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. FILE DECRIPTIONS 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)