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Method Article
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 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.
Mitochondria play important roles in diverse biological processes, such as cellular respiration for energy, nutritional metabolism, and cell death1. The malfunction of mitochondria is related to brain, muscle, and developmental diseases2,3. Therefore, studies on mitochondria are vital to improve human aging and health.
The budding yeast Saccharomyces cerevisiae (S. cerevisiae) has long been used to study the functions of genes in mitochondria4 because yeast mutants defective in respiration can still produce energy for survival by fermentation. However, it is petite-positive and can proliferate without mitochondrial DNA (mtDNA). Consequently, the gene mutants defective in mitochondrial gene expression often lose their mtDNA, which complicates further study. In contrast, the fission yeast Schizosaccharomyces pombe (S. pombe), which is evolutionarily distant from S. cerevisiae, is a petite-negative yeast that requires mtDNA for survival. Moreover, the organization of mtDNA and mitochondrial mRNA of S. pombe is similar to those of higher eukaryotes5. Many (96) essential genes in S. pombe, compared to only six essential genes in S. cerevisiae, are required for mitochondrial gene expression6. Thus, S. pombe is emerging as an attractive model to study novel functions of genes in mitochondria. However, the number of publications studying mitochondria in S. cerevisiae is about 100-fold more than that in S. pombe, and the reported methods and protocols studying mitochondria in S. pombe are also scarce7.
The mitochondrial respiratory chain is crucial for cellular energy production and serves as a core of mitochondria8. It comprises respiratory chain complexes I-V, such as NADH-ubiquinone oxidoreductase (Complex I), succinate dehydrogenase (Complex II), ubiquinone-cytochrome c oxidoreductase or cytochrome bc1 complex (Complex III), cytochrome c oxidase (Complex IV), and ATP synthase (Complex V), together with two intermediary substrates carrying electron, ubiquinone (CoQ) and cytochrome c (Cyt c)9. They also interact and form higher-order supercomplexes, whose assembly mechanisms remain largely unclear10,11. However, S. pombe lacks complex I, which may be replaced by external NADH dehydrogenases Nde1 and internal Ndi112,13,14. The mitochondrial genome in S. pombe encodes a subunit of complex III (Cob1), three subunits of complex IV (Cox1, Cox2, Cox3), and three subunits of complex V (Atp6, Atp8, Atp9)15,16. We have recently reported that the expression and complexes assembly of these proteins are affected by the deletion of RNA helicase Mss116 (Δmss116)16 and assembly factor Shy1 (Δshy1)15 in S. pombe, respectively. To facilitate the discovery of more genes' novel functions in the mitochondrial respiratory chain using these methods, here we provide a detailed protocol for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the expression levels and blue native-PAGE (BN-PAGE) analysis of complexes assembly of the mitochondrial respiratory chain proteins in S. pombe.
The rationale behind the isolation of mitochondria from S. pombe is based on the methods established in S. cerevisiae17. The spheroplasts are first prepared by digesting yeast cell walls. They are mechanically homogenized, and the mitochondria are fractionated by differential centrifugation18. Subsequently, the mitochondrial respiratory chain proteins are solubilized and immunoblotted following SDS-PAGE and BN-PAGE. The BN-PAGE technique was originally developed for the separation of mitochondrial membrane proteins such as respiratory chain complexes19,20,21. The membrane proteins with preserved intact complexes are solubilized by mild nonionic detergents and charged by anionic dye Coomassie G-250. Thus, protein complexes are separated according to their mass in a gradient native-PAGE gel22. This method has been widely used for studying mitochondrial respiratory chain complexes in S. cerevisiae23 and mammalian cells24,25; however, it has not been extensively applied to S. pombe mitochondria.
Collectively, here we present a method in which mitochondria are isolated from S. pombe cells, and the mitochondrial respiratory chain proteins are subjected to SDS-PAGE and BN-PAGE followed by immunoblotting. The experimental flowchart is illustrated in Figure 1. With high-quality mitochondria and antibodies, this method described in S. pombe can also be applied to other organisms to identify more genes with functions in the expression and/or complexes assembly of mitochondrial respiratory chain proteins.
