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Originally published In Press as doi:10.1074/jbc.M300949200 on March 20, 2003

J. Biol. Chem., Vol. 278, Issue 21, 18990-18996, May 23, 2003
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Tpn1p, the Plasma Membrane Vitamin B6 Transporter of Saccharomyces cerevisiae*

Jürgen Stolz {ddagger} and Martin Vielreicher

From the Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Universitätsstrasse 31, D-93040 Regensburg, Germany

Received for publication, January 28, 2003 , and in revised form, March 12, 2003.
   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 B6, and mammalian organisms depend on its uptake from the diet. In addition to the ability to use extracellular vitamin B6, 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 B6. Tpn1p is a member of the purine-cytosine 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 B6 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 B6 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 B6 biosynthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vitamins are essential dietary compounds for many organisms. Most water-soluble vitamins or derivatives thereof function as cofactors in enzymatic reactions. The group of vitamins that is referred to as vitamin B6 consists of pyridoxine (PN),1 pyridoxal (PL), and pyridoxamine (PM). The active intracellular form of vitamin B6, pyridoxal 5-phosphate (PLP), has multiple roles as a versatile cofactor of enzymes that almost exclusively function in the metabolism of amino compounds. PLP-dependent enzymes include {alpha}-, {beta}-, and {gamma}-synthetases, racemases, and decarboxylases of amino acids as well as aminotransferases (1).

Unlike mammals, most unicellular organisms and plants are prototrophic for vitamin B6. The pathway that catalyzes PN biosynthesis is best characterized in Escherichia coli (2, 3). A recently discovered pathway of vitamin B6 biosynthesis depends on the products of the SNZ (also referred to as PDX1) and SNO (also referred to as PDX2) genes (4, 5, 6, 7, 8). Organisms encode either Snz/Sno-like proteins or proteins with homology to E. coli PdxA/PdxJ, but not both (5, 9). The proteins encoded by the SNZ and SNO genes are highly conserved and have members in all three domains of life (9). The Cercospora nicotianae SNZ homolog SOR1 is required for resistance to singlet oxygen-generating photosensitizers (10). This finding was surprising and led to the discovery that PN, PL, PM, and PLP directly act as chemical quenchers of singlet oxygen and are as effective as well known antioxidants such as vitamins C and E (5, 11). Thus, the pyridoxine biosynthesis pathway serves a dual function to provide PLP as a cofactor and to provide protection against active oxygen species.

Three homologs of SNZ genes and three homologs of SNO genes are present in the genome of Saccharomyces cerevisiae. The genes encoding these proteins are arranged into divergently oriented SNZ-SNO gene pairs that are regulated by a common promotor (12). The expression of SNZ1 is induced in stationary phase (13), a feature later also found for SNO1 (12). In S. cerevisiae, deletion of SNZ1 causes PN dependence and deletion of SNO1 slows growth, whereas deletion of SNO2, SNO3, SNZ2, or SNZ3 does not lead to PN requirement (14). Sno1p and Snz1p are present in a high molecular mass complex (12) and show two-hybrid interactions with each other as well as with other proteins, including Snz2/3p and Sno2/3p (12, 14, 15, 16).

In vitamin B6 auxotrophic mutants, uptake of PN, PL, or PM from the extracellular space is required to generate intracellular PLP via a salvage pathway. The biochemistry of vitamin B6 uptake is best studied in Saccharomyces carlsbergensis (17), an allopolyploid hybrid thought to have originated from fusion of S. cerevisiae with an unidentified yeast species (18). Based on experiments with labeled substrates, Shane and Snell (17) concluded that S. carlsbergensis has two high affinity transport systems for vitamin B6. Consistent with an active transport mechanism for vitamin B6 uptake, S. carlsbergensis accumulates PN up to 3000-fold relative to outside concentrations, and transport is sensitive to metabolic inhibitors (17). Accumulation of PN is not a unique feature of S. carlsbergensis, but is commonly found in many yeast species, regardless whether they are prototrophic or auxotrophic for vitamin B6 (19).

Despite much evidence that vitamin B6 uptake across the plasma membrane is a protein-mediated process, not a single sequence of a PN transport protein is known to date. Here, we used S. cerevisiae mutants to identify Tpn1p, a protein from the purine-cytosine permease family that mediates the high affinity uptake of vitamin B6 across the plasma membrane.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Media—The yeast media used were YPD (1% yeast extract, 2% peptone, and 2% dextrose) and SD (synthetic dextrose; 2% glucose and 0.67% yeast nitrogen base without amino acids). SD medium contains 2 µg/liter PN (1.93 µM). For media with varying concentrations of vitamin B6, yeast nitrogen base without amino acids and without vitamins (Bio 101, Inc.) was used. All vitamins were added to the standard concentrations present in yeast nitrogen base, and PN, PL, or PM was added to give the desired concentrations. Only supplements that were required by the strains were added. Growth assays were performed with yeast cell suspensions prepared in water in 96- well plates. Starting from the highest cell density (A600 = 0.6), serial 10-fold dilutions were made and transferred to plates with a replicating tool.

