Converting the yeast arginine can1 permease to a lysine permease.
Amino acid uptake in yeast cells is mediated by about 16 plasma membrane permeases, most of which belong to the amino acid-polyamine-organocation (APC) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino acid permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Although Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H(+)-driven arginine-specific permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a three-dimensional Can1 structural model based on the crystal structure of the bacterial APC family arginine/agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q and E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H(+) coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport.
Laboratoire de Physiologie Moléculaire de la Cellule CP300, Institut de Biologie et de Médecine Moléculaires (IBMM), Université Libre de Bruxelles (ULB), 11 Rue des Pr. Jeener et Brachet, 6041 Gosselies, Belgium.
Kassem
K
Ghaddar
Structure et Fonction des Membranes Biologiques, Centre de Biologie Structurale et de Bioinformatique, Université Libre de Bruxelles (ULB), Bld. du Triomphe, 1050 Brussels, Belgium.
Eva-Maria
EM
Krammer
Laboratoire de Physiologie Moléculaire de la Cellule CP300, Institut de Biologie et de Médecine Moléculaires (IBMM), Université Libre de Bruxelles (ULB), 11 Rue des Pr. Jeener et Brachet, 6041 Gosselies, Belgium.
Natalija
N
Mihajlovic
Machine Learning Group, Computer Science Department, Faculté des Sciences, Université Libre de Bruxelles (ULB), 1050 Brussels, Belgium.
Sylvain
S
Brohée
Laboratoire de Physiologie Moléculaire de la Cellule CP300, Institut de Biologie et de Médecine Moléculaires (IBMM), Université Libre de Bruxelles (ULB), 11 Rue des Pr. Jeener et Brachet, 6041 Gosselies, Belgium. Electronic address: bran@ulb.ac.be.
Bruno
B
André
Structure et Fonction des Membranes Biologiques, Centre de Biologie Structurale et de Bioinformatique, Université Libre de Bruxelles (ULB), Bld. du Triomphe, 1050 Brussels, Belgium. Electronic address: mprevost@ulb.ac.be.
Martine
M
Prévost
D000595:Amino Acid Sequence; D026923:Amino Acid Transport Systems, Basic / Q000737:chemistry* / Q000235:genetics; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D021621:Imaging, Three-Dimensional; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity
D026923:Amino Acid Transport Systems, Basic; C502212:CAN1 protein, S cerevisiae; C430023:LYP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D001120:Arginine
Amino Acid Transport; Ligand Docking; Membrane Proteins; Molecular Modeling; Protein Engineering; Proton Transport; Site-directed Mutagenesis; Transporters; Yeast Physiology