Protein-mediated facilitative glucose transport is vital to cellular metabolism and organismal carbohydrate homeostasis. In spite of extensive study, the mechanism of glucose transport is unknown. The long-range goal of this laboratory is to understand the molecular mechanism of protein-mediated glucose transport. To achieve this goal, we propose to investigate the structure and function of the human glucose transport protein Glut1. This mammalian glucose transport protein is uniquely amenable to analysis being available as a purified protein from red blood cells and as a recombinant protein expressed in, and affinity-purified from Cos-7 cells.
Specific Aims 1, 4, and 5 test the hypothesis that Glut1 functions as a cooperative homo tetramer in the cell membrane.
Specific Aim 1 asks if tetrameric Glut1 is an artifact of isolation by investigating the effects of different detergents and lipids on transporter oligomeric size.
Specific Aim 4 measures Glut1 cooperativity by stopped flow analysis of ligand binding to ask if perturbations of Glut1 oligomeric size by cysteine mutagenesis and by heterocomplex formation also affect Glut1 cooperativity.
Specific Aim 5 exploits an unanticipated and recently discovered result of Glut1 cooperativity (transport stimulation by subsaturating levels of competitive inhibitors) to ask if altered Glut1 oligomeric size affects this cooperative behavior. Carbohydrate libraries will also be screened for potential """"""""kinetic activators"""""""" of transport.
Specific Aims 2 and 3 address Glut1 structure.
Specific Aim 2 proposes tryptophan mutagenesis to identify Glut1 tryptophan residue(s) that mediate Glut1 tryptophan fluorescence quenching when ligands bind specifically at sugar uptake or sugar export sites.
Specific Aim 3 maps Glut1 sugar uptake and export domains by use of site-specific photo-ligands, peptide mapping and peptide sequencing. Identified sites will be subjected to alanine scanning mutagenesis and the recombinant Glut1 will be remapped and tested for activity to confirm or refute loss of function.
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