The lager brewing yeast S. carlsbergensis 4228 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braun-schweig, Germany). FY1679-08a (MATa ura3-52 leu2-{Delta}1 trp1-{Delta}63 his3- {Delta}200) and the isogenic ygl186c{Delta} strain (MATa ura3-52 his3-{Delta}200 trp1- {Delta}63 ygl186c{Delta}::kanMX4) were obtained from EUROSCARF (Frankfurt am Main, Germany). All other yeast strains used in this study were isogenic to W303-1A (MATa leu2-3,112 his3-11,15 trp1-1 ade2-1 ura3-1 can1-100) (20). MW980 (MATa snz1-sno1{Delta}::URA3 snz2-sno2{Delta}::LEU2 snz3-sno3{Delta}::LEU2 trp1-1 ade2-1 his3-1 can1-100) (12) was mutagenized with ethyl methanesulfonate. Two mutants (MW980 tpn1-1 and tpn1-3) with slow growth on 0.02 µM PN were crossed with RKY02-1D (MAT{alpha} ade2-1 ura3-1 trp1-1 leu2-3 his 3-11,15 can1-100 bna1{Delta}::HIS3) (21). A haploid strain derived from the cross of MW980 tpn1-3 that showed slow growth on medium containing 0.02 µM PN was then crossed with W303-1A snz1-sno1{Delta}::his5+ (MATa leu2-3,112 trp1-1 ade2-1 ura3-1 can1-100 snz1-sno1{Delta}::his5+). This cross was performed to substitute the snz1-sno1{Delta}::URA3 deletion for a snz1-sno1{Delta}::his5+ allele. From the progeny, JSX14-3a (MATa leu2-3,112 trp1-1 ade2-1 ura3-1 can1-100 snz1-sno1{Delta}::his5+ tpn1-3) was used for complementation. The linked SNZ1-SNO1 genes were deleted in W303-1A with PCR products using the Schizosaccharomyces pombe his5+ gene as a marker. The ygl186c{Delta}::kanMX4 deletion was transferred from FY1679 into W303-1A and MW980 after PCR with primers flanking the integration site. All deletions were verified by PCR.

Plasmid Constructs—An S. cerevisiae library in the multicopy vector YEp352 (22) was used. It contains genomic DNA that was partially digested with Sau3AI and ligated into the BamHI site. Plasmids that complemented the growth defect of JSX14-3a were characterized by restriction digests to identify SNZ1-SNO1-containing plasmids. All other plasmids were sequenced. To generate a full-length allele of TPN1, the part of the gene that is missing in the library clone was amplified with Vent polymerase and ligated to the part of TPN1 that encodes the C terminus using the HindIII site within the open reading frame (ORF). Overexpression of TPN1 was achieved in pVT100-U, a URA3 multicopy vector with the yeast ADH1 promotor (23).

All other constructs were made in the YCplac (single copy) and YEplac (multicopy) series of plasmids (24). A green fluorescent protein (GFP)-tagged version of TPN1 was generated in YEplac195, comprising the TPN1 promotor (429 bp upstream of the ATG start codon), followed by the GFP sequence, the TPN1 ORF, and 742 bp of TPN1 terminator sequence. The plasmid was transformed into W303-1A wild-type cells, and fluorescence was recorded with a Zeiss LSM 510 META confocal microscope. An identical construct in YCplac33 did not yield any fluorescence above background levels.

To generate the SNZ1-containing plasmids, a library plasmid harboring the SNZ1-SNO1 gene pair was cut with SphI and BspEI to isolate SNZ1 along with 478 bp of promotor and 691 bp of terminator sequences. The SNZ1 gene was ligated into SphI-XmaI-cut YCplac33 or YEplac195. SNO1 was isolated as a ScaI-HindIII fragment containing 427 bp of promotor and 454 bp of terminator sequences. SNO1 was ligated into SmaI-HindIII-cut YCplac22 or YEplac112. All plasmids were transformed into W303-1A snz1-sno1{Delta}::his5+ tpn1{Delta}::kanMX4.

For the analysis of TPN1 alleles from S. cerevisiae and S. carlsbergensis, PCR with Vent polymerase was performed on genomic DNA as a template and primers that allowed amplification of the entire ORF. The products of two independent PCRs were ligated into pCR4-TOPO (Invitrogen) and sequenced.

