The polybasic region of Rho GTPases defines the cleavage by Yersinia enterocolitica outer protein T (YopT).
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.
Fueller
Florian
F
Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, 79104 Freiburg, Germany.
Schmidt
Gudula
G
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D054876:Prenylation; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C116117:YopT protein, Yersinia; D003546:Cysteine Endopeptidases; D020741:rho GTP-Binding Proteins