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 density of the culture at a wavelength of 600 nm (OD600).</li>
	<li>Take the volume corresponding to 0.2 absorbance units in sterile tubes, pellet yeast cells at 9,000 x g for 30 s at room temperature, and discard the supernatant.</li>
	<li>Wash cells with 0.5 mL of sterile water by vortexing for 5 s. Pellet yeast cells at 9,000 x g for 30 s at room temperature and discard the supernatant. Dilute cells in 2 mL of fresh YPGal medium.</li>
	<li>Incubate agitating at 200 rpm and 30 &#176;C until OD600 reaches 1.5&#8211;1.9 values. 
	NOTE: Yeast growth rates vary, so be prepared to wait. It is reasonable to make steps 1.3&#8211;1.6 early in the morning. It usually takes 4&#8211;5 h, but can take longer.</li>
</ol>
2. Radioactive isotope incorporation
<ol>
	<li>Transfer the culture volume equivalent to one optical unit in a microcentrifuge tube. Spin the tubes at 3,000 x g for 1 min and discard the supernatant. Wash with 0.5 mL of sterile water by vortexing for 5 s.
	<ol>
		<li>Pellet yeast cells at 9,000 x g for 30 s at room temperature and discard the supernatant. Resuspend yeast cells in 0.5 mL of sterile translation buffer. Place the suspension in a 15 mL tube. 
		NOTE: Translation buffer is a solution containing 2% galactose (w/v) and 50 mM potassium phosphate with pH 6.0.</li>
	</ol>
	</li>
	<li>Add cycloheximide to cell suspension up to a final concentration of 0.2 mg/mL. Incubate for 5 min agitating at 200 rpm and 30 &#176;C to inhibit cytosolic translation. 
	NOTE: Cycloheximide solution (20 mg/mL, in ethanol) should be prepared fresh before the experiment. Chloramphenicol can be successfully substituted with anisomycin in the same concentration7.</li>
	<li>Add 25&#8211;30 &#956;Ci of 35S-methionine to the cell suspension and incubate for 30 min agitating at 200 rpm and 30 &#176;C. 
	NOTE: The mixture of 35S-methionine and 35S-cysteine can also be used. Pure 35S-methionine results in the strongest signal and the best signal-to-noise ratio. Generally, methionine is more effective than cysteine because the content of this amino acid in mitochondrial proteins is higher. However, the EasyTag mixture of 35S-methionine and cysteine is less expensive and gives comparable results7. 
	CAUTION: 35S-methionine is radioactive. Follow the usual safety practices for handling radioactive materials. 
	NOTE: It is well known that the incorporation of radioactivity reaches a limit due to which the total signals level off over time. Once this limit is reached, it is almost impossible to determine the rates by which the different translation products are synthesized. To analyze the rate of mitochondrial translation, it is imperative to be in a condition in which the signal intensity increases with time of incubation with 35S-methionine in a linear manner. For common laboratory wild-type strains, the signal is already saturated for incubation times far shorter than the 30 min. Translational mutants can behave very differently. A time-course should be performed to establish the kinetics of radioactivity incorporation and thus the rate of translation, at least when working with a new strain. For this, we suggest taking samples with an interval of several minutes (e.g., 2.5, 5.0, 7,5, 10, and 20 min).</li>
	<li>Add unlabeled &#34;cold&#34; methionine (final concentration should be 20 mM) and puromycine (final concentration should be 1&#8211;10 &#956;g/mL) to stop the labeling. Incubate for 10 min agitating at 200 rpm and 30 &#176;C. 
	NOTE: This step is critical to give ribosomes time to finish translating the peptides. Otherwise, all polypeptides shorter than full-length will be detected at a different size. A time-course experiment (pulse-chase) can be done at this step to determine the stability of mitochondrial translation products. For this, continue the incubation taking samples with an interval of 30 min (e.g., 30, 60, 90, and 120 min).</li>
