We are studying the mechanisms of protein translocation into, and across, biological membranes. The process is basic to subcellular compartmentation and is a vital aspect of processes from hormone secretion to bacterial pathogenesis. We have purified and reconstituted E. coli preprotein translocase, the complex protein which transports preproteins across the plasma membrane. We have identified its major subunits and established the fundamentals of its catalytic cycle. Preprotein translocase has a SecA peripheral membrane domain, which is bound to both acidic lipids and to a membrane-embedded, heterohexameric integral membrane domain of SecYEGDFyajC. The SecA subunit carries successive loops of preprotein across the membrane, driven in its insertion and de-insertion cycle by the energy of ATP binding and hydrolysis. We now propose to purify the overproduced, epitope-tagged translocase, collaboratively determine its structure, and explore the environment of the transiting chain, the leader (signal) peptide, and the SecA subunit as it inserts across the membrane with the preprotein. Dynamic questions of translocase function will include the topology of subunits, their exchange among holoenzyme complexes during the catalytic cycle, and the roles of lipid and proton-flux in translocation. Finally, we will study the assembly of integral membrane proteins, whether through this translocase or via spontaneous insertion. This phase of the work can now actually address the fundamental mechanistic questions of secretion and membrane assembly.
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