DOI: 10.5281/zenodo.5163705
title: Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK1

description: 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. <strong>FILE DECRIPTIONS</strong> <strong>Figure 1C: Immunoblots for PINK1 signaling in PINK1 WT and KO mouse cortical neurons.</strong> Scans of X-ray film: GAPDH shown in <strong>Figure1C_GAPDH.tif</strong> Parkin shown in <strong>Figure1C_Parkin.tif</strong> Phospho-Ser65 Parkin shown in <strong>Figure1C_ParkinPSer65.tif</strong> PINK1 shown in <strong>Figure1C_PINK1.tif </strong>(immunoprecipitation) Rab8A shown in <strong>Figure1C_Rab8A.tif</strong> Phospho-Ser111 Rab8A shown in <strong>Figure1C_Rab8APSer111.tif</strong> Ubiquitin shown in <strong>Figure1C_ S10A_Ubiquitin.tif </strong>(same blot used for Figure_S10A, Halo-multiDSK pull-down) Phospho-Ser65 Ubiquitin shown in <strong>Figure1C_S10A_UbiquitinPSer65.tif </strong>(same blot used for Figure_S10A, Halo-multiDSK pull-down) <strong>Figure 4A: Immunoblots for time-course of Parkin-dependent substrates in C57BL/6J neurons.</strong> Scans of X-ray film: CISD1 shown in <strong>Figure4A_CISD1.tif </strong>(Halo-multiDSK pull-down) <strong> Figure4A_INPUT_CISD1.tif</strong> (INPUT) CPT1α shown in <strong>Figure4A_ CPT1</strong><strong>a</strong><strong>.tif</strong> (Halo-multiDSK pull-down) <strong> Figure4A_INPUT_CPT1</strong><strong>a</strong><strong>.tif</strong> (INPUT) Ubiquitin shown in <strong>Figure4A_Ubiquitin.tif </strong>(Halo-multiDSK pull-down) <strong> Figure4A_INPUT_Ubiquitin.tif</strong> (INPUT) Phospho-Ser65 Ubiquitin shown in <strong>Figure4_UbiquitinPSer65.tif </strong>(Halo-multiDSK pull-down) GAPDH Shown in <strong>Figure4A_INPUT_GAPDH.tif </strong>(INPUT) <strong>Figure 4B: Immunoblots for validation of Parkin-dependent substrates in PARKIN WT and KO neurons.</strong> Scans of X-ray film: CISD1 shown in <strong>Figure4B_CISD1.tif </strong>(Halo-multiDSK pull-down) (bottom blot)<strong> Figure4B_INPUT_CPT1a_CISD1.tif</strong> (INPUT) CPT1α shown in <strong>Figure4B_ CPT1a.tif</strong> (Halo-multiDSK pull-down) (top blot)<strong> Figure4B_INPUT_CPT1a_CISD1.tif</strong> ( (INPUT) Ubiquitin shown in <strong>Figure4B_Ubiquitin.tif </strong>(Halo-multiDSK pull-down) <strong> Figure4B_INPUT_Ubiquitin.tif</strong> (INPUT) Phospho-Ser65 Ubiquitin shown in <strong>Figure4B_UbiquitinPSer65.tif </strong>(Halo-multiDSK pull-down) GAPDH Shown in <strong>Figure4B_INPUT_GAPDH.tif </strong>(INPUT) <strong>Figure 5A: Immunoblots for <em>in vitro</em> reconstitution assay in PINK1 WT and KO mouse embryonic fibroblasts.</strong> Scans of X-ray film: CISD1shown in <strong>Figure5A_CISD1.tif</strong> CPT1α shown in <strong>Figure5A_ CPT1a.tif</strong> CYB5B shown in <strong>Figure5A_CYB5B.tif</strong> HK1 shown in <strong>Figure5A_HK1.tif</strong> MFN2 shown in <strong>Figure5A_MFN2.tif</strong> VDAC shown in <strong>Figure5A_VDAC.tif</strong> Phospho-Ser65 Parkin shown in <strong>Figure5A_ParkinPSer65.tif</strong> Parkin shown in <strong>Figure5A_Parkin.tif</strong> Ubiquitin shown in <strong>Figure5A_Ubiquitin.tif</strong> Phospho-Ser65 Ubiquitin shown in <strong>Figure5A_UbiquitinPSer65.tif </strong> <strong>Figure 5B: Immunoblots for validation of Parkin-dependent substrates in<em> in vitro </em>studies.