Publications

• pH-dependent pyridoxine transport by SLC19A2 and SLC19A3: Implications for absorption in acidic microclimates.

  Yamashiro T., Yasujima T., Said H.M., Yuasa H.

  SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, bu ... >> More

  SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent <i>K_m</i> of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate. << Less

  J. Biol. Chem. 295:16998-17008(2020) [PubMed] [EuropePMC]

• Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis.

  Szydlowski N., Buerkle L., Pourcel L., Moulin M., Stolz J., Fitzpatrick T.B.

  Vitamin B6 is a cofactor for more than 140 essential enzymatic reactions and was recently proposed as a potent antioxidant, playing a role in the photoprotection of plants. De novo biosynthesis of the vitamin has been described relatively recently and is derived from simple sugar precursors as wel ... >> More

  Vitamin B6 is a cofactor for more than 140 essential enzymatic reactions and was recently proposed as a potent antioxidant, playing a role in the photoprotection of plants. De novo biosynthesis of the vitamin has been described relatively recently and is derived from simple sugar precursors as well as glutamine. In addition, the vitamin can be taken up from exogenous sources in a broad range of organisms, including plants. However, specific transporters have been identified only in yeast. Here we assess the ability of the family of Arabidopsis purine permeases (PUPs) to transport vitamin B6. Several members of the family complement the growth phenotype of a Saccharomyces cerevisiae mutant strain impaired in both de novo biosynthesis of vitamin B6 as well as its uptake. The strongest activity was observed with PUP1 and was confirmed by direct measurement of uptake in yeast as well as in planta, defining PUP1 as a high affinity transporter for pyridoxine. At the tissue level the protein is localised to hydathodes and here we use confocal microscopy to illustrate that at the cellular level it is targeted to the plasma membrane. Interestingly, we observe alterations in pyridoxine recycling from the guttation sap upon overexpression of PUP1 and in a pup1 mutant, consistent with the role of the protein in retrieval of pyridoxine. Furthermore, combining the pup1 mutant with a vitamin B6 de novo biosynthesis mutant (pdx1.3) corroborates that PUP1 is involved in the uptake of the vitamin. << Less

  Plant J. 75:40-52(2013) [PubMed] [EuropePMC]

• Disrupted in renal carcinoma 2 (DIRC2/SLC49A4) is an H+-driven lysosomal pyridoxine exporter.

  Akino S., Yasujima T., Yamashiro T., Yuasa H.

  Disrupted in renal carcinoma 2 (DIRC2) has gained interest because of its association with the development of renal cancer and cosegregation with a chromosomal translocation. It is a member of the SLC49 family (SLC49A4) and is considered to be an electrogenic lysosomal metabolite transporter; howe ... >> More

  Disrupted in renal carcinoma 2 (DIRC2) has gained interest because of its association with the development of renal cancer and cosegregation with a chromosomal translocation. It is a member of the SLC49 family (SLC49A4) and is considered to be an electrogenic lysosomal metabolite transporter; however, its molecular function has not been fully defined. To perform a detailed functional analysis of human DIRC2, we used a recombinant DIRC2 protein (DIRC2-AA), in which the N-terminal dileucine motif involved in its lysosomal localization was removed by replacing with dialanine for redirected localization to the plasma membrane, exposing intralysosomal segments to the extracellular space. The DIRC2-AA mutant induced the cellular uptake of pyridoxine (vitamin B6) under acidic conditions when expressed transiently in COS-7 cells. In addition, uptake was markedly inhibited by protonophores, indicating its function through an H⁺-coupled mechanism. In separate experiments, the transient overexpression of unmodified DIRC2 (tagged with HA) in human embryonic kidney 293 cells reduced cellular pyridoxine accumulation induced by transiently introduced human thiamine transporter 2/SLC19A3 (tagged with FLAG), a plasma membrane thiamine transporter that also transports pyridoxine. The cellular accumulation of pyridoxine in Caco-2 cells as a cell model was increased by the knockdown of endogenous DIRC2. Overall, the results indicate that DIRC2 is an H⁺-driven lysosomal pyridoxine exporter. Its overexpression leads to a reduction in cellular pyridoxine accumulation associated with reduced lysosomal accumulation and, conversely, its suppression results in an increase in lysosomal and cellular pyridoxine accumulation. << Less

  Life. Sci Alliance 6:0-0(2023) [PubMed] [EuropePMC]

• Amiloride uptake and toxicity in fission yeast are caused by the pyridoxine transporter encoded by bsu1+ (car1+).

  Stolz J., Woehrmann H.J., Vogl C.

  Amiloride, a diuretic drug that acts by inhibition of various sodium transporters, is toxic to the fission yeast Schizosaccharomyces pombe. Previous work has established that amiloride sensitivity is caused by expression of car1+, which encodes a protein with similarity to plasma membrane drug/pro ... >> More

  Amiloride, a diuretic drug that acts by inhibition of various sodium transporters, is toxic to the fission yeast Schizosaccharomyces pombe. Previous work has established that amiloride sensitivity is caused by expression of car1+, which encodes a protein with similarity to plasma membrane drug/proton antiporters from the multidrug resistance family. Here we isolated car1+ by complementation of Saccharomyces cerevisiae mutants that are deficient in pyridoxine biosynthesis and uptake. Our data show that Car1p represents a new high-affinity, plasma membrane-localized import carrier for pyridoxine, pyridoxal, and pyridoxamine. We therefore propose the gene name bsu1+ (for vitamin B6 uptake) to replace car1+. Bsu1p displays an acidic pH optimum and is inhibited by various protonophores, demonstrating that the protein works as a proton symporter. The expression of bsu1+ is associated with amiloride sensitivity and pyridoxine uptake in both S. cerevisiae and S. pombe cells. Moreover, amiloride acts as a competitor of pyridoxine uptake, demonstrating that both compounds are substrates of Bsu1p. Taken together, our data show that S. pombe and S. cerevisiae possess unrelated plasma membrane pyridoxine transporters. The S. pombe protein may be structurally related to the unknown human pyridoxine transporter, which is also inhibited by amiloride. << Less

  Eukaryot. Cell 4:319-326(2005) [PubMed] [EuropePMC]