Identification of the Plasma Membrane H+-Biotin Symporter of Saccharomyces cerevisiae by Rescue of a Fatty Acid-auxotrophic Mutant*

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 ofYGR065C under the control of the constitutiveADH1 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 thatYGR065C encodes the first biotin transporter identified for a non-mammalian organism and designate this gene VHT1 forvitamin H transporter1.


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.
Enzymes depending on enzyme-bound biotin (vitamin H) as a carrier of CO 2 in carboxylation, decarboxylation, and transcarboxylation reactions are found throughout the biological world (1,2). Some organisms, such as Escherichia coli and all higher plants, are able to synthesize biotin de novo (3). However, many other organisms including mammals have lost this capability and are auxotrophic for biotin. The biotin requirement of yeast is well documented for many industrial and laboratory strains of Saccharomyces cerevisiae (4) including strain S288C (5), whose genome has been sequenced. Due to this deficiency, S. cerevisiae has been used in growth assays for the microbiological determination of biotin (6).
In bakers' yeast, the covalent attachment of biotin to biotindependent enzymes is catalyzed by a biotin-protein ligase, which is encoded by the essential BPL1 gene (7). Among the best studied substrates for Bpl1p are acetyl-CoA carboxylase (Acc1p) and the two isoforms of pyruvate carboxylase (Pyc1p and Pyc2p). Acc1p catalyzes the first step of fatty acid biosyn-thesis, i.e. the carboxylation of acetyl-CoA to malonyl-CoA. A loss of functionally active Acc1p produces a lethal phenotype that cannot be rescued by the supply of fatty acids from the growth medium (8,9). This is caused by an impairment of the malonyl-CoA-dependent elongation of long-chain fatty acids to very-long-chain fatty acids, which is assumed to be essential for the nuclear pore structure in yeast (9).
Pyc1p and Pyc2p generate oxaloacetate from pyruvate. This reaction is required for the anaplerotic synthesis of tricarboxylic acid cycle intermediates during gluconeogenesis or amino acids biosynthesis. A pyc1/pyc2 double mutant is no longer able to grow on glucose with ammonia as the sole nitrogen source. Growth of the double mutant is restored when L-aspartate is added to regenerate the oxaloacetate pool (10). A similar phenotype was described for another yeast, Pichia pastoris, after deletion of its single pyruvate carboxylase gene (11).
Pioneering work on biotin transport in S. cerevisiae has been done by Rogers and Lichstein (12,13), who described a carriermediated, high affinity (apparent k m ϭ 0.3 M), and energyrequiring uptake mechanism. They showed that intracellular concentrations of biotin could exceed biotin concentration in the medium Ͼ1000-fold and that this uptake was stimulated by glucose. Rogers and Lichstein also discovered that uptake of biotin is controlled by the biotin level in the growth medium. High levels of biotin reduced the rate of catalyzed biotin uptake. Further support for a specific biotin uptake mechanism in yeast came from the finding that biotin uptake was irreversibly inhibited by the p-nitrophenyl ester of biotin, presumably due to covalent modification of the transport system (14,15).
Despite the extensive biochemical studies on many aspects of biotin transport in yeast, neither the biotin transporter nor its gene has been identified to date. In this paper, we report on the characterization of a yeast mutant (A7-9) that was isolated in a screen for fatty acid auxotrophy. It is demonstrated that the fatty acid auxotrophy in A7-9 results from a defect in biotin uptake from the environment. Complementation of A7-9 with a genomic library led to the identification of the open reading frame YGR065C. The encoded protein exhibits significant structural and sequence homologies to the large family of 12transmembrane helix transporters. We provide evidence that this gene encodes the biotin transporter of S. cerevisiae and named the gene VHT1 for vitamin H transporter 1.
