Mitochondria rely heavily on the nuclear genome, despite having their own DNA. Thus, a highly-regulated mechanism of import is required. This method helps study the process and how it can adapt to external stimuli.
This is the only biochemical technique available to quantitatively evaluate protein import into the various sub-compartments of the mitochondria. The viability of mitochondria is implicated in various disease states, so understanding how protein import is regulated may help contribute to novel therapeutic approaches that target mitochondria. Extra care is required during the isolation to ensure the viability and integrity of the mitochondria.
Thorough mincing of the tissue and gently resuspending all subsequent pellets is necessary. Demonstrating the procedure will be Ashley Oliveira, a PhD student in my laboratory. To begin isolation of mitochondria from skeletal muscle, remove fat and connective tissue from the muscles and mince the tissues on a pre-chilled watch glass until it is a homogenous slurry.
Place the minced tissue in a pre-chilled 50-milliliter plastic centrifugation tube and record the exact weight. Then, dilute the minced tissue tenfold with buffer 1 containing ATP. Use an eight-millimeter twin-blade homogenizer to homogenize the muscle sample at a power output of 9.8 Hertz for 10 seconds, ensuring no visible chunks of muscle remaining.
After spinning the sample at 9, 000x G for 10 minutes at four degrees Celsius, resuspend the pellet carefully in roughly 95 microliters of the resuspension medium. Centrifuge the sample before discarding the supernatant, and resuspend the pellet gently with roughly 180 microliters of resuspension medium using a P-200 pipette. For in vitro translation, prepare enough translation reaction mix to supply 20 microliters per sample of mitochondria to be used in import experiment and for a translation lane.
Place the reaction for incubation at 30 degrees Celsius for 30 minutes. 15 minutes following the start of the translation reaction, aliquot 90 micrograms of mitochondria into sterile 1.5-milliliter tubes and pre-incubate at 30 degrees Celsius for 10 minutes. In a fresh set of sterile 1.5-milliliter tubes, combine 75 micrograms of mitochondria with 18 microliters of the translation reaction, and incubate the tube at 30 degrees Celsius for the desired time.
Keep the remaining volume of the translation reaction on ice. To terminate the import reaction after the appropriate incubation time, remove the tube from 30 degrees Celsius to place it on ice, and then carefully transfer import reaction on top of the tube with the sucrose cushion. Centrifuge the samples with sucrose cushion at 17, 000x G for 15 minutes at four degrees Celsius.
Using a P-1000 pipette, carefully remove the supernatant without disturbing the pellet. To import into the outer membrane, perform the reaction using to Tom40, and resuspend the pellet in 50 microliters of freshly-prepared 0.1-molar sodium carbonate, and incubate on ice for 30 minutes. Following the incubation, centrifuge the sample at 14, 000x G for five minutes at four degrees Celsius.
Next, prepare the samples for SDS-PAGE electrophoresis as described in the manuscript. Prepare the control translation lane by mixing three microliters of the remaining translation reaction with 37 microliters of lysis buffer and five microliters of sample dye. Boil the samples for five minutes at 95 degrees Celsius and spin down gently at a low speed to avoid pelleting mitochondria.
After applying samples to SDS polyacrylamide gel, boil the gel in 5%trichloroacetic acid, or TCA, in a fume hood for five minutes with continuous stirring. Then, place the gel in double-distilled water on a rotating plate for one minute at 50 rotations per minute. Wash the gel in 10-millimolar Tris on a rotating plate for five minutes at 50 rotations per minute.
To precipitate the protein, wash the gel in enough volume of one-molar salicytic acid to cover the gel on a rotating plate for 30 minutes. To dehydrate the gel, place a large sheet of blotting paper down on the porous bed of the gel dryer, and one sheet of a paper towel in the area where the gel will be applied. Then, cut 11 centimeters by 9 centimeters piece of blotting paper and place the paper on the paper towel.
Use a second piece of 11 centimeters by 9 centimeters blotting paper to scoop out the gel from the container and lay the gel down flat on top of the first piece. Place a slightly-larger piece of plastic down on the gel, ensuring no creases or bubbles under the wrap. Then, lay down the plastic cover of the gel dryer over the top of the wrap.
Turn on the vacuum. Ensure that the plastic cover has formed a seal by lifting the corner of the plastic cover and waiting for the cover to reseal. Close the gel dryer to run for 90 minutes, starting at 30 degrees Celsius to reach 80 degrees Celsius gradually, and return to 30 degrees Celsius at the end of the run.
Wrap the gel in the plastic wrap used in the drying process. Once dehydrated, the gel will solidify and feel paper-thin. Place the dehydrated gel in a cassette with a phosphorous film on top.
Expose the film for 24 hours before visualizing the gel using autoradiography with any suitable imager that is capable of phosphorous imaging. The representative analysis shows the normal rates of import for malate dehydrogenase, or MDH, in the subsarcolemmal and intermyofibrillar mitochondria, and the translation product of precursor MDH. The addition of valinomycin inhibited MDH protein import into the matrix of SS and IMF mitochondria.
Similarly, detergent Triton X inhibited MDH import into both sub-fractions due to solubilization of the inner membrane. The estimation of protein import was carried out for increasing time durations, and the consequent data illustrated that import is a time dependent process, and SS and IMF mitochondria have different rates or capacities for import. When rats were subjected to electrical stimulation, Tom 40 import into the outer membrane was higher in muscle from chronically-stimulated animals compared to controls.
Ornithine transcarbamylase, or OCT import, into the mitochondrial matrix was increased at every time point of incubation, resulting in a 1.4-fold increase in mitochondria from chronically-stimulated muscle. The protein import was positively correlated with an index of mitochondrial content and assessed by COX activity. In investigating mitochondrially-mediated apoptosis, Bax and Bak double-knockout animals exhibited reduced protein import into the mitochondrial matrix.
Six weeks of voluntary wheel running rescued the import defect in double-knockout animals. It is important to consider variables such as the duration of the import reaction and which protein is to be imported. Cytosolic fractions can be incorporated into the reaction to understand how cytosolic factors may influence the rate of import.
Alternatively, proteins may be modified before incubation to understand how proteins may be selectively imported. This technique can aid in understanding the complex etiologies of mitochondrial myopathies that may present in various disease states.
