Name: labelling and visualization of mitochondrial genome expression products in baker's yeast saccharomyces cerevisiae
Uploaded: 2021-04-11T14:00:00
Description: Watch this Scientific Journal Video about Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae at JoVE.com

This research was funded by the Russian Foundation for Basic Research, grant number 18-29-07002. P.K. was supported by State Assignment of Ministry of Science and Higher Education of the Russian Federation, grant number AAAA-A16-116021660073-5. M.V.P. was supported by the Ministry of Science and Higher Education of the Russian Federation, grant number 075-15-2019-1659 (Program of Kurchatov Center of Genome Research). The work was partly done on the equipment purchased in the frame of the Moscow State University Program of Development. I.C., S.L., and M.V.B. were additionally supported by Moscow State University grant &#8220;Leading Scientific School Noah&#8217;s Ark&#8221;.

1. Yeast culture preparation
<ol>
	<li>Streak yeast from the frozen stock cultures on fresh plates with the appropriate medium. Put the plates in a culture incubator at 30 &#176;C for 24&#8211;48 h. 
	NOTE: Let the temperature-sensitive mutants grow at the permissive temperature.</li>
	<li>Inoculate yeast cultures in 2 mL of YPGal medium (2% peptone, 1% yeast extract, 2% galactose) from the fresh streak in 15 mL tubes and incubate them overnight agitating at 200 rpm at 30 &#176;C.</li>
	<li>Measure the optical den...

<ol>
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Investigations of gene expression occupy a central part in modern life sciences. Numerous methods providing insights into this complex process have been developed. Here, we described the method allowing to access protein biosynthesis in baker&#39;s yeast S. cerevisiae mitochondria. It is usually applied to compare translation efficiencies of the mRNAs in mitochondria of mutant yeast strain versus wild type to access the consequences of the studied mutation. This is one of the basic experiments the researchers co...

Mitochondria are the organelles deeply involved in the metabolism of a eukaryotic cell. Their electron transfer chain supplies the cell with ATP, the main energetic currency used in multiple biochemical pathways. Besides, they are involved in apoptosis, fatty acid and heme synthesis, and other processes. Dysfunction of mitochondria is a well-known source of human disease1. It can result from mutations in nuclear or mitochondrial genes encoding structural or regulatory components of the organelles2. Baker&#8217;s yeast Saccharomyces cerevisiae is an excellent model organism for studying mitochondrial gene expression due to several reasons. First, their genome is completely sequenced3, well-annotated, and a big sum of data is already available in literature thanks to the long history of investigations carried out with this organism. Second, the manipulations with their nuclear genome are relatively fast and easy because of their fast growth rate and highly efficient homologous recombination system. Third, baker&#8217;s yeast S. cerevisiae is one of the few organisms for which the manipulations with mitochondrial genomes are developed. Finally, baker&#39;s yeast is an aerobe-anaerobe facultative organism, which allows isolation and study of respiratory defective mutants, since they can grow in media containing fermentable carbon sources.
We describe the method to study mitochondrial gene expression of baker&#8217;s yeast S. cerevisiae at the translational level4. Its main principle comes from several observations. First, the yeast mitochondrial genome encodes only eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p5. This number is small, and all of them can be separated by electrophoresis on a single gel in the appropriate conditions. Second, mitochondrial ribosomes belong to the prokaryotic class rather than eukaryotic6, and therefore, the sensitivity to antibiotics is different for yeast cytoplasmic and mitochondrial ribosomes. It allows the inhibition of cytoplasmic translation with cycloheximide, providing the conditions when the labeled amino acid (35S-methionine) is incorporated only in mitochondrial translation products. As a result, the experiment gives information about the rate of amino acid incorporation in mitochondrial proteins synthesized de novo, reflecting the overall efficiency of mitochondrial translation for each of the eight products

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A9099</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>Sigma-Aldrich</td>
			<td>A5306&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Biowave Cell Density Meter CO8000</td>
			<td>BIOCHROM US BE</td>
			<td>80-3000-45</td>
			<td></td>
		</tr>
		<tr>
			<td>BRAND standard disposable cuvettes</td>
			<td>Sigma-Aldrich</td>
			<td>Z330361</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>288306&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C1988&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G5388&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>digital block heater</td>
			<td>Thermo Scientific</td>
			<td>88870001</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyTag L-[35S]-Methionine, 500&#181;Ci (18.5MBq), Stabilized Aqueous Solution</td>
			<td>Perkin Elmer</td>
			<td>NEG709A500UC</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5425</td>
			<td>Thermo Scientific</td>
			<td>13-864-457</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Storage Phosphor Screens</td>
			<td>Sigma-Aldrich</td>
			<td>GE29-0171-33</td>
			<td></td>
		</tr>
		<tr>
			<td>L-methionine</td>
			<td>Sigma-Aldrich</td>
			<td>M9625&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#8242;-Methylenebisacrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>M7279</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Innova 44/44R Shaker Incubator</td>
			<td>New Brunswick Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone from meat, bacteriological</td>
			<td>Millipore</td>
			<td>91249&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>P7626&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660nm Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>22662</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Protean II xi cell</td>
			<td>Bio-Rad</td>
			<td>1651802</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride from Streptomyces alboniger</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Storm 865 phosphor imager</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>93352&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Heated Gel Dryer</td>
			<td>Cleaver Scientific</td>
			<td>CSL-GDVH</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma-Aldrich</td>
			<td>Y1625&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

labelling and visualization of mitochondrial genome expression products in baker's yeast saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Following the protocol described above, we assigned mitochondrial translation products from two S. cerevisiae strains: the wild type (WT) and a mutant bearing deletion of the AIM23 gene (AIM23&#916;), encoding mitochondrial translation initiation factor 3 (Table 1)8. Mitochondrial translation products were radioactively labeled and separated in SDS-PAAG9. The samples were collected every 2.5 min before saturation to build a time course (<strong ...

Watch this Scientific Journal Video about Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae at JoVE.com

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

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.

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