Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK1

How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. Herein we have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activation-dependent diGLY sites in 22 proteins conserved across mouse and human systems. We employ reconstitution assays to demonstrate direct ubiquitylation by Parkin in vitro. We also identified a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation indicating the presence of alternate ubiquitin E3 ligase  pathways that are activated by mitochondrial depolarisation in neurons. Finally we have developed an online resource to search for ubiquitin sites and enzymes in mitochondria of neurons, MitoNUb. These findings will aid future studies to understand Parkin activation in neuronal subtypes.

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Figure 1C: Immunoblots for PINK1 signaling in PINK1 WT and KO mouse cortical neurons.

Scans of X-ray film:

GAPDH shown in Figure1C_GAPDH.tif

Parkin shown in Figure1C_Parkin.tif

Phospho-Ser65 Parkin shown in Figure1C_ParkinPSer65.tif

PINK1 shown in Figure1C_PINK1.tif (immunoprecipitation)

Rab8A shown in Figure1C_Rab8A.tif

Phospho-Ser111 Rab8A shown in Figure1C_Rab8APSer111.tif

Ubiquitin shown in Figure1C_ S10A_Ubiquitin.tif (same blot used for Figure_S10A, Halo-multiDSK pull-down)

Phospho-Ser65 Ubiquitin shown in Figure1C_S10A_UbiquitinPSer65.tif (same blot used for Figure_S10A, Halo-multiDSK pull-down)

Figure 4A: Immunoblots for time-course of Parkin-dependent substrates in C57BL/6J neurons.

Scans of X-ray film:

CISD1 shown in Figure4A_CISD1.tif (Halo-multiDSK pull-down)

  Figure4A_INPUT_CISD1.tif (INPUT)

CPT1α shown in Figure4A_ CPT1a.tif (Halo-multiDSK pull-down)

  Figure4A_INPUT_CPT1a.tif (INPUT)

Ubiquitin shown in Figure4A_Ubiquitin.tif (Halo-multiDSK pull-down)

  Figure4A_INPUT_Ubiquitin.tif (INPUT)

Phospho-Ser65 Ubiquitin shown in Figure4_UbiquitinPSer65.tif (Halo-multiDSK pull-down)

GAPDH Shown in Figure4A_INPUT_GAPDH.tif (INPUT)

Figure 4B: Immunoblots for validation of Parkin-dependent substrates in PARKIN WT and KO neurons.

Scans of X-ray film:

CISD1 shown in Figure4B_CISD1.tif (Halo-multiDSK pull-down)

(bottom blot) Figure4B_INPUT_CPT1a_CISD1.tif (INPUT)

CPT1α shown in Figure4B_ CPT1a.tif (Halo-multiDSK pull-down)

(top blot)  Figure4B_INPUT_CPT1a_CISD1.tif ( (INPUT)

Ubiquitin shown in Figure4B_Ubiquitin.tif (Halo-multiDSK pull-down)

  Figure4B_INPUT_Ubiquitin.tif (INPUT)

Phospho-Ser65 Ubiquitin shown in Figure4B_UbiquitinPSer65.tif (Halo-multiDSK pull-down)

GAPDH Shown in Figure4B_INPUT_GAPDH.tif (INPUT)

Figure 5A: Immunoblots for in vitro reconstitution assay in PINK1 WT and KO mouse embryonic fibroblasts.

Scans of X-ray film:

CISD1shown in Figure5A_CISD1.tif

CPT1α shown in Figure5A_ CPT1a.tif

CYB5B shown in Figure5A_CYB5B.tif

HK1 shown in Figure5A_HK1.tif

MFN2 shown in Figure5A_MFN2.tif

VDAC shown in Figure5A_VDAC.tif

Phospho-Ser65 Parkin shown in Figure5A_ParkinPSer65.tif

Parkin shown in Figure5A_Parkin.tif

Ubiquitin shown in Figure5A_Ubiquitin.tif

Phospho-Ser65 Ubiquitin shown in Figure5A_UbiquitinPSer65.tif 

Figure 5B: Immunoblots for validation of Parkin-dependent substrates in in vitro studies.

Scans of Western blots (LI-COR):

CPT1α shown in Figure5B_ CPT1 α_800nm.tif

Flag-Ub (CPT1α) shown in Figure5B_ CPT1 α_Flag_800nm.tif

His (CPT1α) shown in Figure5B_ CPT1 α_His_800nm.tif

Miro1 shown in Figure5B_Miro1_800nm.tif

Flag-Ub (Miro1) shown in Figure5B_ Miro1_Flag_800nm.tif

His (Miro1) shown in Figure5B_ Miro1_His_800nm.tif

Figure 5C: Immunoblots for time-course of CPT1α ubiquitylation in in vitro studies.

