The proposed research focuses on the role of molecular chaperones in protein targeting and translocation. These cellular processes are of fundamental importance. Targeting of newly synthesized proteins is essential for assembly of new cells and there are numerous examples of diseases arising from defects in targeting. Many physiologically important molecules, such as hormones, undergo translocation across membranes. The long term goal is to understand the role of molecular chaperones in protein translocation across the Escherichia coli inner membrane. The following model serves as a working hypothesis for the proposed research: Nascent precursor proteins are first recognized by chaperone proteins that are dedicated to protein export, such as the E. coli SecB protein. Chaperones recognize an unknown structural feature of the nascent polypeptide. Chaperone binding blocks precursor folding, prevents the loss of export competence and ensures that the signal sequence is available for interaction with the next component of the translocation apparatus, probably the peripheral membrane protein SecA. When the precursor binds SecA, it becomes membrane bound and is able to initiate translocation. To test the steps of this model, the following specific aims are proposed: (l) Identification of residues of SecB that are important for binding of nascent polypeptides and for specificity of binding. These studies will involve crosslinking and mutagenesis. (2) Analysis of the identity and location of amino acid sequences/structures within exported protein precursors that promote SecB binding. Regions of polypeptides that require SecB for export will be analyzed for their ability to bind SecB. (3) Analysis of secA mutations that alter the interaction of SecA protein with the SecB/precursor complex. Biochemical characterization of mutants that improve export in the presence of defective SecB will be conducted. (4) Identification of a cytoplasmic factor that stimulates the export of non- SecB-dependent proteins. An alternative export-dedicated chaperone will be identified genetically and/or biochemically.