Biogenesis of the covalently flavinylated mitochondrial enzyme dimethylglycine dehydrogenase.
Rat dimethylglycine dehydrogenase (Me2GlyDH) was used as model protein to study the biogenesis of a covalently flavinylated mitochondrial enzyme. Here we show that: 1) enzymatically active holoenzyme correlated with trypsin resistance of the protein; 2) folding of the reticulocyte lysate-translated protein into the trypsin-resistant, holoenzyme form was a slow process that was stimulated by the presence of the flavin cofactor and was more efficient at 15 degrees C than at 30 degrees C; 3) the mitochondrial presequence reduced the extent but did not prevent holoenzyme formation; 4) covalent attachment of FAD to the Me2GlyDH apoenzyme proceeded spontaneously and did not require a mitochondrial protein factor; 5) in vitro only the precursor, but not the mature form, of the protein was imported into isolated rat liver mitochondria; in vivo, in stably transfected HepG2 cells, both the precursor and the mature form were imported into the organelle; 6) holoenzyme formation in the cytoplasm did not prevent the translocation of the proteins into the mitochondria in vivo; and 7) lack of vitamin B2 in the tissue culture medium resulted in a reduced recovery of the precursor and the mature form of Me2GlyDH from cell mitochondria, suggesting a decreased efficiency of mitochondrial protein import.
Otto
A
A
Biochemisches Institut, Universitat Freiburg, Germany.
Stoltz
M
M
Sailer
H P
HP
Brandsch
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D050893:Dimethylglycine Dehydrogenase; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis* / Q000378:metabolism; D015870:Gene Expression; D018197:Hepatoblastoma; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D008715:Methionine / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014162:Transfection; D014407:Tumor Cells, Cultured
D024101:Mitochondrial Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D005182:Flavin-Adenine Dinucleotide; D008715:Methionine; D010089:Oxidoreductases, N-Demethylating; C494398:DMGDH protein, human; D050893:Dimethylglycine Dehydrogenase; C494400:Dmgdh protein, rat