Proteins exported via the primary export pathway of the gram negative bacterium Escherichia coli are synthesized as precursors with amino- terminal signal peptides that are thought to have major roles in the export process, including entry into the pathway and translocation across the cytoplasmic membrane. To date, many, perhaps all, of the protein components of the export pathway have been identified. However, the precise role of these components and how they interact with one another, membrane lipids, and the signal peptide and mature moiety of precursor proteins to facilitate the export process have yet to be determined. For the past few years, our laboratory has been intensively investigating the synthesis and secretion of the periplasmic maltose-binding protein (MBP) of Escherichia coli using a combination of genetic and biochemical approaches. Our long term goal is to understand, at the molecular level, the mechanism by which the MBP is exported from its site of synthesis in the cytoplasm to the periplasm. The following are our specific aims for the proposed five year project period: (1) We will continue our analysis of the MBP signal peptide with particular emphasis on the cleavage site and in understanding why certain mutant signal peptides promote unusually rapid MBP export. (2) Studies on the cleavage site of preMBP have provided two new genetic selections that may yield mutants with alterations in SPase I or other known or unknown components of the export machinery. (3) The recognition and translocation of export-defective MBP species in E. coli cells harboring various pr1 suppressor alleles will be investigated further. (4) We will determine if SecB specifically recognizes the mature moiety of precursor MBP, or if such an interaction is not sequence specific and/or is partly determined by the signal peptide or other factors. (5) Experiments are planned to distinguish between the chaperone and targeting functions of SecB, including attempts to isolate mutations in secA or secB that specifically inhibit the targeting activity. (6) In an effort to understand why some proteins are exported in a SecB-independent manner, we will expand our studies addressing the localization of the periplasmic ribose-binding protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI017292-12
Application #
3127114
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-08-01
Project End
1996-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
12
Fiscal Year
1991
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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