Analysis of the Expression and Complexes Assembly of the Mitochondrial Respiratory Chain Proteins in the Fission Yeast Schizosaccharomyces pombe

The scope of our research is mitochondrial protein translation, and we try to elucidate the mechanisms that affect the translation, and assembly of the mitochondrial respiratory chain complex. Our research found that shy1 plays a role in the sustainability of the regular function of mitochondrial by participating in the assembly of complex four infusions. Our laboratory will investigate the mechanism of M=methotrexate infusion.

[Narrator] To begin, measure the wet weight of a cell pellet of Schizosaccharomyces pombe. Re-suspend the cells in eight milliliters of S buffer. Add Dithiothreitol to a final concentration of 10 millimolar, and phenylmethylsulfonyl fluoride to one millimolar, ensuring both reagents are freshly prepared. Add lytic enzymes to the cell suspension to digest the cell wall of Schizosaccharomyces pombe. Rotate the tube on a shaker at 30 degrees Celsius for the duration recommended for the specific lytic enzyme. Use a microscope to observe Spheroplasts formation. Then centrifuge the sample for 10 minutes at 1,000 G, at 4 degrees Celsius to pellet the spheroplasts. Re-suspend the pellet in eight milliliters of ice cold S buffer. After two washes, re-suspend the spheroplasts in eight milliliters of ice cold homogenization buffer containing protease inhibitors. Transfer the mixture to a pre-chilled glass down homogenizer containing a pestle, and a test tube. Homogenize the cells mechanically by performing approximately 15 strokes up and down with the tightly fitting pestle. Then examine the spheroplasts under a microscope to check for membrane breakage. Now transfer the homogenized suspension to centrifuge tubes. Centrifuge for five minutes at 1,000 G at 4 degrees Celsius to pellet unbroken cells and debris. Centrifuge the resulting supernatant for five minutes at 3,000 G at 4 degrees Celsius to pellet the nuclei. Then transfer the supernatant to fresh centrifuge tubes. Centrifuge at 12,000 G for 15 minutes at 4 degrees Celsius to pellet the mitochondria and other organelles, Re-suspend the pellet in one milliliter of ice cold sorbitol EDTA mops buffer after decanting the supernatant. Then centrifuge again for 15 minutes at 12,000 G at 4 degrees Celsius to wash the mitochondria. Re-suspend the final pellet in one milliliter of ice cold sorbitol EDTA mops buffer. Then aliquot the purified mitochondria into storage tubes for future experiments. At SDS page loading buffer into 40 microliters of mitochondrial total protein. Denature the proteins by incubating the mixture for the indicated time at the appropriate temperature. Load approximately 20 micrograms or four microliters of mitochondrial proteins into a 12% SDS page gel before immuno blotting. To prepare the sample for BN page, first pellet mitochondria from previous aliquots by centrifugation. Re-suspend the mitochondria in 200 microliters of three XBN page gel buffer. Next, pipette two microliters of 100 X phenylmethylsulfonyl fluoride and one microliter of one molar magnesium chloride. Centrifuge again for 15 minutes at 12,000 G at 4 degrees Celsius. Re-suspend the mitochondria pellet in 160 microliters of 5% weight by volume digitorum. Incubate on ice for 30 minutes, gently mixing every 10 minutes. Centrifuge the suspension for five minutes at 20,000 G at 4 degrees Celsius. Then transfer the supernatant to a fresh tube. Add 80 microliters of three XBN page sample buffer to the digitorum treated sample. Or 32 microliters to the DDM treated sample. Now assemble the precast native Bis-Tris gels with a 3 to 12% gradient. Load the treated protein samples along with high molecular weight protein markers for native Polyacrylamide gel electrophoresis. Run the gel at a constant voltage of 80 volts, and current of six milliamperes for 30 minutes using cathode buffer containing 0.02% Kumasi G250. Replace the buffer with cathode buffer without Kumasi, and continue running at 10 milliamperes for three hours until the dye front reaches the gel bottom. Cut the gel lane containing the protein marker. Stain the marker with Kumasi R250 buffer for 15 minutes. Then de-stain until the bands become visible. Immerse the remaining part of the gel in BN page transfer buffer for 30 minutes to equilibrate. Rinse 0.45 micrometer PVDF blot membrane with methanol, and equilibrate in transfer buffer for 10 minutes. Transfer proteins from the gel onto the PVDF membrane using a constant current of 300 milliamperes for two hours. Rinse the PVDF membrane with methanol to remove Kumasi dye. Then incubate the membrane in TBS blocking buffer containing 5% skim milk for one hour at 25 degrees Celsius. Incubate the PVDF membrane with the specified primary antibodies against Schizosaccharomyces pombe mitochondrial respiratory chain complexes. After an overnight incubation at four degrees Celsius, wash the membrane using TBST buffer. Then incubate the membrane in the secondary antibody at a dilution of one to 10,000 for one hour at 25 degrees Celsius. After washing the membrane with TBST buffer, expose and scan the PVDF membrane to visualize immuno blot results. Deletion of the shy1 gene, led to a marked reduction in the steady state levels of mitochondrial DNA encoded respiratory chain proteins, Cob1, Cox1, Cox2, Cox3 and Atp6. Blue native page analysis showed that the abundance of DG Solubilized respiratory chain super complexes 3242 and 324 was reduced in Delta shy1 cells. While the levels of super complexes 32.=, and 55N remained unaffected. The level of DDM solubilized diametric complex 3 was unchanged in the Delta shy1 strain.