Scans of Western blots (LI-COR):

CPT1α shown in top blot: Figure5C_Flag_CPT1α_800nm.tif

Flag-Ub shown in bottom blot: Figure5C_Flag_CPT1α_800nm.tif

His shown in Figure5C_ CPT1 α_His_800nm.tif

Figure S3A: Immunoblots for PINK1 signaling in C57BL/6J neurons.

Scans of X-ray film:

GAPDH shown in FigureS3A_GAPDH.tif

Parkin shown in FigureS3A_Parkin.tif

Phospho-Ser65 Parkin shown in FigureS3A_ParkinPSer65.tif

PINK1 shown in FigureS3A_PINK1.tif (immunoprecipitation)

Rab8A shown in FigureS3A_Rab8A.tif

Phospho-Ser111 Rab8A shown in FigureS3A_Rab8APSer111.tif

Ubiquitin shown in FigureS3A_Ubiquitin.tif (same blot used for Figure_4A, Halo-multiDSK pull-down)

Phospho-Ser65 Ubiquitin shown in FigureS3A_UbiquitinPSer65.tif (same blot used for Figure_4A, Halo-multiDSK pull-down)

Phospho-Ser111 Rab8A and Phospho-Ser65 Parkin also shown in FigureS3A_ParkinPSer65_Rab8APSer111.tif

Figure S3B: Immunoblots for comparison of Halo-multiDSK and Halo-TUBE in  C57BL/6J neurons.

Ubiquitin shown in FigureS9_S3_Ubiquitin.tif (same blot used for Figure_S9)

Phospho-Ser65 Ubiquitin shown in FigureS9_S3_UbiquitinPSer65.tif (same blot used for Figure_S9)

Figure S3C: Immunoblots for PINK1 signaling in Parkin WT and KO mouse cortical neurons.

Scans of X-ray film:

GAPDH shown in FigureS3C_GAPDH.tif

Parkin shown in FigureS3C_Parkin.tif

Phospho-Ser65 Parkin shown in FigureS3C_ParkinPSer65.tif

PINK1 shown in FigureS3C_PINK1.tif (immunoprecipitation)

Rab8A shown in FigureS3C_Rab8A.tif

Phospho-Ser111 Rab8A shown in FigureS3C_Rab8APSer111.tif

Ubiquitin shown in FigureS3C_ Ubiquitin.tif (same blot used for Figure_4B, Halo-multiDSK pull-down)

Phospho-Ser65 Ubiquitin shown in FigureS3C_ UbiquitinPSer65.tif (same blot used for Figure_4B, Halo-multiDSK pull-down)

Figure S4: Immunoblots for PINK-Parkin signaling in VPS35 D620N mouse cortical neurons.

Scans of X-ray film:

GAPDH shown in FigureS4_GAPDH.tif

Parkin shown in FigureS4_Parkin.tif

Phospho-Ser65 Parkin shown in FigureS4_ParkinPSer65.tif

Rab8A shown in FigureS4_Rab8A.tif

Phospho-Ser111 Rab8A shown in FigureS4_Rab8APSer111.tif

CISD1 shown in FigureS4_ CISD1.tif (Halo-UBQLN1 pull-down)

Phospho-Ser65 Ubiquitin shown in FigureS4_ UbiquitinPSer65.tif (Halo-UBQLN1 pull-down)

VPS35 shown in FigureS4_VPS35.tif

Scan of Memcode shown in FigureS4_Memcode.tif

Figure S6: Immunoblots for biochemical analysis in C56BL/6J mouse cortical neurons.

Scans of X-ray film:

GAPDH shown in FigureS6_GAPDH.tif

Phospho-Ser65 Ubiquitin shown in FigureS6_ UbiquitinPSer65.tif 

Figure S8: Immunoblots for biochemical analysis in PINK1 WT and KO mouse cortical neurons.

Scans of X-ray film:

GAPDH shown in FigureS8_GAPDH.tif

Phospho-Ser65 Ubiquitin shown in FigureS8_ UbiquitinPSer65.tif

Figure S9: Immunoblots for biochemical analysis of ubiquitylated target in C56BL/6J mouse cortical neurons.

