Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

All animals used in these experiments are maintained in the animal care facility at York University. The experiments are conducted in accordance with the Canadian Council on Animal Care guidelines with approval from the York University Animal Care Committee (Permit: 2017-08).

1. Functional isolation of subsarcolemmal and intermyofibrillar mitochondria from skeletal muscle

1. Reagent preparation:
   1. Prepare all the buffers and media as mentioned in Table 1.
   2. Set the buffers to pH 7.4 and store at 4 °C (up to 2 weeks), except nagrase protease.
   3. Prepare fresh nagarse protease (Table 1) each time.
2. Tissue removal and mincing
   NOTE: A flowchart of the steps below for the isolation of mitochondria is provided in Figure 1.
   1. Fill a glass scintillation vial with ~20 mL of Buffer 1 (Table 1) and place it on ice.
   2. While the mouse is under anesthetic, harvest skeletal muscles (tibialis anterior, quadriceps, gastrocnemius etc.), approximately 500-1000 mg, and place the muscles in the scintillation vial containing chilled Buffer 1.
      NOTE: Gaseous isoflurane is used as an anesthetic at 2.5% at a flow rate of 0.4 L of O₂/min. A pinch test is performed to ensure that the animal is non-responsive.
   3. Following tissue collection, euthanize the animal via cervical dislocation.
   4. Pre-chill a watch glass on ice. Remove any fat or connective tissue from the muscles and mince the tissue on the watch glass until it is a homogenous slurry.
   5. Place the minced tissue in a pre-chilled 50 mL plastic centrifugation tube (see Table of Materials) and record the exact weight.
   6. Dilute the minced tissue 10-fold with Buffer 1 + ATP (Table 1).
   7. Homogenize the muscle sample using an 8 mm twin-blade homogenizer (see Table of Materials) at a power output of 9.8 Hz for 10 s, ensuring no visible chunks of muscle are remaining.
   8. Centrifuge the samples at 800 x g for 10 min using a high-speed centrifuge (see Table of Materials).
      NOTE: The purpose of this step is to separate SS and IMF mitochondrial fractions. The supernate contains SS mitochondria, while the pellet contains IMF mitochondria.
3. SS mitochondrial isolation
   1. Filter the supernate through a single layer of cheesecloth into another set of 50 mL plastic centrifugation tubes to remove any large debris or contaminants.
   2. Centrifuge the supernate at 9,000 x g for 10 min at 4 °C using a high-speed centrifuge.
   3. Discard the supernate and resuspend the pellet in 3.5 mL of Buffer 1 + ATP.
      NOTE: Resuspend with a P1000 and do this carefully. Mitochondria are fragile organelles. Perform gently and avoid touching the pellet with the pipette tip.
   4. Centrifuge the sample at 9,000 x g for 10 min at 4 °C using a high-speed centrifuge.
   5. Discard the supernate unless further processing to isolate a cytosolic fraction devoid of nuclei, mitochondria, and other organelles is desired. Resuspend the pellet carefully in roughly 95 µL of the resuspension medium (Table 1) using a P200 pipette.
      NOTE: Similar to step 1.3.3, avoid touching the pellet with the pipette tip. The volume of the resuspension medium can be altered depending on the size of the pellet. This is the SS mitochondrial fraction, which can be stored on ice until the IMF fraction is isolated. If desired, the cytosolic fraction, devoid of organelles, can be isolated by centrifuging the supernate at 100,000 x g in an ultracentrifuge (see Table of Materials) and thereafter discarding the pellet.
4. IMF mitochondrial isolation
   1. Dilute the pellet from step 1.2.8 by 10-fold in Buffer 1 + ATP. Resuspend using a Teflon pestle by gently mixing the pellet and buffer together until the sample is consistent.
   2. Homogenize the sample using an 8 mm twin-blade homogenizer (see Table of Materials) at a power output of 9.8 Hz for 10 s.
   3. Centrifuge the sample at 800 x g for 10 min at 4 °C using a high-speed centrifuge.
   4. Discard the supernate and dilute the IMF mitochondrial pellet 10-fold using Buffer 2 and resuspend using the Teflon pestle by gently mixing until it is homogeneous.
   5. Add 25 µL/g of tissue to 10 mg/mL nagarse protease (Table 1) and place the centrifugation tube on its side on ice, gently mixing every minute by rocking the tube side to side.
      NOTE: The addition of nagarse helps liberate IMF mitochondria by performing limited digestion of the myofibrils.
