Quantitative proteomic analysis of Parkin substrates in Drosophila neurons

Background: Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson’s Disease (PD).
In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin
activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by
mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs
or proteasome inhibitors, but not in more physiological settings.
Methods: Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies.
Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and
stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state
Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those
substrates in SH-SY5Y cells.
Results: We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression.
These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase
sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another
PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated
Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells.
Conclusions: Collectively our results provide new leads to the possible physiological functions of Parkin activity
that are not overtly biased by acute mitochondrial depolarisation.