Membrane-enriched lysate subjected to ubiquitin capture using UBQLN1(TUBE), multiDSK and mutant multiDSK pull-down (Illustration of sample loading in figures FigureS9_ACSL6,_MFN2, _UbiquitinPSer65, _NAV1.7).

Scans of X-ray film:

Ubiquitin shown in FigureS9_S3_Ubiquitin.tif (same blot used for Figure_S3B)

Phospho-Ser65 Ubiquitin shown in FigureS9_S3_UbiquitinPSer65.tif (same blot used for Figure_S3B)

ABCD3 shown in FigureS9_ABCD3.tif

ACSL1shown in FigureS9_ ACSL1.tif

ACSL6 shown in FigureS9_ACSL6.tif

AGPAT5 shown in FigureS9_AGPAT5.tif

ARHGAP33 shown in FigureS9_ARHGAP33.tif

ATAD1 shown in FigureS9_ATAD1.tif

CAD shown in FigureS9_ CAD.tif

CAMK2A shown in FigureS9_CAMK2A.tif

CAMK2B shown in FigureS9_CAMK2B.tif

CDK16 shown in FigureS9_CDK16.tif

CISD1shown in FigureS9_CISD1.tif

CNN3 shown in FigureS9_CNN3.tif

CPT1α shown in FigureS9_ CPT1A.tif

CYB5B shown in FigureS9_CYB5B.tif

CYB5R3 shown in FigureS9_CYB5R3.tif

DCAKD shown in FigureS9_DCAKD.tif

DCAMKL2 shown in FigureS9_DCAMKL2.tif

FAM213A shown in FigureS9_FAM213A.tif

FBXO41 shown in FigureS9_FBXO41.tif

GK shown in FigureS9_GK.tif

HK1 shown in FigureS9_HK1.tif

HSDL1 shown in FigureS9_HSDL1.tif

MAO-A shown in FigureS9_MAOA.tif

MAO-B shown in FigureS9_MAOB.tif

MAPRE2 shown in FigureS9_MAPRE2.tif

MARC2 shown in FigureS9_MARC2.tif

MFN1 shown in FigureS9_MFN1.tif

MFN2 shown in FigureS9_MFN2.tif

NAV1.7 shown in FigureS9_NAV1.7.tif

P23 shown in FigureS9_p23.tif

PRKCG shown in FigureS9_PRKCG.tif

RAB5C shown in FigureS9_Rab5c.tif

RHOT2 shown in FigureS9_RHOT2.tif

RIMS4 shown in FigureS9_RIMS4.tif

RUFY3 shown in FigureS9_RUFY3.tif

SH3BP4 shown in FigureS9_SH3BP4.tif

SNX3 shown in FigureS9_SNX3.tif

TDRKH shown in FigureS9_TDRKH.tif

TOMM70 shown in FigureS9_TOMM70.tif

Figure S10A: Immunoblots for validation of Parkin-dependent substrates in PINK1 WT and KO neurons.

Scans of X-ray film:

CISD1 shown in Figure S10A _CISD1.tif (Halo-multiDSK pull-down)

  Figure S10A _INPUT_CISD1.tif (INPUT)

CPT1α shown in Figure S10A _ CPT1A.tif (Halo-multiDSK pull-down)

  Figure S10A _INPUT_ CPT1A.tif (INPUT)

Ubiquitin shown in Figure1C_ S10A _Ubiquitin.tif (same blot used for FigureS1C, Halo-multiDSK pull-down)

  Figure S10A _INPUT_Ubiquitin.tif (INPUT)

Phospho-Ser65 Ubiquitin shown in Figure1C_ S10A_UbiquitinPSer65.tif (same blot used for FigureS1C, Halo-multiDSK pull-down)

GAPDH Shown in Figure S10A _INPUT_GAPDH.tif (INPUT)

Figure S10B: Immunoblots for time-course of Parkin-dependent substrates in SH-SY5Y cells.

Scans of X-ray film:

CISD1 shown in Figure S10B _CISD1.tif (Halo-multiDSK pull-down)

CPT1α shown in Figure S10B _ CPT1A.tif (Halo-multiDSK pull-down)

Ubiquitin shown in Figure S10B _Ubiquitin.tif (Halo-multiDSK pull-down)

Phospho-Ser65 Ubiquitin shown in Figure S10B _UbiquitinPSer65.tif (Halo-multiDSK pull-down)

GAPDH Shown in Figure S10B _GAPDH.tif 

Parkin shown in FigureS10B_Parkin.tif

Phospho-Ser65 Parkin shown in FigureS10B_ParkinPSer65.tif

PINK1 shown in FigureS10B_PINK1.tif

OPA1 shown in FigureS10B_OPA1.tif

Figure S11: Immunoblots for in vitro reconstitution assay in HeLa cells.