The YEp24-based genomic library used for complementation was described before (17). The library plasmid pA7-T8, which complemented the mutation of A7-9, was isolated as described in the text. The single copy plasmid pVHT1sc carrying the VHT1 gene under the control of its own promoter was constructed in two steps. The 3.2-kilobase EcoRV fragment of pA7-T8 was ligated into the SmaI site of pUC19, excised with SacI and HindIII, and cloned into the corresponding sites of YCplac33 (18). For construction of the constitutively VHT1-overexpressing plasmid pVHT1oe, the PCR-amplified open reading frame of VHT1 (primers SM15-PstI (5Ј-TGACCTGCAGATGACAATTTCGAAT-AAATCGTGGAGG-3Ј) and SM12-SacI (5Ј-AGTCGAGCTCATAGAAC-GTATAAGGTGACC-3Ј)) was cloned into the PstI and SacI sites of the multicopy plasmid pVT100-U (19). The sequence of the PCR product was verified. Plasmid YEp24 (20) was used as a vector control.
Media for S. cerevisiae were prepared as described (21). Synthetic dextrose (SD) medium contained 0.67% Bacto-yeast nitrogen base without amino acids (Difco, Augsburg, Germany) and 2% glucose and was supplemented with the necessary amino acids to meet the growth requirements of the strains. SCD medium was prepared from SD medium by adding a complete mixture of L-amino acids and the nucleobases adenine and uracil. Fatty acids were added as described previously (16).
The biotin concentration in SD and SCD media prepared with Difco yeast nitrogen base without amino acids is 2 g/liter. Media with other biotin concentrations were made from analytical grade chemicals according to the formula of Difco for yeast nitrogen base without amino acids (omitting biotin), dissolved at 10 times the final concentration, filter-sterilized, and stored at 4°C. Biotin was added to these media from an autoclaved stock (100 mg/liter) to give the desired final concentrations.
Yeast Transformation, Gene Disruption, and Plasmid Isolation-The VHT1 gene was disrupted in JS 91.15-23 by short flanking homologymediated recombination (22). Briefly, the S. cerevisiae HIS3 gene was PCR-amplified using primers 5-YGR065C-HIS3 (5Ј-GGACCGACACA-AATCAATAGATAATGACAATTTCTCTTGGCCTCCTCTAG-3Ј) and 3-YGR065C-HIS3 (5Ј-TATGGTGTCTAAAGCGTAATATACCTATCTA-CTTCGTTCAGAAGTACACG-3Ј), which added 34 bp of VHT1 sequence (underlined) from near the ATG start codon (boldface) and 35 bp of VHT1 sequences (underlined) from a region 15 bp downstream of the stop codon to either side of HIS3, respectively. Yeast cells were transformed with the PCR product and plated on SD plates lacking histidine but containing excess biotin (2 mg/liter). Transformants were replica-plated on SD plates containing 2 g of biotin/liter. Cells unable to grow under these conditions were checked by PCR for correct integration of the HIS3 gene into VHT1. The verified disruptant is referred to as JSY⌬vht1. Yeast transformations (23) and plasmid isolations from S. cerevisiae (24) were performed as described.
Transport Assays-Yeast cells were grown to mid-logarithmic phase (A 600 nm ϳ 1.0 in SD medium or ϳ 3.0 in YPD medium (1% yeast extract, 2% bactopeptone, 2% D-glucose)), harvested by centrifugation, and washed once with ice-cold water. After washing in citrate/phosphate buffer (30 mM citric acid and 40 mM Na 2 HPO 4 , pH 4.2), the cells were resuspended in buffer to give 16 mg (fresh weight)/ml and incubated in a rotatory shaker at 30°C. Cells were energized with glucose (1% final concentration), and the assay was started by the addition of 14 C-labeled biotin (Amersham CFB 253; specific activity ϭ 8.38 mCi/mmol, 10.7 M final concentration). At timed intervals, cells were filtered on nitrocellulose filters and washed with excess water. Incorporated radioactivity was quantified by liquid scintillation counting. Inhibitors were added 1 min before the addition of labeled substrate at the given concentrations. All inhibitors were obtained from Sigma (Deisenhofen, Germany).

