TITLE: Reconstitution of Parkin ubiquitin ligase activity using mouse and human mitochondria
AUTHORS: Odetta Antico, Alban Ordureau, Michael Stevens, J. Wade Harper, Miratul M. K. Muqit

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Analysis of Parkinson’s linked genes PINK1 and Parkin has uncovered a mechanism by which upon loss of mitochondrial membrane potential, Parkin E3 ubiquitin ligase activity is activated by PINK1 kinase activity, to trigger mitochondrial membrane protein ubiquitylation, leading to removal of damaged mitochondria (mitophagy). We and other groups have previously reported in vitro assays of Parkin E3 ligase activity using recombinant Parkin and PINK1 expressed in E. coli. This provided evidence of Parkin activation by PINK1 phosphorylation of Ser65 in both ubiquitin and UBL domain of Parkin. Herein, we report a reconstitution assay in which addition of recombinant Parkin to mitochondria isolated from cells after treatment by combination of Antimycin A and Oligomycin (to induce PINK1 activation on the outer mitochondrial membrane), enables robust ubiquitylation of multiple substrates at the mitochondria. This assay represents a powerful tool to study Parkin E3 ligase activity and the functional interplay between ubiquitylation and phosphorylation mediated by PINK1 and Parkin and their role in reshaping the endogenous mitochondrial proteome.</div></div>

[STEPS]
?. [PROCEDURE TO ISOLATE AND CULTURE MOUSE EMBRYONIC FIBROBLASTS-Dissection of E13.5 mouse embryos ◊TIMING 20 min]
Use sterilized instruments by autoclave or washing them with 70% (vol/vol) ethanol. Dry thoroughly if ethanol is used.
?. [PROCEDURE TO ISOLATE AND CULTURE MOUSE EMBRYONIC FIBROBLASTS-Dissection of E13.5 mouse embryos ◊TIMING 20 min]
Soak dissection tools in 70% ethanol between embryos to prevent contamination.
?. [PROCEDURE TO ISOLATE AND CULTURE MOUSE EMBRYONIC FIBROBLASTS-Dissection of E13.5 mouse embryos ◊TIMING 20 min]
In the hood. Prepare  dishes with cold PBS. Separate embryos from uterus and placenta. Place each embryo into a single dish with cold PBS.
10
?. [PROCEDURE TO ISOLATE AND CULTURE MOUSE EMBRYONIC FIBROBLASTS-Dissection of E13.5 mouse embryos ◊TIMING 20 min]
Number dishes and Eppendorf tubes for tissue collection for genotyping.
?. [PROCEDURE TO ISOLATE AND CULTURE MOUSE EMBRYONIC FIBROBLASTS-Dissection of E13.5 mouse embryos ◊TIMING 20 min]
Euthanize the embryos by decapitation and separate the head from the body.
?. [PROCEDURE TO ISOLATE AND CULTURE MOUSE EMBRYONIC FIBROBLASTS-Dissection of E13.5 mouse embryos ◊TIMING 20 min]
Wash the bodies twice with PBS to minimise contamination and collect a small piece of tail for genotyping.
?. [PROCEDURE TO ISOLATE AND CULTURE MOUSE EMBRYONIC FIBROBLASTS-Dissection of E13.5 mouse embryos ◊TIMING 20 min]
Place the body on a dish with PBS and remove the red spot (bowel) with forceps.
?. [PROCEDURE TO ISOLATE AND CULTURE MOUSE EMBRYONIC FIBROBLASTS-Dissection of E13.5 mouse embryos ◊TIMING 20 min]
Place the body on a clean dish and mince the tissue with a spring scissors (or with a sterile scalpel blades).
?. [Cell dissociation and plating ]
Prepare digestion medium by adding  of DNase I (stock solution 10 mg/mL) to  of Trypsin 0.025% (1:1 Trypsin 0.05%-HBSS).
125 µl
10 mL
?. [Cell dissociation and plating ]
Add  of digestion medium to the tissue and transfer in a  falcon tube.
5 mL
15 mL
?. [Cell dissociation and plating ]
Incubate at  in a water bath for .
37 °C
?. [Cell dissociation and plating ]
Pipette to mechanically dissociate the tissue, gentle and sequential pipetting (using ,  and  pipettes) until cells are completely suspended.
10 mL
5 mL
1 mL
Note: The number of trituration is approximative, it may vary depending on the size of the unbroken tissues.
?. [Cell dissociation and plating ]
Inactivate trypsin digestion by  of culturing medium.
5 mL
?. [Cell dissociation and plating ]
Centrifuge at  for .
