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

Benjamin Clémençon, Benjamin P. Lüscher, Michael Fine, Marc U. Baumann, Daniel V. Surbek, Olivier Bonny, Matthias A. Hediger

Research output: Contribution to journalArticle

18 Citations (Scopus)

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. G LU T9 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.

Original languageEnglish (US)
Article numbere108852
JournalPloS one
Volume9
Issue number10
DOIs
StatePublished - Oct 6 2014
Externally publishedYes

Fingerprint

Xenopus laevis
uric acid
Uric Acid
Oocytes
Purification
transporters
oocytes
Mutagenesis
mutagenesis
Facilitative Glucose Transport Proteins
proteins
Proteins
glucose transporters
proximal tubules
Xenopus Proteins
Xenopus
crystal structure
xylose
Proximal Kidney Tubule
placenta

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis Oocytes System. / Clémençon, Benjamin; Lüscher, Benjamin P.; Fine, Michael; Baumann, Marc U.; Surbek, Daniel V.; Bonny, Olivier; Hediger, Matthias A.

In: PloS one, Vol. 9, No. 10, e108852, 06.10.2014.

Research output: Contribution to journalArticle

Clémençon, Benjamin ; Lüscher, Benjamin P. ; Fine, Michael ; Baumann, Marc U. ; Surbek, Daniel V. ; Bonny, Olivier ; Hediger, Matthias A. / Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis Oocytes System. In: PloS one. 2014 ; Vol. 9, No. 10.
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