</ol>
3. Yeast cell lysis and extraction of proteins
<ol>
	<li>Collect yeast cells by centrifugation at 9,000 x g for 30 s. Wash the cells with 0.5 mL of sterile water by vortexing for 5 s. Pellet yeast cells at 9,000 x g for 30 s at room temperature. Discard the supernatant. 
	NOTE. The protocol can be paused here. Store the samples at -20 &#176;C.</li>
	<li>Add 75 &#956;L of lysis buffer to the pellet and vortex for 5&#8211;10 s. 
	NOTE: Lysis buffer is a solution of 1.8 M NaOH, 1 M &#946;-mercaptoethanol, and 1 mM PMSF in water. Avoid excessive incubation with lysis buffer leading to alkaline hydrolysis of proteins. Immediately proceed to step 3.3.</li>
	<li>Add 500 &#956;L of 0.5 M Tris-HCl buffer with pH 6.8. Vortex briefly.</li>
</ol>
4. Precipitation of proteins
CAUTION: Methanol and chloroform are organic solvents. Follow the usual safety practices for handling organic substances.
<ol>
	<li>Add 600 &#181;L of methanol to the sample. Vortex for 5 s.</li>
	<li>Add 150 &#181;L of chloroform to the sample. Vortex for 5 s.</li>
	<li>Centrifuge the samples for 2 min at 12,000 x g. Carefully discard the upper phase with a sampler.</li>
	<li>Add 600 &#181;L of methanol to the sample. Mix carefully by inverting the tube several times.</li>
	<li>Centrifuge the samples for 2 min at 12,000 x g. Discard the supernatant.</li>
	<li>Air-dry the pellet for 2 min at 80 &#176;C. 
	NOTE: All of the liquid should evaporate. There is a risk to have an aberrant separation of proteins in the gel if the pellet was dried insufficiently. However, it is not possible to over-dry the pellet, so it can even be stored overnight at 4 &#176;C.</li>
	<li>Dissolve precipitated proteins in 60 &#181;L of 1x Laemmli sample buffer.</li>
	<li>Heat for 10 min at 40 &#176;C. 
	NOTE: Avoid boiling the samples at 95 &#176;C, because this causes aggregation. If the aggregates still form, Spin the samples at 12,000 x g for 2 min and collect the supernatant. Samples can be stored at -20 &#176;C. The protocol can be paused here.</li>
</ol>
5. SDS-PAGE
<ol>
	<li>Cast the 17.5% Laemmli SDS-polyacrylamide gel. 
	NOTE: 6 M urea can be added to the gel to resolve better atp8 and atp9. Gradient 15%&#8211;20% gels can also give better resolution.</li>
	<li>Load 15 &#956;L (40&#8211;50 &#181;g) of each sample in the pockets. 
	NOTE: It is not necessary to measure protein concentration if OD600 values were close in step 1.6. If not, measure protein concentrations using the assay with detergent-compatible reagents.</li>
	<li>Run the gel in a cold room until the blue dye reaches approximately 65% of the gel length. 
	NOTE: For the system used (e.g., Protean II xi cell), we use either of the two modes: 16&#8211;17 h at 5 V/cm or 5 h at 15 V/cm. No proteins run faster than the bromophenol blue dye, but long runs can result in blurring of atp8 and atp9 signals.</li>
	<li>Stain the gel with Coomassie brilliant blue and make a scan or photo, which is required as a loading control. 
	NOTE: An alternative way to make a loading control is immunoblotting with an antibody to mitochondrial &#8220;house-keeping&#8221; gene, e. g., porin 1.</li>
</ol>
6. Autoradiography
<ol>
	<li>Dry the gel in a gel-dryer. Keep it in the cassette with a storage phosphor screen for 3&#8211;5 days. 
	NOTE: An alternative to screening the dried gel is the transfer of proteins to a nitrocellulose membrane by electro-blotting and screening it thereafter. It results in stronger signals and sharper bands.</li>
	<li>Scan the screen on a phosphorimager. 
	NOTE: As an alternative to phosphor imaging, X-ray film can also be used to reveal the signals.</li>
</ol>