Characterization of a Yeast Mutant Defective in High Affinity
Biotin Uptake-Fatty acid-auxotrophic mutants have been assigned to four distinct chromosomal loci, i.e. the fatty acid synthase genes FAS1 and FAS2 and the ACC1 and BPL1 genes encoding the acetyl-CoA carboxylase and the biotin-protein ligase, respectively (16,26,27). In addition, several fatty acidauxotrophic yeast strains that are non-allelic to any of the above-mentioned loci 2 were available to us. We speculated that a defective biotin transport system in a biotin-dependent organism such as S. cerevisiae should cause fatty acid auxotrophy, i.e. a phenotype similar to that observed in bpl1 mutants. The fatty acid-auxotrophic phenotype of one of the unassigned mutants, strain A7-9, is demonstrated in Fig. 1. Virtually no growth was observed when A7-9 cells were supplemented with saturated medium-chain fatty acids (caprylate (C 8 ) or caprinate (C 10 )). However, this growth defect was suppressed in the presence of long-chain fatty acids (laurate (C 12 ), myristate (C 14 ), or palmitate (C 16 )).
Analysis of [ 14 C]biotin transport across the plasma membrane revealed that A7-9 is completely defective in biotin uptake ( Fig. 2A). The transport rates in A7-9 were Ͼ100-fold below wild-type levels. Although the intracellular concentrations of biotin in wild-type cells clearly exceeded the extracellular concentration, the biotin level in A7-9 cells was far below the concentration equilibrium. Increased extracellular biotin concentration suppressed the fatty acid-auxotrophic phenotype in A7-9 and restored normal growth (Fig. 2B), probably due to increased passive diffusion of biotin into the cells. Thus, A7-9 is a conditionally fatty acid-auxotrophic mutant that requires long-chain fatty acids or biotin for growth. Compared with wild-type cells, A7-9 needs ϳ100-fold higher vitamin H concentrations for optimal growth. As evident from Fig. 2B, already 2 g of biotin/liter allowed full growth of the wild type, whereas growth of the mutant was barely supported. On YPD complete medium with no extra biotin added, A7-9 showed almost wildtype growth rates (data not shown).
Streptavidin-conjugated peroxidase was used to determine the degree of protein biotinylation in strains A7-9 and BF 89.4-36. A7-9 cells were grown on SD medium supplemented with both long-chain fatty acids and L-aspartate to suppress deficiencies possibly caused by a reduced biotinylation of Acc1p, Pyc1p, and Pyc2p carboxylases. As a control, the mutant was also grown on SCD medium supplemented with excess biotin (1 mg/liter). After separation of cellular proteins by SDSpolyacrylamide gel electrophoresis and subsequent transfer to 1 The abbreviations used are: SCD, synthetic complete dextrose; SD, synthetic dextrose; PCR, polymerase chain reaction; bp, base pair(s). 2 E. Schweizer, unpublished data.
FIG. 1. Fatty acid-dependent growth of the yeast mutant A7-9. Cells were grown in SCD medium supplemented with 0.5% aspartic acid, 0.5% Brij 58, and 0.02% of the indicated fatty acid. Cell densities were determined after 3 days of growth at 30°C. nitrocellulose, three biotinylated bands corresponding to Acc1p, the pyruvate carboxylase isoforms Pyc1p and Pyc2p, and a 47-kDa protein of unknown function (28) were detected in wild-type and mutant cells grown on excess biotin (Fig. 3). On medium supplemented with fatty acids and L-aspartate, biotinylation of these proteins was strongly reduced in A7-9 cells, but not in wild-type cells. The reduced biotinylation of Acc1p in A7-9 was paralleled by a loss of acetyl-CoA carboxylase activity (data not shown).
The biochemical characteristics of A7-9 suggest that a high affinity biotin transporter is affected in this mutant. As a consequence of reduced biotin uptake, protein biotinylation and activity of Acc1p are decreased, and growth depends on the external supply of long-chain fatty acids. Biotin at high external concentrations can restore wild-type levels of protein biotinylation and supports growth in a fatty acid-independent fashion.
