCherry-Parkin and 2HA-RAB7A were treated with DMSO (A) or valinomycin (B) for 3 hr, and subjected to immunostaining. The magnified images of the cells treated with valinomycin were shown in a-f. Bars, 10 μm. (C) TBC1D15/17 DKO cells stably expressing mCherry-Parkin and YFP-RAB7A were treated with DMSO (a and b) or valinomycin (c–f) for 3 hr and then subjected to immunoelectron microscopy with anti-GFP antibody. Panels b and d are the magnified images of boxes in panels a and c, respectively. Bars, 500 nm.

https://doi.org/10.7554/eLife.31326.006
Figure 3 with 1 supplement
RAB7A is required for ATG9A recruitment to damaged mitochondria and encapsulation by autophagic membranes.

(A) siRNA-treated HeLa cells stably expressing mCherry-Parkin were treated with DMSO or valinomycin (Val) for 3 hr. Total cell lysates were analyzed by immunoblotting. (B) siRNA-treated HeLa cells stably expressing mCherry-Parkin and YFP-LC3B were treated with DMSO or valinomycin for 3 hr. The fixed cells were subjected to immunostaining. Images are displayed as z-stacks of five confocal slices. The magnified pictures of the cells treated with valinomycin were shown. Bars, 10 μm. (C) The number of autophagosomes containing PDHA1 inside in each cell was counted. Error bars represent mean ±SE of at least two independent experiments. Statistical differences were determined by student’s t-test. ***p<0.001. (D) The fixed cells as in (A) were subjected to immunostaining. Images are displayed as z-stacks of five confocal slices. Magnified images are shown for cells treated with valinomycin. Bars, 20 μm. (E) Quantification of ATG9A recruitment to damaged mitochondria in (D). Overlapped ATG9A signals with mitochondria-localized mCherry-Parkin per total ATG9A signals were measured. Total ATG9A signal in each cell set to 100%. Error bars represent mean ±SE. Cells from at least two independent experiments were quantified. Statistical difference was determined by student’s t-test. ***p<0.001. (F) Quantification of ATG9A localization on Golgi apparatus (see the Materials and methods for the detail). Error bars represent mean ±SE. Cells from at least two independent experiments were quantified. Statistical difference was determined by student’s t-test ***p<0.001; n.s., not significant.

https://doi.org/10.7554/eLife.31326.007
Figure 3—source data 1

The number of autophagosomes during mitophagy in cells treated with control or RAB7A siRNA.

https://doi.org/10.7554/eLife.31326.009
Figure 3—source data 2

This excel file contains quantification of ATG9A recruitment to damaged mitochondria.

https://doi.org/10.7554/eLife.31326.010
Figure 3—source data 3

Quantification of ATG9A localization.

The ratio of ATG9A on the Golgi apparatus and in the cytosol was quantified.

https://doi.org/10.7554/eLife.31326.011
Figure 3—figure supplement 1
Recruitment of autophagy-related proteins to mitochondria during mitophagy.

siRNA-treated HeLa cells stably expressing mCherry-Parkin alone or with indicated GFP-tagged proteins were treated with valinomycin for 3 hr. The fixed cells were subjected to immunostaining with anti-TOMM20 or anti-ATG16L1 antibodies. Images are displayed as z-stacks of six confocal slices. Bars, 20 μm. Percentages of cells having the indicated number of GFP-foci or ATG16L1 signal were quantified.

https://doi.org/10.7554/eLife.31326.008
Figure 3—figure supplement 1—source data 1

source data1 This excel file contains quantification of recruitment/assembly of autophagy-related proteins, GFP-ULK1, GFP-ATG13, GFP-DFCP1, GFP-WIPI1 and ATG16L1 during mitophagy.

https://doi.org/10.7554/eLife.31326.012
Figure 4 with 1 supplement
Loss of mitochondrial Rab-GAPs induced excess amounts of ATG9A on damaged mitochondria.

