20] Analysis of transporter topology using deletion and epitope tagging

Abstract

Publisher Summary This chapter describes the analysis of transporter topology using deletion and epitope tagging. Several strategies have been employed to elucidate the structure and transmembrane topology of transport proteins. An understanding of transporter structure is crucial for determining the correlation between transporter structure and function. In addition, structural knowledge aids in the design of more specific transporter reagents, which may prove therapeutically useful. Current studies of the transmembrane topology of integral membrane proteins using deletion analysis and epitope tagging derive from gene fusion methods designed to study secretion and topology of bacterial proteins. In these original experiments, a modified form of the Escherichia coli phoA gene, which encodes the secreted periplasmic enzyme alkaline phosphatase, was fused to the C-terminal portion of a protein of interest. Therefore, the level of alkaline phosphatase activity is a direct indication of the location of the enzyme in bacteria and a useful tool for discerning the secretion or topogenic properties of bacterial proteins. The phoA gene fusion method has been used to determine the transmembrane topology of the C-terminal half of the bacterial glutamate transporter in E. coli .