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Journal of Bacteriology, November 2003, p. 6540-6547, Vol. 185, No. 22
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.22.6540-6547.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Assembly of TolC, a Structurally Unique and Multifunctional Outer Membrane Protein of Escherichia coli K-12

John Werner,1 Anne Marie Augustus,2,{dagger} and Rajeev Misra1,3*

Molecular and Cellular Biology Graduate Program,1 Department of Chemistry and Biochemistry,2 Department of Microbiology, Arizona State University, Tempe, Arizona 852873

Received 13 May 2003/ Accepted 20 August 2003

TolC is a multifunctional outer membrane protein of Escherichia coli that folds into a novel {alpha}-ß-barrel conformation absent in the other model outer membrane proteins used in assembly studies. The data presented in this work show that the unique folded structure of TolC reflects a unique assembly pathway. During its assembly, the newly translocated nascent TolC monomers are released in the periplasm. Maturation of these nascent monomers, and possibly their oligomerization, in the periplasm precedes their insertion in the outer membrane. The completion of the assembly process is signaled by the development of a characteristic proteinase K-resistant fragment generated by cleavage at a single, periplasmically exposed, protease-sensitive site of the membrane-anchored trimer. None of the assembly steps of TolC is affected by known folding factors, such as SurA, Skp, and lipopolysaccharide, which have profound effects on the assembly of other model trimeric outer membrane proteins. Two assembly-defective TolC mutants were isolated and characterized. One of the mutants (TolCI106N) was defective in the folding of nascent monomers, while the other (TolCS350F) was impaired in steps involving trimerization and membrane insertion of folded monomers.

* Corresponding author. Mailing address: Department of Microbiology, Arizona State University, LSE407, Tempe, AZ 85287-2701. Phone: (480) 965-3320. Fax: (480) 965-6899. E-mail: rajeev.misra{at}asu.edu.

{dagger} Present address: Cell and Molecular Biology Program, Duke University, Durham, NC 27708.

Journal of Bacteriology, November 2003, p. 6540-6547, Vol. 185, No. 22
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.22.6540-6547.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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