Membrane transport systems are involved in a large number of fundamental cellular activities, and yet most are poorly characterized at the molecular level. The Glut1 glucose transporter is the prototype facilitative transport protein. Facilitative glucose transport is a critical function carried out by virtually all mammalian cells. This process is mediated by members of the Glut (or SLC2a) family of membrane glycoproteins that includes 14 known transporters. The Glut family belongs to the Major Facilitator Superfamily (MFS), which comprises the largest group of membrane transport proteins. The Gluts are responsible for the exchange of glucose between the blood and the cytoplasm of cells, supplying glucose for energy metabolism and biosynthetic reactions. Additionally, glucose transport in certain tissues plays a critical role in whole body glucose homeostasis and is associated with several disease states, including type 2 diabetes, Glut1-deficiency syndrome, Fanconi-Bickel syndrome, tumor progression, and cellular invasion by Human T-Cell Leukemia Virus. Despite the physiologic and clinical importance of glucose transport, relatively little is known concerning the structure and function of the Glut proteins. Recently, high-resolution structures were reported for two bacterial members of the MFS, which has enabled the development of a low-resolution structural model for Glut1 based on homology modeling. The long-term goals of this project are to delineate the structure of Glut1 and the molecular mechanism of facilitative sugar transport. To make further progress towards accomplishing these long-term goals, the following specific aims are proposed for the next project period: 1. Nine of the 12 predicted transmembrane helices and approximately 41% of the 492 residues comprising Glut1 have been analyzed thus far by site-directed mutagenesis. The 3 remaining transmembrane helices will be analyzed by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method (SCAM). 2. Proximity relationships between pairs of transmembrane helices will be determined by chemical crosslinking of di-cysteine mutants constructed in novel functional cysteine-less Glut1 reporter constructs. 3. Procedures will be developed for the purification of milligram quantities of recombinant Glut1 and Glut4 proteins in an attempt to produce crystals for X-ray diffraction analysis.
This aim will be conducted in collaboration with Dr. Da-Neng Wang at NYU.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Sechi, Salvatore
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Washington University
Anatomy/Cell Biology
Schools of Medicine
Saint Louis
United States
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Mueckler, Mike; Thorens, Bernard (2013) The SLC2 (GLUT) family of membrane transporters. Mol Aspects Med 34:121-38
Mueckler, Mike; Makepeace, Carol (2012) Ligand-induced movements of inner transmembrane helices of Glut1 revealed by chemical cross-linking of di-cysteine mutants. PLoS One 7:e31412
Alisio, Arturo; Mueckler, Mike (2010) Purification and characterization of mammalian glucose transporters expressed in Pichia pastoris. Protein Expr Purif 70:81-7
Mueckler, Mike; Makepeace, Carol (2009) Model of the exofacial substrate-binding site and helical folding of the human Glut1 glucose transporter based on scanning mutagenesis. Biochemistry 48:5934-42
Song, Xiao Mei; Hresko, Richard C; Mueckler, Mike (2008) Identification of amino acid residues within the C terminus of the Glut4 glucose transporter that are essential for insulin-stimulated redistribution to the plasma membrane. J Biol Chem 283:12571-85
Mueckler, Mike; Makepeace, Carol (2006) Transmembrane segment 12 of the Glut1 glucose transporter is an outer helix and is not directly involved in the transport mechanism. J Biol Chem 281:36993-8
Mueckler, Mike; Makepeace, Carol (2005) Cysteine-scanning mutagenesis and substituted cysteine accessibility analysis of transmembrane segment 4 of the Glut1 glucose transporter. J Biol Chem 280:39562-8
Mueckler, Mike; Makepeace, Carol (2004) Analysis of transmembrane segment 8 of the GLUT1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility. J Biol Chem 279:10494-9
Mueckler, Mike; Roach, William; Makepeace, Carol (2004) Transmembrane segment 3 of the Glut1 glucose transporter is an outer helix. J Biol Chem 279:46876-81
Alisio, Arturo; Mueckler, Mike (2004) Relative proximity and orientation of helices 4 and 8 of the GLUT1 glucose transporter. J Biol Chem 279:26540-5

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