Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis oocytes system

PLoS One. 2014 Oct 6;9(10):e108852. doi: 10.1371/journal.pone.0108852. eCollection 2014.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Membrane / metabolism
  • Chromatography, Affinity
  • Chromatography, Gel
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression Regulation
  • Glucose Transport Proteins, Facilitative / chemistry*
  • Glucose Transport Proteins, Facilitative / isolation & purification*
  • Glucose Transport Proteins, Facilitative / metabolism
  • Humans
  • Models, Molecular
  • Oocytes / metabolism*
  • Organic Anion Transporters / chemistry*
  • Organic Anion Transporters / isolation & purification*
  • Organic Anion Transporters / metabolism
  • Phylogeny
  • Silver Staining
  • Structural Homology, Protein
  • Xenopus laevis


  • Glucose Transport Proteins, Facilitative
  • Organic Anion Transporters
  • SLC2A9 protein, human
  • urate transporter

Grant support

This study was supported by the Swiss National Science Foundation (SNSF) through the National Centre of Competence in Research (NCCR) TransCure and The TransCure International Fellowship Program (IFP TransCure), funded in part by an FP7 European Marie Curie Actions grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.