SummaryIn this paper the cloning of a full-length cDNA clone encoding the PmSUC2 sucrose-H + symporter from Invertase null mutants of yeast expressing PmSUC2accumulate sucrose more than 200-fold. This transport was sensitive to uncouplers or SH-group inhibitors. Plasma membranes from yeast cells expressing the PmSUC2 protein were purified and fused to proteoliposomes containing cytochrome-coxidase. In this system sucrose is accumulated only when proton motive force is generated, indicating that PmSUC2 is a sucrose-H + symporter. The apparent molecular weight of the PmSUC2 protein is 35 kDa on 10% SDS-polyacrylamide gels. The presented data strongly support the theory of phloem loading from the apoplastic space by a sucrose-H + symporter.

Riboflavin (vitamin B 2 ) is the direct precursor of the flavin cofactors flavin mononucleotide and flavin adenine dinucleotide, essential components of cellular biochemistry. In this work we investigated the unrelated proteins YpaA from Bacillus subtilis and PnuX from Corynebacterium glutamicum for a role in riboflavin uptake. Based on the regulation of the corresponding genes by a riboswitch mechanism, both proteins have been predicted to be involved in flavin metabolism. Moreover, their primary structures suggested that these proteins integrate into the cytoplasmic membrane. We provide experimental evidence that YpaA is a plasma membrane protein with five transmembrane domains and a cytoplasmic C terminus. In B. subtilis, riboflavin uptake was increased when ypaA was overexpressed and abolished when ypaA was deleted. Riboflavin uptake activity and the abundance of the YpaA protein were also increased when riboflavin auxotrophic mutants were grown in limiting amounts of riboflavin. YpaA-mediated riboflavin uptake was sensitive to protonophors and reduced in the absence of glucose, demonstrating that the protein requires metabolic energy for substrate translocation.In addition, we demonstrate that PnuX from C. glutamicum also is a riboflavin transporter. Transport by PnuX was not energy dependent and had high apparent affinity for riboflavin (K m 11 M). Roseoflavin, a toxic riboflavin analog, appears to be a substrate of PnuX and YpaA. We propose to designate the gene names ribU for ypaA and ribM for pnuX to reflect that the encoded proteins function in riboflavin uptake and that the genes have different phylogenetic origins.Riboflavin consists of a ribityl side chain linked to an aromatic isoalloxazine ring structure. It is the precursor of the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which both are essential components of cellular metabolism. Riboflavin is phosphorylated to give FMN by flavokinase (EC 2.7.1.26), and FMN is subsequently converted to FAD by FAD synthetase (EC 2.7.7.2). Whereas free riboflavin does not have biological activity, the mostly noncovalently bound flavin cofactors FMN and FAD are the active groups of a large number of flavoproteins. These are involved in a wide range of redox reactions and catalyze the dehydrogenation of metabolites, one-and two-electron transfer reactions from and to redox centers, and hydroxylation reactions (9). Flavins are also known to act as chromophores in photoreceptors, such as the plant blue light sensors cryptochrome and phototropin (reviewed in reference 3). Moreover, flavins are the ligands of dodecin, a recently identified flavoprotein that has the highest binding affinity to lumichrome, a lightinduced degradation product of riboflavin with an alloxazine ring structure lacking a ribityl side chain (13, 48).Whereas vertebrates can generate FMN and FAD from riboflavin, they lack the enzymes to synthesize riboflavin, making this compound a vitamin (vitamin B 2 ). In contrast, plants and most microorganisms are capable of ...

No 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 ...

