cDNA cloning and mitochondrial import of the β‐subunit of the human electron‐transfer flavoprotein

• Published in: European journal of biochemistry Vol. 213; no. 3; pp. 1003 - 1008
• Main Authors: FINOCCHIARO, Gaetano, COLOMBO, Irma, GARAVAGLIA, Barbara, GELLERA, Cinzia, VALDAMERI, Giulio, GARBUGLIO, Nicoletta, DIDONATO, Stefano
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-05-1993; Blackwell
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Blotting, Northern; Cloning, Molecular; DNA - chemistry; DNA - genetics; DNA - isolation & purification; Electron-Transferring Flavoproteins; Flavoproteins - chemistry; Flavoproteins - genetics; Flavoproteins - metabolism; Fundamental and applied biological sciences. Psychology; Genes. Genome; Humans; Mitochondria - metabolism; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Rabbits; Sequence Analysis; Swine; Transcription, Genetic; Human; Intracellular transport; Mitochondria; Beta-Peptide chain; Primary structure; Flavoprotein; Molecular cloning
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1993.tb17847.x

We have isolated a cDNA clone which encodes the entire β‐subunit of human electron‐transferring flavoprotein (ETF) by screening an expression library from human liver using polyclonal antibodies against porcine ETF. This cDNA encodes a protein of 255 amino‐acid residues with a predicted molecular mass of 27 877 Da which shows a high degree of similarity with partial amino acid sequences obtained from both rat liver and Paracoccus denitrificansβ‐ETF. Northern‐blot analysis shows that the human β‐ETF mRNA is approximately 1 kb in size and is abundant in liver, heart and skeletal muscle.
Incubation with intact mitochondria indicates that the cDNA‐encoded β‐ETF polypeptide contains the information necessary to reach the mitochondrial matrix. These data are in agreement with previous experiments suggesting that β‐ETF, unlike the majority of nuclear‐encoded mitochondrial matrix proteins, does not have a cleavable leader peptide. Furthermore, when valinomycin is added to the incubation mixture, the import is abolished, thus demonstrating that it is an energy‐dependent process.
Interestingly, the sequence analysis of β‐ETF protein identifies a 26.3% identity with the Fix A gene product of the nitrogen‐fixing bacterium Azorhizobium caulinodans.