A protein factor of rat liver mitochondrial matrix involved in flavinylation of dimethylglycine dehydrogenase

The involvement of rat liver mitochondria in the flavinylation of the mitoc hondrial matrix flavoenzyme dimethylglycine dehydrogenase (Me(2)GlyDH) has been investigated. Me(2)GlyDH was synthesized as an apoenzyme in the rabbit reticulocyte lysate (RL) transcription/translation system and its flavinyl ation was monitored by virtue of the trypsin resistance of the holoenzyme. The rate of holoenzyme formation in the presence of FAD was stimulated with increasing efficiency by the addition of solubilized mitoplasts, mitochond rial matrix and DEAE-purified matrix fraction. Apo-Me(2)GlyDH was also conv erted into holoenzyme when the solubilized mitoplasts were supplemented wit h FMN and ATP. This observation is consistent with the existence of a mitoc hondrial FAD synthetase generating the FAD needed for holoenzyme formation from its precursors. Holoenzyme formation in the presence of FAD increased linearly with the concentration of matrix protein in the assay, and depende d on the amount of externally added Me(2)GlyDH with saturation characterist ics. These findings suggest the presence of a protein factor in the mitocho ndrial matrix which stimulates Me(2)GlyDH flavinylation. This factor was di fferent from both mitochondrial heat shock protein (Hsp)70, as shown by imm unodepletion experiments, and mitochondrial Hsp60, as demonstrated by the c apability of a DEAE-purified matrix fraction devoid of Hsp60 to accelerate flavinylation of both RL translated and purified Me(2)GlyDH.