title: Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis oocytes system.
abstract: The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.
authors.affiliation: Institute of Biochemistry and Molecular Medicine (IBMM), and NCCR TransCure, University of Bern, Bern, Switzerland.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Clémençon
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Bern, Bern, Switzerland; Department of Clinical Research, University of Bern, Bern, Switzerland.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Lüscher
authors.affiliation: Institute of Biochemistry and Molecular Medicine (IBMM), and NCCR TransCure, University of Bern, Bern, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Fine
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Bern, Bern, Switzerland; Department of Clinical Research, University of Bern, Bern, Switzerland.
authors.forename: Marc U
authors.initials: MU
authors.lastname: Baumann
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Bern, Bern, Switzerland; Department of Clinical Research, University of Bern, Bern, Switzerland.
authors.forename: Daniel V
authors.initials: DV
authors.lastname: Surbek
authors.affiliation: Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland.
authors.forename: Olivier
authors.initials: O
authors.lastname: Bonny
authors.affiliation: Institute of Biochemistry and Molecular Medicine (IBMM), and NCCR TransCure, University of Bern, Bern, Switzerland.
authors.forename: Matthias A
authors.initials: MA
authors.lastname: Hediger
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D051246:Glucose Transport Proteins, Facilitative / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009865:Oocytes / Q000378:metabolism*; D027361:Organic Anion Transporters / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010802:Phylogeny; D016622:Silver Staining; D040681:Structural Homology, Protein; D014982:Xenopus laevis
chemical_list: D051246:Glucose Transport Proteins, Facilitative; D027361:Organic Anion Transporters; C411811:SLC2A9 protein, human; C103862:urate transporter