Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein-ubiquinone oxidoreductase.
Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) in the inner mitochondrial membrane accepts electrons from electron-transfer flavoprotein which is located in the mitochondrial matrix and reduces ubiquinone in the mitochondrial membrane. The two redox centers in the protein, FAD and a [4Fe4S]+2,+1 cluster, are present in a 64-kDa monomer. We cloned several cDNA sequences encoding the majority of porcine ETF-QO and used these as probes to clone a full-length human ETF-QO cDNA. The deduced human ETF-QO sequence predicts a protein containing 617 amino acids (67 kDa), two domains associated with the binding of the AMP moiety of the FAD prosthetic group, two membrane helices and a motif containing four cysteine residues that is frequently associated with the liganding of ferredoxin-like iron-sulfur clusters. A cleavable 33-amino-acid sequence is also predicted at the amino terminus of the 67-kDa protein which targets the protein to mitochondria. In vitro transcription and translation yielded a 67-kDa immunoprecipitable product as predicted from the open reading frame of the cDNA. The human cDNA was expressed in Saccharomyces cerevisiae, which does not normally synthesize the protein. The ETF-QO is synthesized as a 67-kDa precursor which is targeted to mitochondria and processed in a single step to a 64-kDa mature form located in the mitochondrial membrane. The detergent-solubilized protein transfers electrons from ETF to the ubiquinone homolog, Q1, indicating that both the FAD and iron-sulfur cluster are properly inserted into the heterologously expressed protein.
Goodman
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Department of Pediatrics, University of Colorado School of Medicine, Denver 80262.
Axtell
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Bindoff
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Beard
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Gill
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Frerman
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism*; D044927:Electron-Transferring Flavoproteins; D004926:Escherichia coli; D044943:Fatty Acid Desaturases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005333:Fetus; D005420:Flavoproteins / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000587:Oxidoreductases Acting on CH-NH Group Donors; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D013552:Swine
D017931:DNA Primers; D018076:DNA, Complementary; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases; D000587:Oxidoreductases Acting on CH-NH Group Donors; C023120:electron-transferring-flavoprotein dehydrogenase