MECHANISM OF PROTEIN LOCALIZATION IN ESCHERICHIA COLI

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Our overall goal is to elucidate the molecular details of preprotein targeting to and translocation through biological membranes using the simple bacterium, Escherichia coli, as a facile genetic and biochemical system in this undertaking. Although rapid progress has been made in elucidating some of the basic features of E. coli protein secretion, most steps in this pathway are not understood in sufficient detail to construct a coherent biochemical scheme. Towards this goal we will focus on a central component of this system, SecA ATPase, which interacts with preproteins, the SecB chaperone, anionic phospholipids, integral membrane proteins SecY/SecE, as well as its own mRNA, and is the translocation ATPase required for preprotein insertion and passage across the inner membrane. Many of these interactions will be defined further, their molecular basis determined, their regulation by nucleotide binding and hydrolysis investigated, and their real purpose in the catalysis of protein secretion deciphered. In order to understand the function(s) of this complex ATPase, the distinct biochemical activities catalyzed by the two separate ATP-binding domains of SecA will be determine. The basis of SecA recognition of preproteins will be investigated employing a combined genetic and biochemical strategy to elucidate the structural features of the signal peptide and mature region of the preprotein that allow SecA recognition, and regions of SecA important in promoting these interactions will be determined. The basis of SecA's peripheral and integral associations with the inner membrane will be investigated further by identifying the SecA receptor and defining components that promote and regulate SecA's integral association with the membrane. A novel mapping technique employing cysteine-specific cleavage will be used to locate with high precision the ATP-binding, preprotein-binding, lipid- binding, and mRNA-binding sites on SecA's primary structure. A refined structural knowledge of SecA protein will be gained through collaborative studies to crystallize SecA protein or an important SecA substructure and determine its three dimensional structure at atomic resolution. Finally, the basis for SecA regulation and its coordination with the protein secretion status of the cell will be determine using genetic and biochemical approaches to locate the secretion-responsive element on geneX-secA mRNA, assess the importance of geneX translation termination and SecA protein binding in the function of this element, and identify any additional factors important in secA regulation. These studies should be of broad significance to understanding these basic problems in other protein secretion systems in normal and abnormal cells states as well as of practical importance in attempting to modify the export pathway to accommodate novel substrates.