REDESIGN OF STRUCTURAL REGIONS OF ALKALINE PHOSPHATASE

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Many proteins which are synthesized in the cytoplasm of cells are ultimately found in noncytoplasmic locations. The correct targeting and transport of proteins must occur across the endoplasmic reticulum membrane, the peroxisomal membrane, the membranes of mitochondria and chloroplasts and those of bacterial cells. The unifying feature between secreted proteins in all systems is the requirement for a signal peptide. The long-term objectives of this work are to determine the structural requirements of signal peptides that are necessary for protein secretion and to elucidate how these physical properties interface a protein to be exported with the secretion pathway. The earmark of a signal peptide is a cluster of hydrophobic residues in its central, hydrophobic core region. This structural feature plays a role in the membrane insertion and translocation steps of secretion and may provide a critical recognition element linking the preprotein to the secretion pathway. The specific aims of the proposed research are to determine the characteristics of an optimal signal peptide hydrophobic core unit and how the properties of this domain interface with other signal peptide subsegments during different stages of the secretion process. The role of this domain in SecA and SecY interactions and in differentiating SecB-dependent and independent pathways will then be examined. For this purpose, a systematic series of mutants of the Escherichia coli alkaline phosphatase gene will be produced. The mutant signal sequences will be designed to amplify certain physical traits to test the roles of conformation, length, hydrophobicity and overall topology. These will be evaluated in vivo for the extent to which different steps of the secretion process are accomplished. Representative series will then 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 synthetic signal peptides are designed with the aim of establishing the same hierarchy for binding in vitro as we observe for function in vivo. The features of the hydrophobic core domain which are amenable to change in response to environment and those which are universally critical for secretion and are thus, conserved, will be highlighted through a comparative analysis between E. coli and the thermophile, Thermotoga neapolitana. The movement of proteins across membranes is vital to the health of all cells. Understanding the structural features of signal peptides which enhance correct compartmentalization in bacteria will be useful for probing their eukaryotic counterparts. The principles which evolve can be applied to the tissue-specific targeting of therapeutic agents and the design of vehicles to transport other proteins, including eukaryotic proteins, into the E. coli periplasm for subsequent isolation.