Centrifuge: 1200 33
?. [Cell dissociation and plating ]
Remove media and resuspend in  culturing media.
5 mL
?. [Cell dissociation and plating ]
Filter the cells through a  filter.
70
Note: The cell suspension should be filtered in order to exclude any undigested tissue pieces or aggregates from the newly prepared cell suspension.
?. [Cell dissociation and plating ]
Take a  aliquot, add 1:1 ratio Trypan Blu and determine the density of cells and cell viability to the cell counter.
15 µl
?. [Cell dissociation and plating ]
Plate the 3.0 × 106 cells/well plates out on  dishes, containing  of pre-warmed culturing media.
10
10 mL
?. [Cell dissociation and plating ]
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).
Note: In this assay we use primary MEFs between 8-10 passages.
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial depolarisation ◊TIMING 4h, day of experiment]
To depolarize or uncouple mitochondrial membrane potential in MEFs, treat the cultures for  with a combination of  Antimycin A and  Oligomycin dissolved in DMSO at .
37 °C
Note: Before the experiment, MEFs were plated in 15 cm dishes and stimulated at 80-90% confluence.
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Gently aspirate the medium from wells.
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Wash twice by adding  of warmed DPBS (room temperature) containing protease inhibitors and phosphatase inhibitors.
5 mL
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Place the  dish  and add  of Hypotonic Buffer. Carefully scrape the cells and collect the cells in a  microcentrifuge tube. Add  of Hypotonic Buffer in each tube (for a total of ).
15
on ice
1 mL
15 mL
2 mL
3 mL
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Stand  for  in the cold room.
on ice
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Homogenise cells using a stainless steel Dounce homogeniser with 45 strokes.
Note: Check cell disruption with light microscope, 80-90 % cells should be disrupted.
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Add to the disrupted cells 2.5X MSH buffer and mix.
Note: Mixing 2.5X MSH volume to the initial volume of hypotonic buffer will give 1X MSH.
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Centrifuge the homogenate at  in a refrigerated centrifuge for , to remove cell debris and nuclei.
Centrifuge: 700 34
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Transfer supernatant into a new  tube and centrifuge at  x  at  to remove residual nuclei and cell debris.
15 mL
Centrifuge: 700 34
4 °C
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Centrifuge at  x  at  to pellet mitochondria.
Centrifuge: 9000 34
4 °C
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Resuspend mitochondria in  of 1X MSH buffer and centrifuge at  x  at  to pellet mitochondria. Repeat twice to remove any cytosolic proteins.
1 mL
Centrifuge: 9000 34
4 °C
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Centrifuge at  x  at  to pellet mitochondria and resuspend in  of MUB Buffer.
Centrifuge: 9000 34
4 °C
150 µl
?. [MITOCHONDRIAL ISOLATION FOR MEFs-Mitochondrial isolation ◊TIMING 1-1.5h]
Protein quantification: take a small aliquot of mitochondria (10 µL), add 1% Triton, vortex and estimate protein concentration by using the Coomassie Protein Assay.
Usually from a 15 cm dish of MEFs at 90% confluence it is possible to isolate 200µg of crude mitochondria.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial depolarisation ◊TIMING 2h, day  of experiment]
To depolarize or uncouple mitochondrial membrane potential in MEFs, treat the cultures for  with a combination of  Antimycin A and  Oligomycin dissolved in DMSO at .
37 °C
Note: Before the experiment Hela were plated in 15 cm dishes and stimulated to 80- 90% confluence.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Gently aspirate the medium from wells.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Wash twice by adding  of warmed DPBS () containing protease inhibitors and phosphatase inhibitors.
5 mL
4 Room temperature
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Place the  and add  of Hypotonic Buffer. Carefully scrape the cells and collect the cells in a  microcentrifuge tube. Add  of Hypotonic Buffer in each tube (for a total of ).
15
on ice
1 mL
15 mL
2 mL
3 mL
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Stand  for  in the cold room.
on ice
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Homogenise cells using a stainless steel Dounce homogeniser with 25 strokes.
Note: Check cell disruption with light microscope, 80-90 % cell should be disrupted.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Add to the disrupted cells 2.5X MSH buffer and mix.
Note: Mixing 2.5X MSH volume to the initial volume of hypotonic buffer will give 1X MSH.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Centrifuge the homogenate at  in a refrigerated centrifuge for , to remove cell debris and nuclei.
Centrifuge: 700 34
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Transfer supernatant into a new  tube and centrifuge at  x  at  to remove residual nuclei and cell debris.
15 mL
Centrifuge: 700 34
4 °C
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Centrifuge at  x  at  to pellet mitochondria.
