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

Abstract

To select the gene coding for an isoleucine permease, an isoleucine dependent strain (ilv1 cha1) 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-alpha-amino acids was unaltered 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.

Genetic Interaction between theero1-1andleu2Mutations inSaccharomyces cerevisiae

“…We wondered whether the inability of an ero1-1 leu2 strain to grow on 30 mg/l leucine can be explained by a dysfunction of Bap2p. Previous studies indicate that the lack of the C-terminal 29 residues of Bap2p causes increased capacity for uptake of isoleucine, leucine, and valine, 17) due to increased stability of the permease. 20) Hence we transformed the ero1-1 leu2 strain with a plasmid carrying the truncated allele (BAP2Á29), and observed that this caused a moderate gain in the ability to grow on SC medium at 30 C (Fig.…”

“…Bap2p has decreased stability in an ero1-1 strain On preferred nitrogen sources, the main leucine import system in yeast is the Bap2 protein, 17,30) an integral protein in the plasma membrane (PM). We wondered whether the inability of an ero1-1 leu2 strain to grow on 30 mg/l leucine can be explained by a dysfunction of Bap2p.…”

“…The synthetic complete medium (SC) consisted of a 0.67% yeast nitrogen base without amino acids (DIFCO, Tokyo, Japan), 2% glucose, a mixture of succinic acid (10 g/l) and sodium hydroxide (6 g/l) as buffer (pH 5.5), and other nutrients as specified. 17) The standard concentration of L-leucine in this medium is 30 mg/l. The ero1-1 leu2 strain was enabled to grow by supplementation of the medium with additional L-leucine (to 180 mg/l) to yield the SC + 5xleu medium.…”

Amino acids mediate colony and cell differentiation in the fungal pathogen Candida parapsilosis

“…After incubating this mixture at 37 uC, amino acid uptake was stopped by addition of 5 ml ice-cold PBS. The cells were collected on a Whatman GF/C glass microfibre filter, washed three times with cold PBS, air-dried and their radioactivity measured in a scintillation counter (Grauslund et al, 1995). All uptake studies were done in triplicate, and mean values were determined.…”

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

“…Many of these have been identified previously through mutant studies and their substrate specificity is known; others are open reading frames, identified in the Yeast Genome Project as homologous to other members of the family (Nelissen et al, 1995). The known amino acid permeases show several common features: a length of approximately 600 amino acids, a central hydrophobic section which contains 12 putative membrane-spanning domains, no N-terminal signal sequence and a C-terminal which is fairly hydrophilic and may be involved in permease regulation (Grausland et al, 1995;Zhu et al, 1996).…”