Protein Import Assay into Mitochondria Isolated from Human Cells

Materials and Reagents

1. Cultivation of HEK293 cells

   1. Cell culture dishes 100 × 20 mm (Sarstedt, catalog number: 83.3902)

   2. Cell culture dishes 150 × 20 mm (Sarstedt, catalog number: 83.3903)

   3. Autoclaved disposable glass Pasteur pipets without cotton pad (VWR, catalog number: HECH40567001)

   4. Serological pipettes: Sterile individually packed,

      2 ml pipettes (Sarstedt, catalog number: 83.3903)

      5 ml pipettes (Sarstedt, catalog number: 86.1252.001)

      10 ml pipettes (Sarstedt, catalog number: 86.1254.001)

      25 ml pipettes (Sarstedt, catalog number: 86.1685.001)

   5. Flp-In T-REx HEK293 cells (Human embryonic kidney 293, Invitrogen, catalog number: R78007)

   6. Dulbecco’s modified Eagle medium (DMEM) medium-complete (see Recipes)

      10% Fetal calf serum (FCS, Sigma-Aldrich, catalog number: F0804) and

      1% penicillin/streptomycin (P/S, Sigma-Aldrich, catalog number: P0781-100ML)

      DMEM high glucose (Thermo Fisher, catalog number: 41965-062)

   7. Dulbecco's Phosphate Buffered Saline (PBS) (see Recipes)

      PBS powder (Sigma-Aldrich, catalog number: D5652)

   8. Trypsin-EDTA solution (see Recipes)

      10× Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T4174)

      
      

2. Isolation of mitochondria from HEK293 cells

   1. Cell scraper 16 mm (Sarstedt, catalog number: 831832)

   2. 15 ml Falcon tubes (VWR, catalog number: 734-0451)

   3. 3 × 150 mm confluent dishes plated with HEK293 cells

      Note: Cells are seeded 3-4 days prior to the mitochondrial isolation. Cells are grown to 90% confluency. See Note 2.

   4. Complete^TM Protease Inhibitor Cocktail EDTA-free tablets (Roche, catalog number: 11873580001). Add freshly before use. Aliquots can be stored at -20°C

   5. PBS, store at 4°C before use

   6. Bradford Reagent ROTI^® Quant (Roth, catalog number: K015.1)

   7. 1× buffer M (see Recipes)

      Mannitol (Merck, catalog number: 1.05983)

      Sucrose (Sigma-Aldrich, catalog number: S0389)

      HEPES-KOH, pH 7.4 (VWR, catalog number: 441487)

      EGTA-KOH, pH 7.4 (Roth, catalog number: 3054.1)

      
      

3. Synthesis of [³⁵S]-labeled precursor

   1. TNT^® Quick Coupled Transcription/Translation System SP6 Promoter (Promega, catalog number: L2080). See Note 3.

   2. EasyTag^TM EXPRESS ³⁵S Protein Labeling Mix, [³⁵S]-, 7 mCi, 1175 Ci/mmol (PerkinElmer, catalog number: NEG772007MC). See Note 4.

   3. DNA template (1 µg ml^-1) containing the gene of the mitochondrial precursor inserted downstream of the SP6 promoter. Plasmid vector: pGEM-3/4 (Promega). Store at -20°C.

   4. Nuclease-free double-distilled water

   5. 200 mM L-methionine (Merck, catalog number: 1057070100) in nuclease-free double-distilled water. Store at 4°C

   6. 50 mM ethylene diamine tetra acidic acid (EDTA) disodium salt 2-hydrate, pH 8 (AppliChem, catalog number: A2937) in nuclease-free double-distilled water

   7. 1× sample buffer (see Recipes)

      Tris(hydroxymethyl)aminomethane (Tris, VWR, catalog number: 0497)

      Glycerol (Sigma-Aldrich, catalog number: G7757)

      Bromophenol blue (Roth, catalog number: A512.1)

      Dithiothreitol (DTT, AppliChem, catalog number: A1101)

      
      

4. Import of [³⁵S]-labeled precursors into isolated mitochondria

   1. Blotting membrane, nitrocellulose, Amersham^TM Protran^® (VWR, catalog number: 10600001), 0.2 µm pore size

   2. 20 mM Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma-Aldrich, catalog number: C2759)

   3. 200 mM phenylmethylsulfonyl fluoride (PMSF, AppliChem, catalog number: A0999.0100)

   4. Crushed ice

   5. Triton X-100 (AppliChem, catalog number: A1388)

   6. 1× buffer M (see Recipes)

   7. 20 µg/import reaction mitochondria (see: Recipes)

   8. 20 µg ml^-1 peqGOLD proteinase K (see Recipes)

   9. 1× sample buffer (see Recipes)

   10. SEH buffer (see Recipes)

Equipment

1. Cultivation of HEK293 cells

   1. Laminar flow hood class II (ENVAIR eco)

   2. CO₂ incubator

   3. Vacuum pump

   4. Microscope

   5. Centrifuge (e.g., Eppendorf, model: 5702R)

   Equipped cell culture laboratory containing, e.g., a laminar flow hood class II (ENVAIR eco), a CO₂ incubator for cell cultivation set at 37°C and 5% CO₂, a vacuum pump for removal of medium using sterile glass Pasteur pipets, a microscope, and a cell counter and a cooling centrifuge.

   
   

2. Isolation of mitochondria from HEK293 cells

   1. Electric stirrer (Heidolph RZR 2020)

   2. Potter S homogenizer (15 ml) with tight-fitting PTFE (polytetrafluorethylen) plungers (Sartorius, catalog number: 8542406)

      The range of clearance (i.e., the average distance between the pestle and the cylinder walls) optimal for mitochondria isolation is thereby from 0.045-0.065 mm.

   3. Centrifuge (Eppendorf, model: 5702R)

   4. Centrifuge (Eppendorf, model: 5417R)

   5. Vacuum pump (KNF laboport minipump) for removal of medium using sterile glass Pasteur pipets.

      
      

3. [³⁵S]-labeled precursor synthesis

   Hypoxic Glove Box and Cabinet (Coy Laboratory Products, “Coy 1 Person O₂ Control Glove Box”, catalog number: 031615); not necessary if precursor protein is not prone to air oxidation (e.g., does not contain or contains only few cysteine residues).

   
   

4. Import of [³⁵S]-labeled precursors into isolated mitochondria

   1. ThermoMixer Eppendorf (F1.5, #EP5384000012)

   2. Centrifuge (Eppendorf, model: 5417R)

   3. System for SDS-PAGE electrophoresis and (semidry) blotting (Bio-Rad, MiniPROTEAN Tetra Cell, catalog number: 1658000)

   4. Imaging System Typhoon FLA 9500 (GE Healthcare, catalog number: 9351515)

   5. Image Eraser FLA (GE Healthcare, catalog number: 12999750)

Software

1. ImageQuant TL 8.1 (GE Healthcare Life Science).

Procedure