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

Abstract

The involvement of rat liver mitochondria in the flavinylation of the mitochondrial 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 flavinylation 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, mitochondrial matrix and DEAE-purified matrix fraction. Apo-Me 2 GlyDH was also converted into holoenzyme when the solubilized mitoplasts were supplemented with FMN and ATP. This observation is consistent with the existence of a mitochondrial 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 depended on the amount of externally added Me 2 GlyDH with saturation characteristics. These findings suggest the presence of a protein factor in the mitochondrial matrix which stimulates Me 2 GlyDH flavinylation. This factor was different from both mitochondrial heat shock protein (Hsp)70, as shown by immunodepletion experiments, and mitochondrial Hsp60, as demonstrated by the capability of a DEAE-purified matrix fraction devoid of Hsp60 to accelerate flavinylation of both RL translated and purified Me 2 GlyDH.Keywords: dimethylglycine flavinylation; dehydrogenase; flavinylation stimulating factor; mitochondria.Although mitochondrial protein biogenesis in mammals has been intensively investigated over the last 20 years (reviewed in [1]) the mechanisms which secure and regulate the assembly of newly synthesized enzymes with vitamin-derived cofactors essential for catalysis inside organelles, are still incompletely understood. The majority of mitochondrial enzymes are imported from the cytoplasm as precursor molecules. During passage through the import complexes of the outer and inner mitochondrial membrane the preproteins are in an unfolded state [1]. It is generally assumed that only after removal of the presequence by the mitochondrial processing peptidase (MPP) does the protein fold in the matrix space into the native conformation of the holoenzyme as a result of the incorporation of the cofactor. Thus, a co-ordination of protein import and cofactor supply in the mitochondrial matrix is indispensable.Mitochondria are the cellular site at which many flavoproteins are located, where they act as dehydrogenases and oxidases in co-operation with the riboflavin-derived redox cofactors FMN and FAD (reviewed in [2]). The mechanism(s) by which mitochondria obtain their flavin cofactors has been recently investigated both in rat liver mitochondria (RLM) and in Saccharomyces cerevisiae mitochondria [3±7]. As far as RLM are concerned, it has previously been shown that mitochondria can synthesize FAD from FMN taken up in a carrier-mediated process [3].Moreover, several inv...

Flavinylation of the precursor of mitochondrial dimethylglycine dehydrogenase by intact and solubilised mitochondria

“…It has been also recently shown that FAD newly synthesised starting from FMN and ATP inside the mitochondrial matrix can be e⁄-ciently incorporated into the apo-£avoenzyme dimethylglycine dehydrogenase (Me 2 GlyDH) (EC 1.5.99.2), a mitochondrial matrix enzyme involved in choline degradation. Moreover, the attachment of FAD to Me 2 GlyDH via histidyl(N3)( 8K)FAD covalent linkage, is assisted by a mitochondrial £a-vinylation stimulating factor (mtFSF) localised in the matrix [9]. These ¢ndings corroborate the notion that cofactor binding occurs inside the organelles.…”

“…The biogenesis of holo-Me 2 GlyDH starting from the apo form of its precursor was studied here in a model system consisting of the in vitro synthesised protein added to solubilised mitoplasts in the presence of FAD, under the experimental conditions optimised before, when £avinylation was started from the mature form of the protein [9].…”

“…1: the £avinylation of [ 35 S]Met-labelled pMe 2 GlyDH synthesised in the transcription-translation rabbit RL system was determined by assaying the trypsin resistance of the protein, which was correlated with the £avinylated form [9,10]. The densitometric analysis of the autoradiograms in Fig.…”

“…The £avinylation of the [ 35 S]Met-labelled RL translated pMe 2 -GlyDH was assayed by trypsin digestion, followed by SDS^PAGE and autoradiography, according to [9,10].…”

“…Indeed, we have partially puri¢ed the mtFSF as acting on the mature form of Me 2 GlyDH (mMe 2 GlyDH) synthesised in vitro [9]. The question about which the form of the protein (mature or precursor) is the in vivo substrate for £avinylation, that is to say whether once inside the organelle apo-protein £avinylation precedes or follows precursor processing, is still unsolved.…”