Complementation Cloning of the Biotin Transporter Gene-The wild-type allele of the gene affected in strain A7-9 was isolated by complementing the mutant with a yeast genomic library constructed in the high copy vector YEp24 (17). Among 11,000 uracil-prototrophic colonies, 15 were able to grow on SCD plates lacking fatty acids. Two of three isolated plasmids (pA7-T8 and pA7-T10) had identical inserts of ϳ8 kilobases, and the third harbored a larger, overlapping DNA fragment. Fig. 4 shows that full complementation was also achieved with plasmid pVHT1sc, which contains a 3.2-kilobase EcoRV fragment of pA7-T8 subcloned into the single copy plasmid YC-plac33 (18).
Sequence analysis of pVHT1sc identified a 3233-bp fragment from chromosome VII covering the open reading framesYGR063C, YGR064W, and YGR065C. The open reading frame YGR063C has previously been identified as SPT4, a gene coding for a zinc-finger protein involved in regulation of chromatin structure and gene expression (29,30). The open reading frame YGR064W codes for a putative protein of 122 amino acids with one potential transmembrane domain. It overlaps with SPT4 and thus is unlikely to be transcribed. Therefore, only YGR065C was likely to be responsible for the complementation of A7-9.
Characterization of the Open Reading Frame YGR065C-A DNA fragment containing only the YGR065C reading frame was generated by PCR and subcloned into pVT100-U (19) downstream of the strong and constitutive ADH1 promoter to give plasmid pVHT1oe. After transformation with this construct, A7-9 was able to grow in the absence of fatty acids (Fig.  4), indicating that YCR065C indeed complements the mutation in A7-9.
YGR065C, which has no functional assignment in public data bases, codes for a protein of 593 amino acids (Fig. 5A). A hydrophobicity analysis of this sequence (Fig. 5B) (31) suggested 12 transmembrane domains, a number that has been found in numerous plasma membrane transporters (32). According to the prediction, Ygr065p starts with a long hydrophilic domain of 120 amino acids, and both the N and C termini are on the cytoplasmic side of the membrane. Six asparagine residues are part of consensus sequences for N-glycosylation. Two of these residues are located on the extracellular surface (Asn 146 , between transmembrane domains 1 and 2, and Asn 406 , between transmembrane domains 7 and 8) and might thus be accessible for glycosylation during protein secretion.
Ygr065p belongs to a family of eight putative yeast transporter proteins (33) that is part of the major facilitator superfamily (34). So far, a function has been attributed to only two members of this group: Dal5p, the plasma membrane-localized permease for allantoate and ureidosuccinate (35,36), and Fen2p, the plasma membrane-localized pantothenate transporter (37  Extracts from the biotin transport-deficient mutant A7-9 and from the control strain BF 89.4-36 were separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Experimental conditions were as described previously (27). A7-9 was grown in SCD medium containing 0.5% aspartic acid and 1 mg of biotin/liter (ϩbio) or 0.5% Brij 58 and 0.03% fatty acids (1:1 mixture of myristate and palmitate; ϩFA). BF 89.4-36 cells were grown in the same fatty acid-containing SCD medium with (ϩbio) or without (ϩFA) biotin supplementation (1 mg/liter). As was confirmed by Coomassie Blue staining of a reference gel, identical amounts of protein were applied to each lane (data not shown). Biotinylated reference proteins were purchased from Bio-Rad.
FIG. 4. Complementation of the biotin transport-deficient mutant A7-9 by YGR065C. Strain A7-9 was transformed with plasmid pA7-T8 (carrying the initially obtained genomic fragment), pVHT1sc (a single copy plasmid carrying VHT1 under control of the VHT1 promoter), or pVHT1oe (a multicopy plasmid with VHT1 under control of the ADH1 promoter) or with the empty control vector YEp24 (20). Transformants were streaked on SD plates containing 2 g or 2 mg of biotin/liter. Growth of the strains was scored after 3 days at 29°C.
