cDNA cloning and mitochondrial import of the beta-subunit of the human electron-transfer flavoprotein.
We have isolated a cDNA clone which encodes the entire beta-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 denitrificans beta-ETF. Northern-blot analysis shows that the human beta-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 beta-ETF polypeptide contains the information necessary to reach the mitochondrial matrix. These data are in agreement with previous experiments suggesting that beta-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 beta-ETF protein identifies a 26.3% identity with the Fix A gene product of the nitrogen-fixing bacterium Azorhizobium caulinodans.
Istituto Nazionale Neurologico, Divisione di Biochimica e Genetica, Milano, Italy.
G
G
Finocchiaro
I
I
Colombo
B
B
Garavaglia
C
C
Gellera
G
G
Valdameri
N
N
Garbuglio
S
S
Didonato
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D011817:Rabbits; D017421:Sequence Analysis; D013552:Swine; D014158:Transcription, Genetic
D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D004247:DNA