Riboflavin is a water-soluble vitamin (vitamin B 2 ) required for the production of the flavin cofactors FMN and FAD. Mammals are unable to synthesize riboflavin and need a dietary supply of the vitamin. Riboflavin transport proteins operating in the plasma membrane thus have an important role in the absorption of the vitamin. However, their sequences remained elusive, and not a single eukaryotic riboflavin transporter is known to date. Here we used a genetic approach to isolate MCH5, a Saccharomyces cerevisiae gene with homology to mammalian monocarboxylate transporters, and characterize the protein as a plasma membrane transporter for riboflavin. This conclusion is based on the suppression of riboflavin biosynthetic mutants (rib mutants) by overexpression of MCH5 and by synthetic growth defects caused by deletion of MCH5 in rib mutants. We also show that cellular processes in multiple compartments are affected by deletion of MCH5 and localize the protein to the plasma membrane. Transport experiments in S. cerevisiae and Schizosaccharomyces pombe cells demonstrate that Mch5p is a high affinity transporter (K m ‫؍‬ 17 M) with a pH optimum at pH 7.5. Riboflavin uptake is not inhibited by protonophores, does not require metabolic energy, and operates by a facilitated diffusion mechanism. The expression of MCH5 is regulated by the cellular riboflavin content. This indicates that S. cerevisiae has a mechanism to sense riboflavin and avert riboflavin deficiency by increasing the expression of the plasma membrane transporter MCH5. Moreover, the other members of the MCH gene family appear to have unrelated functions.The proteins that require FMN or FAD as cofactors are termed flavoproteins. Mostly, they contain noncovalently bound flavin cofactors and are specific for either FAD or FMN. Some of them contain auxiliary groups such as pteridin, heme, iron-sulfur centers, molybdenum, or other metal ions or contain disulfides in their active site. Flavoproteins are involved in a wide range of biochemical reactions. They play a pivotal role in the dehydrogenation of metabolites in one-and two-electron transfer reactions from and to redox centers, in the activation of oxygen for oxidation, and in hydroxylation reactions (1).The flavin cofactor FMN is produced from riboflavin by the action of riboflavin kinase. FAD derives from FMN and ATP, a reaction catalyzed by FAD synthetase. Riboflavin is a vitamin (vitamin B 2 ) for mammals and many other organisms. Thus, dietary riboflavin has to be taken up from the gut and then provided to every single cell in a multicellular organism. Plasma membrane riboflavin transporters are thought to play an important role in the distribution of riboflavin. However, their existence in many cell types up to now has only been demonstrated biochemically (reviewed in Ref. 2). Whereas passive uptake of riboflavin is commonly observed in riboflavin-sufficient conditions, riboflavin uptake at low concentrations follows saturation kinetics and displays high affinity for the substrate (K m ϭ 1 nM to 1 M ...

Like most microorganisms, the yeast Saccharomyces cerevisiae is prototrophic for riboflavin (vitamin B 2 ). Riboflavin auxotrophic mutants with deletions in any of the RIB genes frequently segregate colonies with improved growth. We demonstrate by reporter assays and Western blots that these suppressor mutants overexpress the plasma-membrane riboflavin transporter MCH5. Frequently, this overexpression is mediated by the transcription factor Put3, which also regulates the proline catabolic genes PUT1 and PUT2. The increased expression of MCH5 may increase the concentrations of FAD, which is the coenzyme required for the activity of proline oxidase, encoded by PUT1. Thus, Put3 regulates proline oxidase activity by synchronizing the biosynthesis of the apoenzyme and the coenzyme FAD. Put3 is known to bind to the promoters of PUT1 and PUT2 constitutively, and we demonstrate by gel-shift assays that it also binds to the promoter of MCH5. Put3-mediated transcriptional activation requires proline as an inducer. We find that the increased activity of Put3 in one of the suppressor mutants is caused by increased intracellular levels of proline. Alternative PUT3-dependent and -independent mechanisms might operate in other suppressed strains.