Reconstitution of Parkin ubiquitin ligase activity using mouse and human mitochondria

Use sterilized instruments by autoclave or washing them with 70% (vol/vol) ethanol. Dry thoroughly if ethanol is used.

Soak dissection tools in 70% ethanol between embryos to prevent contamination.

In the hood. Prepare 10cm dishes with cold PBS. Separate embryos from uterus and placenta. Place each embryo into a single dish with cold PBS.

Number dishes and Eppendorf tubes for tissue collection for genotyping.

Euthanize the embryos by decapitation and separate the head from the body.

Wash the bodies twice with PBS to minimise contamination and collect a small piece of tail for genotyping.

Place the body on a dish with PBS and remove the red spot (bowel) with forceps.

Place the body on a clean dish and mince the tissue with a spring scissors (or with a sterile scalpel blades).

Prepare digestion medium by adding 125µL of DNase I (stock solution 10 mg/mL) to 10mL of Trypsin 0.025% (1:1 Trypsin 0.05%-HBSS).

Add 5mL of digestion medium to the tissue and transfer in a 15mL falcon tube.

Incubate at 37°C in a water bath for 0h 15m 0s.

Pipette to mechanically dissociate the tissue, gentle and sequential pipetting (using 10mL, 5mL and 1mL pipettes) until cells are completely suspended.

Inactivate trypsin digestion by 5mL of culturing medium.

Centrifuge at 1200rpm,0h 0m 0s for 0h 5m 0s.

Remove media and resuspend in 5mL culturing media.

Filter the cells through a 70μm filter.

Take a 15µL aliquot, add 1:1 ratio Trypan Blu and determine the density of cells and cell viability to the cell counter.

Plate the 3.0 × 10⁶ cells/well plates out on 10cm dishes, containing 10mL of pre-warmed culturing media.

Change media every 5 days, grow to 90% confluence and split (minimum 25% confluence to keep cells within the range to promote growth). The growth rate progressively declines when transformation occurs and the cells become immortal, at approximately after 18 passages (it can be variable).

To depolarize or uncouple mitochondrial membrane potential in MEFs, treat the cultures for 4h 0m 0s with a combination of 10micromolar (µM) Antimycin A and 1micromolar (µM) Oligomycin dissolved in DMSO at 37°C.

Gently aspirate the medium from wells.

Wash twice by adding 5mL of warmed DPBS (room temperature) containing protease inhibitors and phosphatase inhibitors.

Place the 15cm dish 37On ice and add 1mL of Hypotonic Buffer. Carefully scrape the cells and collect the cells in a 15mL microcentrifuge tube. Add 2mL of Hypotonic Buffer in each tube (for a total of 3mL).

Stand 37On ice for 0h 15m 0s in the cold room.

Homogenise cells using a stainless steel Dounce homogeniser with 45 strokes.

Add to the disrupted cells 2.5X MSH buffer and mix.

Centrifuge the homogenate at 700x g,0h 0m 0s in a refrigerated centrifuge for 0h 10m 0s, to remove cell debris and nuclei.

Transfer supernatant into a new 15mL tube and centrifuge at 700x g,0h 0m 0s x 0h 10m 0s at 4°C to remove residual nuclei and cell debris.

Centrifuge at 9000x g,0h 0m 0s x 0h 10m 0s at 4°C to pellet mitochondria.

Resuspend mitochondria in 1mL of 1X MSH buffer and centrifuge at 9000x g,0h 0m 0s x 0h 10m 0s at 4°C to pellet mitochondria. Repeat twice to remove any cytosolic proteins.

Centrifuge at 9000x g,0h 0m 0s x 0h 10m 0s at 4°C to pellet mitochondria and resuspend in 150µL of MUB Buffer.

Protein quantification: take a small aliquot of mitochondria (10 µL), add 1% Triton, vortex and estimate protein concentration by using the Coomassie Protein Assay.

To depolarize or uncouple mitochondrial membrane potential in MEFs, treat the cultures for 2h 0m 0s with a combination of 10micromolar (µM) Antimycin A and 1micromolar (µM) Oligomycin dissolved in DMSO at 37°C.

Gently aspirate the medium from wells.

Wash twice by adding 5mL of warmed DPBS (4Room temperature) containing protease inhibitors and phosphatase inhibitors.