</strong> Scans of Western blots (LI-COR): CPT1α shown in <strong>Figure5B_ CPT1 </strong><strong>α_800nm</strong><strong>.tif</strong> Flag-Ub (CPT1α) shown in <strong>Figure5B_ CPT1 </strong><strong>α_Flag_800nm</strong><strong>.tif</strong> His (CPT1α) shown in <strong>Figure5B_ CPT1 </strong><strong>α_His_800nm</strong><strong>.tif</strong> Miro1 shown in <strong>Figure5B_Miro1_800nm.tif</strong> Flag-Ub (Miro1) shown in <strong>Figure5B_ Miro1</strong><strong>_Flag_800nm</strong><strong>.tif</strong> His (Miro1) shown in <strong>Figure5B_ Miro1</strong><strong>_His_800nm</strong><strong>.tif</strong> <strong>Figure 5C: Immunoblots for time-course of CPT1</strong><strong>α</strong><strong> ubiquitylation in<em> in vitro </em>studies.</strong> Scans of Western blots (LI-COR): CPT1α shown in top blot: <strong>Figure5C_Flag_CPT1</strong><strong>α_800nm</strong><strong>.tif</strong> Flag-Ub shown in bottom blot: <strong>Figure5C_Flag_CPT1</strong><strong>α_800nm</strong><strong>.tif</strong> His shown in <strong>Figure5C_ CPT1 </strong><strong>α_His_800nm</strong><strong>.tif</strong> <strong>Figure S3A: Immunoblots for PINK1 signaling in C57BL/6J neurons.</strong> Scans of X-ray film: GAPDH shown in <strong>FigureS3A_GAPDH.tif</strong> Parkin shown in <strong>FigureS3A_Parkin.tif</strong> Phospho-Ser65 Parkin shown in <strong>FigureS3A_ParkinPSer65.tif</strong> PINK1 shown in <strong>FigureS3A_PINK1.tif </strong>(immunoprecipitation) Rab8A shown in <strong>FigureS3A_Rab8A.tif</strong> Phospho-Ser111 Rab8A shown in <strong>FigureS3A_Rab8APSer111.tif</strong> Ubiquitin shown in <strong>FigureS3A_Ubiquitin.tif </strong>(same blot used for Figure_4A, Halo-multiDSK pull-down) Phospho-Ser65 Ubiquitin shown in <strong>FigureS3A_UbiquitinPSer65.tif </strong>(same blot used for Figure_4A, Halo-multiDSK pull-down) Phospho-Ser111 Rab8A and Phospho-Ser65 Parkin also shown in <strong>FigureS3A_ParkinPSer65_Rab8APSer111.tif</strong> <strong>Figure S3B: Immunoblots for comparison of Halo-multiDSK and Halo-TUBE in C57BL/6J neurons.</strong> Ubiquitin shown in <strong>FigureS9_S3_Ubiquitin.tif </strong>(same blot used for Figure_S9) Phospho-Ser65 Ubiquitin shown in <strong>FigureS9_S3_UbiquitinPSer65.tif </strong>(same blot used for Figure_S9) <strong>Figure S3C: Immunoblots for PINK1 signaling in Parkin WT and KO mouse cortical neurons.</strong> Scans of X-ray film: GAPDH shown in <strong>FigureS3C_GAPDH.tif</strong> Parkin shown in <strong>FigureS3C_Parkin.tif</strong> Phospho-Ser65 Parkin shown in <strong>FigureS3C_ParkinPSer65.tif</strong> PINK1 shown in <strong>FigureS3C_PINK1.tif </strong>(immunoprecipitation) Rab8A shown in <strong>FigureS3C_Rab8A.tif</strong> Phospho-Ser111 Rab8A shown in <strong>FigureS3C_Rab8APSer111.tif</strong> Ubiquitin shown in <strong>FigureS3C_ Ubiquitin.tif </strong>(same blot used for Figure_4B, Halo-multiDSK pull-down) Phospho-Ser65 Ubiquitin shown in <strong>FigureS3C_ UbiquitinPSer65.tif </strong>(same blot used for Figure_4B, Halo-multiDSK pull-down) <strong>Figure S4: Immunoblots for PINK-Parkin signaling in VPS35 D620N mouse cortical neurons.</strong> Scans of X-ray film: GAPDH shown in <strong>FigureS4_GAPDH.tif</strong> Parkin shown in <strong>FigureS4_Parkin.tif</strong> Phospho-Ser65 Parkin shown in <strong>FigureS4_ParkinPSer65.tif</strong> Rab8A shown in <strong>FigureS4_Rab8A.tif</strong> Phospho-Ser111 Rab8A shown in <strong>FigureS4_Rab8APSer111.tif</strong> CISD1 shown in <strong>FigureS4_ CISD1.tif </strong>(Halo-UBQLN1 pull-down) Phospho-Ser65 Ubiquitin shown in <strong>FigureS4_ UbiquitinPSer65.tif</strong> (Halo-UBQLN1 pull-down) VPS35 shown in <strong>FigureS4_VPS35.tif</strong> Scan of Memcode shown in <strong>FigureS4_Memcode.tif</strong> <strong>Figure S6: Immunoblots for biochemical analysis in C56BL/6J mouse cortical neurons.