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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI017292-11
Application #
3127119
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-08-01
Project End
1991-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
11
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Barkocy-Gallagher, G A; Cannon, J G; Bassford Jr, P J (1994) Beta-turn formation in the processing region is important for efficient maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo. J Biol Chem 269:13609-13
Barkocy-Gallagher, G A; Cannon, J G; Bassford Jr, P J (1994) Thirty-three amino acids of the mature moiety of an unprocessed maltose-binding protein are sufficient for export in Escherichia coli. J Bacteriol 176:3397-9
Strobel, S M; Cannon, J G; Bassford Jr, P J (1993) Regions of maltose-binding protein that influence SecB-dependent and SecA-dependent export in Escherichia coli. J Bacteriol 175:6988-95
Barkocy-Gallagher, G A; Bassford Jr, P J (1992) Synthesis of precursor maltose-binding protein with proline in the +1 position of the cleavage site interferes with the activity of Escherichia coli signal peptidase I in vivo. J Biol Chem 267:1231-8
Puziss, J W; Harvey, R J; Bassford Jr, P J (1992) Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP. J Bacteriol 174:6488-97
Puziss, J W; Strobel, S M; Bassford Jr, P J (1992) Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations. J Bacteriol 174:92-101
Fikes, J D; Barkocy-Gallagher, G A; Klapper, D G et al. (1990) Maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo. Sequence requirements for efficient processing and demonstration of an alternate cleavage site. J Biol Chem 265:3417-23
Weiss, J B; Bassford Jr, P J (1990) The folding properties of the Escherichia coli maltose-binding protein influence its interaction with SecB in vitro. J Bacteriol 172:3023-9
Bassford Jr, P J (1990) Export of the periplasmic maltose-binding protein of Escherichia coli. J Bioenerg Biomembr 22:401-39
Collier, D N; Strobel, S M; Bassford Jr, P J (1990) SecB-independent export of Escherichia coli ribose-binding protein (RBP): some comparisons with export of maltose-binding protein (MBP) and studies with RBP-MBP hybrid proteins. J Bacteriol 172:6875-84

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