cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein  

作　　者：HUANG Shengbing SONG Wei LIN Qishui 

机构地区：[1]Key Laboratory of Molecular Cell Biology,Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200031,China

出　　处：《Science China(Life Sciences)》2005年第4期357-367,共11页中国科学(生命科学英文版)

基　　金：This project was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences.

摘　　要：A membrane-bound protein was purified from rat liver mitochondria.After being di-gested 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 re-ductase activity)of ETF-QO.Results demonstrated that the 32 amino acid peptide was a mito-chondrial 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 fu-sion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.

关 键 词：electron transfer flavoprotein ubiquinone oxidoreductase PRECURSOR rat protein purification gene clone tissuespecificity. 

分 类 号：N[自然科学总论]