BIOCHEMICAL STUDIES OF ERYTHROCYTE TRANSPORT PROTEINS

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The long-term goal of this project is to understand the molecular mechanisms of protein-mediated transport across biological membranes. The proposal is focused on three red blood cell membrane proteins: the C1--HCO3- exchanger (band 3 or AE1); a 43 kDa lactic acid transporter; and the water channel protein, CHIP 28. All three of these proteins are abundant enough to make it possible to study structure-function relations in the native membrane. The first aim is to use biochemical techniques to refine the topographic model of the arrangement of the band 3 polypeptide in the membrane and to define arginine, lysine and aspartate or glutamate residues of functional importance. Recent data indicate that red cell band 3 polypeptide may be heterogeneous as a result of alternately spliced RNA; the nature of this heterogeneity will be characterized in detail, using both biochemical and molecular biological approaches. Further advances in the understanding of band 3 will require higher resolution structure. One of the goals of the proposed work is to prepare two-dimensional crystals of band 3 in native membranes; these crystals will provide material for structural studies in other laboratories. Lactate transport is of significance in several pathophysiological situations, including diabetes, ischemic heart disease, and neoplasia. However, almost nothing is known about the transport protein itself. The cDNA for the protein will be cloned and sequenced from an erythroid library, using probes derived from the sequence of isolated protein. The cDNA sequence will then be used as a guide for further studies of structure-function relations in this protein. The function of the protein will also be studied in intact cells under pre-steady state conditions, to test a kinetic model for the catalytic cycle for transport. The water channel (CHIP 28) will be characterized with respect to the topography of the protein, using a monoclonal antibody recently raised in this laboratory. The goal of the studies is to test a simple structural model for the folding of the protein in the membrane. The locations of functionally important amino acid residues will also be determined, using methods that have been developed for band 3. Finally, the rate of proton or hydroxyl ion transport through the water channel will be quantified.