Scans of X-ray film:

CISD1shown in FigureS11_CISD1.tif

CPT1α shown in FigureS11_ CPT1a.tif

CYB5B shown in FigureS11_CYB5B.tif

HK1 shown in FigureS11_HK1.tif

MFN2 shown in FigureS11_MFN2.tif

VDAC shown in FigureS11_VDAC.tif

Phospho-Ser65 Parkin shown in FigureS11_ParkinPSer65.tif

Parkin shown in FigureS11_Parkin.tif

Ubiquitin shown in FigureS11_Ubiquitin .tif

Phospho-Ser65 Ubiquitin shown in FigureS11_UbiquitinPSer65 .tif

FigureS12A: Purification of CPT1α protein.

FigureS12A_ Akta_Purifier_Curves.txt.

 Tab delimited text of data from the AKTA system, plotted in figure S12A. Columns are laid out as volume/measured parameter    for each parameter from the Akta. The columns plotted in figure S12A were mAU (columns A and B in excel) and fractions (columns M and N in excel).

Figure_S12A_Coomassie_Gel.tif. 

Coomassie stained gels of fractions from the AKTA system in figure S12A.

Top: Left hand Coomassie gel, figure S12A

Bottom: Right hand Coomassie gel, figure S12A

FigureS12B-C: Purification of recombinant Parkin targets 

Blots shown in the paper are highlighted in bold.

FigureS12B_Ub_Targets_High_800nm.tif

Coomassie stained gels of the parkin targets alongside a BSA curve.

Left: Lane: 1: Mwt Marker, 2: Fam213A, 3: MAO-B, 4: CAMK2α, 5: MAO-A (figure S12B), 6: GST-MAPRE2, 7: MBP-CYB5R3, 8: MBP-CYB5B, 9: MBP-CYB5B, 10: SNX3, 11: His-SUMO-MAO-B, 12: GST-MAO-B, 13: 0.03125 ug BSA, 14: 0.0625 ug BSA, 15: 0.125 ug BSA, 16: 0.25 ug BSA, 17: 0.5 ug BSA, 18: 1 ug BSA

Right: Lane: 1: Mwt Marker, 2: GST-MAO-A, 3: GST-FAM213A, 4: MBP-CAMK2α (figure S12B), 5: GST-TDRKH, 6: MBP-CPT1α, 7: MBP-CYB5B, 8: CAMK2β, 9:, 10: His-SUMO-MAO-B, 11: GST-MAO-B, 12: 0.03125 ug BSA, 13: 0.0625 ug BSA, 14: 0.125 ug BSA, 15: 0.25 ug BSA, 16: 0.5 ug BSA, 17: 1 ug BSA

Figure_S12B_Ub_Targets_Low_800nm.tif

Coomassie stained gels of the parkin targets alongside a BSA curve.

Lane: 1: Mwt Marker, 2: GST-Miro1 (figure S12B), 3: His-SUMO-Fam213A, 4: His-SUMO-MAPRE2, 5: His-SUMO-MAO-A, 6: His-SUMO-MAO-B, 7: His-SUMO-SNX26, 8: His-SNX3, 9: His-MAPRE2, 10: GST-FAM213A, 11: His-SUMO-CAMK2α, 12: His-SUMO-CAMK2β, 13: GST-FAM213A, 14: 0.03125 ug BSA, 15: 0.0625 ug BSA, 16: 0.125 ug BSA, 17: 0.25 ug BSA, 18: 0.5 ug BSA, 19: 1 ug BSA

Figure_S12C_Ub_Targets_800nm.tif

Coomassie stained gels of the parkin targets in duplicate alongside a BSA curve.

Top: Lane: 1: Mwt Marker, 2+3: ACSL1 45-end (Figure S12C), 4+5: SNX3 (Figure S12C), 6+7: MFN1, 8+9: MFN2, 10+11: 0.125 ug BSA, 12+13: 0.25 ug BSA, 14+15: 0.5 ug BSA, 16+17: 1 ug BSA

Middle: Lane: 1: Mwt Marker, 2+3: CAMK2α, 4+5: CAMK2β (Figure S12C), 6+7: MAO-A, 8+9: MAO-B (Figure S12C), 10+11: 0.125 ug BSA, 12+13: 0.25 ug BSA, 14+15: 0.5 ug BSA, 16+17: 1 ug BSA

Bottom: Lane: 1: Mwt Marker, 2+3: SRCIN, 4+5: His-MAPRE2, 6+7: FAM213A (Figure S12C), 8+9: CISD1, 10+11: 0.125 ug BSA, 12+13: 0.25 ug BSA, 14+15: 0.5 ug BSA, 16+17: 1 ug BSA

FigureS13-S14: Immunoblots for validation of Parkin-dependent substrates in in vitro studies.