Place the 15cm 4On ice and add 1mL of Hypotonic Buffer. Carefully scrape the cells and collect the cells in a 15mL microcentrifuge tube. Add 2mL of Hypotonic Buffer in each tube (for a total of 3mL).

Stand 4On ice for 0h 15m 0s in the cold room.

Homogenise cells using a stainless steel Dounce homogeniser with 25 strokes.

Add to the disrupted cells 2.5X MSH buffer and mix.

Centrifuge the homogenate at 700x g,0h 0m 0s in a refrigerated centrifuge for 0h 10m 0s, to remove cell debris and nuclei.

Transfer supernatant into a new 15mL tube and centrifuge at 700x g,0h 0m 0s x 0h 10m 0s at 4°C to remove residual nuclei and cell debris.

Centrifuge at 9000x g,0h 0m 0s x 0h 10m 0s at 4°C to pellet mitochondria.

Resuspend mitochondria in 1mL 1X MSH buffer and centrifuge at9000x g,0h 0m 0s x 0h 10m 0s at 4°C to pellet mitochondria. Repeat twice to remove any cytosolic proteins.

Centrifuge at 9000x g,0h 0m 0s x 0h 10m 0s at 4°C to pellet mitochondria and resuspend in 150µL-250µL of MUB Buffer.

Protein quantification: take a small aliquot of mitochondria (10µL), add 1% Triton, vortex and estimate protein concentration by using the Coomassie Protein Assay.

Resuspend isolated mitochondria in MUB buffer at concentration ~1mg/mL, in order to use a volume of mitochondria < 10% of reaction volume.

Use 5µg of mitochondria for a total volume reaction of 50µL.

Defrost proteins °C and prepare a master mix considering that for one single reaction it is required 1micromolar (µM) Parkin, 0.1micromolar (µM) His-UbE1, 1micromolar (µM) UBE2L3 and 30micromolar (µM) Ubiquitin (as negative control prepare a master mixer without Parkin).

Prepare reaction buffer with 50millimolar (mM) Tris 7.5, 5millimolar (mM) MgCl2 and 0.5millimolar (mM) TCEP.

Combine reaction buffer, master mix of proteins and mitochondria.

Aliquot in order to distribute 50µL in 1.5mL Eppendorf tube and start the ubiquitylation reaction by adding 2millimolar (mM) ATP in each Eppendorf tube.

Place the eppendorf tubes in a thermomixer and incubate the reaction at 30°C, shaking for 2h 0m 0s (MEFs) or 1h 30m 0s (HeLa) at 1000rpm,0h 0m 0s.

Stop the reaction by the addition of 4X LDS loading buffer containing 10% of 2- mercaptoethanol.

Boil the samples for 0h 3m 0s at97°C.

Analyse samples by running 20µL of reaction on Nu-page Bis-Tris 4-12% gels for a better resolution of ubiquitin chains, at 120 V for ~2h. Use MES SDS Running Buffer or MOPS SDS Running Buffer according to the size of protein to be analysed.

Transfer gel on PVDF membrane for phospho-ubiquitin and ubiquitin signal and nitrocellulose membrane for phospho-Parkin and Parkin signal. Transfer in Towbin buffer at 80 V for 1h 30m 0s 97On ice or in cold room (for HK1 it is recommended to transfer at 90 V for 1.5h).

Incubate membrane with blocking buffer 5% milk in 0.1% TBS-Tween for 1h 0m 0s at 97Room temperature

Remove blocking buffer, if primary antibodies are in 5% BSA, rinse twice with 0.1% TBS-Tween to remove any traces of milk, add primary antibodies and incubate 1h 0m 0s at 4°C.

Remove primary antibody and wash 3 times with 0.1%TBS-Tween for 0h 10m 0s.

Add secondary antibodies, HRP-conjugate for 1h 0m 0s at 4Room temperature diluted 1:5000 in 1% BSA (0.1% TBS-Tween). Use 1:10000 dilution in 1% BSA for Parkin antibody and 1:10000 dilution in 5% milk for and phospho-Parkin antibody.

Remove secondary antibody and wash 3 times with 0.1%TBS-Tween for 0h 10m 0s.

Develop signal using ECL western Blotting reagents and analysing with Chemidoc.