Centrifuge: 9000 34
4 °C
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Resuspend mitochondria in  1X MSH buffer and centrifuge at x  at  to pellet mitochondria. Repeat twice to remove any cytosolic proteins.
1 mL
Centrifuge: 9000 34
4 °C
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Centrifuge at  x  at  to pellet mitochondria and resuspend in - of MUB Buffer.
Centrifuge: 9000 34
4 °C
150 µl
250 µl
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Mitochondrial isolation ◊TIMING 1-1.5h]
Protein quantification: take a small aliquot of mitochondria (), add 1% Triton, vortex and estimate protein concentration by using the Coomassie Protein Assay.
10 µl
Usually from one 15 cm dish of HeLa at 80-90% confluence it is possible to isolate 600-800µg of crude mitochondria.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-UBIQUITYLATION ASSAY ◊TIMING 2.5h-3h]
Resuspend isolated mitochondria in MUB buffer at concentration ~, in order to use a volume of mitochondria
?. [MITOCHONDRIAL ISOLATION FOR HeLa-UBIQUITYLATION ASSAY ◊TIMING 2.5h-3h]
Use  of mitochondria for a total volume reaction of .
5 µg
50 µl
?. [MITOCHONDRIAL ISOLATION FOR HeLa-UBIQUITYLATION ASSAY ◊TIMING 2.5h-3h]
Defrost proteins  and prepare a master mix considering that for one single reaction it is required  Parkin,  His-UbE1,  UBE2L3 and  Ubiquitin (as negative control prepare a master mixer without Parkin).
0 °C
?. [MITOCHONDRIAL ISOLATION FOR HeLa-UBIQUITYLATION ASSAY ◊TIMING 2.5h-3h]
Prepare reaction buffer with  Tris ,  MgCl2 and  TCEP.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-UBIQUITYLATION ASSAY ◊TIMING 2.5h-3h]
Combine reaction buffer, master mix of proteins and mitochondria.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-UBIQUITYLATION ASSAY ◊TIMING 2.5h-3h]
Aliquot in order to distribute  in  Eppendorf tube and start the ubiquitylation reaction by adding  ATP in each Eppendorf tube.
50 µl
1.5 mL
?. [MITOCHONDRIAL ISOLATION FOR HeLa-UBIQUITYLATION ASSAY ◊TIMING 2.5h-3h]
Place the eppendorf tubes in a thermomixer and incubate the reaction at , shaking for  (MEFs) or  (HeLa) at .
30 °C
Centrifuge: 1000 33
?. [MITOCHONDRIAL ISOLATION FOR HeLa-UBIQUITYLATION ASSAY ◊TIMING 2.5h-3h]
Stop the reaction by the addition of 4X LDS loading buffer containing 10% of 2- mercaptoethanol.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d]
Boil the samples for  at.
97 °C
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d]
Analyse samples by running  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.
20 µl
For phospho-parkin and Parkin blots, it is recommended to dilute the final reaction 1 in 25 with 1X LDS containing 2.5 % 2- mercaptoethanol.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d]
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  or in cold room (for HK1 it is recommended to transfer at 90 V for 1.5h).
on ice
Note: Prepare only 1 membrane per transfer tank –avoid multiple membranes for transfer in same tank as this reduces ubiquitin transfer.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d]
Incubate membrane with blocking buffer 5% milk in 0.1% TBS-Tween for  at
97 Room temperature
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d]
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  at .
4 °C
Note: Prepare phospho-Ubiquitin Antibody (1:2000), Ubiquitin Antibody (1:1000), CISD1 Antibody (1:1000), CPT1α Antibody (1:1000), CYB5B Antibody (1:1000), HK1 Antibody (1:1000), MFN2 Antibody (1:1000), VDAC Antibody (1:1000) and Parkin Antibody (1:1000) in 5% BSA (TBS-Tween). Prepare phospho-Parkin Antibody (1:2000) in 5% milk (TBS-Tween). To avoid non-specific signal, it is recommended to preincubate phospho-Parkin antibody with a membrane for 2 days before using it.
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d]
Remove primary antibody and wash 3 times with 0.1%TBS-Tween for .
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d]
Add secondary antibodies, HRP-conjugate for  at  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.
4 Room temperature
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d]
Remove secondary antibody and wash 3 times with 0.1%TBS-Tween for .
?. [MITOCHONDRIAL ISOLATION FOR HeLa-Immunoblotting of ◊TIMING 5h-2d]
Develop signal using ECL western Blotting reagents and analysing with Chemidoc.
Note: Depending on signal, film can be best for sensitivity. To improve detection of HK1 and VDAC ubiquitylation, it is recommended to develop signal using Super signal West Dura reagents.