Uptake Experiments—Cells were grown to mid-logarithmic phase in SD medium, washed with water, and suspended in citric acid/phosphate buffers containing 1% D-glucose to energize the cells. 500 µl of cells (0.25 A600 units/ml for cells overexpressing TPN1 and 10 A units/ml for others) were incubated at 30 °C and stirred with a magnetic stirrer. The substrate, a mixture of tritiated PN (20 Ci/µmol; American Radiolabelled Chemical, Inc., St. Louis, MO) and unlabeled PN, was added to give a final concentration of 10 µM. Competitors or inhibitors were tested at pH 4.0 and added 60 s before starting the experiment. At given times, 60-µl aliquots were withdrawn, diluted with 5 ml of water, and filtered. After washing with excess water, the radioactivity associated with the filters was quantified by scintillation counting.

RNA Analyses—MW980 cells were cultured overnight in media with 2, 0.2, and 0.02 µM PN and diluted into fresh media with the same concentrations of PN. Cells from the 0.02 µM PN culture were additionally used to inoculate media with 0, 2, and 0.2 nM PN. RNA was prepared from cells (40 A600 units), separated on formaldehyde gels, and transferred to nitrocellulose membranes. The blots were incubated with 32P-labeled probes corresponding to the entire ORF of TPN1. RNA prepared from strains carrying a tpn1{Delta} deletion showed no signals with the TPN1 probe (data not shown). Blots were exposed to phosphorimaging screens for quantification. Signals obtained with an actin (ACT1) probe were used for normalization.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Mutants with Defects in PN Uptake—The yeast S. cerevisiae has two pathways for the synthesis of the essential cofactor PLP. One pathway requires the de novo synthesis of pyridoxine. The other pathway is the salvage pathway, which involves the transport of external sources of vitamin B6 across the plasma membrane. Because intracellular PN can derive from these two pathways, we reasoned that mutation of a putative PN transporter alone would not give rise to a phenotype. We therefore used strain MW980, which lacks all three SNZ-SNO gene pairs, for a mutational approach to identify the PN transporter.

As expected, S. cerevisiae wild-type strains were capable of growing on a wide variety of PN concentrations and also grew in the absence of PN (Fig. 1A). MW980 grew like the wild-type strain when PN was present at concentrations of 0.02 µM or higher, but grew poorly at lower concentrations or when PN was absent (Fig. 1A).



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  		FIG. 1.
Characterization of pyridoxine transport mutants. A, W303-1A (wild-type), MW980 (deleted for all members of the SNZ and SNO gene families), and two mutants that were generated in MW980 (tpn1-1 and tpn1-3) were cultured in SD medium, and serial dilutions of the cells were spotted on plates containing the indicated concentrations of PN. Growth was recorded after 3 days at 30 °C. B, uptake of PN was measured with a standard protocol. The yeast strains used were those listed in A and were grown in medium containing 2 µM PN prior to the assay. {blacksquare}, MW980; •, W303-1A; {square}, tpn1-3; {circ}, tpn1-1.

 

To identify mutants with defects in PN uptake, MW980 was treated with ethyl methanesulfonate and plated on YPD medium. Colonies were replicated on medium containing 2 or 0.02 µM PN. Two mutant strains carrying recessive and allelic mutations (tpn1-1 and tpn1-3) were isolated. Both mutants were growth-restricted on plates containing 20 nM PN or less, but grew like wild-type cells on plates with higher concentrations of PN (Fig. 1A). Compared with MW980, both mutants also had growth deficits on plates containing PL or PM (data not shown).

Using [3H]PN as a substrate in a whole cell uptake experiment, we found that both mutants were defective in PN transport compared with W303-1A (wild-type) or MW980 (Fig. 1B). The amount of PN taken up by cells (1 A600 unit) in the 4-min experiment was 2.4 pmol for tpn1-1, 4.2 pmol for tpn1-3, 12.3 pmol for the wild-type strain, and 13.7 pmol for MW980. The higher activity of tpn1-3 relative to tpn1-1 is consistent with its slightly better growth (Fig. 1A). Moreover, the vitamin B6 auxotrophic mutant MW980 possessed higher activity for PN transport compared with wild-type cells. We conclude that both tpn1 mutants are defective in growth on low concentrations of vitamin B6 because of a defect in PN uptake.

Complementation Cloning of YGL186C—To eliminate unwanted mutations, we next crossed both mutants with a wild-type strain and tested the progeny from this cross for the presence of the mutation. All strains that showed growth defects possessed the snz1-sno1{Delta}::URA3 deletion, ruling out a role of SNZ2-SNO2 and SNZ3-SNO3 in PN biosynthesis. Strain JSX14-3a, which carries the tpn1-3 mutation, resulted after a second cross that exchanged the snz1-sno1{Delta}::URA3 deletion for a snz1-sno1{Delta}::his5+ allele. JSX14-3a was transformed with a yeast genomic library generated in a multicopy vector and plated on medium containing 20 nM PN.

