Branched-chain Amino Acid Permease Research Articles

Novel function of transcription factor Uga3 as an activator of branched-chain amino acid permease BAP2 gene expression

Gene regulation in yeast occurs at the transcription level, i.e. the basal level of expression is very low and increased transcription requires gene-specific transcription factors allowing the recruitment of basal transcriptional machinery. Saccharomyces cerevisiae BAP2 gene encodes the permease responsible for most uptake of leucine, valine and isoleucine, amino acids that this yeast can use as nitrogen sources. Moreover, BAP2 expression is known to be induced by the presence of amino acids such as leucine. In this context, the results presented in this paper show that BAP2 is an inducible gene in the presence of nitrogen-non-preferred source proline but exhibits high constitutive non-inducible expression in nitrogen-preferred source ammonium. BAP2 expression is regulated by the SPS sensor system and transcription factors Leu3, Gcn4 and Dal81. This can be achieved or not through a direct binding to the promoter depending on the quality of the nitrogen source. We further demonstrate here that an interaction occurs in vivo between Uga3 ‒ the transcriptional activator responsible for γ-aminobutyric acid (GABA)-dependent induction of the GABA genes ‒ and the regulatory region of the BAP2 gene, which leads to an increase in BAP2 transcription.

Modulating acetate ester and higher alcohol production in Saccharomyces cerevisiae through the cofactor engineering.

Flavor production by esters or by higher alcohols play a key role in the sensorial quality of fermented alcoholic beverages. In Saccharomyces cerevisiae cells, the syntheses of esters and higher alcohols are considerably influenced by intracellular CoA levels catalyzed by pantothenate kinase. In this work, we examined the effects of cofactor CoA and acetyl-CoA synthesis on the metabolism of esters and higher alcohols. Strains 12α-BAP2 and 12α+ATF1 where generated by deleting and overexpressing BAP2 (encoded branched-chain amino acid permease) and ATF1 (encoded alcohol acetyl transferases), respectively, in the parent 12α strains. Then, 12α-BAP2+CAB1 and 12α-BAP2+CAB3 strains were obtained by overexpressing CAB1 (encoded pantothenate kinase Cab1) and CAB3 (encoded pantothenate kinase Cab3) in the 12α-BAP2 strain, and 12α-BAP2+CAB1+ATF1 and 12α-BAP2+CAB3+ATF1 were generated by overexpressing ATF1 in the pantothenate kinase overexpression strains. The acetate ester level in 12α-BAP2 was slightly changed relative to that in the control strain 12α, whereas the acetate ester levels in 12α-BAP2+CAB1, 12α-BAP2+CAB3, 12α-BAP2+CAB1+ATF1, and 12α-BAP2+CAB3+ATF1 were distinctly increased (44-118% for ethyl acetate and 18-57% for isoamyl acetate). The levels of n-propanol, methyl-1-butanol, isopentanol, isobutanol, and phenethylol levels were changed and varied among the six engineered strains. The levels of acetate esters and higher alcohols can be modulated by changing the CoA and acetyl-CoA levels. The method proposed in this work supplies a practical means of breeding yeast strains by modulating acetate ester and higher alcohol production.

Intermediate Levels of Bacillus subtilis CodY Activity Are Required for Derepression of the Branched-Chain Amino Acid Permease, BraB.

The global transcriptional regulator, CodY, binds strongly to the regulatory region of the braB gene, which encodes a Bacillus subtilis branched-chain amino acid (BCAA) permease. However, under conditions that maximize CodY activity, braB expression was similar in wild-type and codY null mutant cells. Nonetheless, expression from the braB promoter was significantly elevated in cells containing partially active mutant versions of CodY or in wild-type cells under growth conditions leading to intermediate levels of CodY activity. This novel pattern of regulation was shown to be due to two opposing mechanisms, negative and positive, by which CodY affects braB expression. A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY. Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY. ScoC-mediated repression of braB in codY null mutants cells was as efficient as direct, CodY-mediated repression in wild-type cells under conditions of high CodY activity. However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression. We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d’être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter. The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

Genome sequence analysis of potential probiotic strain Leuconostoc lactis EFEL005 isolated from kimchi.

