This Article Full Text Full Text (PDF) Other Versions of this Article: JB.01103-06v1 189/2/446    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ureta, A. R. Articles by Silhavy, T. J. Search for Related Content PubMed PubMed Citation Articles by Ureta, A. R. Articles by Silhavy, T. J.

Kinetic Analysis of the Assembly of the Outer Membrane Protein LamB in Escherichia coli Mutants Each Lacking a Secretion or Targeting Factor in a Different Cellular Compartment

Alejandro R. Ureta, Robert G. Endres, Ned S. Wingreen, and Thomas J. Silhavy^*

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

Received 25 July 2006/ Accepted 16 October 2006

Outer membrane ß-barrel proteins in gram-negative bacteria, such as Escherichia coli, must be translocated from their site of synthesis in the cytoplasm to the periplasm and finally delivered to the outer membrane. At least a dozen proteins located in the cytoplasm, the periplasm, and both the inner and outer membranes are required to catalyze this complex assembly process. At normal growth temperatures and conditions the transport and assembly processes are so fast that assembly intermediates cannot be detected. Using cells grown at a low temperature to slow the assembly process and pulse-chase analysis with immunodetection methods, we followed newly synthesized LamB molecules during their transit through the cell envelope. The quality and reproducibility of the data allowed us to calculate rate constants for three different subassembly reactions. This kinetic analysis revealed that secB and secD mutants exhibit nearly identical defects in precursor translocation from the cytoplasm. However, subsequent subassembly reaction rates provided no clear evidence for an additional role for SecD in LamB assembly. Moreover, we found that surA mutants are qualitatively indistinguishable from yfgL mutants, suggesting that the products of both of these genes share a common function in the assembly process, most likely the delivery of LamB to the YaeT assembly complex in the outer membrane.

Published ahead of print on 27 October 2006.

Present address: Laboratory for Vaccine Research, Department of Biotechnology, Instituto de Higiene, Facultad de Medicina, Avda. A. Navarro 3051, Montevideo, CP 11600 Uruguay.

This article has been cited by other articles:

• Vuong, P., Bennion, D., Mantei, J., Frost, D., Misra, R. (2008). Analysis of YfgL and YaeT Interactions through Bioinformatics, Mutagenesis, and Biochemistry. J. Bacteriol. 190: 1507-1517 [Abstract] [Full Text]  
• Sklar, J. G., Wu, T., Kahne, D., Silhavy, T. J. (2007). Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Genes Dev. 21: 2473-2484 [Abstract] [Full Text]  
• Jain, S., Goldberg, M. B. (2007). Requirement for YaeT in the Outer Membrane Assembly of Autotransporter Proteins. J. Bacteriol. 189: 5393-5398 [Abstract] [Full Text]  
• Sklar, J. G., Wu, T., Gronenberg, L. S., Malinverni, J. C., Kahne, D., Silhavy, T. J. (2007). Lipoprotein SmpA is a component of the YaeT complex that assembles outer membrane proteins in Escherichia coli. Proc. Natl. Acad. Sci. USA 104: 6400-6405 [Abstract] [Full Text]