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Published on: July 20, 2022
cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer
Shengbing Huang1, Wei Song, Qishui Lin
1Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Abstract:
A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5'-terminal and 3'-terminal were obtained by RACE technique. The full-length cDNA that encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-QOp. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.
Insights
Researchers identified and cloned the full-length cDNA for rat Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase (ETF-QO). This protein is crucial for mitochondrial electron transport and is highly expressed in key rat organs.
Area of Science:
- Biochemistry
- Molecular Biology
- Mitochondrial Function
Background:
- Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase (ETF-QO) is an essential mitochondrial inner membrane protein involved in electron transport.
- ETF-QO contains FAD and a [4Fe-4S] redox center, facilitating crucial metabolic processes.
Purpose of the Study:
- To clone and characterize the full-length cDNA of rat ETF-QO.
- To investigate the function and localization of the expressed rat ETF-QO protein.
Main Methods:
- Purification of a membrane-bound protein from rat liver mitochondria.
- Peptide sequencing, RACE (Rapid Amplification of cDNA Ends) technique for cDNA cloning.
- Expression in Saccharomyces cerevisiae and COS-7 cells, NBT reductase activity assay, GFP fusion protein localization.
Main Results:
- Cloned a 616-amino acid full-length cDNA highly homologous to human ETF-QO, identified as rat ETF-QOp.
- Expressed ETF-QO in yeast, showing mitochondrial localization and electron transfer activity.
- Demonstrated that the N-terminal 32 amino acids serve as a mitochondrial targeting peptide.
- Confirmed proper insertion of FAD and iron-sulfur clusters and high expression in rat heart, liver, and kidney.
Conclusions:
- The cloned cDNA represents rat ETF-QOp, a functional mitochondrial protein.
- The N-terminal 32-amino acid sequence acts as a mitochondrial targeting signal.
- Rat ETF-QO is properly folded and functional when expressed, with significant expression in vital organs.
