REDESIGN OF STRUCTURAL REGIONS OF ALKALINE PHOSPHATASE

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The correct transport of proteins must occur across the membranes of all prokaryotic and eukaryotic cells. The unifying feature among secreted proteins in all systems is the requirement for a signal peptide. The long-term goals of this work are to identify the physical properties of signal peptides which are necessary for protein secretion, and to delineate the interactions between the signal peptide and components of the protein transport apparatus which serve to direct a protein to its final destination. Using Escherichia coli as a model system, the specific aims of the proposed research are to: (1)determine the characteristics of model preproteins, including the modulatory factors of signal peptides, which confer SecB dependence; (2)determine the substrate specificity of SecA with regard to the physical properties of signal peptides and preproteins; (3)determine the properties of SecA required for signal peptide binding; (4) determine how SecYEG modulates signal peptide interactions; (5)determine how interactions between the signal peptide and components of the secretion machinery are integrated to achieve transport overall. For these studies we will use alkaline phosphatase as a prototype, redesigning its signal peptide or mature regions with model sequences designed to amplify certain traits to test the roles of hydrophobicity, conformation, and charge. Mutants with these sequences are evaluated in vivo for the extent to which different steps of the secretion process are accomplished in wild type and Sec- deficient host strains. Representatives will be used for in vitro analyses to establish direct interactions between signal peptides with particular properties and the Sec machinery. Biochemical analyses and direct binding studies with the corresponding synthetic signal peptides are designed with the aim of establishing the same hierarchy for binding in vitro as we observe for function in vivo. Knowledge of how signal peptides enhance correct compartmentalization in bacteria will be useful in understanding secretion in other normal and diseased cells. The principles which evolve can be applied to the tissue-specific targeting of therapeutic agents, the design of vehicles to transport other proteins, and the development of transport inhibitors.