Leuconostoc lactis EFEL005 (KACC 91922) isolated from kimchi showed promising probiotic attributes; resistance against acid and bile salts, absence of transferable genes for antibiotic resistance, broad utilization of prebiotics, and no hemolytic activity. To expand our understanding of the species, we generated a draft genome sequence of the strain and analyzed its genomic features related to the aforementioned probiotic properties. Genome assembly resulted in 35 contigs, and the draft genome has 1,688,202 base pairs (bp) with a G+C content of 43.43%, containing 1,644 protein-coding genes and 50 RNA genes. The average nucleotide identity analysis showed high homology (≥ 96%) to the type strain L. lactis KCTC3528, but low homology (≤ 95%) to L. lactis KCTC3773 (formerly L. argentinum). Genomic analysis revealed the presence of various genes for sucrose metabolism (glucansucrases, invertases, sucrose phosphorylases, and mannitol dehydrogenase), acid tolerance (F1F0 ATPases, cation transport ATPase, branched-chain amino acid permease, and lysine decarboxylase), vancomycin response regulator, and antibacterial peptide (Lactacin F). No gene for production of biogenic amines (histamine and tyramine) was found. This report will facilitate the understanding of probiotic properties of this strain as a starter for fermented foods.

Efficient Overproduction of Membrane Proteins in Lactococcus lactis Requires the Cell Envelope Stress Sensor/Regulator Couple CesSR

BackgroundMembrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein.Methods and FindingsWe describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold.ConclusionsThe results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins.

The role of oxygen in yeast metabolism during high cell density brewery fermentations

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.

Identification and Functional Characterization of theLactococcus lactisCodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

Transcriptome analyses have previously revealed that a gene encoding the putative amino acid transporter CtrA (YhdG) is one of the major targets of the pleiotropic regulator CodY in Lactococcus lactis and Bacillus subtilis. The role of ctrA in L. lactis was further investigated with respect to both transport activity as well as CodY-mediated regulation. CtrA is required for optimal growth in media containing free amino acids as the only amino acid source. Amino acid transport studies showed that ctrA encodes a secondary amino acid transport system that is specific for branched-chain amino acids (BCAAs) (isoleucine, leucine, and valine) and methionine, which is in disagreement with its previously proposed function (a cationic amino acid transporter), which was assigned based on homology. We propose to rename CtrA BcaP, for branched-chain amino acid permease. BcaP is a member of a group of conserved transport systems, as homologs are widely distributed among gram-positive bacteria. Deletion of bcaP resulted in the loss of most of the BCAA uptake activity of L. lactis, indicating that BcaP is the major BCAA carrier of this organism. Deletion of bcaP together with a second (putative) BCAA permease, encoded by brnQ, further reduced the viability of the strain. DNA microarray analysis showed that deletion of bcaP predominantly affects genes belonging to the regulons of the transcriptional regulator CodY, which is involved in global nitrogen metabolism and needs BCAAs for its activation, and of CmbR, which is involved in sulfur amino acid metabolism.

The N-terminal domain of yeast Bap2 permease is phosphorylated dependently on the Npr1 kinase in response to starvation

The Saccharomyces cerevisiae branched-chain amino acid permease Bap2p plays a major role in leucine, isoleucine, and valine transport, and its synthesis is regulated transcriptionally. Bap2p undergoes a starvation-induced degradation depending upon ubiquitination and the functions of N- and C-terminal domains of Bap2p. Here we show that the N-terminal domain of Bap2p is phosphorylated in response to rapamycin treatment when both the N- and C-termini of Bap2p are fused to glutathione S-transferase. The phosphorylation is dependent on Ser/Thr kinase Npr1p. In npr1 cells, Bap2p becomes slightly more susceptible to the rapamycin-induced degradation, suggesting that Npr1p counteracts the degradation system for Bap2p.

A monocarboxylate permease of Rhizobium leguminosarum is the first member of a new subfamily of transporters.

Amino acid transport by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra). However, mutation of these transporters does not prevent this organism from utilizing alanine for growth. An R. leguminosarum permease (MctP) has been identified which is required for optimal growth on alanine as a sole carbon and nitrogen source. Characterization of MctP confirmed that it transports alanine (K(m) = 0.56 mM) and other monocarboxylates such as lactate and pyruvate (K(m) = 4.4 and 3.8 micro M, respectively). Uptake inhibition studies indicate that propionate, butyrate, alpha-hydroxybutyrate, and acetate are also transported by MctP, with the apparent affinity for solutes demonstrating a preference for C3-monocarboxylates. MctP has significant sequence similarity to members of the sodium/solute symporter family. However, sequence comparisons suggest that it is the first characterized permease of a new subfamily of transporters. While transport via MctP was inhibited by CCCP, it was not apparently affected by the concentration of sodium. In contrast, glutamate uptake in R. leguminosarum by the Escherichia coli GltS system did require sodium, which suggests that MctP may be proton coupled. Uncharacterized members of this new subfamily have been identified in a broad taxonomic range of species, including proteobacteria of the beta-subdivision, gram-positive bacteria, and archaea. A two-component sensor-regulator (MctSR), encoded by genes adjacent to mctP, is required for activation of mctP expression.

