THe lactose carrier of Escherichia coli is a member of a class of membrane proteins known as cation-substrate cotransporters. These types of proteins are widely found in bacteria, fungi, plant, and animal cells. Examples of human genetic diseases are known in which the primary lesion involves a defect in a cation- substrate cotransporter. The primary aim of the proposed research is a molecular understanding of the relationship between the protein structure of the lactose carrier and its function of cotransporting H+ and lactose into the bacterial cytoplasm. The sugar recognition site will be analyzed by isolating and sequencing several novel classes of lactose carrier """"""""sugar- specificity"""""""" mutants which have alterations in their ability to recognize sugars. It is expected that most """"""""sugar-specificity"""""""" mutants will involve amino acid substitutions which are at, or close to, the sugar recognition site. Further information concerning the importance of particular amino acid side chains for sugar recognition and transport will also be provided by site-directed mutagenesis. In order to obtain a better understanding of the H+ recognition site, """"""""H+-coupling"""""""" mutants will be isolated from parental strains which have aberrant H+ coupling. Some of these mutants may involve interesting changes at, or close to, the H+ recognition site and provide important information concerning the mechanism of H+ coupling. In addition, the existence of possible ionic interactions which may be important for H+ coupling will be investigated via site- directed mutagenesis. Finally, models pertaining to the secondary structure of the lactose carrier within the membrane have relied primarily on the degree of segment hydropathicity. As an alternative, the topology of the lactose carrier within the membrane will be analyzed by isolating and sequencing a collection of lac Y/pho A fusions. Overall, this work should provide important insights into the structure/function relationships within the lactose carrier. Moreover, it is hoped that the general features which are learned about the molecular mechanism of the lactose carrier will ultimately apply to other cation-substrate cotransport systems found in bacteria, fungi, plant, and animal cells.
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