Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2.
Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition.
Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Sagamihara, Japan.
Yuki
Y
Usami
Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Sagamihara, Japan.
Satsohi
S
Uemura
Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Sagamihara, Japan.
Takahiro
T
Mochizuki
Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Sagamihara, Japan.
Asami
A
Morita
Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai, Japan.
Fumi
F
Shishido
Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai, Japan.
Jin-ichi
J
Inokuchi
Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Sagamihara, Japan; Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan. Electronic address: abef@chem.aoyama.ac.jp.
Fumiyoshi
F
Abe
D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D026905:Amino Acid Transport Systems; C097094:BAP2 protein, S cerevisiae; D026901:Membrane Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D007930:Leucine
Homology modeling; Leucine permease Bap2; Saccharomyces cerevisiae; logP