The VHT1 gene was disrupted in strain JS 91.15-23; transformants were selected on SD plates containing 2 mg of biotin/ liter; and disruptants were identified on plates containing 2 g of biotin/liter. These cells had the HIS3 marker gene correctly integrated at the VHT1 locus and are referred to as JSY⌬vht1. By genetic complementation analysis, JSY⌬vht1 proved to be allelic to the A7-9 mutant. Moreover, all biochemical parameters investigated, such as biotin-or fatty acid-dependent growth, biotin uptake, and protein biotinylation, were the same in both mutants (data not shown).
Biotin Uptake by Vht1p and VHT1 mRNA Levels Are Modulated by the Biotin Content of the Medium-Biotin transport activity in S. cerevisiae is modulated by the biotin content of the media. The activity increases with decreasing biotin concentrations, and protein biosynthesis is necessary for this increase in activity (13). We analyzed the biotin transport activities of A7-9 cells expressing VHT1 under the control of its own promoter from the centromere plasmid pVHT1sc and, alternatively, of A7-9 cells that carried the VHT1 coding region downstream of the strong and constitutive ADH1 promoter in the multicopy plasmid pVHT1oe.
After growth in SD medium with 0.2, 2, or 20 g of biotin/ liter, biotin uptake was determined (Fig. 6). In agreement with previous data (13), we observed a pronounced effect of the extracellular biotin concentration on biotin uptake in pVHT1sc transformants expressing VHT1 from its genuine promoter. The biotin transport rate was maximal at 0.2 g of biotin/liter and became very low at 2 and 20 g of biotin/liter (Fig. 6A). As expected, biotin transport rates in pVHT1oe transformants were increased and independent of extracellular biotin concentrations (Fig. 6B), suggesting that regulatory sequences in the VHT1 promoter are responsible for the biotin responsiveness seen in Fig. 6A.
For further analyses, RNA was prepared from JS 91.15-23 wild-type cells grown on 0.2, 2, or 20 g of biotin/liter and probed with a radiolabeled VHT1 fragment on a Northern blot. A single hybridization signal corresponding to a mRNA of ϳ1900 bp was observed in each of the three samples (Fig. 7). VHT1 mRNA was most abundant at 0.2 g of biotin/liter. VHT1 mRNA was less abundant at 2 g/liter, and the level was even further reduced at 20 g of biotin/liter. These results are in good agreement with the biotin transport activities determined above (Fig. 6A) and show that extracellular biotin concentrations modulate VHT1 mRNA levels and thereby Vht1p transport activity.
Transport by Vht1p Is Specific for Biotin and Sensitive to Protonophores-A recently identified mammalian vitamin transporter, SMVT, transports the three vitamins biotin, pantothenate, and lipoate with similar affinities by means of a Na ϩ symport mechanism (38,39). Transport of [ 14 C]biotin by Vht1p was not inhibited when pantothenate was added at 10 times the concentration of biotin (data not shown). Also other compounds with structural similarity to biotin, such as allantoin, allantoate, xanthine, uric acid, and urea, had no effect on biotin transport (data not shown), suggesting that Vht1p is specific for biotin.
Vht1p-dependent [ 14 C]biotin transport was further characterized by measuring its activity in the presence of protonophores (such as 2,4-dinitrophenol and carbonyl cyanide mchlorophenylhydrazone), the membrane-impermeable SHmodifying agent p-chloromercuribenzenesulfonic acid, or the above-mentioned inhibitor of biotin transport (biotinyl-p-nitrophenyl ester) (Fig. 8). In accordance with published data (14), biotinyl-p-nitrophenyl ester strongly reduced biotin transport to values Ͻ4% of the uninhibited activity. The protonophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone inhibited transport rates by 81 and 86%, respectively, suggesting a proton symport mechanism for biotin uptake. p-Chloromercuribenzenesulfonic acid had no effect on biotin transport.

