Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization

The PARKIN ubiquitin ligase (also known as PARK2) and its regulatory kinase PINK1 (also known as PARK6), often mutated in familial early-onset Parkinson’s disease, have central roles in mitochondrial homeostasis and mitophagy. Whereas PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate porin, mitofusin and Miro proteins on the MOM, the full repertoire of PARKIN substrates—the PARKIN-dependent ubiquitylome—remains poorly defined. Here we use quantitative diGly capture proteomics (diGly) to elucidate the ubiquitylation site specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization. Hundreds of dynamically regulated ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, we found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of the PARKIN active site residue C431, which has been found mutated in Parkinson’s disease patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and Drosophila melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1–PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis.

[1]  Rachel E. Klevit,et al.  UbcH7 reactivity profile reveals Parkin and HHARI to be RING/HECT hybrids , 2011, Nature.

[2]  S. Gygi,et al.  Defining the Human Deubiquitinating Enzyme Interaction Landscape , 2009, Cell.

[3]  Steven P Gygi,et al.  A probability-based approach for high-throughput protein phosphorylation analysis and site localization , 2006, Nature Biotechnology.

[4]  T. Dawson,et al.  The role of parkin in familial and sporadic Parkinson's disease , 2010, Movement disorders : official journal of the Movement Disorder Society.

[5]  Steven P Gygi,et al.  The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry , 2008, Nature Protocols.

[6]  Miratul M. K. Muqit,et al.  PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65 , 2012, Open Biology.

[7]  Maki Maeda,et al.  Fis1 acts as a mitochondrial recruitment factor for TBC1D15 that is involved in regulation of mitochondrial morphology , 2013, Journal of Cell Science.

[8]  R. Youle,et al.  Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin. , 2012, Developmental cell.

[9]  W. Saxton,et al.  Parkinson's Disease–Associated Kinase PINK1 Regulates Miro Protein Level and Axonal Transport of Mitochondria , 2012, PLoS genetics.

[10]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[11]  Xinnan Wang,et al.  PINK1 and Parkin Target Miro for Phosphorylation and Degradation to Arrest Mitochondrial Motility , 2011, Cell.

[12]  Hyeseong Cho,et al.  Mitofusin 1 is degraded at G2/M phase through ubiquitylation by MARCH5 , 2012, Cell Division.

[13]  Edward L. Huttlin,et al.  A Tissue-Specific Atlas of Mouse Protein Phosphorylation and Expression , 2010, Cell.

[14]  Fabienne C. Fiesel,et al.  PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1 , 2010, Nature Cell Biology.

[15]  R. Youle,et al.  Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin , 2010, The Journal of cell biology.

[16]  Edward L. Huttlin,et al.  Systematic and quantitative assessment of the ubiquitin-modified proteome. , 2011, Molecular cell.

[17]  Michael Lazarou,et al.  PINK1 drives Parkin self-association and HECT-like E3 activity upstream of mitochondrial binding , 2013, The Journal of cell biology.

[18]  R. Youle,et al.  Mechanisms of mitophagy , 2010, Nature Reviews Molecular Cell Biology.

[19]  N. Mizushima,et al.  Parkin Mediates Proteasome-dependent Protein Degradation and Rupture of the Outer Mitochondrial Membrane*♦ , 2011, The Journal of Biological Chemistry.

[20]  J. Yates,et al.  An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database , 1994, Journal of the American Society for Mass Spectrometry.

[21]  Angela C. Poole,et al.  The Mitochondrial Fusion-Promoting Factor Mitofusin Is a Substrate of the PINK1/Parkin Pathway , 2010, PloS one.

[22]  A. Vashisht,et al.  Voltage-dependent Anion Channels (VDACs) Recruit Parkin to Defective Mitochondria to Promote Mitochondrial Autophagy* , 2012, The Journal of Biological Chemistry.

[23]  A. Whitworth,et al.  Drosophila Parkin requires PINK1 for mitochondrial translocation and ubiquitinates Mitofusin , 2010, Proceedings of the National Academy of Sciences.

[24]  R. Youle,et al.  Mitochondrial quality control mediated by PINK1 and Parkin: links to parkinsonism. , 2012, Cold Spring Harbor perspectives in biology.

[25]  Brendan K Faherty,et al.  Optimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics*S , 2006, Molecular & Cellular Proteomics.

[26]  Sonja Hess,et al.  Broad activation of the ubiquitin–proteasome system by Parkin is critical for mitophagy , 2011, Human molecular genetics.

[27]  H. Walden,et al.  Autoregulation of Parkin activity through its ubiquitin‐like domain , 2011, The EMBO journal.

[28]  S. Bloor,et al.  LC3C, Bound Selectively by a Noncanonical LIR Motif in NDP52, Is Required for Antibacterial Autophagy , 2012, Molecular cell.

[29]  N. Hattori,et al.  Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin‐like domain , 2003, EMBO reports.

[30]  L. Chin,et al.  Phosphorylation of parkin by Parkinson disease-linked kinase PINK1 activates parkin E3 ligase function and NF-kappaB signaling. , 2010, Human molecular genetics.

[31]  Sarah Sonnay,et al.  Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1 , 2011, Journal of neurochemistry.

[32]  R. Youle,et al.  p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both , 2010, Autophagy.

[33]  Y. Yoon,et al.  Control of Mitochondrial Morphology Through Differential Interactions of Mitochondrial Fusion and Fission Proteins , 2011, PloS one.

[34]  David S. Park,et al.  ROS-dependent regulation of Parkin and DJ-1 localization during oxidative stress in neurons. , 2012, Human molecular genetics.

[35]  Sebastian A. Wagner,et al.  A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles* , 2011, Molecular & Cellular Proteomics.

[36]  R. Youle,et al.  Parkin is recruited selectively to impaired mitochondria and promotes their autophagy , 2008, The Journal of cell biology.