Library transformants were tested on plates containing no vitamin B6 to identify clones that were able to synthesize PN. All of these transformants contained plasmids with the linked SNZ1-SNO1 genes. We did not isolate a single plasmid with the SNZ2-SNO2 or SNZ3-SNO3 gene pair from >20 PN prototrophic strains. Plasmids that did not confer vitamin B6 prototrophy were sequenced, and eight of them contained a gene coding for a protein with homology to plasma membrane transporters. The identified ORF, YGL186C, codes for a protein with homology to Fcy2p, the S. cerevisiae transporter for adenine, guanine, and cytosine (25, 26). In S. cerevisiae, Ygl186cp, Fcy2p, and the Fcy21 and Fcy22 proteins that are similar to Fcy2p form the family of purine-cytosine permeases within the major facilitator superfamily (27). Ygl186cp is 29% identical to Fcy2p and 58% identical to Pcpl3p, a protein from Kluyveromyces marxianus whose function is unknown (28). An alignment of these three proteins is presented in Fig. 5. Interestingly, cytosine, one of the substrates of Fcy2p, and vitamin B6 compounds show similarities in their chemical structures (Fig. 2A).



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  		FIG. 5.
Alignment of pyridoxine and purine-cytosine transporters. The K. marxianus Pcpl3 protein is aligned with the S. cerevisiae vitamin B6 transporter Tpn1p and the purine-cytosine transporter Fcy2p. Black boxes indicate identical residues, and gray boxes indicate conservative exchanges. The transmembrane domains of Tpn1p as predicted with TMpred (29) are indicated with black bars and Roman numerals as described for Fig. 2A. Amino acid positions that are changed in S. carlsbergensis (V123L) and in the proteins encoded by tpn1-1 (G203E) and tpn1-3 (V207M) are indicated. START marks Met66, which is likely to be used as a start codon in the truncated allele of TPN1 identified in the screening.

 


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  		FIG. 2.
Structural similarity between vitamin B6 and cytosine and hydropathy analysis of Tpn1p. A, chemical structures of pyridoxal, pyridoxine, pyridoxamine, and cytosine. Note that the structures shown are those for the neutral compounds, whereas the forms of vitamin B6 are a mixture of ionic species whose charge depends on the pH of the medium. B, hydrophobicity analysis of the Tpn1p primary structure with TMpred (29). Default settings were used to calculate the hydropathy profile. The position of Met66 (M66), the putative start site of the protein encoded by the truncated TPN1 allele, is indicated. Roman numerals are given for hydrophobic protein segments that are likely to represent transmembrane domains. aa, amino acids.

 

Analysis with the TMpred program (29) showed that the 579-amino acid protein encoded by YGL186C is a membrane protein with 12 transmembrane domains (Fig. 2B), a feature typical of plasma membrane permeases for various substrates (30). However, the YGL186C ORF was contained only partially on all eight plasmids we isolated. The promotor and N terminus of the protein were missing due to the presence of a Sau3AI restriction site that had been cut during the generation of the library. The protein encoded by the truncated gene putatively starts at methionine 66 within the hydrophilic N-terminal part of the protein (Fig. 2B), thus leaving intact the 12 hydrophobic domains that presumably are essential for activity. Although tpn1-3 mutants carrying multicopy plasmids with the truncated gene did not display elevated levels of plasma membrane PN transport relative to controls (data not shown), the plasmids reproducibly complemented the growth phenotype of the mutants. It seemed possible that the truncated gene was expressed at levels that were undetectable by transport assays, but were sufficient to rescue the growth phenotype of tpn1-3 cells. Moreover, we were encouraged by the structural similarity of PN, PL, and PM to cytosine and the homology of Ygl186cp to Fcy2p and characterized YGL186C in more detail.

YGL186C Encodes a Plasma Membrane Transport Protein for Pyridoxine—We performed uptake experiments with FY1679 wild-type cells and the isogenic ygl186c{Delta} deletion mutant to determine whether Ygl186cp functions in PN transport (Fig. 3A). Deletion of YGL186C caused a strong reduction in plasma membrane transport of PN, with the activity dropping from 27 pmol of PN taken up within 4 min in the wild-type cells to 3 pmol of PN in the ygl186c{Delta} mutant (Fig. 3A). This low level of PN uptake is comparable to the activity of the tpn1-1 and tpn1-3 mutants (Fig. 1B) and shows that YGL186C is involved in the plasma membrane transport of PN. In light of this observation and also data described below, we will refer to YGL186C as TPN1 (transport of pyridoxine-1).