</strong> Scans of X-ray film: GAPDH shown in <strong>FigureS6_GAPDH.tif</strong> Phospho-Ser65 Ubiquitin shown in <strong>FigureS6_ UbiquitinPSer65.tif </strong> <strong>Figure S8: Immunoblots for biochemical analysis in PINK1 WT and KO mouse cortical neurons.</strong> Scans of X-ray film: GAPDH shown in <strong>FigureS8_GAPDH.tif</strong> Phospho-Ser65 Ubiquitin shown in <strong>FigureS8_ UbiquitinPSer65.tif</strong> <strong>Figure S9: Immunoblots for biochemical analysis of ubiquitylated target in C56BL/6J mouse cortical neurons.</strong> 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 <strong>FigureS9_S3_Ubiquitin.tif </strong>(same blot used for Figure_S3B) Phospho-Ser65 Ubiquitin shown in <strong>FigureS9_S3_UbiquitinPSer65.tif </strong>(same blot used for Figure_S3B) ABCD3 shown in <strong>FigureS9_ABCD3.tif</strong> ACSL1shown in <strong>FigureS9_ ACSL1.tif</strong> ACSL6 shown in <strong>FigureS9_ACSL6.tif</strong> AGPAT5 shown in <strong>FigureS9_AGPAT5.tif</strong> ARHGAP33 shown in <strong>FigureS9_ARHGAP33.tif</strong> ATAD1 shown in <strong>FigureS9_ATAD1.tif</strong> CAD shown in <strong>FigureS9_ CAD.tif</strong> CAMK2A shown in <strong>FigureS9_CAMK2A.tif</strong> CAMK2B shown in <strong>FigureS9_CAMK2B.tif</strong> CDK16 shown in <strong>FigureS9_CDK16.tif</strong> CISD1shown in <strong>FigureS9_CISD1.tif</strong> CNN3 shown in<strong> FigureS9_CNN3.tif</strong> CPT1α shown in <strong>FigureS9_ CPT1A.tif</strong> CYB5B shown in <strong>FigureS9_CYB5B.tif</strong> CYB5R3 shown in <strong>FigureS9_CYB5R3.tif</strong> DCAKD shown in <strong>FigureS9_DCAKD.tif</strong> DCAMKL2 shown in <strong>FigureS9_DCAMKL2.tif</strong> FAM213A shown in <strong>FigureS9_FAM213A.tif</strong> FBXO41 shown in <strong>FigureS9_FBXO41.tif</strong> GK shown in <strong>FigureS9_GK.tif</strong> HK1 shown in <strong>FigureS9_HK1.tif</strong> HSDL1 shown in <strong>FigureS9_HSDL1.tif</strong> MAO-A shown in <strong>FigureS9_MAOA.tif</strong> MAO-B shown in <strong>FigureS9_MAOB.tif</strong> MAPRE2 shown in <strong>FigureS9_MAPRE2.tif</strong> MARC2 shown in <strong>FigureS9_MARC2.tif</strong> MFN1 shown in <strong>FigureS9_MFN1.tif</strong> MFN2 shown in <strong>FigureS9_MFN2.tif</strong> NAV1.7 shown in <strong>FigureS9_NAV1.7.tif</strong> P23 shown in <strong>FigureS9_p23.tif</strong> PRKCG shown in <strong>FigureS9_PRKCG.tif</strong> RAB5C shown in <strong>FigureS9_Rab5c.tif</strong> RHOT2 shown in <strong>FigureS9_RHOT2.tif</strong> RIMS4 shown in <strong>FigureS9_RIMS4.tif</strong> RUFY3 shown in <strong>FigureS9_RUFY3.tif</strong> SH3BP4 shown in <strong>FigureS9_SH3BP4.tif</strong> SNX3 shown in <strong>FigureS9_SNX3.tif</strong> TDRKH shown in <strong>FigureS9_TDRKH.tif</strong> TOMM70 shown in <strong>FigureS9_TOMM70.tif</strong> <strong>Figure S10A: Immunoblots for validation of Parkin-dependent substrates in PINK1 WT and KO neurons.