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The polybasic region of Rho GTPases defines the cleavage by Yersinia enterocolitica outer protein T (YopT)

Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, 79104 Freiburg, Germany

(RECEIVED March 18, 2008; FINAL REVISION May 21, 2008; ACCEPTED May 27, 2008)

Pathogenic Yersinia strains evade the innate immune responses of the host by producing effector proteins (Yersinia outer proteins [Yops]), which are directly injected into mammalian cells by a type III secretion system (TTSS). One of these effector proteins (YopT) disrupts the actin cytoskeleton of the host cell resulting in cell rounding. YopT is a cysteine protease that cleaves Rho proteins directly upstream of the post-translationally modified cysteine. Thereby, it releases the GTPases from the membrane leading to inactivation. Small GTPases are modified by isoprenylation of the cysteine of the CAAX box, cleavage of the –AAX tripeptide, and methylation of the cysteine. We have shown that isoprenylation and the endoproteolytic cleavage of the tripeptide of Rho GTPases are essential for YopT-induced cleavage, whereas carboxyl methylation is not required. In the present study, we post-translationally modified RhoA, Rac, Cdc42, and several mutants in vitro and characterized the YopT-induced cleavage with recombinant YopT. We show that farnesylated RhoA is a preferred substrate of YopT compared with the geranylgeranylated GTPase. Geranylgeranylated RhoA, however, is the preferred substrate for YopT-catalyzed cleavage with a threefold faster turnover rate over Rac and Cdc42. Moreover, our data indicate that the composition of the polybasic region of the GTPases defines the specificity and efficiency of the YopT-induced cleavage, and that a space between the polybasic stretch of amino acids at the C terminus and the CAAX box enhances the turnover rate of YopT-catalyzed cleavage.

Keywords: bacterial toxin; YopT; Rho GTPases; post-translational modification; polybasic region

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.035386.108.

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