Riboflavin Uptake and FAD Synthesis in Saccharomyces cerevisiae Mitochondria

Abstract

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1⌬ mutant strain was constructed. Coupled mitochondria isolated from flx1⌬ mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1⌬ mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.The mechanism by which mitochondria obtain their own flavin cofactors is an interesting point of investigation because FMN and FAD are mainly located in mitochondria, where they act as redox cofactors of a number of dehydrogenases and oxidases that play a crucial role in both bioenergetics and cellular regulation (for reviews see Refs. 1 and 2).As far as mammalian mitochondria are concerned, we have demonstrated that in rat liver the main source of intramitochondrial flavin cofactors is riboflavin (Rf) 1 taken up from the cytosol. FAD synthesis occurs inside the organelle from imported Rf and mitochondrial ATP, consistent with the presence of a mitochondrial riboflavin kinase (EC 2.7.1.26) and an FAD synthetase (EC 2.7.7.2) (3, 4). Newly synthesized FAD can be either efficiently incorporated into newly imported apo-flavoproteins (5, 6) or can be exported into the outer mitochondrial compartments, where it is reconverted to Rf by FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2) in a recycling pathway, i.e. the Rf-FAD cycle (4, 7). This novel mitochondrial pathway is assumed to play a central role in cellular Rf homeostasis and in flavoprotein biogenesis (5,8).The origin of flavin cofactors in yeast mitochondria is still controversially discussed. It has been reported that yeast mitochondria do not contain their own FAD synthetase activity an...

FAD Synthesis and Degradation in the Nucleus Create a Local Flavin Cofactor Pool

“…However, using cell fractionation procedures and enzymatic activity measurements, we have demonstrated the presence of FADS activity in mitochondria from rat liver (18,19), Saccharomyces cerevisiae (20,21) and Nicotiana tabacum Yellow Bright-2 (22). More recently, the existence of different natural FADS isoforms with distinct features regarding molecular mass and subcellular localization has been confirmed by biochemical and immunohistochemical approaches (23).…”

SdhE Is a Conserved Protein Required for Flavinylation of Succinate Dehydrogenase in Bacteria

“…For FAD-UV assays, His-tagged purified proteins were separated by SDS-PAGE (15% polyacrylamide gels for SdhA and 20% gels for SdhE), destained, and exposed to UV light (FAD-UV) as described previously (18). Aliquots of the same samples were separated on additional gels for visualization of proteins by Coomassie Blue staining or Western blotting.…”

“…The presence of covalently bound FAD can be determined by running purified proteins on a denaturing gel as noncovalently linked FAD is released from the protein, whereas covalently linked FAD remains bound (31,32). Visualization by UV light (FAD-UV) produces an illuminated band at the same molecular weight as the protein of interest (18,31). To test FAD insertion, N-terminally His-tagged SdhA was overexpressed in WT or ⌬sdhE Serratia, purified, and examined by FAD-UV.…”

Cytochrome c Is Released in a Reactive Oxygen Species-Dependent Manner and Is Degraded via Caspase-Like Proteases in Tobacco Bright-Yellow 2 Cells en Route to Heat Shock-Induced Cell Death

“…Hence, we can deduce that the low level of cyt c, as well as of mADK and mGDH, in the cytosolic fraction of control cells is a result of manipulation during the cell fractionation procedure. As a further control, the intactness of the mitochondria was also checked both in control and in 2-h HS cells by measuring the activity of both mADK (Bobba et al, 1999) and fumarase (Balk et al, 1999;Bafunno et al, 2004), a mitochondrial matrix enzyme (Table I). Negligible mADK activity was found in the cytosolic fraction of either control or HS cells and the assay of fumarase activity showed a mitochondrial intactness ranging from 94% to 96%.…”