Rhizobium leguminosarum has a second general amino acid permease with unusually broad substrate specificity and high similarity to branched-chain amino acid transporters (Bra/LIV) of the ABC family.

Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra(Rl)). Characterization of the solute specificity of Bra(Rl) shows it to be the second general amino acid permease of R. leguminosarum. Although Bra(Rl) has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (L-glutamate, L-arginine, and L-histidine), in addition to neutral amino acids (L-alanine and L-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be alpha-amino acids. Consistent with this, Bra(Rl) is the first ABC transporter to be shown to transport gamma-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by Bra(Rl) does not appear to be stereospecific as D amino acids cause significant inhibition of uptake of L-glutamate and L-leucine. Unlike all other solutes tested, L-alanine uptake is not dependent on solute binding protein BraC(Rl). Therefore, a second, unidentified solute binding protein may interact with the BraDEFG(Rl) membrane complex during L-alanine uptake. Overall, the data indicate that Bra(Rl) is a general amino acid permease of the HAAT family. Furthermore, Bra(Rl) has the broadest solute specificity of any characterized bacterial amino acid transporter.

Control of Higher Alcohol Production by Manipulation of the BAP2 Gene in Brewing Yeast

We studied whether the production of higher alcohols during wort fermentation is responsive to branched-chain amino acid metabolism. Addition of valine, isoleucine, and leucine to the brewing wort resulted in increased assimilation of each amino acid by yeast cells and caused increased production of the corresponding higher alcohol, suggesting that production of a higher alcohol is correlated with the assimilation of the corresponding amino acid. Therefore, we attempted the constitutive expression of the gene (BAP2) for the branched-chain amino acid permease in brewing yeast and investigated the effects on assimilation of branched-chain amino acids and production of higher alcohols during wort fermentation. Constitutive expression of the BAP2 gene resulted in accelerated rates of assimilation for leucine, valine, and isoleucine. This caused increased production of isoamyl alcohol derived from leucine, while an increase of isobutyl alcohol derived from valine or active amyl alcohol from isoleucine was not observed. These results suggest that there are distinct but interrelated mechanisms for the production of each higher alcohol.

Isolation and characterization of a gene specific to lager brewing yeast that encodes a branched-chain amino acid permease.

We found two types of branched-chain amino acid permease gene (BAP2) in the lager brewing yeast Saccharomyces pastorianus BH-225 and cloned one type of BAP2 gene (Lg-BAP2), which is identical to that of Saccharomyces bayanus (by-BAP2-1). The other BAP2 gene of the lager brewing yeast (cer-BAP2) is very similar to the Saccharomyces cerevisiae BAP2 gene. This result substantiates the notion that lager brewing yeast is a hybrid of S. cerevisiae and S. bayanus. The amino acid sequence homology between S. cerevisiae Bap2p and Lg-Bap2p was 88%. The transcription of Lg-BAP2 was not induced by the addition of leucine to the growth medium, while that of cer-BAP2 was induced. The transcription of Lg-BAP2 was repressed by the presence of ethanol and weak organic acid, while that of cer-BAP2 was not affected by these compounds. Furthermore, Northern analysis during beer fermentation revealed that the transcription of Lg-BAP2 was repressed at the beginning of the fermentation, while cer-BAP2 was highly expressed throughout the fermentation. These results suggest that the transcription of Lg-BAP2 is regulated differently from that of cer-BAP2 in lager brewing yeasts.

Solute-binding protein-dependent ABC transporters are responsible for solute efflux in addition to solute uptake.

