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Biochemistry

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

Published: April 11th, 2021

DOI:

10.3791/62020

1Department of Biology, M.V. Lomonosov Moscow State University, 2Institute of Functional Genomics, M.V. Lomonosov Moscow State University, 3National Research Centre "Kurchatov Institute"

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.

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

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’s yeast Saccharomyces cerevisiae is an excellent model organism for studying mitochondrial gene express....

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1. Yeast culture preparation

  1. Streak yeast from the frozen stock cultures on fresh plates with the appropriate medium. Put the plates in a culture incubator at 30 °C for 24–48 h.
    NOTE: Let the temperature-sensitive mutants grow at the permissive temperature.
  2. 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 °C.
  3. Measure the optical den.......

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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 (AIM23Δ), 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 (

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Investigations of gene expression occupy a central part in modern life sciences. Numerous methods providing insights into this complex process have been developed. Here, we described the method allowing to access protein biosynthesis in baker'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.......

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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 “Leading Scientific School Noah’s Ark&#....

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

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