Mitochondria are essential organelles of eukaryotic cells capable for aerobic respiration. Their dysfunction is a well known source of human disease. Baker's yeast mitochondria contains circular genome encoding eight proteins.
In this video, we described the protocol used to assay protein biosynthesis in yeast mitochondria by means of radioactive labeling of translational products and subsequent separation by gel electrophoresis. The procedure includes several major steps. Streak yeast from frozen stock cultures on fresh plates with appropriate medium.
Put the plates in the culture incubator at 30 degrees for 24 to 48 hours. Inoculate these cultures in two milliliters of medium from the fresh streak in a 15 milliliter tube and incubate them overnight agitating at 200 RPM at 30 degrees. Measure the optical density of the culture at the wavelength of 600 nanometers.
Take the volume corresponding to 0.2 absorbency units in sterile tubes Palette yeast cells at 9, 000 G for 30 seconds at room temperature. Discard this supernatant. Wash the cells with 0.5 milliliters of sterile water by vortexing for five seconds.
Pallette yeast cells at 9, 000 G for 30 seconds at room temperature. Discard the supernatant. Dilute cells in two milliliters of fresh wipe agar medium.
Incubate edge taken at 200 RPM and 30 degrees until the optical density reaches from 1.5 to 1.9 from 1.5 to 1.9 values. Transfer the culture volume equivalent to one optical unit in micro centrifuge tube. Spin the tubes at 3000 G for one minute.
Discard the supernatant. Wash the cells with 0.5 milliliters of sterile water by vortexing for five seconds. Pallette yeast cells at 9, 000 G for 30 seconds at room temperature Discard the supernatant.
Re-suspend yeast cells in 0.5 milliliters of sterile translation buffer. Place the suspension in 15 milliliter tube. Add cycloheximide to cell suspension up to final concentration of 0.2 milligrams per milliliter Incubate for five minutes edge taken at 200 RPM and 30 degrees to inhibit cytosolic translation.
Add from 25 to 30 Add from 25 to 30 microcurie of S35 methionine of S35 methionine to cell suspension and incubate for 30 minutes edge taken at 200 RPM and 30 degrees. Add unlabeled cold methionine and borymycin to stop the labeling Incubate for 10 minutes edge taken at 200 RPM and 30 degrees. Collect yeast cells by centrifugation at 9, 000 G for 30 seconds.
Wash the cells with 0.5 milliliters of sterile water by vortexing for five seconds. Pallette yeast cells at 9, 000 G for 30 seconds at room temperature. Discard the supernatant.
Add 75 microliters of lysis buffer to the palette and vortex for five to 10 seconds. Add 500 microliters of 0.5 molar tris buffer of 0.5 molar tris buffer with pH 6.8. With pH 6.8.
Vortex briefly. Add 600 microliters of methanol to the sample. Vortex for five seconds.
Add 150 microliters of chloroform to the sample. Vortex for five seconds. Centrifuge the samples for two minutes at 12, 000 G.Carefully discard opaface with a sampler.
Add 600 microliters of methanol to the sample. Mix carefully by inverting the tube several times. Centrifuge samples for two minutes at 12, 000 G.Discard supernatant.
Air dry the pallette for two minutes at 80 degrees. Dissolve precipitated proteins in 60 microliters of Laemmli sample buffer. Heat the samples for 10 minutes at 14 degrees.
Cause the 17.5%Laemmli SDS polyacrylamide gel. Load 15 microliters of each sample in the pockets. Run the gel in the cold room until the blue dye reaches approximately 65%of the gel lymph.
Stain the gel with Kumasi brilliant blue and make scan or photo which is required as loading control. Dry the gel in the gel dryer. Keep it in the cassette with storage phosphor screen for three to five days.
Scan the screen on phosphor imager. Following the protocol described above, we assigned mitochondrial translation products from two Saccharomyces cerevisiae strains. The wild type and the mutant variant deletion of the Aim23 gene encoding mitochondrial translation initiation factor three.
Mitochondrial translation products were already actively labeled and separated in the denaturing polyacrylamide gel. The samples were collected every two and a half minutes before saturation to build the time course. The gel was stained, dried and screened after the five day exposition.
In the case of a successful experiment, the picture demonstrates eight bands assigned according to the standard pattern. However, the intensities of individual bands can be highly variable depending on the strain and experimental conditions. Each band corresponds to one translation product.
The data suggests that the mutant strain is capable of mitochondrial protein synthesis because all products appearing in the wild type are visible in this mutant. However, the intensities of the bands are different from the wild type, meaning that this deletion of Aim23 affects mitochondrial gene expression. Kumasi brewed in blue stain and serves as a loading control.
Their result in data can be quantified to identify differences between strains or experimental conditions using Image G or Image Quan software. For these, the ratio of the signal corresponding to every product is calculated to the total signal. Mean values and standard deviations are calculated in at least three independent experiments.
The kinetics of synthesized protein turned over is studied in a past year's experiment. Samples are collected at the indicated time points after the labeling reaction is stopped by cold methionine and borymycin. This control is necessary to estimate the stability of the product.
Immuno-staining with Anti-Porin one antibodies is a loading control. We described the method, which is often used to study protein biosynthesis in yeast mitochondria. This protocol is relatively simple and fast to perform.
At the same time, it allows to get valuable information about the translation rate of four yeast mitochondrial mRNAs, which is highly advantageous. However, it has several limitations requiring additional experiments and lies in this interstate levels of proteins and mRNAs. It can provide the information about these functions in mitochondrial translation system, but more advanced techniques should be used to discover the mechanism.
