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Determination of a region in SecA that interacts with presecretory proteins in Escherichia coli
E Kimura1, M Akita, S Matsuyama
1Institute of Applied Microbiology, University of Tokyo, Japan.
Insights
SecA protein fragments were reconstituted to identify the region responsible for binding presecretory proteins. The study found this crucial SecA region is located between amino acid residues 267 and 340.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein-protein interactions
Background:
- SecA protein is essential for protein transport across the bacterial membrane.
- SecA recognizes the signal peptide of presecretory proteins, particularly its positive charge.
- Understanding SecA's interaction domain is key to elucidating protein translocation mechanisms.
Purpose of the Study:
- To map the specific region of SecA responsible for cross-linking with presecretory proteins.
- To investigate the structural requirements of SecA fragments for interaction with signal peptides.
- To determine the influence of ATP and other presecretory proteins on SecA-substrate binding.
Main Methods:
- Preparation and reconstitution of various amino- and carboxyl-terminal SecA fragments.
- Cross-linking assays using [35S]proOmpF-Lpp and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
- Analysis of cross-linked fragments by SDS-PAGE to identify binding domains.
Main Results:
- Reconstituted SecA analogues cross-linked with proOmpF-Lpp when fragments structurally complemented.
- Cross-linking was signal peptide-dependent, enhanced by ATP, and reduced by competing proteins.
- The primary cross-linking site was on amino-terminal fragments, with a minimal size requirement.
- The interaction domain was localized to residues 267-340 of SecA.
Conclusions:
- The SecA region between residues 267 and 340 is critical for binding presecretory proteins.
- Structural integrity and specific domains of SecA are necessary for substrate recognition.
- These findings provide insights into the molecular mechanism of protein translocation by SecA.
Abstract:
SecA interacts with presecretory proteins through recognition of the positive charge at the amino terminus of the signal peptide (Akita, M., Sasaki, S., Matsuyama, S., and Mizushima, S. (1989) J. Biol. Chem. 265, 8164-8169). A large variety of amino-terminal and carboxyl-terminal fragments of SecA were prepared in 6 M guanidine hydrochloride. SecA analogues were then reconstituted from them and examined for their ability to cross-link with [35S]proOmpF-Lpp, a presecretory protein, in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The reconstituted SecA analogues were active in the cross-linking with proOmpF-Lpp when the SecA fragments used were large enough to structurally complement each other. The cross-linking was signal peptide-dependent and suppressed in the presence of other presecretory proteins. The cross-linking was enhanced in the presence of ATP. The SecA fragments that cross-linked with proOmpF-Lpp were then analyzed on sodium dodecyl sulfate-polyacrylamide gels. The cross-linking preferentially took place on fragments possessing the amino terminus of SecA. Weak cross-linking was also observed with carboxyl-terminal fragments when they were large enough. The smallest amino-terminal and carboxyl-terminal fragments with which the cross-linking was observed were 39 and 72 kDa, respectively. From these results, the region responsible for the cross-linking with presecretory proteins was deduced to be located between amino acid residues 267 and 340 from the amino terminus of SecA. These results are discussed in relation to the structure and function of SecA.