The Branched-Chain Amino Acid Permease Gene ofSaccharomyces cerevisiae,BAP2, Encodes the High-Affinity Leucine Permease (S1)

Schreve, James; Garrett, Jinnie M.

doi:10.1002/(sici)1097-0061(199704)13:5<435::aid-yea95>3.0.co;2-t

Cited by 21 publications

(10 citation statements)

References 14 publications

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“…Since BAP2 encodes a high-anity permease that catalyzes transport of leucine, isoleucine and valine, it may well be an allele of LET1. The recent report of Schreve and Garrett (1997) supports this idea. These authors present evidence that Bap2p is the permease responsible for transport system S1.…”

Section: Discussionmentioning

confidence: 78%

“…By employing an alternative approach, Grauslund et al (1995) have cloned a gene, designated BAP2, that encodes a branched-chain amino acid permease. The BAP2 gene has been cloned independently by Schreve and Garrett (1997) as a multicopy suppressor of the aat1 gene, which is involved in regulation of amino acid transport. These authors also present kinetic evidence indicating that BAP2 encodes the high-anity permease responsible for S1.…”

Section: Introductionmentioning

confidence: 99%

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The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport

et al. 1999

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Leucine uptake by Saccharomyces cerevisiae is mediated by three transport systems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine and valine. A new mutant defective in both group-specific transport activities was isolated by employing a gap1 leu4 strain and selecting for trifluoroleucine-resistant mutants which also showed greatly reduced ability to utilize L-leucine as sole nitrogen source and very low levels of [14C]L-leucine uptake. A multicopy plasmid containing a DNA fragment which complemented the leucine transport defect was isolated by selecting for transformants that grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying [14C]L-leucine uptake. Transformation of one such mutant, lep1, restored sensitivity to trifluoroleucine. The complementing gene, designated LEP1, was subcloned and sequenced. The LEP1 ORF encodes a large protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association). Instead, Lep1p is a very basic protein (pI of 9.2) that contains a putative bipartite signal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein. A database search revealed that LEP1 encodes a polypeptide that is identical to Sac3p except for an N-terminal truncation. The original identification of SAC3 was based on the isolation of a mutant allele, sac3-1, that suppresses the temperature-sensitive growth defect of an actin mutant containing the allele act1-1. Sac3p has been previously shown to be localized in the nucleus. When a lep1 mutant was crossed with a sac3 deletion mutant, no complementation was observed, indicating that the two mutations are functionally allelic.

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Section: Discussionmentioning

confidence: 78%

Section: Introductionmentioning

confidence: 99%

The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport

et al. 1999

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“…It is known that the addition of NH 4 + to yeast cells causes nitrogen catabolite inactivation and repression of several enzymes and permeases involved in the utilization of secondary nitrogen sources [26]. Leucine has been shown to be transported by at least three systems in S. cerevisiae : GAP (general amino acid permease), S1 (high-affinity permease) and S2 (low-affinity permease) [27]. In NH 4 + -containing media the activity of GAP is inhibited [28, 29] and the activity of S1 and S2 proteins is strongly reduced [30].…”

Section: Resultsmentioning

confidence: 99%

Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

et al. 2017

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BackgroundA commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug-resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele.ResultsA K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth.ConclusionsIn this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0715-8) contains supplementary material, which is available to authorized users.

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“…BAP2 (S1) [3] and S2 are group-specific systems which transport all three branched-chain amino acids (leucine, valine and isoleucine). S1 has a high affinity for the substrate, but transports at a relatively low velocity, while S2 has a low affinitiy and a high velocity.…”

Section: Cellular and Molecular Biology Letters 257mentioning

confidence: 99%

Brefeldin a decreases the activity of the general amino acid permease (GAP1) and the more specific systems for L-leucine uptake in Saccharomyces cerevisiae

Alonso

Burgos

Pannunzio

et al. 2006

Cellular and Molecular Biology Letters

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Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the endoplasmic reticulum to the Golgi complex. In this study, we aimed to characterize L-leucine uptake in Saccharomyces cerevisiae in the presence of brefeldin A. For this purpose, we used a synthetic medium, containing L-proline and the detergent SDS, which allows the agent to permeate into the yeast cell. The results obtained with a wild type strain and a gap1 mutant indicate that BFA causes either direct or indirect modification of the transport and/or processing of L-leucine permeases. The presence of BFA affects the kinetic parameter values for L-leucine uptake and decreases not only the uptake mediated by the general system (GAP1), but also that through the specific BAP2 (S1) and/or S2 systems.

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The Branched-Chain Amino Acid Permease Gene ofSaccharomyces cerevisiae,BAP2, Encodes the High-Affinity Leucine Permease (S1)

Cited by 21 publications

References 14 publications

The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport

The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport

Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Brefeldin a decreases the activity of the general amino acid permease (GAP1) and the more specific systems for L-leucine uptake in Saccharomyces cerevisiae

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