(A) HeLa cells stably expressing YFP-LC3B and mCherry-Parkin were treated with valinomycin for 3 hr followed by immunostaining. Magnified images of boxes (a) and (b) are shown in the right. Bars, 10 μm. (B) The indicated HCT116 cells stably expressing YFP-LC3B and mCherry-Parkin were treated with valinomycin for 3 hr followed by immunostaining. Bars, 10 μm. (C) YFP-LC3B and ATG9A signals prepared as in (B) were processed, and overlapped ATG9A signal with YFP-LC3B per total ATG9A signals in each cell were measured. Total ATG9A signal in each cell set to 100%. Error bars represent mean ±SE. Statistical difference was determined by student’s t-test. ***p<0.001.

https://doi.org/10.7554/eLife.31326.013
Figure 4—source data 1

Quantification of colocalization of ATG9A and YFP-LC3B signals during mitophagy.

https://doi.org/10.7554/eLife.31326.015
Figure 4—figure supplement 1
ATG9A and ATG16L1 recruitment to mitochondria in TBC1D15/17 DKO cells.

(A) Low-magnified images in Figure 4B were shown. Bars, 20 μm. Colocalization of the accumulation of YFP-LC3B and ATG9A in TBC1D15/17 DKO cells was indicated as arrowheads. (B) The indicated cells stably expressing mCherry-Parkin and YFP-LC3B were treated with valinomycin for 3 hr. The fixed cells were subjected to immunostaining. Bars, 20 μm. ATG16L1 dots colocalized with YFP-LC3B were indicated as arrowheads.

https://doi.org/10.7554/eLife.31326.014
RAB7A is required for mitophagy.

(A) siRNA-treated HeLa cells stably expressing YFP-Parkin were treated with valinomycin (Val) for the indicated times and total cell lysates were analyzed by immunoblotting. I and II denote unmodified and lipidated LC3B, respectively. (B) Cells in (A) were subjected to immunostaining. DAPI was used for nuclei staining since anti-mtDNA antibody non-specifically stains nuclei of the cells having no mtDNA. Magnified pictures were shown for mtDNA degradation in cells treated with valinomycin for 24 hr. Bars, 20 μm. (C) Quantification of YFP-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial and complete denote that Parkin translocates to some of or all mitochondria, respectively. Error bars represent mean ±SE and over 100 cells were counted in each of three separate wells. (D - F) Percentages of cells having the indicated amount of TOMM20 (D), PDHA1 (E) and mtDNA (F) after 24 hr of valinomycin treatment were shown. Error bars represent mean ±SE from three independent replicates. Over 100 cells were counted in each of three separate wells.

https://doi.org/10.7554/eLife.31326.016
Figure 5—source data 1

This excel file contains quantification of YFP-Parkin recruitment to damaged mitochondria, degradation of TOMM20, and degradation of PDHA1 upon mitophagy.

https://doi.org/10.7554/eLife.31326.017
Figure 5—source data 2

Quantification of mtDNA degradation upon mitophagy.

https://doi.org/10.7554/eLife.31326.018
MON1/CCZ1 complex is required for RAB7A recruitment to damaged mitochondria.

(A) Lysates of TBC1D15/17 DKO HCT116 cells stably expressing mCherry-Parkin and 2HA-RAB7A (T22N) were subjected to HA-IP, followed by trypsin digestion and mass spectrometric analysis. High-confidence candidate interacting protein (HCIPs) partners of RAB7A (T22N) are color-coded: untreated (green outline) and 3 hr of valinomycin (magenta outline). Line quality as described in figure key indicates criteria used for inclusion. (B) EGFP-MON1B and untagged CCZ1 (or EGFP-CCZ1 and untagged MON1B) were transiently expressed with DsRed-RAB7A (WT or T22N) in HeLa cells. Bars, 10 μm. (C) siRNA-treated TBC1D15/17 DKO cells stably expressing mCherry-Parkin and 2HA-RAB7A were treated with valinomycin for 3 hr and subjected to immunostaining. Bars, 10 μm. (D) RAB7A recruitment to mitochondria in (C) was quantified. Total signals of 2HA-RAB7A in each cell set to 100%. Error bars represent mean ±SE of at least two independent experiments. Statistical differences were determined by one-way ANOVA with Dunnett’s multiple comparisons test. ***p<0.001; n.s., not significant. (E) HCT116 cells or those stably expressing GFP-MON1B were treated with the indicated siRNAs. Total cell lysates were analyzed by immunoblotting. GFP-MON1B was detected by anti-GFP antibody.

https://doi.org/10.7554/eLife.31326.019
Figure 6—source data 1

This excel file contains quantification of 2HA-RAB7A recruitment to mitochondria in TBC1D15/17 DKO cells.

https://doi.org/10.7554/eLife.31326.020
Figure 7 with 1 supplement
RAB5 is recruited to damaged mitochondria during mitophagy.

