TITLE: Human in vitro Parkin ubiquitin E3 ligase assay
AUTHORS: Michael Stevens, Miratul M. K. Muqit

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mutations in PARK2 encoding Parkin are causal for early-onset Parkinson’s disease. Parkin is a ubiquitin E3 ligase and is activated by the PINK1 kinase to stimulate ubiquitin-dependent removal of damaged mitochondria via autophagy (mitophagy). Previous studies have identified Miro1 as a Parkin substrate in cells and in vitro. This protocol describes a facile direct assay of Parkin E3 ligase activity using recombinant untagged Parkin and GST-Miro1 as a substrate. This assay can be employed to assess the effect of small molecule on Parkin activity as well as the impact of Parkinson’s disease mutations of Parkin.</div><br/><div class = "text-block">In cells the removal of malfunctioning organelles is important for cellular survival. Damaged mitochondria are removed via mitophagy upon activation of the PTEN induced kinase 1 (PINK1) / Parkin pathway. In healthy mitochondria PINK1 is cleaved by PARL and released into the cytoplasm where it is degraded by the N-end rule. In damaged mitochondria depolarisation of the mitochondrial membrane stabilises PINK1, causing it to accumulate on the outer mitochondrial membrane. Stabilised PINK1 phosphorylates Ser65 on both ubiquitin and the ubiquitin like (Ubl) domain of Parkin, this activated Parkin. Active Parkin ubiquitinates outer mitochondrial membrane proteins, leading to ubiquitin chains containing pSer65 ubiquitin. The formation of these chains on the mitochondria identifies it for removal by mitophagy. Loss of activity in either Parkin or PINK1 have been observed in cases of early-onset Parkinson’s disease. Miro1 is a mitochondrial GTPase involved in mitochondrial morphology and translocation. Previous studies have shown Miro1 to be efficiently ubiquitylated by Parkin at multiple sites.</div><div class = "text-block">The following protocol outlines a substrate-based catalytic ubiquitin ligase assay for human Parkin that employs GST-Miro1. Parkin is pre-incubated with either wild type (wt) PINK1 from Tribolium castaneum (TcPINK1) to activate it; the kinase inactive (KI, D359A) version to maintain Parkin’s inactive state; or is left untreated. In each case the proteins, UbE1, UbE2L3, Flag-Ub and GST-Miro1 are then added to start the ubiquitylation reaction. The reaction is terminated by the addition of SDS-loading dye. Once the assay is complete, western blotting is performed to demonstrate Parkin activation via multi-monoubiquitylation of GST-Miro1 as seen by 3 or 4 additional bands in the sample treated with wt TcPINK1, but not in the samples treated with KI TcPINK1 or the untreated sample.</div></div>

[STEPS]
?. [Preamble]
The set of reactions performed are where Parkin is phosphorylated by wt MBPTcPINK1, kinase inactive (KI) MBP-TcPINK1, and no MBP-TcPINK1, with all reactions performed in duplicate.
?. [Preamble]
and  are required per reaction.
[kinase master mix]
[ubiquitylation master mix]
?. [Preamble]
The volume of master mix made in each case is the volume required for the number of reactions + 10 % rounding up, for instance if 6 reactions are going to be performed then enough master mix should be made for 6 + 1 (0.6 rounding up) reactions.
?. [Making Master Mixes]
Prior to starting reactions keep reagents and master mixes  .
on ice
?. [Making Master Mixes]
Combine non-protein reagents for each master mix, vortex the master mix, and settle the master mix to the bottom of the microcentrifuge tube.
?. [Making Master Mixes]
Defrost proteins   and add to kinase and ubiquitin master mixes, mix master mixes by pipetting.
on ice
?. [Making Master Mixes]
Just before starting kinase reactions dilute wt and KI MBP-PINK1 to 2.16 uM with deionised water, aliquot  to two different microcentrifuge tube per MBP-PINK1 mix along with deionised water.
[of the mixes]
?. [Ubiquitylation Reactions]
Begin the kinase reaction by adding  to each reaction tube, and incubate the reaction  .
[kinase master mix]
Centrifuge: 1000 33, 30 min, 37 10
?. [Ubiquitylation Reactions]
Add  to each reaction and incubate the ubiquitylation reaction . Stop the reaction by the addition of  and incubate at  for  to denature the proteins and make SDS-PAGE samples.
[ubiquitylation master mix]
Centrifuge: 1000 33, 30 min, 37 10
[4x LDS loading buffer]
95 °C
?. [Resolving Target Proteins by Western Blot]
Analyse assays by immunoblotting for the protein read outs GST-Miro1 to observe target ubiquitylation, His-Ube1, and Flag-Ub.
?. [Resolving Target Proteins by Western Blot]
Dilute the final reaction sample 1 in 3 with 1x LDS loading buffer and resolve   by SDS-PAGE using two 4-20 % polyacrylamide gel.
[final sample]
?. [Resolving Target Proteins by Western Blot]
Transfer proteins to nitrocellulose membranes and immunoblot against anti-Miro1, anti-Flag, and anti-6His.
?. [Resolving Target Proteins by Western Blot]
If ubiquitylation has worked there should be ubiquitin chains observed with the flag antibody only in reactions with MBP-wt TcPINK1 added.
?. [Resolving Target Proteins by Western Blot]
In addition, Miro 1 ubiquitylation should only be observed in reactions with wt TcPINK1 added.