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  		FIG. 3.
Tpn1p transports pyridoxine and localizes to the plasma membrane. A, uptake assays were performed with FY1679-08a (wild-type; {square}) and the isogenic ygl186c{Delta}::kanMX4 mutant ({triangleup}). B, W303-1A expressing full-length TPN1 from a multicopy plasmid ({blacksquare}) and W303-1A (wild-type; {square}) were assayed for PN transport. C, shown are a bright-field image (left panel) and a confocal image (right panel)of cells expressing the N-terminally GFP-tagged Tpn1 protein from a multicopy plasmid.

 

A full-length allele of TPN1 was generated and overexpressed using pVT100-U. Overexpression of TPN1 increased PN uptake activity to 700 pmol of PN/A600 units of cells within 5 min (Fig. 3B). This corresponds to a >20-fold increase in PN transport relative to wild-type cells. The increase in PN transport upon overexpression and the decrease in activity following deletion of TPN1, as well as the homology to Fcy2p, made it very likely that Tpn1p is a PN permease. This was further substantiated by the localization of the N-terminally GFP-tagged protein (Fig. 3C). Whereas W303-1A control cells produced only weak vacuolar fluorescence (presumably due to the accumulation of a red pigment in ade2 mutants), the GFP-TPN1-expressing cells showed fluorescence at the plasma membrane (Fig. 3C, right panel). Some intensely fluorescent spots were seen in cells that had high expression levels, and these might correspond to artifacts originating from overexpression. The uptake experiments and data on the localization of Tpn1p thus provide strong evidence that TPN1 encodes a plasma membrane-localized transporter for PN.

Tpn1p Is a Proton-PN Symporter with a Broad Substrate Specificity—Cells overexpressing TPN1 from a multicopy plasmid were used for a detailed characterization of the PN transport activity. We found that PN transport was maximal when the uptake experiments were performed at pH 4.0, and 50% of this activity was detectable at pH 7.0. The activity decreased more rapidly toward acidic pH values, falling to 45% at pH 3.0. The Km value for PN uptake is 0.55 µM (data not shown), classifying Tpn1p as a high affinity transporter for PN.

We next performed uptake experiments at pH 4.0 to determine whether other vitamin B6 compounds compete with PN uptake (Fig. 4A). We found that PM had only a weak effect when present at a 10-fold molar excess, but PN uptake was reduced to 50% at a 50-fold excess of PM. PL was more potent in competing PN uptake when present at a 10-fold excess. PN and 4-deoxypyridoxine both were equally effective competitors and reduced the PN uptake to 5% of control levels. It is possible that the weak competition of PM relative to PL, PN, and 4-deoxypyridoxine is caused by its amino group, which introduces an additional positive charge.



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  		FIG. 4.
Tpn1p transports PN, PL, and PM. A, transport assays were performed at pH 4.0 with cells overexpressing TPN1. The activity in the absence of competitors is given as 100%. Competitors (PM, PL, PN, and 4-deoxypyridoxine (dPN)) were added at a 10- or 50-fold excess relative to the concentration of labeled PN. The protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added at a final concentration of 50 µM. B, MW980 (a strain deleted for all six SNZ and SNO homologs) and TPN1 deletion mutants in MW980 (MW980 tpn1{Delta}) or the wild-type background (W303-1A tpn1{Delta}) were grown in SD medium; diluted in water; and spotted on plates containing the indicated concentrations of PN, PL, or PN. Growth was recorded after 3 days at 30 °C.

 

PN uptake was also strongly reduced in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone, and the remaining activity amounted to 9% of uninhibited cells. This indicates that, similar to the homologous Fcy2 protein (31, 32, 33, 34), Tpn1p acts as a proton symporter. Thus, the PN transporter Tpn1p has a broad specificity for all three forms of vitamin B6.

Tpn1p Is the Only Pyridoxine Transporter in S. cerevisiae— Experiments with S. carlsbergensis had provided evidence that two activities catalyze PN uptake (17). We investigated whether the situation in S. cerevisiae is similar and analyzed the effect of deletion of TPN1 in a wild-type strain and in MW980 (Fig. 4B). A tpn1{Delta} strain grew like the wild-type strain under all conditions tested (Fig. 4B), although it possessed only residual levels of PN transport activity (Fig. 3A). This shows that wild-type cells derive most of their PN from biosynthesis. In contrast, growth was severely compromised when TPN1 was deleted in MW980. The concentrations that were necessary to support growth of MW980 tpn1{Delta} were 2 µM for PN and PL, whereas PM was at least 10-fold less potent. At high outside concentrations, sufficient amounts of PL and PN might enter the cells by passive diffusion, whereas PM might diffuse more slowly because it is positively charged. Because MW980 tpn1{Delta} failed to grow on low concentrations of PN, PL, or PM and because PM and PL acted as competitors of PN transport, we conclude that S. cerevisiae has only one transport protein for all three forms of vitamin B6 and that this protein is Tpn1p.

