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

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...

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Published inPloS one Vol. 9; no. 10; p. e108852
Main Authors Clémençon, Benjamin, Lüscher, Benjamin P, Fine, Michael, Baumann, Marc U, Surbek, Daniel V, Bonny, Olivier, Hediger, Matthias A
Format Journal Article
LanguageEnglish
Published United States Public Library of Science 06.10.2014
Public Library of Science (PLoS)
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Summary: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.
Bibliography:Conceived and designed the experiments: BC BPL MF OB MAH. Performed the experiments: BC BPL. Analyzed the data: BC BPL MF MAH. Contributed reagents/materials/analysis tools: BC BPL. Wrote the paper: BC BPL MF MUB DVS OB MAH.
Competing Interests: The authors have declared that no competing interests exist.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0108852