The Monocarboxylate Transporter Homolog Mch5p Catalyzes Riboflavin (Vitamin B2) Uptake in Saccharomyces cerevisiae

Abstract

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 ...

Transport and cytotoxicity of the anticancer drug 3-bromopyruvate in the yeast Saccharomyces cerevisiae

“…Additionally, the yeast genome encodes five members of the Monocarboxylate Porter (MCP) Family (De Hertogh et al 2002), but their role in importing monocarboxylic acids has not been proven (Makuc et al 2001;Reihl and Stolz 2005). Casal et al (Casal et al 1999).…”

Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

“…Its homologues were identified among bacterial genes but not among mammalian genes (26). The Mch5p protein of Saccharomyces cerevisiae is an ortholog of the mammalian monocarboxylate transporter and has been reported to transport riboflavin; the transport of riboflavin by the mammalian monocarboxylate transporter has, however, not been assessed (33). hRFT1 and rRFT1 exhibit no significant similarity to RibU, Mch5p, or SLC families; therefore, mammalian riboflavin transporters (hRFT1 and rRFT1) appear to belong to a novel mammalian riboflavin transporter family and not to the bacterial riboflavin transporter family.…”

The Proline-Dependent Transcription Factor Put3 Regulates the Expression of the Riboflavin Transporter MCH5 in Saccharomyces cerevisiae

“…Strains with a deletion of MCH5 and of a RIB gene show synthetic growth defects and a reduced efficiency in catalyzing FAD-dependent cellular processes. Moreover, the expression of MCH5 is regulated according to the riboflavin supply (Reihl and Stolz 2005). Most recently, a mammalian riboflavin transporter has been characterized, which again is not related to the previously known riboflavin transporters and was earlier mistaken as a G-protein coupled receptor (Yonezawa et al 2008).…”

“…Yet another type of plasma-membrane riboflavin transporter is present in fungi. We used a multicopy suppressor screen of S. cerevisiae riboflavin auxotrophic strains to identify MCH5, the first known eukaryotic riboflavin transporter gene (Reihl and Stolz 2005). Riboflavin transport in yeast is not significantly stimulated by glucose or ethanol and not inhibited by proton ionophors, indicating that Mch5 acts as a facilitator.…”

“…Haploid rib2D, rib3D, and rib7D strains were generated from heterozygous diploids by sporulation and tetrad dissection on YPD plates with 200 mg/liter added riboflavin. rib4D strains were not included in the analysis because they are capable of riboflavin synthesis (Kis et al 2001;Reihl and Stolz 2005). Alternatively, strains based on W303 (Thomas and Rothstein 1989) or CEN.PK113-13D (Makuc et al 2001;Reihl and Stolz 2005) were used.…”