   6. After 5 min, add 20 mL of Buffer 2 (Table 1) to dilute the nagarse protease to the point of inactivity and stop the digestion.
   7. Immediately centrifuge the sample at 5,000 x g for 5 min at 4 °C using a high-speed centrifuge.
   8. Discard the supernate and dilute the pellet 10-fold using Buffer 2 and resuspend using a Teflon pestle by gently mixing.
   9. Centrifuge the sample at 800 x g for 15 min at 4 °C to pellet the fibrous material.
   10. Pour the supernate in a new set of 50 mL plastic centrifuge tubes, careful not to disrupt the pellet, which can be discarded.
   11. Centrifuge the sample at 9,000 x g for 10 min at 4 °C as in step 1.4.7.
   12. Discard the supernate, add 3.5 mL of Buffer 2 and gently resuspend the pellet using a P1000 pipette.
   13. Centrifuge the sample at 9,000 x g for 10 min at 4 °C as in step 1.4.7.
   14. Discard the supernate and resuspend the pellet gently with roughly 180 µL of resuspension medium using a P200 pipette.
       ​NOTE: The volume of the resuspension medium can be altered depending on the size of the pellet. This is the IMF mitochondrial fraction, which can be stored on ice until it is to be used in the import assay.

2. Mitochondrial protein import

1. In vitro transcription
   NOTE: These subsequent steps outline how to prepare a radiolabelled protein for import. The import pathway under investigation may dictate the choice of target protein, for example, to evaluate import into the mitochondrial matrix, ornithine carbamyl transferase, OCT, and malate dehydrogenase, MDH, are commonly used, while Tom40 is commonly used as an indication of import into the outer mitochondrial membrane. For the following steps, plasmid DNA encoding the protein of choice is required. The transcription of plasmid DNA can be done prior to the mitochondrial isolation on a separate day, and the mRNA collected from this experiment can be stored and used in future translation and import experiments.
   1. Linearize the DNA: Combine 40 µL of plasmid DNA (5 µg/µL), 5 µL of 10x enzyme buffer and 5 µL of restriction enzyme and incubate at 37 °C for 30 min.
      NOTE: The restriction enzymes utilized may vary based on the plasmid, which may require a different enzyme buffer, and experimental conditions DNA can be used in any amount/volume, as the template can be scaled up or down.
   2. Purify and precipitate the linearized DNA using phenol and ethanol. Carry out all the subsequent steps (2.1.3-2.1.15) in sterile 1.5 mL tubes, and use a tabletop centrifuge (see Table of Materials) for the centrifugation steps.
   3. Add the appropriate volume of sterile H₂O so that the final volume equals 400 µL. Then, add 400 µL of phenol.
   4. Mix vigorously by inversion for ~10 s, and centrifuge at 17,000 x g for 1 min at 4 °C. Withdraw and save the upper phase.
   5. Add 400 µL of phenol:chloroform:isoamylalcohol (25:24:1, v:v:v).
   6. Mix vigorously by inversion for ~10 s, and centrifuge at 17,000 g for 1 min at 4 °C. Withdraw and save the upper phase.
   7. Add 400 µL of chloroform:isoamylalcohol (24:1, v:v).
   8. Mix vigorously by inversion for ~10 s, and centrifuge at 17,000 g for 1 min at 4 °C. Withdraw and save the upper phase.
   9. Add 40 µL of 3M sodium acetate (pH 7.0), and add 1 mL of 95% ethanol.
   10. Place sterile 1.5 mL tubes in -80 °C freezer on their side for 15 min.
   11. Centrifuge the samples at 17,000 x g for 10 min at 4 °C.
   12. Discard the supernate and wash the pellet with 300 µL of 80% ethanol.
   13. Centrifuge the samples at 17,000 x g for 2 min at 4 °C.
   14. Discard the supernate and air-dry the pellet. Once the pellet is dry, it should be clear.
   15. Resuspend the pellet in 20 µL of sterile H₂O
   16. Measure the DNA concentration using a spectrophotometer (see Table of Materials) and ensure that the A_260:280 ratio is above 1.8.