(A) WT or TBC1D15/17 DKO cells stably expressing mCherry-Parkin and 3HA-mRAB5C were treated with DMSO or valinomycin for 3 hr. The cells were subjected to immunostaining. The magnified pictures were shown in the right. Bars, 10 μm. (B) HCT116 cells were subjected to immunostaining. Bars, 20 μm. (C) The indicated HCT116 cells stably expressing mCherry-Parkin were treated with DMSO or valinomycin for 3 hr followed by immunostaining. The magnified pictures were shown in the right. Bars, 10 μm. (D) siRNA-treated TBC1D15/17 DKO cells stably expressing mCherry-Parkin and 3HA-mRAB5C were treated with valinomycin for 3 hr and subjected to immunostaining. Bars, 10 μm. (E) Quantification of mRAB5C recruitment to damaged mitochondria in (D). Total signals of 3HA-mRAB5C in each cell set to 100%. Error bars represent mean ±SE of at least two independent experiments. Statistical differences were determined by one-way ANOVA with Dunnett’s multiple comparisons test. ***p<0.001; n.s., not significant.

https://doi.org/10.7554/eLife.31326.021
Figure 7—source data 1

This excel file contains quantification of 3HA-RAB5C recruitment to mitochondria in TBC1D15/17 DKO cells.

https://doi.org/10.7554/eLife.31326.023
Figure 7—figure supplement 1
Localization of RAB5B, RAB29, and RAB17 during mitophagy.

(A and B) WT and TBC1D15/17 DKO HCT116 cells stably expressing 3HA-mRAB5C (A) or 3HA-mRAB5B (B) were subjected to immunostaining. Images are displayed as z-stacks of four confocal slices. Bars, 20 μm. (C) The indicated cells stably expressing 3HA-mRAB5B and mCherry-Parkin were treated with DMSO or valinomycin for 3 hr followed by immunostaining. Magnified images are shown in the right. Bars, 10 μm. (D) The indicated cells stably expressing 3HA-mRAB29 were subjected to immunostaining. Images are displayed as z-stacks of four confocal slices. Bars, 20 μm. (E and F) The indicated cells stably expressing mCherry-Parkin, and 3HA-mRAB29 (E) or 3HA-mRAB17 (F) were treated with DMSO or valinomycin for 3 hr followed by immunostaining. Magnified images are shown in the right. Bars, 10 μm.

https://doi.org/10.7554/eLife.31326.022
Figure 8 with 3 supplements
RABGEF1 is recruited to the damaged mitochondria in a ubiquitin-binding dependent manner.

(A) HeLa cells transiently expressing mChery-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr followed by immunostaining. The magnified pictures were shown in the right. Bars, 10 μm. (B) Total cell lysates of (A) were analyzed by immunoblotting. Anti-GFP antibody was used for the GFP-mRABGEF1 detection. * and # denote ubiquitinated forms and truncated forms, respectively. (C) Quantification of RABGEF1 recruitment to damaged mitochondria in (A). None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. (D) Recombinant ubiquitin (Ub) pre-treated with or without GST-TcPINK1 was subjected to pull-down assay with GST-mRABGEF1. W and E indicate wash and eluted fractions, respectively. 10%, 10% of input. (E) Percentages of the amount of ubiquitin in the eluted fraction in (D) were shown. The error bars represent mean ±SE from three independent experiments. (F) K48-linked and K63-linked Ub chains pre-treated with or without GST-TcPINK1 were subjected to pull-down assay with GST-mRABGEF1. (G) Interactions between GST-mRABGEF1 (WT or Y26A/A58D) and ubiquitin or phosphorylated ubiquitin were measured by ITC. N, stoichiometry of binding.

https://doi.org/10.7554/eLife.31326.024
Figure 8—source data 1

Quantification of RABGEF1 recruitment to damaged mitochondria during mitophagy.

https://doi.org/10.7554/eLife.31326.028
Figure 8—source data 2

Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin.

https://doi.org/10.7554/eLife.31326.029
Figure 8—source data 3

Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin.

https://doi.org/10.7554/eLife.31326.030
Figure 8—figure supplement 1
RABGEF1 recruitment to mitochondria during mitophagy.