Sequencing of tpn1-1 and tpn1-3 and the TPN1 Gene from S. carlsbergensis—A diploid strain constructed by mating tpn1-3 and tpn1{Delta} strains possessed growth and PN transport characteristics that were indistinguishable from those of the haploid tpn1-3 strain (data not shown). This indicated that the TPN1 gene was mutated in MW980 tpn1-3 and, by inference, also in MW980 tpn1-1. To identify these mutations, the gene was amplified from wild-type and mutant strains and sequenced. TPN1 from W303-1A was identical to the sequence in public data bases that was derived from strain S288C. The mutant alleles contained transitions from G to A that affected G608 in tpn1-1 and G619 in tpn1-3. Thus, in the protein encoded by tpn1-3, Val207 of the protein was changed to methionine (Fig. 5). Most Tpn1p homologs either contain valine or isoleucine at this position, with the only known exception being a protein from Neurospora crassa (GenBankTM/EBI Data Bank accession number CAD37047 [GenBank] ) that contains methionine. The tpn1-1 mutation affected the codon for Gly203, which was changed to encode glutamate (Fig. 5). Gly203 is a highly conserved amino acid in all known members of the purine-cytosine permease family (data not shown). We conclude that both point mutations affect residues in transmembrane domain 4 of Tpn1p, with the tpn1-1 mutation adding a negative charge and the tpn1-3 mutation being relatively mild.

To address whether S. carlsbergensis has a gene with homology to TPN1, we used primers directed against the S. cerevisiae sequence in a PCR with S. carlsbergensis genomic DNA. The sequence of the PCR product (S. carlsbergensis 4228 TPN1 gene, GenBankTM/EBI Data Bank accession AY216467 [GenBank] ) shows that, despite the presence of 15 base exchanges relative to TPN1, the primary structure of the encoded protein is identical, with the exception of a single amino acid: the S. carlsbergensis protein has leucine at position 123, where Tpn1p from S. cerevisiae has valine (Fig. 5). Because leucine is present in a similar position in Pcpl3p from K. marxianus (Fig. 5) and in other sequenced yeast species (data not shown), the S. carlsbergensis protein is highly likely to be a pyridoxine transporter.

TPN1 Expression Is Induced by Pyridoxine Depletion—To analyze whether expression of TPN1 responds to the vitamin B6 concentrations in the medium, we performed Northern blot experiments with RNA isolated from MW980 that had been grown on a variety of PN concentrations (Fig. 6). TPN1 was expressed under all conditions tested, but the strongest signals were detected in cells that had been grown on 2 nM PN or below. These concentrations were not sufficient to support maximal growth of MW980 (Figs. 1A and 4B). Similar to the amount of rRNAs detected in the gel, the signals obtained with the ACT1 probe decreased with lower concentrations of PN (Fig. 6 and data not shown). When the ACT1 signals were used for normalization, we found that expression of TPN1 was increased 15-fold in cells grown in PN-free medium relative to PN-sufficient medium (2 µM PN). We did not observe significant changes in the expression of TPN1 when W303-1A was used in parallel experiments (data not shown). Together, these data show that expression of TPN1 is modulated and highly increased when PN becomes limiting for growth.



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  		FIG. 6.
Expression of TPN1 increases with decreasing PN concentrations. The PN auxotrophic strain MW980 was grown in media with the indicated concentrations of PN. RNA was prepared from these cells, separated on gels, and blotted onto nitrocellulose filters. Hybridizations were performed with probes corresponding to the entire ORF of TPN1 or ACT1.

 

SNO1 Is Not Required for Pyridoxine Biosynthesis—Our experiments started with MW980, a PN auxotrophic strain deleted for all SNZ-SNO genes. In the course of the experiments, we noted that mutants that lacked SNZ1-SNO1 but that contained wild-type alleles of SNZ2-SNO2 and SNZ3-SNO3 were auxotrophic for PN. Moreover, only plasmids containing SNZ1-SNO1 were found to restore JSX14-3a to PN prototrophy. We were interested to investigate whether both SNZ1 and SNO1 are required for PN biosynthesis and generated single copy and multicopy plasmids containing only one of the ORFs.

