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

Abstract

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.

Figure 1.

Farnesylated RhoA is the preferred substrate of YopT. RhoA wild type and RhoA(L193S) (a mutant that is farnesylated) was post-translationally modified and ¹⁴C-labeled. After determination of the concentration of radiolabeled, isoprenylated RhoA (2.1 μM ± 0.2), the proteolytic cleavage of farnesylated RhoA (∇, n = 3) and geranylgeranylated RhoA (●, n = 6) was initiated by addition of YopT in a 1:10 ratio (YopT: GTPase) at 37°C. The reaction was terminated by the addition of 5% SDS. The proteins were precipitated with 30% TCA and ¹⁴C-labeled proteins were quantified using a filter assay. Efficiency of YopT cleavage was illustrated as a decrease of radiolabeled protein in percent over time.

Figure 2.

(A) RhoA is the preferred substrate for YopT-catalyzed proteolysis. The GTPases RhoA, Rac, and Cdc42 were post-translationally modified and radiolabeled (1.6 μM ± 0.2). Following quantification of ¹⁴C-labeled RhoA (●, n = 6), Rac (▼, n = 6), and Cdc42 (□, n = 6) the proteolytic cleavage of the C-terminal cysteine was initiated by adding YopT in a 1:10 ratio (YopT: GTPase) at 37°C. At the indicated time the reaction was stopped by the addition of 5% SDS. Proteins were precipitated with 30% TCA. The radiolabeled GTPases were measured using a filter assay and scintillation counting. (B) Proteolytic processing of RhoA, RhoA(Δ189), and RhoA(Δ188/189) by YopT. RhoA and the deletion mutants RhoA(Δ189) and RhoA(Δ188/189) were post-translationally modified and then radiolabeled. After determination of the concentration of ¹⁴C-labeled RhoA (●, n = 6), RhoA(Δ189) (▽, n = 9), and RhoA(Δ188/189) (■, n = 3) (2.1 μM ± 0.2) the proteolytic processing of the GTPases was initiated by addition of YopT in 1:10 ratio (YopT: GTPase) at 37°C. The reaction was terminated by adding 5% SDS. The proteins were precipitated by the addition of 30% TCA. Radiolabeled GTPases were determined using a filter assay and a scintillation counter. The proteolytic activity of YopT was illustrated in percent of remaining ¹⁴C-labeled GTPases (t ₀ = 100%).

Figure 3.

(A) Proteolytic cleavage of RhoA, Rac(188+S), Rac(188+SG), Cdc42(187+S), and Cdc42(187+SG) by YopT. RhoA and the insertion mutants Rac(188+S), Rac(188+SG), Cdc42(187+S), and Cdc42(187+SG) were post-translationally modified and radiolabeled with ¹⁴C-SAM. After determination of the concentration of radiolabeled GTPases (2.5 μM ± 0.1) the proteolytic cleavage of RhoA (●, n = 3), Rac(188+S) (▽, n = 3), GST-Rac(188+SG) (■, n = 3), Cdc42(187+S) (◇, n = 3), and GST-Cdc42(187+SG) (▲, n = 3) was initiated by addition of YopT in a ratio of 1:10 (YopT: GTPase) at 37°C. The reaction was stopped by the addition of 5% SDS and the proteins were precipitated with 30% TCA. Radiolabeled GTPases were determined using a filter assay. The efficiency of YopT cleavage was demonstrated by the amount of remaining radiolabeled GTPases in percent (t ₀ = 100%). (B) Proteolytic cleavage of RhoA, RhoA(S188R), RhoA(S188R/G189K), Rac(188+S/R185G), and Rac(188+S/polyRhoA) by YopT. GST-RhoA, GST-RhoA(S188R), GST-RhoA(S188R/G189K), GST-Rac(188+S/R185G), and GST-Rac(188+S/polyRhoA), were post-translationally modified and radiolabeled. After determination of the concentration of radiolabeled GTPases (2.5 μM ± 0.3) the proteolytic cleavage of GST-RhoA (●, n = 3), GST-RhoA(S188R) (▽, n = 3), GST-RhoA(S188R/G189K) (■, n = 3), GST-Rac(188+S/R185G) (◇, n = 3), and GST-Rac(188+S/polyRhoA) (▲, n = 3), was initiated by addition of YopT in a ratio of 1:10 (YopT: GTPase) at 37°C. The reaction was terminated by adding 5% SDS. The proteins were precipitated by the addition of 30% TCA. Radiolabeled GTPases were determined using a filter assay and a scintillation counter. The proteolytic activity of YopT was illustrated by the remaining ¹⁴C-labeled GTPases in percent (t ₀ = 100%).