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	<li>Taylor, R. W., Turnbull, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+mutations+in+human+disease.">Mitochondrial DNA mutations in human disease.</a> Nature Reviews. Genetics. 6 (5), 389-402 (2005).</li><li>Park, C. B., Larsson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+mutations+in+disease+and+aging.">Mitochondrial DNA mutations in disease and aging.</a> The Journal of Cell Biology. 193 (5), 809-818 (2011).</li><li>Goffeau, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+with+6000+genes.">Life with 6000 genes.</a> Science. 274 (5287), 546-563 (1996).</li><li>Westermann, B., Herrmann, J. M., Neupert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+translation+products+in+vivo+and+in+organello+in+yeast.">Analysis of mitochondrial translation products in vivo and in organello in yeast.</a> Methods in Cell Biology. 65, 429-438 (2001).</li><li>Foury, F., Roganti, T., Lecrenier, N., Purnelle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complete+sequence+of+the+mitochondrial+genome+of+Saccharomyces+cerevisiae.">The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae.</a> FEBS Letters. 440 (3), 325-331 (1998).</li><li>Desai, N., Brown, A., Amunts, A., Ramakrishnan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+yeast+mitochondrial+ribosome.">The structure of the yeast mitochondrial ribosome.</a> Science. 355 (6324), 528-531 (2017).</li><li>Sasarman, F., Shoubridge, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioactive+labeling+of+mitochondrial+translation+products+in+cultured+cells.">Radioactive labeling of mitochondrial translation products in cultured cells.</a> Methods in Molecular Biology. 837, 207-217 (2012).</li><li>Kuzmenko, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aim-less+translation:+loss+of+Saccharomyces+cerevisiae+mitochondrial+translation+initiation+factor+mIF3/Aim23+leads+to+unbalanced+protein+synthesis.">Aim-less translation: loss of Saccharomyces cerevisiae mitochondrial translation initiation factor mIF3/Aim23 leads to unbalanced protein synthesis.</a> Science Reports. 6, 18749 (2016).</li><li>Laemmli, U. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Keil, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxa1-ribosome+complexes+coordinate+the+assembly+of+cytochrome+c+oxidase+in+mitochondria.">Oxa1-ribosome complexes coordinate the assembly of cytochrome c oxidase in mitochondria.</a> Journal of Biological Chemistry. 287 (41), 34484-34493 (2012).</li><li>Singhal, R. 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U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coa3+and+Cox14+are+essential+for+negative+feedback+regulation+of+COX1+translation+in+mitochondria.">Coa3 and Cox14 are essential for negative feedback regulation of COX1 translation in mitochondria.</a> The Journal of Cell Biology. 191 (1), 141-154 (2010).</li><li>Bietenhader, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+relocation+of+the+mitochondrial+ATP9+gene+to+the+nucleus+reveals+forces+underlying+mitochondrial+genome+evolution.">Experimental relocation of the mitochondrial ATP9 gene to the nucleus reveals forces underlying mitochondrial genome evolution.</a> PLoS Genetics. 8 (8), e1002876 (2012).</li><li>Couvillion, M. T., Churchman, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ribosome+(mitoribosome)+profiling+for+monitoring+mitochondrial+translation+in+vivo.">Mitochondrial ribosome (mitoribosome) profiling for monitoring mitochondrial translation in vivo.</a> Current Protocols in Molecular Biology. 119, 4.28.1-4.28.25 (2017).</li><li>Suhm, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+system+to+monitor+mitochondrial+translation+in+yeast.">A novel system to monitor mitochondrial translation in yeast.</a> Microbial Cell. 5 (3), 158-164 (2018).</li></ol>