   17. Dilute the DNA to a concentration of 0.8 µg/µL in sterile H₂O.
   18. Transcribe the DNA: Combine plasmid (0.8 µg/µL, 60.8 µL), sterile H₂O (8.4 µL), 10 mM NTP (5.2 µL), 10 mM ATP ( 10.0 µL), 1 mM M-7-G (11.6 µL), the mix as mentioned step 2.1.19 (15.6 µL), RNAsin (5.2 µL) and an appropriate RNA polymerase (4.8 µL) to form "one reaction mix" (total volume = 121.6 µL) and incubate for 90 min at the optimum temperature for the polymerase (37 °C for T7 polymerase; 40 °C for SP6 polymerase)
   19. To prepare the mix add 200 µL of 1M HEPES (pH 7.9), 100 µL of 1 M MgAc₂, 500 µL of 4 M KAc, 100 µL of 20 mM spermidine, 100 µL of 0.5 M DTT, and 200 µL of sterile H₂0.
       NOTE: The reaction can be scaled up or down in volume as long as the provided proportions of reagents are maintained. The RNA polymerase used is dictated by the bacterial promoter sequence within the plasmid being used.
   20. Then, purify and precipitate the mRNA using phenol and ethanol as in steps 2.1.2-2.1.15. Bring the volume up to 400 µL with sterile H₂O (add 280 µL) and proceed as described in steps 2.1.2-2.1.15. Resuspend the final pellet in 20 µL of sterile H₂O.
   21. Measure the concentration of mRNA using a spectrophotometer, ensure that the A_260:280 ratio is ~2.0.
   22. Dilute the mRNA to 2.8 µg/µL in sterile H₂O, and store at -20 °C in 50 µL aliquots.
2. In vitro translation
   NOTE: The translation of the desired DNA must be carried out on the same day of the import experiment. This experiment requires the use of radiolabeled amino acids, and therefore the user must have a permit for the use of radioisotopes, and all handling and disposal must be in accordance with the institution's policies/requirements. Steps involving radioisotope safety measures have been omitted or described briefly, as these will likely differ based on the institution.
   1. Prepare enough translation reaction mix (Table 2) to supply ~20 µL per sample of mitochondria to be used in the import experiment and include the same volume for a translation lane.
   2. Incubate the reaction for 30 min at 30 °C (time may vary with mRNA, 25-60 min).
   3. Record the ³⁵S-methionine use as per the institution's radioactive safety requirements and dispose of anything that has come into contact with the radioisotope per the institution's guidelines.
3. Protein import
   NOTE: Freshly isolated mitochondria are required for the next steps. Refer to the mitochondrial isolation protocol. Other isolation methods can be used as long as the mitochondria are viable and functional. For the subsequent steps, the mitochondrial samples utilized are resuspended in a resuspension buffer as described above (Table 1). The protein concentration of the mitochondrial sample must also be measured prior to starting the import assay.
   1. Roughly 15 min following the start of the translation reaction, aliquot 90 µg of mitochondria into sterile 1.5 mL tubes and pre-incubate at 30 °C for ~10 min.
      NOTE: Aliquot more mitochondria than what will actually be used in the experiment; only 75 µg will be actually used. However, aliquoting in excess is not a requirement.
   2. In a fresh set of sterile 1.5 mL tubes, combine 75 µg of mitochondria with 18 µL of the translation reaction and incubate at 30 °C for desired time.
   3. Keep the remaining volume of the translation reaction on ice; this will be utilized later.
      NOTE: Import is a time-dependent process, so it may be prudent to start with a time-course experiment ranging in incubation times from 1-60 min. A typical reaction time is 30 min.
   4. During this time, prepare the sucrose cushion by combining 1 g of sucrose, 200 µL of 2.5 M KCl, 10 µL 1 M MgCl₂, 100 µL of 1 M HEPES (pH 7.4), and bring the volume to 5 mL using sterile H₂O.
   5. Prepare a new set of sterile 1.5 mL tubes corresponding to each tube in the import reaction. Aliquot 600 µL of the sucrose cushion into each tube and keep on ice until the import reaction is over.
   6. To terminate the import reaction after the appropriate incubation time, remove the tube from 30 °C, place it on ice and then carefully transfer the import reaction on top of the tube with the sucrose cushion.
      NOTE: This step needs to be done very slowly to ensure that the mitochondria are not damaged/ruptured. The sucrose cushion helps slow down the migration of the mitochondria and "cushions" its descent to the bottom of the tube during the subsequent centrifugation step.
   7. Centrifuge the samples + sucrose cushion at 17,000 x g for 15 min at 4 °C.
   8. Prepare the breaking buffer by combining 25 mL of 2.4 M sorbitol, 2 mL of 1 M HEPES (pH 7.4), and 72 mL of double-distilled H₂O.