VHT1 Encodes the Plasma Membrane H ϩ -Biotin
Symporter of S. cerevisiae-The gene responsible for the fatty acid-auxotrophic phenotype of the yeast mutant A7-9 was identified, and the encoded protein was characterized as a plasma membrane-localized biotin transporter. The gene was named VHT1 for vitamin H transporter 1. Additional phenotypic effects associated with a vht1 mutation include the inability to grow at low biotin concentrations sufficient for wild-type yeast strains. Furthermore, the mutant exhibited drastically reduced protein biotinylation and hence a very low activity of the biotin-dependent enzyme Acc1p. All of these effects can be suppressed with high extracellular concentrations of biotin when the passive diffusion of this vitamin across the plasma membrane is sufficient to meet the biotin demand of the cells. Also in E. coli, high external concentrations of biotin are reported to allow import of biotin by diffusion (40).
VHT1 was identified by complementation of the mutant A7-9 with a yeast genomic library. It encodes a membrane protein from the allantoate permease family. Several lines of evidence confirm that VHT1 codes for the biotin transporter of the S. cerevisiae plasma membrane. (i) Vht1p shows sequence homology to Dal5p, a yeast plasma membrane transporter specific for allantoate and ureidosuccinate, which are structurally related to biotin; and similarly, Vht1p is related to Fen2p, a yeast plasma membrane transporter for pantothenate. (ii) Disruption of VHT1 results in the inability to grow on SD medium at low biotin concentrations (2 g/liter). The complete loss of biotin uptake activity (Fig. 1) shows that Vht1p is the only high affinity biotin transporter encoded in the yeast genome. (iii) Overexpression of VHT1 under the control of the ADH1 promoter causes a dramatic increase in biotin transport activity irrespective of the biotin content of the medium (Fig. 6B). (iv) VHT1 mRNA levels and biotin uptake rates are modulated by extracellular biotin and increase as a consequence of biotin limitation (Figs. 6A and 7). (v) Biotinyl-p-nitrophenyl ester, a strong inhibitor of biotin transport in S. cerevisiae, inhibits Vht1p-dependent biotin transport by Ͼ90%. The last two findings confirm results previously obtained from biochemical and kinetic analyses of yeast biotin transport (12)(13)(14).
Vht1p Shares No Homology with a Mammalian Na ϩ -dependent Vitamin Transporter-The sensitivity of biotin uptake to uncouplers of transmembrane proton gradients (such as 2,4dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) suggests that Vht1p mediates the H ϩ -coupled symport of biotin. Consistent with this interpretation, biotin transport leads to intracellular accumulation of biotin and is stimulated by glucose (12), a sugar that directly activates the proton-extruding ATPase of the yeast plasma membrane (41).
Biotin uptake studies performed with intact mammalian cells (42) or with tissue-derived membrane vesicles (43) clearly indicated a Na ϩ symport mechanism for biotin. Most remarkably, uptake of biotin was competitively inhibited by pantothenic acid and lipoate, suggesting a single protein for the uptake of all three vitamins (38,44). This was confirmed after the recent cloning and heterologous expression of the rat SMVT gene in Xenopus laevis oocytes (39). The Na ϩ -dependent SMVT protein, which belongs to the Na ϩ -dependent glucose transporter family, exhibits no structural homology to the H ϩ -dependent Vht1p protein characterized in this study.
E. coli, like most bacteria, is able to synthesize biotin, and indeed, most of the biotin required by humans is not derived from the diet, but rather from the intestinal microflora (45). Although being prototrophic for biotin, E. coli also takes up this vitamin by a carrier-mediated, saturable, high affinity (k m ϭ 0.14 M), and energy-dependent process (46). As in S. cerevisiae, biotin transport in E. coli is suppressed by the biotin concentration in the media (46,47). To date, no information is available on bacterial biotin transporters. A BLAST search (48) in publicly available data banks using Vht1p as query sequence yielded no match.
In conclusion, plants and most bacteria are able to synthesize biotin and do not depend on biotin uptake. In contrast, mammals and yeast are unable to synthesize biotin, but efficiently accumulate biotin from their environment. In yeast, the biotin transporter is only synthesized when required, i.e. at very low extracellular biotin concentrations. The biotin transporters of mammals and yeast are clearly different, both structurally and functionally. Thus, the yeast biotin transporter may represent a suitable target for new antifungal drugs with applications in agriculture and medicine.