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J. Biol. Chem., Vol. 282, Issue 17, 13022-13032, April 27, 2007

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Intracellular Localization of Type III-delivered Pseudomonas ExoS with Endosome Vesicles^*

From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

ExoS (453 amino acids) is a bi-functional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96-219 include the Rho GTPase-activating protein (RhoGAP) domain, and residues 234-453 include the 14-3-3-dependent ADPribosyltransferase domain. Earlier studies also identified an N-terminal domain (termed the membrane localization domain) that comprises residues 51-77 and includes a novel leucine-rich motif that targets ExoS to the perinuclear region of cultured cells. There is limited information on how ExoS or other type III cytotoxins enter and target intracellular host proteins. Type III-delivered ExoS localized to both plasma membrane and perinuclear region, whereas ExoS(MLD) was localized to the cytosol. Plasma membrane localization of ExoS was transient and had a half-life of 20 min. Type III-delivered ExoS co-immunoprecipitated 14-3-3 proteins and Rab9, Rab6, and Rab5. Immunofluorescence experiments showed that ExoS colocalized with Rab9, Rab6, and Rab5. Fluorescent energy transfer was detected between ExoS and 14-3-3 proteins but not between ExoS and Rabs proteins. Together, these results indicate that type III-delivered ExoS localizes on the host endosomes and utilizes multiple pathways to traffic from the plasma membrane to the perinuclear region of intoxicated host cells.

Received for publication, July 3, 2006 , and in revised form, February 15, 2007.

^* This work was supported by National Institutes of Health Grant AI030162. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Medical College of Wisconsin, Dept. of Microbiology and Molecular Genetics, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8412; Fax: 414-456-6535; E-mail: jtb01{at}mcw.edu.

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