title: Asymmetry in inward- and outward-affinity constant of transport explain unidirectional lysine flux in Saccharomyces cerevisiae.
abstract: The import of basic amino acids in Saccharomyces cerevisiae has been reported to be unidirectional, which is not typical of how secondary transporters work. Since studies of energy coupling and transport kinetics are complicated in vivo, we purified the major lysine transporter (Lyp1) of yeast and reconstituted the protein into lipid vesicles. We show that the Michaelis constant (KM) of transport from out-to-in is well in the millimolar range and at least 3 to 4-orders of magnitude higher than that of transport in the opposite direction, disfavoring the efflux of solute via Lyp1. We also find that at low values of the proton motive force, the transport by Lyp1 is comparatively slow. We benchmarked the properties of eukaryotic Lyp1 to that of the prokaryotic homologue LysP and find that LysP has a similar KM for transport from in-to-out and out-to-in, consistent with rapid influx and efflux. We thus explain the previously described unidirectional nature of lysine transport in S. cerevisiae by the extraordinary kinetics of Lyp1 and provide a mechanism and rationale for previous observations. The high asymmetry in transport together with secondary storage in the vacuole allow the cell to accumulate basic amino acids to very high levels.
authors.affiliation: Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Frans
authors.initials: F
authors.lastname: Bianchi
authors.affiliation: Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Joury S van 't
authors.initials: JS
authors.lastname: Klooster
authors.affiliation: Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Stephanie J
authors.initials: SJ
authors.lastname: Ruiz
authors.affiliation: Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Katja
authors.initials: K
authors.lastname: Luck
authors.affiliation: Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Tjeerd
authors.initials: T
authors.lastname: Pols
authors.affiliation: Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
authors.forename: Ina L
authors.initials: IL
authors.lastname: Urbatsch
authors.affiliation: Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands.|Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman
mesh_terms: D026923:Amino Acid Transport Systems, Basic / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active / Q000502:physiology; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D026923:Amino Acid Transport Systems, Basic; C430023:LYP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008239:Lysine