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  • Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics.

Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics.

Molecular cell (2018-04-17)
Alban Ordureau, Joao A Paulo, Wei Zhang, Tim Ahfeldt, Jiuchun Zhang, Erin F Cohn, Zhonggang Hou, Jin-Mi Heo, Lee L Rubin, Sachdev S Sidhu, Steven P Gygi, J Wade Harper
ABSTRACT

Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains.

MATERIALS
Product Number
Brand
Product Description

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Carbonyl cyanide 3-chlorophenylhydrazone, ≥97% (TLC), powder
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PR-619, ≥95% (HPLC)
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Benzamidine, ≥95.0%
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Triethylammonium bicarbonate buffer, 1.0 M, pH 8.5±0.1
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Antimycin A from Streptomyces sp.
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Oligomycin A, ≥99% (HPLC)
Supelco
Empore SPE Disks, matrix active group C18, diam. 47 mm, pk of 20
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