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Journal of Bacteriology, February 2000, p. 742-748, Vol. 182, No. 3
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Two Regions of EpsL Involved in Species-Specific Protein-Protein Interactions with EpsE and EpsM of the General Secretion Pathway in Vibrio cholerae

Maria Sandkvist,1,* Jerry M. Keith,2 Michael Bagdasarian,3 and S. Peter Howard4

Department of Biochemistry, American Red Cross, Holland Laboratory, Rockville, Maryland 208551; Vaccine and Therapeutic Development Section, Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892-43502; Department of Microbiology, Michigan State University, East Lansing, Michigan 488243; and Department of Biology, University of Regina, Regina, Saskatchewan, Canada S4S 0A24

Received 24 August 1999/Accepted 8 November 1999

Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from Aeromonas hydrophila together with either EpsE or its Aeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.

* Corresponding author. Mailing address: Department of Biochemistry, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Phone: (301) 738-0604. Fax: (301) 738-0794. E-mail: sandkvis{at}usa.redcross.org.

Journal of Bacteriology, February 2000, p. 742-748, Vol. 182, No. 3
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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