The long-term objective of this proposal is to understand the physiological and structural basis for the function of membrane transport processes.
The specific aim i s to study two sugar-cation cotransport carriers of E. coli as """"""""model system"""""""" for similar carriers in animal cells (this laboratory has had a longstanding interest in the glucose-Na+ cotransport system of the small intestine). We postulate that the earliest cell in evolution utilized a """"""""proton economy"""""""", using proton pumps and utilizing the proton elec- trochemical gradient for cotransport processes. Later in evolution animal cells evolved Na+-K+ pumps and the Na+ gradient was used for cotransport processes. One example of a possible intermediate stage in evolution is the melibiose carrier which can use either H+ or Na+, for cotransport. One experimental approach will be to determine the amino acid sequence of several carrier proteins to see the relationship between carriers of different cell types. The lactose carrier or melibiose carrier from several microorganisms will be cloned and sequenced by the Sanger dideoxynucleotide method. A correlation will be made between the conserved regions of the transport protein and the physiological parameters common to each. A second approach will be to determine the amino acids in the carrier protein that normally recognize the sugar on the one hand and the cation on the other. Sugar recognition and cation recognition mutants will be isolated with several techniques which we have successfully utilized in the past. The gene for the carrier protein will be isolated and the DNA sequenced. Analysis of the amino acid substitutions found by this method should provide useful information concerning the binding sites. A third approach will be a study of the orientation of various segments of the molecule with respect to the membrane. Both genetic as well as immunological methods will be utilized.
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