The authors have nothing to disclose.

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 conduct when they study the mitochondrial function of yeast cells bearing the mutation suggested to influence mitochondria8,11,12,13. It is often combined with the measurements of oxygen consumption rate and mitochondrial membrane potential. However, the information it provides is not sufficient to distinguish what stage of gene expression is affected. A set of additional experiments is required to find it out. First, northern blot or RT-qPCR evaluation of mitochondrial mRNAs is necessary to assess the transcriptional step. Second, a Western blot of total protein extracts with specific antibodies should be done to assess the protein level. Third, the pulse of labeled 35S-methionine (hot) should be continued with the addition of unlabeled (cold) methionine (chase) and several time points should be collected and analyzed on the gel to investigate the stability of the proteins.
Accurate analysis of mitochondrial gene expression using 35S pulse labeling requires control reactions, especially when the researcher lacks the experience handling it or works with a new yeast strain or a mutant. In these cases, good negative control is a rho0 strain devoid of mitochondrial DNA. It shows efficient cycloheximide inhibition of cytosolic translation and confirms that the banding pattern is mitochondrial translation specific. If the rho0 strain is not available, then we suggest including chloramphenicol along with cycloheximide to inhibit all protein synthesis to confirm cycloheximide efficiency and specificity of the banding pattern.
The closest modification of the protocol is the pulse-chase when the culture is incubated in the shaker (step 2.4) longer than suggested in the pulse experiment (Figure 1C). It is used to study the turnover and stability of mitochondrial translation products. There is another modification of the method when the radioactive labeling is done in organello, not in vivo4. It suggests the isolation of mitochondria from yeast cells. This modification is faster if frozen mitochondria were previously stocked in aliquots. Another advantage is the absence of cycloheximide treatment, which affects different aspects of cellular metabolism. However, isolation of mitochondria and freeze-thawing them can perturb the translation complexes in the organelles providing an artificial picture. Another important modification of the protocol can be done after the separation of mitochondrial translation products in polyacrylamide gel (step 5). Instead of Coomassie Brilliant Blue staining and drying the gel, the protein can be transferred to the nitrocellulose membrane by electro-blotting. This results in stronger and sharper signals. The main reason is that 35S decays by emission of beta particles with very short penetration, so the signal is easily screened in this approach. Electrophoretic conditions can also be modified to provide better resolution. One point is to add 6M urea in the gel, which improves the separation of atp8 and atp914. Another way is using gradient 15%&#8211;20% gels.
Translational profiling15 is the method used to dive deeper into the alterations of mitochondrial translation. As compared to radioactive labeling, it allows establishing positions of mitochondrial ribosomes on the mRNA, which makes it possible to find the exact step (initiation, elongation, or termination) being affected. However, profiling is much more expensive, complicated, and time-consuming. Rationally it can be done after the label incorporation experiment, not vice versa. Recently, a novel approach to monitoring yeast mitochondrial translation has been developed16. It avoids treatment with cycloheximide, which is advantageous because such treatment affects cellular metabolism and signaling pathways. Instead of radioactively labeled amino acid incorporation, it utilizes the insertion of a recoded gene for super-folded GFP (sfGFP) in yeast mitochondrial genome, which allows direct measurement of mitochondrial translation using flow cytometry. However, the application of this approach requires the special yeast strain with modified mitochondrial DNA containing sfGFP coding sequence placed between 5&#8217;- and 3&#8217;- flanking sequences of a certain mitochondrial gene.
The label incorporation experiment includes several critical steps, which cannot be compromised in a successful experiment. First, fresh yeast cultures should be used (step 1.1). Keeping yeasts on the plates longer than 1 month is not recommend; otherwise, they can behave unpredictably in this assay. Second, the cycloheximide solution should be prepared fresh before the experiment and stored frozen no longer than 1 week (step 2.1). The old solution loses its ability to inhibit cytoplasmic translation resulting in a completely aberrant band pattern in the radioautography. Third, 35S-methionine should be fresh and active (step 2.2), otherwise, the intensities of the bands will be weak (Figure 1D). Using the reagent that passed four half-lives (4 x 87.4 days) is not recommended. Avoid boiling the protein samples at 95 &#176;C as standard sample preparation guides suggest (step 4.8) because mitochondrial proteins are highly hydrophobic and prone to aggregation.
There are several common issues one can experience dealing with this method. The first one is the weak intensity of the bands on the radioautography. To fix it make sure, that fresh 35S-methionine is used, a sufficient amount of yeast cells is taken, and the proteins do not aggregate in the pockets on the gel, which can be controlled by Coomassie staining. Keep the dried gel with the screen for no less than 3 days. The second issue is the incorrect band pattern. If it is encountered, make sure that fresh yeast plates and freshly prepared cycloheximide are used. Keep in mind that relative intensities of the bands can vastly vary in different yeast strains and electrophoresis conditions. It makes little sense to load the protein molecular weight ladder on the gel since mitochondrial translation products are never separated according to their molecular masses in this procedure because they are highly hydrophobic. Thus, Cox I (58 kDa) migrates faster than Var 1 (47 kDa). Depending on the buffer conditions, the proteins can even switch positions with one of the neighbors. The third common issue is the blurred picture with no sharp bands observed after Coomassie staining. It indicates the mistakes in the casting of the gel, incorrect buffer composition, or degradation of proteins in the samples. It is recommended to prepare new gel buffers and running buffer carefully checking the composition and the pH values.

