EXPORT OF PROTEINS IN ESCHERICHIA COLI

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Translocation of specific polypeptides across or into biological membranes occurs during eukaryotic secretion, organelle biogenesis and prokaryotic protein export. The research proposed here is aimed at elucidation of this widespread and important phenomenon through studies of the export of the soluble maltose-binding protein from the cytoplasm into the periplasm of Escherichia coli. Strong emphasis is put on understanding the role of protein folding during the process, including both the intrinsic folding properties of the proteins to be exported and the modulation of that folding by the interaction of those polypeptides with components of the apparatus particularly SecB and SecA. SecB plays a crucial role in maintaining precursor maltose-binding protein in a loosely folded state that is competent for export. SecB displays a remarkable ability to recognize many different polypeptide ligands as nonnative. Several of the specific aims of this proposal are directed toward testing a working model for the mechanism of recognition of nonnative structure by SecB. To this end the regions of the polypeptide ligand and the regions of SecB that interact will be characterized. Proteolytic treatment of the SecB.ligand complex followed by isolation of the peptides that remain bound to SecB will allow the determination of the sequences within the ligand that define the binding motif. The sites on SecB that are in contact with ligands will be characterized using chemical crosslinkers and fluorescent probes coupled to peptides that have been shown previously to bind SecB. SecB is a member of a broad family of proteins, the chaperones, that are found from bacteria to mammalian cells and that interact with loosely structured polypeptides to facilitate a wide range of processes including protein folding and localization. Thus what is learned about recognition of nonnative structure by SecB will be of wide significance. During export the precursor interacts with SecB, SecA and the membrane translocase. The contribution of the leader peptide and the folding properties of precursor maltose-binding protein to each of these interactions will be assessed in three systems. Interactions between purified components will be studied by ultrasensitive titration calorimetry to establish the binding parameters. Calorimetry will also be used in studies of interaction of model peptide ligands and SecB. A cell free system will be employed to analyze the complexes characterized by calorimetry for efficiency in promoting translocation of precursors into inverted membrane vesicles. The pathway of export in vivo will be delineated through analyses of exponentially growing cultures of E. coli. The cultures will be radiolabelled and the passage of precursors from one complex to the next will be followed by co-immunoprecipitation of the various components. All three systems will make use of a series of well- characterized species of maltose-binding proteins with altered folding properties to assess the importance of conformation in each of the steps of export. The proposed research spans the spectrum from biophysical and biochemical studies of purified proteins to analyses of exponentially growing cells. It is from the integration of data obtained in all of these systems that we shall learn the most.