The ATP-binding cassette (ABC) transporter superfamily is one of the most widespread of all gene families and currently has in excess of 1100 members in organisms ranging from the Archaea to manQ1. The movement of the diverse solutes of ABC transporters has been accepted as being strictly unidirectional, with recent models indicating that they are irreversible. However, contrary to this paradigm, we show that three solute-binding protein-dependent (SBP) ABC transporters of amino acids, i.e. the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra) of Rhizobium leguminosarum and the histidine permease (His) of Salmonella typhimurium, are bidirectional, being responsible for efflux in addition to the uptake of solutes. The net solute movement measured for an ABC transporter depends on the rates of uptake and efflux, which are independent; a plateau is reached when both are saturated. SBP ABC transporters promote active uptake because, although the Vmax values for uptake and efflux are not significantly different, there is a 103-104 higher affinity for uptake of solute compared with efflux. Therefore, the SBP ABC transporters are able to support a substantial concentration gradient and provide a net uptake of solutes into bacterial cells.

The basal turnover of yeast branched-chain amino acid permease Bap2p requires its C-terminal tail

The branched-chain amino acid permease Bap2p is a transport system for leucine, isoleucine, and valine in Saccharomyces cerevisiae, and its synthesis is regulated transcriptionally. However, the downregulation mechanisms of Bap2p have not been established. Here we demonstrate that the C-terminal region of Bap2p plays a pivotal role in its basal turnover. Truncation of the C-terminal 29 residues caused the stabilization and accumulation in the plasma membrane of Bap2p. Furthermore, when the Bap2p C-terminal region was fused to green fluorescent protein, the fusion protein localized to the plasma membrane, suggesting the existence of a possible degradation-related acceptor site for the C-terminal tail of Bap2p.

The basal turnover of yeast branched-chain amino acid permease Bap2p requires its C-terminal tail.

The branched-chain amino acid permease Bap2p is a transport system for leucine, isoleucine, and valine in Saccharomyces cerevisiae, and its synthesis is regulated transcriptionally. However, the downregulation mechanisms of Bap2p have not been established. Here we demonstrate that the C-terminal region of Bap2p plays a pivotal role in its basal turnover. Truncation of the C-terminal 29 residues caused the stabilization and accumulation in the plasma membrane of Bap2p. Furthermore, when the Bap2p C-terminal region was fused to green fluorescent protein, the fusion protein localized to the plasma membrane, suggesting the existence of a possible degradation-related acceptor site for the C-terminal tail of Bap2p.

Stp1p, Stp2p and Abf1p are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae

Expression of the BAP3 gene of Saccharomyces cerevisiae, encoding a branched chain amino acid permease, is induced in response to the availability of several naturally occurring amino acids in the medium. This induction is mediated via an upstream activating sequence (called UAS(aa)) in the BAP3 promoter, and dependent on Stp1p, a nuclear protein with zinc finger domains, suggesting that Stp1p is a transcription factor involved in BAP3 expression. In this paper, we show that Stp2p, a protein with considerable similarity to Stp1p, is also involved in the induction of BAP3 expression. To gain more insight into the roles of STP1 and STP2, we have overexpressed both Stp1p and Stp2p in yeast cells. Gel shift assays with the UAS(aa)as a probe show that the UAS(aa)can form two major complexes. One complex is dependent on Stp2p overexpression and the other is formed independently of STP1 or STP2, suggesting that the UAS(aa)is also bound by another factor. Here we show that the other factor is Abf1p, which binds specifically to the UAS(aa)of BAP3.

Mutations in five loci affecting GAP1-independent uptake of neutral amino acids in yeast.

In order to identify genes involved in uptake of isoleucine, leucine and valine in Saccharomyces cerevisiae we isolated mutants that, on a complex medium, were sensitive to an inhibitor of the biosynthesis of the branched-chain amino acids. Mutants that in a secondary screen showed reduced uptake of isoleucine, leucine and valine when growing in synthetic complete medium were further characterized. Genetic analysis identified five loci, named ssy1 through ssy5. ssy2 corresponds to the previously characterized bap1 mutation, which we recently have found to be allelic to stp1. ssy1, ssy3 and ssy5 exhibit a reduced uptake of phenylalanine, methionine and threonine, as well. Furthermore, they are resistant to several neutral amino acid analogs. ssy4 only affects uptake of few neutral amino acids and is as sensitive as the wild type to the amino acid analogs tested. It was previously found that a C-terminal truncation of 29 codons of BAP2, which encodes a branched-chain amino acid permease, results in increased uptake of the branched-chain amino acids. We find epistasis of the C-terminally truncated BAP2 gene over the ssy4 mutation, while the other ssy mutations are epistatic over the truncated BAP2 gene. SSY1, SSY3 and SSY5 were cloned from a low-copy genomic library by complementation of the mutants. The SSY3 gene and the SSY5 gene show no significant homology to any sequence in the databases. SSY1 is a member of the major family of genes encoding amino acid permeases in yeast. We discuss possible roles of Ssy1p in amino acid uptake.