1. Preparation of S. pombe spheroplasts
2. Mechanical homogenization of S. pombe spheroplasts
3. Isolation of S. pombe mitochondria by centrifugation
4. SDS-PAGE and immunoblotting of mitochondrial respiratory chain proteins
5. Mitochondrial sample preparation for BN-PAGE
6. BN-PAGE and immunoblotting of mitochondrial respiratory chain complexes
We previously used this protocol to investigate the effects of the deletion of shy1, the S. pombe homolog of human SURF1, on the expression of mtDNA-encoded respiratory chain proteins and the assembly of mitochondrial respiratory chain complexes. We found that the steady-state levels of Cob1, Cox1, Cox2, Cox3, and Atp6 were significantly reduced in the Δshy1 strain (Figure 3), indicating that Shy1 is required for the expression of respiratory chain pr...
In this study, we present a detailed protocol for isolating S. pombe mitochondria and performing SDS-PAGE and BN-PAGE to analyze the expression and complexes assembly of mitochondrial respiratory chain proteins.
Co-immunoprecipitation (co-IP) has been commonly used to detect the assembly of protein complexes; however, it is challenging for co-IP to detect the assembly of mitochondrial membrane proteins. Instead, BN-PAGE has several advantages: (i) some antibodies cannot recognize the ...
We have no conflicts of interest.
We thank Dr. Ying Huang and Dr. Ying Luo for their support. This work was supported by grants (32270048 to J. S. and 31900403 to G. F.) from the National Natural Science Foundation of China.
Name | Company | Catalog Number | Comments |
6-Aminocaproice acid | Sangon | A430196 | |
Acrylamide/Bis (37.5: 1) 40% | Biolab | GS1374 | |
Ammonium persulfate | Sangon | A100486 | |
Anti-Atp6 antibody | Bioworld | in-house | IB: 1:200 |
Anti-Cob1 antibody | Bioworld | in-house | IB: 1:1000 |
Anti-Cox1 antibody | Bioworld | in-house | IB: 1:2000 |
Anti-Cox2 antibody | Bioworld | in-house | IB: 1:1000 |
Anti-Cox3 antibody | Bioworld | in-house | IB: 1:200 |
Anti-Hsp60 antibody | Bioworld | in-house | IB: 1:3000 |
BCA Protein Quantification Kit | LEAGENE | PT0001 | |
Bis-Tris | YEASEN | 60105ES25 | |
Coomassie Brilliant Blue G-250 | SERVA | 17524.01 | |
Coomassie Brilliant Blue R-250 | Sangon | A100472 | |
Digitonin | Sigma | D141 | |
D-Sorbitol | Sangon | A100691 | |
EDTA | Solarbio | E8030 | |
Gradient mixer | Millet scientific | MGM-50 | |
HEPES | Solarbio | H8090 | |
High molecular weight protein marker for native PAGE | Real Times | RTD6142 | |
IgG-Alexa Fluor Plus 800 (anti-rabbit secondary antibody) | Thermo Fisher | A32735 | IB: 1:10000 |
Immobilon-FL 0.45 μm PVDF membrane | Millipore | IPFL00005 | |
Kimble Kontes Dounce tissue grinder | Thermo Fisher | K8853000015 | |
KOH | Sangon | A610441 | |
Lallzyme MMX | Lallemand | N.A. | |
Lysing enzyme | Sigma | L3768 | |
Lyticase | Sigma | L4025 | |
MgCl2 | Sangon | A601336 | |
MOPS | Sangon | A421333 | |
Native Bis-Tris Gels (precast) | Solarbio | PG41510-N | |
PMSF | Sangon | A610425 | |
Precast Gel Running buffer for Native-PAGE | Solarbio | PG00020-N | |
Protease inhibitor cocktail for yeast extracts | Beyotime | P1020 | |
Sucrose | Sangon | A610498 | |
TEMED | Sigma | T9281 | |
Tricine | Sigma | T0377 | |
Tween-20 | Solarbio | T8220 | |
Vinotaste | Novozymes | N.A. | |
Zymolyase | MP Biomedicals | 320921 |
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