   9. Prepare lysis buffer for SDS-PAGE gel electrophoresis: Combine 50 mL of heated glycerol, 11.5 g of SDS, 31.25 mL of 1 M Tris (pH 6.8) and bring the volume to 500 mL with double-distilled H₂O.
   10. For sample preparation, mix 475 µL of lysis buffer with 25 µL of β-mercaptoethanol to be used in a later step.
       NOTE: Lysis buffer can be made in advance and stored at room temperature; however, the addition of β-mercaptoethanol is to be done fresh.
   11. Using a P1000 pipette, carefully remove the supernate and do not disturb the pellet. Dispose of the supernate in an appropriate radioactive waste container.
   12. For import into the outer membrane, perform this using Tom40, resuspend the pellet in 50 µL of freshly prepared 0.1 M sodium carbonate (pH 11.5) and incubate on ice for 30 min.
   13. Following the incubation, centrifuge the sample 14,000 x g for 5 min at 4 °C. This step is only done for outer membrane import reactions.
   14. To prepare the samples for SDS-PAGE electrophoresis, add 20 µL of breaking buffer, 20 µL of lysis buffer, and β-mercaptoethanol and resuspend well. Add 5 µL of sample dye.
   15. Prepare the control/translation lane by combining 3 µL of the remaining translation reaction from step 2.3.3 with 37 µL of lysis buffer and 5 µL of sample dye. Mix well.
   16. Boil the samples for 5 min at 95 °C and spin down gently at a low speed to avoid pelleting mitochondria.
   17. Apply the samples to a SDS polyacrylamide gel.
       NOTE: The gel percentage and subsequent run time will depend on which protein is being imported. For example, OCT is 37 kDa, and its mature processed band is slightly smaller; therefore, a 12% gel allows good separation of these bands.
   18. Once electrophoresis is complete, remove the gel and place it in double-distilled H₂O.
   19. Boil the gel in 5% TCA in a fume hood for 5 min, continuously stirring/moving the gel to avoid cracking.
       NOTE: TCA helps solidify the gel for visualization; this can be done in a metal tray over a Bunsen burner. Use enough TCA to generously cover the gel, ~½ full.
   20. Remove the gel and place it back in double-distilled H₂O. Agitate it on a rotating plate for 1 min at ~50 rpm.
   21. Wash the gel in 10 mM Tris on a rotating plate for 5 min at ~50 rpm, again using enough to generously cover the gel, ~½ of the container.
   22. Precipitate the protein by washing the gel in 1 M Salicyclic acid on a rotating plate for 30 min, again using enough Salicyclic acid to generously cover the gel, ~½ of the container being used.
   23. Dehydrate the gel as described in steps 2.3.24-2.3.31.
       NOTE: This can be done in a number of ways. A gel dryer (see Table of Materials) provides a time-efficient option (described below); however other commercially available methods/kits can be used that may be more cost-effective but more time-consuming. Gel drying kits can be utilized as an alternative that does not require the application of heat or suction but instead allows the gel to air-dry between cellophane sheets to prevent distortion.
   24. Place a large sheet of blotting paper down on the porous bed of the gel dryer. Place one sheet of paper towel in the area where the gel will be applied.
   25. Cut a 15 cm x 20 cm piece of blotting paper and place it on the paper towel.
   26. Scoop out the gel from the container using a second piece of 15 cm x 20 cm blotting paper and lay it down flat on top of the first piece.
   27. Cut a slightly larger piece of plastic wrap, ~20 cm x 25 cm, and place it down on gel, ensuring no creases or bubbles under the wrap.
       NOTE: This may take a few attempts, but it is imperative that there are no trapped air pockets.
   28. Gently lay down the plastic cover of the gel dryer over top of the wrap, again ensuring that there are no bubbles or creases.
   29. Turn on the pump/vacuum and ensure that the plastic cover has formed a seal. Test the seal by lifting a corner of the plastic cover and wait for it to re-seal.
   30. Close the gel dryer and run for 90 min. Set the temperature to start at 30 °C, gradually reach 80 °C, and then return to 30 °C at the end of the run.
   31. Once dehydrated, the gel will solidify and feel paper-thin. Wrap the gel in the plastic wrap used in the drying process.
   32. Place the dehydrated gel in a film cassette with a phosphorus film (see Table of Materials), with the white side facing the gel. Exposure times vary but can be 24-48 h for visualization of bands.
   33. Visualize using autoradiography using any suitable imager that is capable of phosphorus imaging.