(A) The indicated cells were treated with DMSO or valinomycin for 3 hr followed by immunostaining. Bars, 10 μm. Graphs for quantification of RABGEF1 recruitment to mitochondria were shown below the images. None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. The error bars represent mean ±SE and over 100 cells were counted in each of three separate wells. (B) WT and TBC1D15/17 DKO HCT116 cells stably expressing mCherry-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr. GFP-mRABGEF1 signals were enhanced by immunostaining with anti-GFP antibody. Bars, 10 μm. (C) Total cell lysates in (B) were analyzed by immunoblotting. * and # denote ubiquitinated forms and truncated forms, respectively.

https://doi.org/10.7554/eLife.31326.025
Figure 8—figure supplement 1—source data 1

This excel file contains quantification of RABGEF1 (WT and Y26A/A58D mutant) recruitment to mitochondria in HCT116 (WT and TBC1D15/17 DKO) cells.

https://doi.org/10.7554/eLife.31326.031
Figure 8—figure supplement 2
Mitochondrial recruitment of RABGEF1 is not affected by the downstream Rabs and Rab-related factors.

(A) GFP-mRABGEF1 was transiently expressed in siRNA-treated HeLa cells. The cells were then treated with valinomycin for 3 hr followed by immunostaining. Bars, 20 μm. (B) Quantification of mitochondrial recruitment of GFP-mRABGEF1 in HeLa cells. (C) Quantification of mitochondrial recruitment of GFP-mRABGEF1 in HCT116 cells. None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. The error bars represent mean ± SE and over 100 cells were counted in each of three separate wells.

https://doi.org/10.7554/eLife.31326.026
Figure 8—figure supplement 2—source data 2

Quantification of RABGEF1 recruitment to mitochondria in HeLa cells treated with the indicated siRNA during mitophagy.

https://doi.org/10.7554/eLife.31326.032
Figure 8—figure supplement 2—source data 3

Quantification of RABGEF1 recruitment to mitochondria in HCT116 cells treated with the indicated siRNA during mitophagy.

https://doi.org/10.7554/eLife.31326.033
Figure 8—figure supplement 3
Preparation of recombinant RABGEF1 and ubiquitin.

(A) CBB staining of purified recombinant GST-mRABGEF1. (B) CBB staining of purified recombinant tandem linear ubiquitin (1×, 2×, 3×, and 4×) each and the mixture. (C) Linear ubiquitins were incubated with or without GST-TcPINK1 followed by SDS-PAGE and immunoblotting. (D) CBB staining of purified recombinant GST-mRABGEF1 used for ITC. (E) Ubiquitin and phosphorylated ubiquitin purified from bacterial cells (see the Materials and methods for details) were analyzed by SDS-PAGE or Phos-tag PAGE followed by CBB staining.

https://doi.org/10.7554/eLife.31326.027
RABGEF1 is important for mitochondrial clearance.

(A) WT and RABGEF1-mAID HCT116 cells were treated with or without IAA for 16 hr. Total cell lysates were analyzed by immunoblotting. (B) Quantification of Parkin recruitment to mitochondria in WT and RABGEF1-mAID HCT116 cells after 3 hr of valinomycin treatment. Partial and complete denote that YFP-Parkin signals were overlapped with some of and all mitochondria, respectively. (C) YFP-Parkin stably expressing WT and RABGEF1-mAID HCT116 cells pre-treated with IAA were treated with valinomycin for the indicated times. Total cell lysates were analyzed by immunoblotting. (D) WT and RABGEF1-mAID HCT116 cells stably expressing YFP-Parkin and mt-mKeima were treated with IAA for 16 hr followed by DMSO or OAQ for 6 hr and subjected to FACS analysis. Plots are representative of n = 3 experiments. (E) Quantification of mitophagy in (D). Error bars represent mean ±SE of three independent experiments. Statistical differences were determined by student’s t-test. *p<0.05.

https://doi.org/10.7554/eLife.31326.034
Figure 9—source data 1

Quantification of YFP-Parkin recruitment to mitochondria in RABGEF1-mAID HCT116 and the corresponding WT cells during mitophagy.

https://doi.org/10.7554/eLife.31326.035
Figure 9—source data 2

Quantification of mitophagy using mt-mKeima and FACS analysis.

https://doi.org/10.7554/eLife.31326.036
Mitochondrial localization of TBC1D15.