Fig. 4B shows that MW980 was capable of producing some growth on medium lacking vitamin B6, whereas growth of this strain was abolished upon deletion of TPN1. This shows that pyridoxine transport-proficient cells have supplies of vitamin B6 that allow for some growth in the absence of PN biosynthesis. To eliminate this interference, we used W303-1A snz1-sno1{Delta} tpn1{Delta} to analyze the contribution of SNZ1 and SNO1 to PN biosynthesis.

On plates that contained no source of vitamin B6, expression of SNZ1 alone conferred growth (Fig. 7). The growth-promoting effect of SNZ1 was weak when present on a single copy plasmid and stronger when a multicopy plasmid was used. In contrast, SNO1 did not confer vitamin B6-independent growth even when present in multiple copies. Interestingly, single copy expression of SNO1 stimulated growth in cells that carried a single copy of SNZ1. This shows that the SNO1 gene present on the plasmids is functional. Thus, expression of SNZ1 (but not of SNO1) restores PN prototrophy to a strain that is deleted for both genes.



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  		FIG. 7.
Sno1p is not required for PN biosynthesis. Single copy (sc) or multicopy (mc) plasmids harboring either SNZ1 or SNO1 were analyzed for complementation of W303-1A snz1-sno1{Delta}::his5+ tpn1{Delta}:: kanMX4. The cells were grown overnight in SD medium and washed with water, and dilutions were spotted on plates with or without 2 µM PN as indicated. The plates were scanned after 3 days at 30 °C.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
PLP, the active form of vitamin B6, is an essential cofactor for many enzymatic reactions. Moreover, PN, PL, PM, and PLP directly act in active oxygen resistance (5, 11), a biological function not previously assigned to vitamin B6. Whereas many organisms have the capability to synthesize vitamin B6, auxotrophic unicellular and mammalian organisms depend on the uptake of vitamin B6 across the plasma membrane. To identify a PN transporter, we generated S. cerevisiae mutants that were unable to utilize external sources of vitamin B6. By genetic complementation, we identified TPN1, a gene that encodes the first known plasma membrane transporter for vitamin B6.

The protein encoded by the tpn1-3 mutant allele has a conservative exchange (V207M) that allows for a higher activity for PN uptake and slightly better growth compared with tpn1-1 (G203E). Although both mutations reduce the transport activity to levels similar to those of tpn1{Delta} strains, the amino acid exchanges do not abolish Tpn1p function. This is evident from growth assays (Figs. 1A and 4B) that show that tpn1{Delta} strains need 10-fold higher concentrations of PN relative to the tpn1-1 and tpn1-3 mutants.

The use of the weaker tpn1-3 strain in the complementation might have facilitated the discovery of the library plasmid containing a truncated allele of TPN1. Although this allele does not increase PN uptake when present in multiple copies, it is likely to give rise to a functional protein, indicating that the 65 N-terminal amino acids of the protein are dispensable for activity and targeting to the plasma membrane. We previously noted that growth assays are more sensitive than uptake assays to determine vitamin transport activities (35), and similar results were obtained in this study of vitamin B6 transport. We attribute this to the fact that only little vitamin B6 has to be taken up to satisfy the demand for PLP, which has catalytic functions and is lost only as a consequence of cell divisions. Moreover, the longer time scale of growth assays may enable the detection of activities that remain undetectable in uptake experiments.

Tpn1p is a high affinity transporter for PN (Km = 0.55 µM) with an acidic pH optimum (pH 4.0). This, together with the reduction of PN transport in the presence of protonophores, strongly argues for a proton symport mechanism for PN uptake. The related purine-cytosine transporter Fcy2p, which, like Tpn1p, displays micromolar affinities for its substrates, also mediates substrate translocation by a proton symport mechanism (31, 32, 33, 34). Homologs of TPN1 can be identified in many fungal genomes, and in a recent analysis, PN was accumulated in the majority of the yeast species tested (19).

Vitamin B6 transport has been intensively studied in S. carlsbergensis, in which two high affinity uptake systems exist: system 1 has affinity for PN and PL and a pH optimum of 3.5, whereas system 2 is specific for PM and PN and has a pH optimum of 6.0 (17). The activity of Tpn1p is very similar to that of system 1. Moreover, S. carlsbergensis contains a protein that is almost identical to Tpn1p (Fig. 5) and thus is very likely to function in PN uptake. The protein that corresponds to system 2 of S. carlsbergensis is not known. For S. cerevisiae, it is clear that Tpn1p is the only functional transport protein for PN encoded in the genome and that this protein also mediates the uptake of PL and, to a lesser extent, of PM.

