Search

The flavinylation and the presequence processing of the mitochondrial matrix enzyme dimethylglycine dehydrogenase (Me(2)GlyDH) were investigated with the reticulocyte lysate translated precursor (pMe(2)GlyDH) added to solubilised mitoplasts of rat liver mitochondria. The flavinylation of pMe(2)GlyDH was strictly dependent on the addition of mitochondrial protein(s), among which the mitochondrial flavinylation stimulating factor [Brizio C., et al. (2000) Eur. J. Biochem 267, 4346-4354], that actively promotes holo-Me(2)GlyDH formation. The precursor processing, that accompanies the biogenesis of the enzyme, was not required to allow the flavinylation to proceed. The comparison of the time course of the flavinylation and the processing of pMe(2)GlyDH demonstrated that the covalent attachment of the flavin moiety preceded the presequence processing by mitochondrial processing peptidase.

We show here that TPP→TMP conversion can take place in rat liver mitochondria. This occurs via the novel, putative TPP pyrophosphatase localised in the mitochondrial matrix, as shown both by digitonin titration and by an HPLC enzyme assay carried out on the mitochondrial matrix fraction. Certain features of the reaction, including the substrate and pH dependence, are reported. Additional evidence is given that externally added TMP can cross the mitochondrial membrane in a manner consistent with the occurrence of a carrier-mediated process. This can occur both via the TPP translocator and via a novel translocator, inhibited by CAT but different from the ADP/ATP carrier.