(A) HA-TBC1D15 (upper) and HA-TBC1D17 (lower) with or without YFP-FIS1 were transiently expressed in HeLa cells. The cells were subjected to immunostaining. Bars, 20 μm. (B) HeLa cells stably expressing GST-Parkin were treated with the indicated siRNA. After 3 hr of valinomycin treatment, cells were subjected to immunostaining. Bars, 10 μm.

https://doi.org/10.7554/eLife.31326.037
Proposed model of mitophagy regulated by endosomal Rab cycles.

(1) Through phosphorylation by PINK1, Parkin and ubiquitin ubiquitinate damaged mitochondria. (2) RABGEF is recruited to mitochondria and (3) endosomal Rab cycles including RAB5 and MON1/CCZ1 complex direct RAB7A to the mitochondria. (4) ATG9A vesicles are recruited to the autophagosome formation sites, in a RAB7A-dependent manner, where ATG9A vesicles and LC3-labeled autophagic membranes are assembled. (5) Mitochondrial Rab-GAPs, TBC1D15 and TBC1D17, dissociate RAB7A from the mitochondrial membranes to complete the Rab cycles.

https://doi.org/10.7554/eLife.31326.038

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line
(Homo sapiens)
HeLaATCCCVCL_0030
Cell line
(H. sapiens)
HCT116ATCCCVCL_0291
Cell line
(H. sapiens)
FIS1-/-Otera et al. (2010)
Cell line
(H. sapiens)
TBC1D15/17 DKOYamano et al. (2014)
Cell line
(H. sapiens)
HCT116-OsTIR1Natsume et al. (2016)
Cell line
(H. sapiens)
RABGEF1-mAIDthis papermAID sequence were inserted into HCT116-OsTIR1 cell line to produce RABGEF1-mAID.
Cell line
(H. sapiens)
HEK293TATCCCVCL_0063
AntibodyRabbit anti-GFP
(polyclonal)
Abcamab6556
AB_305564
1:1000 (WB),
1:1000 (IF)
AntibodyMouse anti-MFN2
(monoclonal)
Abcamab56889
AB_2142629
1:500(WB)
AntibodyRabbit anti-TOMM20
(polyclonal)
Santa Cruz Biotechnologysc-11415
AB_2207533
1:2000 (WB),
1:1000 (IF)
AntibodyRabbit anti-LC3BSigmaL7543
AB_796155
1:1000 (WB)
AntibodyMouse anti-MT-CO2
(monoclonal)
Abcamab110258
AB_10887758
1:500 (WB)
AntibodyMouse anti-Actin
(monoclonal)
MilliporeMAB1501R
AB_2223041
1:2000 (WB)
AntibodyMouse anti-RAB7
(monoclonal)
Abcamab50533
AB_882241
1:1000 (WB)
AntibodyRabbit anti-RABGEF1
(polyclonal)
NOVUS BIOLOGICALSNBP1-49938
AB_10012128
1:500 (WB)
AntibodyMouse anti-CCZ1
(monoclonal)
Santa Cruz Biotechnologysc-5142901:100 (WB)
AntibodyMouse anti-ubiquitin
(monoclonal)
Santa Cruz Biotechnologysc-8017
AB_628423
1:1000 (WB)
AntibodyRabbit anti-S65
phosphorylated ubiquitin
Koyano et al. (2014)1:500 (WB)
AntibodyRabbit anti-GFP
(polyclonal)
InvitrogenA-11122
AB_221569
1:1000 (IF)
AntibodyMouse anti-GFP
(monoclonal)
InvitrogenA-11120
AB_221568
1:1000 (IF)
1:500 (immuno-EM)
AntibodyMouse anti-TOMM20
(monoclonal)
Santa Cruz Biotechnologysc-17764
AB_628381
1:200 (IF)
AntibodyMouse anti-HA
(monoclonal)
MBL Life scienceM180-3
AB_10951811
1:2000 (IF)
AntibodyMouse anti-HA
(monoclonal)
COVANCEMMS-101R-500
AB_10063630
1:500 (IF)
AntibodyMouse anti-LAMP2
(monoclonal)
Santa Cruz Biotechnologysc-18822
AB_626858
1:100 (IF)
AntibodyMouse anti-EEA1
(monoclonal)
BD Biosciences610457
AB_397830
1:200 (IF)
AntibodyMouse anti-GM130
(monoclonal)
BD Biosciences610822
AB_398141
1:1000 (IF)
