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 expres sion library from human liver using polyclonal antibodies against porc ine ETF. This cDNA encodes a protein of 255 amino-acid residues with a predicted molecular mass of 27877 Da which shows a high degree of sim ilarity with partial aminoacid sequences obtained from both rat liver and Paracoccus denitrificans beta-ETF. Northern-blot analysis shows th at the human beta-ETF mRNA is approximately 1 kb in size and is abunda nt in liver, heart and skeletal muscle. Incubation with intact mitocho ndria indicates that the cDNA-encoded beta-ETF polypeptide contains th e information necessary to reach the mitochondrial matrix. These data are in agreement with previous experiments suggesting that beta-ETF, u nlike the majority of nuclear-encoded mitochondrial matrix proteins, d oes not have a cleavable leader peptide. Furthermore, when valinomycin is added to the incubation mixture, the import is abolished, thus dem onstrating 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.