SUGAR TRANSPORTER OLIGOMERIC STRUCTURE AND FUNCTION

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Cellular nutrient and electrolyte transport is a fundamental but incompletely characterized biological process mediated by two broad classes of transport mechanism - channels and carriers. All mammalian cells transport sugars by a carrier-mediated transport mechanism called glucose uniport or glucose transport. This crucial cellular process provides sugars for ATP synthesis, for maintenance of reducing potential and for the biosynthesis of sugar-containing macromolecules such as glycoproteins, glycolipids and nucleic acids. Sugar transport in muscle, liver and the blood brain barrier are also critically important to organismal energy homeostasis. Impaired transport by these tissues is manifest in diseases that include diabetes, glycogen storage diseases and blood brain barrier disorders. Glucose transport involves catalytic steps common to all carrier-mediated transport mechanisms. Because it is abundantly expressed, the red blood cell and blood brain barrier glucose transport system is amenable to biophysical, biochemical and molecular analysis. This system has thus become a prototype not only for studies of the biologically important sugar transport process but also for understanding the wider family of carrier mechanisms. Despite extensive analysis, the structural basis of protein mediated sugar transport process is unknown. Our long term goal is to understand the molecular mechanism of sugar transport. We propose the following aims in our continuing efforts towards this goal: 1) We have demonstrated that the erythrocyte glucose transporter is an allosteric complex of four identical subunits - the GLUT1 protein. This structure is present in all GLUT1-expressing mammalian cells examined to date. Tetrameric GLUT1 is stabilized by non-covalent interactions between subunits which require a subunit-fold promoted by an intra-subunit disulfide bridge. Disulfide disruption causes transporter dissociation into dimers. Subunits of dimeric and tetrameric GLUT1 appear to interact through N-terminal domains (GLUT1 residues 1-199). Precisely what subsequences mediate these interactions and how these interactions lead to oligomerization are unknown. We outline a co-immunoprecipitation strategy using chimeric transporters to broadly map these domains and to begin to understand their role in GLUT1 oligomerization. 2) Tetrameric GLUT1 transports sugars 15-fold more rapidly than does dimeric GLUT1 and, unlike dimeric GLUT1, is characterized by heterotropic, cooperative interactions between sugar import and export sites. Our recent studies show that cooperative ligand binding is not always required for rapid sugar transport and that other, uncharacterized subunit interactions promote rapid substrate translocation by tetrameric GLUT1. We describe sugar transport and ligand binding experiments that exploit chimeric transporters from Aim 1 to map GLUT1 domains required for rapid transport function and/or cooperative ligand binding. We thereby determine whether these domains are separate from or identical to oligomerization domains.