BackgroundThe bacterium Bacillus subtilis, which is not a natural riboflavin overproducer, has been converted into an excellent production strain by classical mutagenesis and metabolic engineering. To our knowledge, the enhancement of riboflavin excretion from the cytoplasm of overproducing cells has not yet been considered as a target for (further) strain improvement. Here we evaluate the flavin transporter RibM from Streptomyces davawensis with respect to improvement of a riboflavin production strain.ResultsThe gene ribM from S. davawensis, coding for a putative facilitator of riboflavin uptake, was codon optimized (ribMopt) for expression in B. subtilis. The gene ribMopt was functionally introduced into B. subtilis using the isopropyl-β-thiogalactopyranoside (IPTG)-inducible expression plasmid pHT01: Northern-blot analysis of total RNA from IPTG treated recombinant B. subtilis cells revealed a ribMopt specific transcript. Western blot analysis showed that the his6-tagged heterologous gene product RibM was present in the cytoplasmic membrane. Expression of ribM in Escherichia coli increased [14C]riboflavin uptake, which was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of ribMopt supported growth of a B. subtilis ΔribB::Ermr ΔribU::Kanr double mutant deficient in riboflavin synthesis (ΔribB) and also deficient with respect to riboflavin uptake (ΔribU). Expression of ribMopt increased roseoflavin (a toxic riboflavin analog produced by S. davawensis) sensitivity of a B. subtilis ΔribU::Kanr strain. Riboflavin synthesis by a model riboflavin B. subtilis production strain overproducing RibM was increased significantly depending on the amount of the inducer IPTG.ConclusionsThe energy independent flavin facilitator RibM could in principle catalyze riboflavin export and thus may be useful to increase the riboflavin yield in a riboflavin production process using a recombinant RibM overproducing B. subtilis strain (or any other microorganism).

Bakers' yeast is auxotrophic for biotin (vitamin H) and depends on the efficient uptake of this compound from the environment. A mutant strain with strongly reduced biotin uptake and with reduced levels of protein biotinylation was identified. The strain was auxotrophic for long-chain fatty acids, and this auxotrophy could be suppressed with high levels of biotin in the medium. After transformation of this mutant with a yeast genomic library, the unassigned open reading frame YGR065C was identified to complement this mutation. This gene codes for a protein with 593 amino acids and 12 putative transmembrane helices. Northern blot analysis revealed that, in wild-type cells, the corresponding mRNA levels were increased at low biotin concentrations. Likewise, cellular biotin uptake was increased with decreasing biotin availability. Expression of YGR065C under the control of the constitutive ADH1 promoter resulted in very high biotin transport rates across the plasma membrane that were no longer regulated by the biotin concentration in the growth medium. We conclude that YGR065C encodes the first biotin transporter identified for a non-mammalian organism and designate this gene VHT1 for vitamin H transporter 1.

Pyridoxine (PN) is a metabolic precursor of pyridoxal phosphate that functions as a cofactor of many enzymes in amino acid metabolism. PN, pyridoxal, and pyridoxamine are collectively referred to as vitamin B 6 , and mammalian organisms depend on its uptake from the diet. In addition to the ability to use extracellular vitamin B 6 , most unicellular organisms are also capable of synthesizing PN to generate pyridoxal phosphate. Here, we report the isolation of Saccharomyces cerevisiae mutants that have lost the ability to transport PN across the plasma membrane. We used these mutants to isolate TPN1, the first known gene encoding a transport protein for vitamin B 6 . Tpn1p is a member of the purinecytosine permease family within the major facilitator superfamily. The protein functions as a proton symporter, localizes to the plasma membrane, and has high affinity for PN. TPN1 mutants lost the ability to utilize extracellular PN, pyridoxal, and pyridoxamine, showing that there is no other transporter for vitamin B 6 encoded in the genome. Amino acid substitutions that led to a loss of Tpn1p function localized to transmembrane domain 4 within the 12-transmembrane domain protein. Moreover, expression of TPN1 was regulated and increased with decreasing concentrations of vitamin B 6 in the medium. We also provide evidence that of the highly conserved SNZ and SNO genes in S. cerevisiae, only the protein encoded by SNZ1 is required for vitamin B 6 biosynthesis.

The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.