Blots shown in the paper are highlighted in bold.

Scans of X-ray film:

FigureS13A-B_S14C-D_a6His_Film.tif

Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1

Membranes from left to right:

Top: 1: FAM213A (Figure S13B), 2:  MAO-B (Figure S13A), 3: MAPRE2, 4: CAMK2β (Figure S14D)

Top: 1: CAMK2α (Figure S14C), 2: Miro1, 3: MAO-A (Figure S13A)

FigureS13A-B_S14C-D_aFLAG_Film.tif

Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1

Membranes from left to right:

Top: 1: FAM213A (Figure S13B), 2: MAPRE2, 3: MAO-A (Figure S13A)

Top: 1: MAO-B (Figure S13A), 2: CAMK2α (Figure S14C), 3: CAMK2β (Figure S14D), 4: Miro1  

FigureS13A-B_S14C-D_aTarget_05m00s_Film.tif

Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1

Membranes from left to right:

Top: 1: FAM213A, 2: MAPRE2, 3: MAO-A

Top: 1: MAO-B, 2: CAMK2α (Figure S14C), 3: CAMK2β (Figure S14D), 4: Miro1  

FigureS13A-B_S14C-D_aTarget_06m00s_Film.tif

Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1

Membranes from left to right:

Top: 1: FAM213A (Figure S13B), 2: MAPRE2, 3: MAO-A (Figure S13A)

Top: 1: MAO-B (Figure S13A), 2: CAMK2α, 3: CAMK2β, 4: Miro1  

FigureS13C: Immunoblots for validation of Parkin-dependent substrates in in vitro studies.

Scans of Western blots (LI-COR):

ACSL1 shown in FigureS13C_ACSL1_800nm.tif

Flag-Ub (ACSL1) shown in Figure_S13C_ACSL1_Flag_800nm.tif

His (ACSL1) shown in Figure_S13C_ACSL1_His_800nm.tif

FigureS14B: Immunoblots for validation of Parkin-dependent substrates in in vitro studies.

Blots shown in the paper are highlighted in bold.

Scans of Western blots (LI-COR):

SNX3 shown in FigureS14B_SNX3_800nm.tif

Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1

Membranes, left to right: SNX3 (Figure S14B), FAM213A, Miro1, Miro1

Flag-Ub (SNX3) shown in Figure_S14B_Flag_SNX3_800nm.tif

Gel loading: lanes: 1+2: – PINK1, 3+4:WT PINK1, 5+6: KD PINK1

Membranes, left to right: Miro1, FAM213A, Miro1, SNX3 (Figure S14B)

His (SNX3) shown in Figure_S14B_His_SNX3_700nm.tif

Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1

Membranes, left to right: CPT1α, MFN1, SNX3 (Figure S14B), FAM213A

FigureS1: Immunocytochemistry of mouse cortical neurons.

FigureS1_TILE_maximun intensity projection.tif

Original acquisition: MAP2 neuronal marker (green-488); GFAP astrocytic marker (red-594); Hoechst nuclear marker (blue 361/497). 

FigureS1MGB (RGB).tif

Modified from FigureS1_TILE_maximun intensity projection.tif: MAP2 (green); GFAP (magenta); Hoechst (blue).

Table for Fig1DE.xlsx

Numerical data for the charts shown in Figure1D and Figure1E.

Table for FigS1A.xlsx

      Numerical data for quantification of Map2 and GFAP in mouse cortical neurons shown in FigureS1A.

Table for FigS1B (Cortical neurons_DIA_Report).xlsx

      Numerical data for DIA proteomic analysis of AO and DMSO treated cortical neurons shown in FigureS1B.

Table for FigS2B_C_Oxygraph.xlsx

Numerical data for the charts shown in FigureS2B and FigureS2C.

Table for Fig3A.xlsx

Numerical data for total protein abundance in PINK1 WT and KO shown in Figure3A.

Table for Fig3B.xlsx

Numerical data of abundance of phosphorylated-Ser65 or Ser57  shown in Figure3B.