STRUCTURE AND MECHANISM OF THE E COLI MANNITOL PERMEASE

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Our laboratory is actively investigating the structure and mechanism of the enzyme permease responsible for the tightly coupled transport and phosphorylation of the hexito: D-mannitol in Escherichia coli. This protein is an integral membrane component of the bacterial phosphotransferase system (PTS) as well as serving as the primary chemotactic receptor for D-mannitol in E. coli. For the past 8 years, its structure and its catalytic mechanism have been studied in several laboratories, including our own. The broad objective of these studies is to determine in as much molecular detail as possible the mechanism by which the mannitol permease carries out its receptor and transport function. This is not completely known for any integral membrane transport protein, and our results should therefore add fundamental knowledge to the study of membrane protein structure and function. Since all cells rely on proper functioning of integral membrane proteins for interaction with their environment, and some diseased cells may lack one or more of theses functions, the study of these fundamental processes is essential in understanding the principles of growth and metabolism in both normal and diseased cells. In the proposed project continuation our aims are to: 1) continue our studies on the intramembrane structure of the membrane-bound domain of the mannitol permease; 2) study the structure of the cytoplasmic domain of the protein; 3) continue our studies on the mechanism of transport and phosphorylation catalyzed by this protein; 4) study structure-function relationships in this protein using in vitro mutagenesis techniques; and 5) isolate and characterize mutants in specific functions of the mannitol permease. A combination of biochemical, biophysical, molecular biological and genetic techniques will be used in these studies. Successful completion of this work will lead to significant progress toward our overall goal of understanding the molecular basis of transport in this system.