Uptake of vitamin B6 has also been demonstrated in mammalian cells that are incapable of synthesizing vitamin B6. Whereas passive diffusion followed by metabolic trapping is responsible for the accumulation of PN in brush-border membrane vesicles (36), the mechanism of renal transport is different. In isolated renal cells, uptake of PN is saturable (Km = 1.3 µM), competed by non-phosphorylated PN analogs as well as by PM (37). Cultured renal epithelial cells show a similar activity (Km = 2.4 µM), and uptake of labeled PN is competed by PL and 4-deoxypyridoxine, but not by PLP (38). Data base searches with Tpn1p as a query sequence show that the purine-cytosine permease family is restricted to unicellular organisms. It seems feasible, however, that mammalian vitamin B6 permeases can be identified by heterologous complementation of tpn1{Delta} mutants.

We found that expression of TPN1 is transcriptionally regulated and highly induced when PN drops below the threshold concentration needed to support maximal growth (Figs. 1A and 4B). Changes in the expression of TPN1 have been found in many genome-wide expression profiling experiments. TPN1 is induced by nitrogen depletion and amino acid starvation and in stationary phase (39) and after treatment with 3-amino-1,2,4-triazole (3-AT), an agent that blocks histidine biosynthesis (40). The response to 3-AT is mediated by the transcription factor Gcn4p, and gcn4 mutants are unable to increase TPN1 expression following 3-AT treatment (40). In support of this observation, the TPN1 promotor contains a Gcn4p-binding motif (–218TGACTC–213) (41), a feature also present in the promotor of PCPL3 from K. marxianus (28). In addition to increasing vitamin B6 uptake, 3-AT also increases the expression of SNZ1 and SNO1 (but not of SNZ2-SNO2 and SNZ3-SNO3) in a GCN4-dependent manner (40). SNZ1 and SNO1 are also induced in stationary phase, after nitrogen depletion, and when amino acids are scarce (12, 39). Thus, in addition to its well characterized function as a transcriptional activator of amino acid biosynthesis genes (42), Gcn4p increases the expression of genes that are necessary for the generation of PLP, a cofactor needed by many amino acid biosynthesis enzymes (1). Although TPN1 is embedded in the regulatory network of amino acid biosynthesis genes, expression of TPN1 is increased only 4-fold following administration of 3-AT (40). We observed a 15-fold increase in the level of TPN1 mRNA following PN depletion (Fig. 6), making it very likely that additional inputs exist that influence the expression of TPN1.

C. nicotianae and N. crassa mutants in SNO homologs are auxotrophic for PN (6, 8); but in S. cerevisiae, the role of SNO1 is less clear. Whereas loss of SNZ1 abolishes growth, some growth is observed in the absence of SNO1, showing that Sno1p is not required for PN biosynthesis (Fig. 7) (14). The catalytic activities of Sno1p and Snz1p are not known, but it has been argued that Sno1p might catalyze an early step in PN biosynthesis, whereas Snz1p might catalyze a later step (14). In this scenario, intermediates of the vitamin B6 biosynthesis pathway would be generated by a Sno1p-independent pathway that has only weak activity. Because SNZ1 overexpression confers better growth than single copy expression (Fig. 7), our data show that the activity of Snz1p, rather than the activity of an unknown pathway, is the limiting step in PN biosynthesis when Sno1p is absent. The stimulation of PN biosynthesis in a strain that has growth-limiting levels of Snz1p (Fig. 7) suggests that Sno1p is required to activate or stabilize Snz1p. This is consistent with Snz1p and Sno1p being present in a multiprotein complex (12, 14, 15, 16) and does not exclude an enzymatic role for Sno1p that was suggested based on sequence homology to glutamine aminotransferases (43).


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY216467 [GenBank] .

* This work was supported by Grants SFB521/C7 and STO434/1-2 from the Deutsche Forschungsgemeinschaft and by a grant from the Bavarian Ministry for Science, Research, and Arts (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 49-941-943-3005; Fax: 49-941-943-3352; E-mail: juergen.stolz{at}biologie.uni-regensburg.de.

1 The abbreviations used are: PN, pyridoxine; PL, pyridoxal; PM, pyridoxamine; PLP, pyridoxal 5'-phosphate; ORF, open reading frame; GFP, green fluorescent protein; 3-AT, 3-amino-1,2,4-triazole. Back


   ACKNOWLEDGMENTS
 
We thank Manuela Reich and Petra Schitko for expert technical assistance and Tim Levine and Jan Malinsky for assistance with confocal microscopy. Margaret Werner-Washburne and Roza Kucharczyk provided some of the yeast strains used in this study, and Heike Wöhrmann performed the experiment presented in Fig. 7. We also thank Widmar Tanner, Alison Gillingham, and Manfred Gahrtz for critically reading the manuscript.



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 RESULTS
 DISCUSSION
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