Amino acids induce expression of BAP2, a branched-chain amino acid permease gene in Saccharomyces cerevisiae.

Branched-chain amino acid uptake in Saccharomyces cerevisiae is mediated by at least three transport systems: the general amino acid permease Gap1p, the branched-chain amino acid permease Bap2p, and one or more so far unknown permeases. Regulation of the transcription of BAP2 is mainly subject to the presence of certain amino acids in the medium. The level of transcription is low during growth on a minimal medium with proline as the sole nitrogen source. As assayed with a lacZ fusion, the level of transcription is slightly higher (3-fold) on a minimal medium with ammonium ions as a nitrogen source, and transcription is induced about 20-fold by addition of leucine (0.2 mM). As little as 10 microM leucine causes a fivefold induction. Addition of (L)-leucine to minimal proline medium, on the other hand, has no effect on BAP2 transcription. The two known permeases for transport of branched-chain amino acids, Gap1p and Bap2p, are thus not active at the same time. The BAP2 promoter contains one or two putative Gcn4p binding sites and one putative Leu3p binding site. None of the three is needed for induction by leucine. Induction of BAP2 transcription by leucine is accompanied by an increase in branched-chain amino acid uptake. This elevation is interpreted to be partly the result of an increased level of the Bap2p permease in the plasma membrane, because deletion of BAP2 slightly decreases the induction of uptake. There is still a leucine-inducible increase in branched-chain amino acid uptake in a delta gap1 delta bap2 strain, indicating that BAP2 shares leucine induction with at least one remaining branched-chain amino acid-transporting permease.

BAP2, a gene encoding a permease for branched-chain amino acids in Saccharomyces cerevisiae

To select the gene coding for an isoleucine permease, an isoleucine dependent strain ( ilv 1 chal) was transformed with a yeast genomic multicopy library, and colonies growing at a low isoleucine concentration were selected. Partial sequencing of the responsible plasmid insert revealed the presence of a previously sequenced 609 codon open reading frame of chromosome II with homology to known permeases. Deletion, extra dosage and C-terminal truncation of this gene were constructed in a strain lacking the general amino acid permease, and amino acid uptake was measured during growth in synthetic complete medium. The following observations prompted us to name the gene BAP2 (branched-chain amino acid permease). Deletion of BAP2 reduced uptake of leucine, isoleucine and valine by 25–50%, while the uptake of 8 other l-α-amino acids was unlatered or slightly increased. Introduction of BAP2 on a centromere-based vector, leading to a gene dosage of two or slightly more, caused a 50% increase in leucine uptake and a smaller increase for isoleucine and valine. However, when the 29 C-terminal codons of the plasmid-borne copy of BAP2 were substituted, the cells more than doubled the uptake of leucine, isoleucine and valine, while no or little increase in uptake was observed for the other 8 amino acids.

A high‐affinity uptake system for branched‐chain amino acids in Saccharomyces cerevisiae

In order to isolate mutants with impaired uptake of branched-chain amino acids, mutants were induced which on complex medium were sensitive to an inhibitor of branched-chain amino acid biosynthesis. Eighteen mutants of independent origin were found. Ten of them were assayed for branched-chain amino acid uptake. Three of these were impaired in the uptake of L-valine, L-isoleucine and L-leucine, while the rest were unaffected in uptake of any of the three amino acids. Kinetics of the uptake by one selected mutant and the parental strain S288C were compared to models for one or two systems obeying Michaelis-Menten kinetics. This analysis suggested that a high-affinity system for all three amino acids is absent in the mutant, whereas low-affinity uptake of L-isoleucine and L-leucine by one or more systems remains unaffected. Moreover, medium-affinity uptake components for L-valine and L-leucine, not originally seen in the wild type, were identified in the mutant. In the wild type, 10 mM of any of the amino acids L-alanine, L-cysteine, L-isoleucine, L-leucine, L-tryptophan and L-valine reduce uptake of any of the three branched-chain amino acids. We propose that a permease responsible for high-affinity uptake of the branched-chain amino acids in strain S288C is partially or completely inactive in the mutant. Tetrad analysis shows that the phenotype can be ascribed to a single Mendelian gene. The wild-type allele is denoted BAP1 for branched-chain amino acid permease. The BAP1-dependent system is different from the general amino acid permease.