AntibodyMouse anti-pryruvate dehydrogenase E1-alpha subunit (PDHA1) (monoclonal)Abcamab110334
AB_10866116
1:500 (IF)
AntibodyMouse anti-DNA
(monoclonal)
MilliporeCBL186
AB_11213573
1:500 (IF)
AntibodyRabbit anti-RAB5
(monoclonal)
Cell Signaling
Technology
3547
AB_2300649
1:200 (IF)
AntibodyRabbit anti-RAB7
(monoclonal)
Cell Signaling
Technology
9367
AB_1904103
1:100 (IF)
AntibodyRabbit anti-TBC1D15
(clonal)
A kind gift from N.
Ishihara, Kurume
University, Japan
1:50 (IF)
AntibodyRabbit anti-ATG9A
(clonal)
A kind gift from N.
Mizushima, University of
Tokyo,Japan
1:100 (IF)
AntibodyRabbit anti-ATG16L1A kind gift from N.
Mizushima, University of
Tokyo,Japan
1:200 (IF)
AntibodyGoat anti-Rabbit IgG, Alexa Fluor 488 conjugatedThermo Fisher ScientificA-11034
AB_2576217
1:500 (IF)
AntibodyGoat anti-Rabbit IgG, Alexa Fluor 568 conjugatedThermo Fisher ScientificA-11036
AB_10563566
1:500 (IF)
AntibodyGoat anti-Rabbit IgG, Alexa Fluor 647 conjugatedThermo Fisher ScientificA-21245
AB_2535813
1:500 (IF)
AntibodyGoat anti-Mouse IgG,
Alexa Fluor 488 conjugated
Thermo Fisher ScientificA-11029
AB_138404
1:500 (IF)
AntibodyGoat anti-Mouse IgG,
Alexa Fluor 568 conjugated
Thermo Fisher ScientificA-11031
AB_144696
1:500 (IF)
AntibodyGoat anti-Mouse IgG,
Alexa Fluor 647 conjugated
Thermo Fisher ScientificA-21236
AB_2535805
1:500 (IF)
AntibodyNanogold-conjugated
anti-mouse IgG antibody
Nanoprobes2002
AB_2637031
1:200 (IF)
AntibodyAnti-rabbit IgG horseradish peroxidase-linked secondary
antibodies
GE HealthcareNA934
AB_772206
1:5000 (WB)
AntibodyAnti-HA beadsSigma-aldrichA2095
AB_257974
Chemical compound,
drug
Lpofectamine RNAiMAXInvitrogenInvitrogen:
13778–150
Chemical compound,
drug
FuGENE6PromegaPromega:
E2692
Chemical compound,
drug
FuGENE HDPromegaPromega:
E2311
Chemical compound,
drug
DMEMLife TechnologiesLife Technologies:
31053–028
Chemical compound,
drug
DMEMSigma-aldrichSigma-aldrich:
D5796-500ML
Chemical compound,
drug
Sodium pyruvateLife TechnologiesLife Technologies:
11360–070
Chemical compound,
drug
GlutamineLife TechnologiesLife Technologies:
25030–081
Chemical compound,
drug
GlutaMAXLife TechnologiesLife Technologies:
35050–061
Chemical compound,
drug
Nonessential amino acidsLife TechnologiesLife Technologies:
11140–050
Chemical compound,
drug
McCoy's 5ALife TechnologiesLife Technologies:
16600–082
Chemical compound,
drug
PolybreneSigma-aldrichSigma-aldrich:
H9268
Chemical compound,
drug
ValinomycinSigma-aldrichSigma-aldrich:
V0627-10MG
Chemical compound,
drug
OligomycinCalbiochemCalbiochem:
495455–10 MG
Chemical compound,
drug
Antimycin ASigma-aldrichSigma-aldrich:
A8674-25MG
Chemical compound,
drug
Q-VD-OPHSM BiochemicalsSM Biochemicals:
SMPH001
Chemical compound,
drug
Q-VD-OPHSigma-aldrichSigma-aldrich:
SML0063-1MG
Chemical compound,
drug
Indole-3-acetic acid (IAA)WakoWako:
090–07123
Chemical compound,
drug
G418Sigma-aldrichSigma-aldrich:
G8168
Chemical compound,
drug
Hygromycin BInvitrogenInvitrogen:
10687–010
Chemical compound,
drug
DAPIThermo Fisher ScientificThermo Fisher Scientific:
D3571
Chemical compound,
drug
