The long-term aim of this proposal is to determine how the Na+/glucose cotransporter actually transports Na+ and glucose across the membrane. To achieve this aim it is necessary to unravel the molecular architecture of the transport protein. The short-term goal is to identify the Na+ and sugar binding sites of the protein and the long-term goal is to delineate the structural topology and conformations of the isolated protein. The Na+, sugar and inhibitor (phlorizin) binding sites on the cotransporter will be identified using three strategies: The first, is to label the Na+/glucose cotransport in brush border membrane vesicles at tyrosine residues occurring at the Na+ binding site, and lysine residues at the glucose and phlorizin binding sites. The protein will be cleaved by proteolysis, purified, and sequenced; The second, is to employ molecular engineering techniques to mutate tyrosine and lysine residues thought to be at the active sites. We will then determine whether or not these are the residues labeled by group specific reagents, and establish whether or not the tyrosine and/or lysine residues are critically involved in Na+/glucose cotransport; and third, we will use molecular techniques to test the hypothesis that Gly43 and Arg300 are involved in Na+ binding and transport. Transport properties of mutants will be assayed in Xenopus oocytes, and immunoprecipitation techniques will be utilized to confirm the presence of the mutant proteins in the oocyte plasma membrane. Our initial strategy toward achieving the long-term goal of purifying the Na+/glucose cotransporter will involve overexpression in E. coli and in cultured insect cells. Fusion proteins will be expressed in bacteria, and baculovirus constructs will be used to overexpress the cotransporter in sf9 cells. With high expression levels, at least two orders of magnitude greater than in enterocytes, we will be well positioned to purify the protein by electrophoresis and chromatography. The protein will first be used to raise antibodies, but the long-term objective is to use the protein for structure and functional studies.
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