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><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&amp;nbsp;</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&amp;nbsp;</td><td></td></tr><tr><td>cycloheximide</td><td>Sigma-Aldrich</td><td>C1988&amp;nbsp;</td><td></td></tr><tr><td>D-(+)-Galactose</td><td>Sigma-Aldrich</td><td>G5388&amp;nbsp;</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7021&amp;nbsp;</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&amp;micro;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&amp;nbsp;</td><td></td></tr><tr><td>methanol</td><td>Sigma-Aldrich</td><td>34860&amp;nbsp;</td><td></td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime;-Tetramethylethylenediamine</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>N,N&amp;prime;-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&amp;nbsp;</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride</td><td>Sigma-Aldrich</td><td>P7626&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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 (Figure 1A). The gel was stained, dried, and screened after the 5-day exposition (Figure 1A).
In the case of a successful experiment, the picture demonstrates eight bands assigned according to the standard pattern4. 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 (Figure 1A) suggest that the AIM23&#916; strain is capable of mitochondrial protein synthesis because all products appearing in the WT are visible in this mutant. However, the intensities of the bands are different from the WT, meaning that the deletion of AIM23 affects mitochondrial gene expression8. Coomassie Brilliant Blue staining serves as a loading control.
The resulting data can be quantified (Figure 1B) to identify differences between strains or experimental conditions using ImageJ10 or ImageQuant software. For this, the ratios of the signal corresponding to every product to the total signal are calculated. Mean values and standard deviations are calculated in at least three independent experiments.
The kinetics of synthesized protein turnover is studied in a pulse-chase experiment (Figure 1C). Samples are collected at the indicated time points after the labeling reaction is stopped by cold methionine and puromycine in step 2.4. This control is necessary to estimate the stability of the products because the intensity of the signal is a result of two opposite processes: synthesis of new chains and protein degradation. Immunostaining with anti-porin 1 antibodies is a loading control.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62020/62020fig01.jpg" /> 
Figure 1: Representative radioactive labeling of yeast mitochondrial translation products. (A) Time course of 35S-methionine incorporation in mitochondrially synthesized proteins in live yeast cells of WT and AIM23&#916; strains. Coomassie Brilliant Blue staining is a loading control. (B) Levels of mitochondrially-encoded proteins after 5 min labeling with 35S-methionine. The relative expression is normalized to the total expression of mitochondrially encoded protein genes. Error bars indicate the standard deviation of the mean of at least three independent experiments. (C) Turnover of mitochondrially synthesized proteins in wild type and Aim23&#916; strains. The labeling was stopped and the samples were collected at the indicated time points. Immunostaining with anti-porin 1 antibody is a loading control. (D) Sub-optimal experiment with old 35S-methionine, radioautography. Figure&#160;1A,B,C are adapted from8 with minor modifications. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62020/62020fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>strain</td>
			<td>genotype</td>
		</tr>
		<tr>
			<td>WT</td>
			<td>MAT&#945; mal</td>
		</tr>
		<tr>
			<td>AIM23&#916;</td>
			<td>MAT&#945; mal, AIM23::KanMX4</td>
		</tr>
	</tbody>
</table>
Table 1. Genotypes of S. cerevisiae strains

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 ...

labelling-visualization-mitochondrial-genome-expression-products

Nanyang Technological University

Research

JoVE Journal

Biochemistry

4.4K Views.  M.V. Lomonosov Moscow State University. Mitochondriën zijn essentiële organellen van eukaryotische cellen die in staat zijn tot aerobe ademhaling. Hun disfunctie is een bekende bron van menselijke ziekten. Baker's gist mitochondriën bevat circulair genoom dat codeert voor acht eiwitten.In deze video beschreven we het protocol dat wordt gebruikt om eiwitbiosynthese in gist mitochondriën te analyseren door middel van radioactieve etikettering van translationele producten en daaropvolgende scheiding door gel-elektroforese. ...

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

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Genetics

Biolistic Transformation of Yeast Mitochondria for Mutagenetic Analysis

Mitochondrial Transformation in Baker&#39;s Yeast to Study Translation and Respiratory Complex Assembly

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

Author Spotlight: Advancing Techniques and Discoveries in Protein Synthesis and Assembly Through Innovative Mitochondrial Research

This work explains how to transform yeast mitochondria using a biolistic method. We also show how to select and purify the transformants and how to introduce the desired mutation in the target position within the mitochondrial genome.

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Biology

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

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

The mitochondrial respiratory chain is crucial for cellular energy metabolism, serving as the core of oxidative phosphorylation. The mitochondrial respiratory chain comprises five enzyme complexes and their interacting supercomplexes. Analysis of the expression and complexes assembly of these proteins is vital to understanding mitochondrial function. This can be studied by combining biochemical and genetic methods in an excellent model organism fission yeast Schizosaccharomyces pombe (S. pombe), which provides a compensatory system to budding yeast for studies of mitochondrial biology. Here, we present a detailed protocol for the isolation of S. pombe mitochondria and analysis of expression levels and complexes assembly of the mitochondrial respiratory proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blue native-PAGE (BN-PAGE). Briefly, mitochondria from the wild-type and gene mutants are purified, and then their complexes are solubilized and subjected to SDS-PAGE/BN-PAGE and immunoblotting. This method enables the characterization of a gene's novel function in the mitochondrial respiratory chain.

The fission yeast Schizosaccharomyces pombe is emerging as an attractive model for studying mitochondria. Here, we describe a protocol for analyzing the abundance and assembly of the mitochondrial respiratory complexes in S. pombe. This enables the characterization of conserved genes' novel functions in the mitochondrial respiratory chain.

The mitochondrial respiratory chain is crucial for cellular energy metabolism, serving as the core of oxidative phosphorylation. The mitochondrial ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

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

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