</strong> Scans of X-ray film: CISD1 shown in <strong>Figure S10A _CISD1.tif </strong>(Halo-multiDSK pull-down) <strong> Figure S10A _INPUT_CISD1.tif </strong>(INPUT) CPT1α shown in <strong>Figure S10A _ CPT1A.tif</strong> (Halo-multiDSK pull-down) <strong> Figure S10A _INPUT_ CPT1A.tif</strong> (INPUT) Ubiquitin shown in <strong>Figure1C_ S10A _Ubiquitin.tif </strong>(same blot used for FigureS1C, Halo-multiDSK pull-down) <strong> Figure S10A _INPUT_Ubiquitin.tif</strong> (INPUT) Phospho-Ser65 Ubiquitin shown in <strong>Figure1C_ S10A_UbiquitinPSer65.tif </strong>(same blot used for FigureS1C, Halo-multiDSK pull-down) GAPDH Shown in <strong>Figure S10A _INPUT_GAPDH.tif </strong>(INPUT) <strong>Figure S10B: Immunoblots for time-course of Parkin-dependent substrates in SH-SY5Y cells.</strong> Scans of X-ray film: CISD1 shown in <strong>Figure S10B _CISD1.tif </strong>(Halo-multiDSK pull-down) CPT1α shown in <strong>Figure S10B _ CPT1A.tif</strong> (Halo-multiDSK pull-down) Ubiquitin shown in <strong>Figure S10B _Ubiquitin.tif </strong>(Halo-multiDSK pull-down) Phospho-Ser65 Ubiquitin shown in <strong>Figure S10B _UbiquitinPSer65.tif </strong>(Halo-multiDSK pull-down) GAPDH Shown in <strong>Figure S10B _GAPDH.tif </strong> Parkin shown in <strong>FigureS10B_Parkin.tif</strong> Phospho-Ser65 Parkin shown in <strong>FigureS10B_ParkinPSer65.tif</strong> PINK1 shown in <strong>FigureS10B_PINK1.tif</strong> OPA1 shown in <strong>FigureS10B_OPA1.tif</strong> <strong>Figure S11: Immunoblots for <em>in vitro</em> reconstitution assay in HeLa cells.</strong> Scans of X-ray film: CISD1shown in <strong>FigureS11_CISD1.tif</strong> CPT1α shown in <strong>FigureS11_ CPT1</strong><strong>a</strong><strong>.tif</strong> CYB5B shown in <strong>FigureS11_CYB5B.tif</strong> HK1 shown in <strong>FigureS11_HK1.tif</strong> MFN2 shown in <strong>FigureS11_MFN2.tif</strong> VDAC shown in <strong>FigureS11_VDAC.tif</strong> Phospho-Ser65 Parkin shown in <strong>FigureS11_ParkinPSer65.tif</strong> Parkin shown in <strong>FigureS11_Parkin.tif</strong> Ubiquitin shown in <strong>FigureS11_Ubiquitin .tif</strong> Phospho-Ser65 Ubiquitin shown in <strong>FigureS11_UbiquitinPSer65 .tif</strong> <strong>FigureS12A: Purification of CPT1</strong><strong>α protein.</strong> <strong>FigureS12A_</strong><strong> Akta_Purifier_Curves.txt</strong>. 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). <strong>Figure_S12A_Coomassie_Gel.tif</strong>. 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 <strong>FigureS12B-C: Purification of recombinant Parkin targets </strong> Blots shown in the paper are highlighted in bold. <strong>FigureS12B_Ub_Targets_High_800nm.tif</strong> Coomassie stained gels of the parkin targets alongside a BSA curve. Left: Lane: 1: Mwt Marker, 2: Fam213A, 3: MAO-B, 4: CAMK2α, <strong>5: MAO-A (figure S12B)</strong>, 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, <strong>4: MBP-CAMK2</strong><strong>α</strong><strong> (figure S12B)</strong>, 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 <strong>Figure_S12B_Ub_Targets_Low_800nm.tif</strong> Coomassie stained gels of the parkin targets alongside a BSA curve. Lane: 1: Mwt Marker, <strong>2: GST-Miro1 (figure S12B</strong>), 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 <strong>Figure_S12C_Ub_Targets_800nm.tif</strong> Coomassie stained gels of the parkin targets in duplicate alongside a BSA curve. Top: Lane: 1: Mwt Marker, <strong>2+3: ACSL1 45-end (Figure S12C)</strong>, <strong>4+5: SNX3 (Figure S12C),</strong> 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, <strong>4+5: CAMK2</strong><strong>β</strong><strong> (Figure S12C)</strong>, 6+7: MAO-A, <strong>8+9: MAO-B (Figure S12C)</strong>, 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: <strong>FAM213A (Figur</strong>