Protease inhibitor cocktailRocheRoche:
11 873 580 001
Chemical compound,
drug
Phos-tagWakoWako: 304–93521
Chemical compound,
drug
DTBP (dimethyl 3,3'-dithiobispropionimidate)PiercePierce: 20665
Chemical compound,
drug
TCEP (Tris(2-carboxylethyl)phosphine)Sigma-aldrichSigma-aldrich:
C4706-10G
Chemical compound,
drug
GSH (L-glutathione reduced)Sigma-aldrichSigma-aldrich:
G4251-25G
Chemical compound,
drug
PhosSTOP phosphatase
inhibitor cocktail
RocheRoche:
04 906 845 001
Chemical assay or kitBCIP-NBT solution kitNacalai TesqueNacalai Tesque:
03937–60
Chemical assay or kitWestern Lightning
Plus-ECL
PerkinElmerPerkinElmer:
NEL105001EA
Peptide, recombinant
protein
HA peptideSigma-aldrichSigma-aldrich:
I2149
Peptide, recombinant
protein
Ubiquitin from
bovine erythrocytes
Sigma-aldrichSigma-aldrich:
U6253
Peptide, recombinant
protein
1x ubiquitinthis paper1x human ubiquitin (C-terminal His-tagged)
Peptide, recombinant
protein
2x ubiquitinthis paper2x tandem linear human ubiquitin (C-terminal His-tagged)
Peptide, recombinant
protein
3x ubiquitinthis paper3x tandem linear human ubiquitin (C-terminal His-tagged)
Peptide, recombinant
protein
4x ubiquitinthis paper4x tandem linear human ubiquitin (C-terminal His-tagged)
Peptide, recombinant
protein
GST-mRABGEF1 (WT)this paperGST-tagged mouse RABGEF1 (WT) 1-74aa
Peptide, recombinant
protein
GST-mRABGEF1 (Y26A)this paperGST-tagged mouse RABGEF1 (Y26A) 1-74aa
Peptide, recombinant
protein
GST-mRABGEF1 (A58D)this paperGST-tagged mouse RABGEF1 (A58D) 1-74aa
Peptide, recombinant
protein
GST-mRABGEF1 (Y26A/A58D)this paperGST-tagged mouse RABGEF1 (Y26A/A58D) 1-74aa
Peptide, recombinant
protein
GST-TcPINK1Yamano et al. (2015)
OtherNi-NTA agaroseQIAGENQIAGEN:
30230
OtherPD MidiTrap G-25GE HealthcareGE Healthcare:
28-9180-08
OtherGlutathione-Sepharose 4BGE HealthcareGE Healthcare:
17-0756-01
OtherSuperdex 75
10/300 column
GE HealthcareGE Healthcare:
17-5174-01
OtherAmicon Ultra
centrifugal filters
MilliporeMillipore: UFC800308 for 3K
Millipore: UFC800308 for 10K
Software, algorithmPhotoshopAdobeSCR_014199
Software, algorithmVolocityPerkinElmerSCR_002668
Software, algorithmZEN microscope
software
Carl ZeissSCR_013672
Software, algorithmGraphPad Prism v6.0dGraphPad SoftwareSCR_002798

Additional files

Supplementary file 1

Proteomic analysis of 2HA-RAB7A (T22N)-associated proteins during mitophagy.

This files contains all raw and analyzed mass spectrometric data and analysis parameters. Proteomic analysis of 2HA-RAB7A (T22N)-associated proteins in TBC1D15/17 DKO HCT116 cells stably expressing mCherry-Parkin after 3 hr of valinomycin treatment using CompPASS. The tab labeled 'Analysis' contains information regarding cell lines used, experimental conditions, descriptions of all worksheets including raw data that contain the complete lists of all proteins identified, WDN-scores, Z-scores, and APSMs, and details of each subsequent analysis performed.

https://doi.org/10.7554/eLife.31326.039
Transparent reporting form
https://doi.org/10.7554/eLife.31326.040

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