Rat Liver Mitochondrial Choline Dehydrogenase And Electron Transfer Flavoprotein - Ubiquinone Oxidoreductase

Rat liver mitochondria choline dehydrogenase (CHDH, EC1.1.99.1) located in the mitochodria inner membrane is an iron sulfur flavoprotein. CHDH is a momomer consisting of two redox centers, FAD and iron sulfur cluster. The amino terminal sequence of purified CHDH from rat liver mitochondria was determined, and its full length cDNA was thus cloned. The deduced open reading frame encodes a protein precursor of 599 amino acids (64Kda). It is hard to specify the precursor's processing site in mitochondria and its signal peptide sequence directly from the cDNA sequence. The processing site meet the requirements of IMP since there was Alanine at –1 and Serine at -3 according the determined N-terminal sequence of mature CHDH of rat, which suggested that the precursor might be processed at intermembrane space. Confocal imaging showed that CHDH precursor gene expressed in NIH-3T3 cells targeted to mitochondria. The final product of recombinant CHDH precursor gene expressed in Saccharomyces cerevisiae was enriched in mitochondria inner membrane and expressed choline dehydrogenase activity, suggested that both FAD and iron sulfur cluster had been assembled correctly to the protein and the mature CHDH located in the inner membrane of mitochondria. Immunoblotting showed that the protein had a high level expression...