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>

<ol>
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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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. 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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. 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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 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 ...

labelling-visualization-mitochondrial-genome-expression-products

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Biochemistry

4.3K Views.  M.V. Lomonosov Moscow State University. Baker’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.

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

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

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 ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.