Studies with the gram-negative bacterium Escherichia coli have indicated that proteins are exported to the periplasm and outer membrane of this organism by a mechanism similar to that proposed for the initial steps of protein secretion in eukaryotic cells. For the past several years, our laboratory has been extensively investigating the synthesis and secretion of the periplasmic maltose-binding protein (MBP) of E. Coli using a combination of genetic and biochemical approaches. Our long term goal is to fully understand, at the molecular level, the mechanism by which the MBP is exported from its site of synthesis in the cytoplasm, through the cytoplasmic membrane, to the periplasm. The following are our specific aims for the proposed five-year project period: (1) We would like to further define the functional determinants of the MBP signal peptide. For example, we hope to determine the absolute minimal requirements for a signal peptide that can facilitate MBP export at a rate and efficiency comparable to the wild-type structure. (2) An attempt will be made to directly demonstrate the existence of an """"""""internal export signal"""""""" (IES) that resides in the mature MBP, far removed from the signal peptide, and to determine its exact role in the export process. (3) The possibility that important export information resides at the extreme amino-terminus of the mature MBP also will be investigated. (4) The pr1D gene and other genes that we identify that are thought to encode specific components of the cell's protein export machinery will be cloned, and the protein products identified and characterized. (5) We hope to use various E. coli mutants to help us to dissect the MBP export pathway. By blocking MBP export at different steps, such mutants could allow us to more easily demonstrate a direct interaction between the MBP and known components of the export machinery at various points along this pathway. (6) We have recently developed an in vitro system in which precursor MBP is efficiently synthesized, imported into inverted E. coli cytoplasmic membrane vesicles, and processed to the mature form. We believe that we can use this system to gain additional insight into the MBP export process.
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