Biochemical characterization of the Yersinia YopT protease: cleavage site and recognition elements in Rho GTPases

Abstract

The Gram-negative bacterial pathogen Yersinia delivers six effector proteins into the host cells to thwart the host innate immune response. One of the effectors, YopT, causes the disruption of the actin cytoskeleton and contributes to the inhibition of phagocytosis of the pathogen. YopT functions as a cysteine protease to cleave Rho family GTPases. We have analyzed the YopT cleavage products of Rho GTPases by TLC and determined their chemical structure by MS. Amino acid labeling experiments were performed to locate the exact site in RhoA where the YopT cleavage occurs. Our data unambiguously demonstrate that YopT cleaves N-terminal to the prenylated cysteine in RhoA, Rac, and Cdc42 and that the cleavage product of the GTPases is geranylgeranyl cysteine methyl ester. YopT cleaves GTP- and GDP-bound forms of RhoA equally, suggesting that the cleavage does not depend upon the conformation status of the GTPases. YopT also cleaves both farnesylated and geranylgeranylated forms of RhoA. The polybasic sequence in the C terminus of RhoA is essential for YopT substrate recognition and cleavage.

Figure 1

Identification of YopT cleavage products by TLC. (A) TLC separation of lipid cleavage products. [³H]Mevalonate-labeled RhoAL63 (lanes 1 and 2), Rac1L61 (lanes 3 and 4), and Cdc42L61 (lanes 5 and 6) were incubated with either wild-type (lanes 1, 3, and 5) or YopT C139S mutant protein (lanes 2, 4, and 6) in vitro. The chloroform extracts of each cleavage reaction were spotted on a TLC plate. The lipid cleavage products were visualized by autoradiography of the TLC plate. GGCM refers to the ³H-labeled synthetic GGCM standard (lane 7). (B) The C-terminal sequence of Rho family GTPases RhoA, Rac1, and Cdc42.

Figure 2

Determination of the YopT cleavage site in RhoA. (A) The prenylated [³⁵S]Cys, but not the [¹⁴C]Gly (N-terminal to the prenylated cysteine), in RhoA was cleaved off by YopT. (Upper) Scintillation counting of the chloroform-extracted YopT cleavage product of RhoA with either [¹⁴C]Gly or [³⁵S]Cys labeling. (Lower) Levels of radiolabeled RhoA by autoradiography (Left) and total RhoA protein by Western blot (Right) present in each cleavage reaction. (B) Nanoelectrospray MS/MS analysis of the lipid YopT cleavage product from RhoA. Shown is the fragmentation pattern for the GGCM standard (Bottom) and for a single charged parent ion (m/z 408.7) from the RhoA sample after a cleavage reaction with YopT (Middle). (Top) Chemical structure of the GGCM standard. The highlighted fragments correspond to the fragment ions observed in the MS/MS spectrum of the standard and the cleavage product from RhoA.

Figure 3

YopT cleaves both GDP- and GTP-bound forms of Rho GTPases. (A) In vitro YopT cleavage assay with RhoAL63 and RhoAN19. GST-tagged RhoA variants were expressed and purified from HEK293T cells labeled with [³H]mevalonate and incubated with YopT. Cleavage products were separated by SDS/PAGE and analyzed by autoradiography. Cleavage efficiency was assessed by loss of the ³H-geranylgeranyl group from RhoA (Upper). Total RhoA proteins were measured by anti-RhoA Western blot analysis (Lower). (B) In vitro YopT cleavage assay with RhoA-GTP[γS] and RhoA-GDP[βS]. Wild-type GST-RhoA was purified from HEK293T cells labeled with [³H]mevalonate and loaded with either GTP[γS] or GDP[βS] in vitro and used in the YopT cleavage assay described in the text.

Figure 4

The C-terminal polybasic sequence in RhoA is essential for YopT recognition and cleavage. (A) Cleavage of the polybasic RhoA mutant by YopT in vivo (Left) and in vitro (Right). RhoA[K(R)5Q] represents the polybasic RhoA mutant with the five basic residues in the C terminus (two arginines and three lysines) substituted to glutamine. Cleavage assays were performed as described in the text. (B) GST-pulldown assay of the polybasic RhoA mutant. GST-RhoA variants were coexpressed with Flag-tagged YopT C139S in HEK293T cells and purified by GST affinity chromatography. The amounts of GST-RhoA on the glutathione beads and the associated YopT were determined by anti-GST (Lower) and anti-Flag (Upper) Western blot, respectively. RhoAΔCaaX refers to the CaaX deletion mutant of RhoA and was used as a negative control. (C) YopT cleavage assay with chimeric GST fusion proteins. GST-CLVL and GST-ARRGKKKSGCLVL correspond to fusion proteins consisting of RhoA CaaX box (CLVL) or the last 13 residues in the C terminus of RhoA (ARRGKKKSGCLVL) and the N-terminal GST tag. GST-H-Ras (1–166)-ARRGKKKSGCLVL is a chimeric construct generated by replacing the carboxyl 23-residue sequence in H-Ras with the 13-residue sequence from the C terminus of RhoA. These GST fusion proteins were expressed in HEK293T cells labeled with [³H]mevalonate. The resulting prenylated GST fusion proteins with ³H-geranylgeranyl labeling were then subjected to YopT cleavage assay as described in the text.

Figure 5

The role of the prenylated cysteine methyl ester in YopT recognition. (A) YopT cleavage assay with farnesylated RhoA. The cleavage assay was carried out in vivo by coexpressing YopT and RhoA L193M (a RhoA mutant that undergoes farnesylation instead of geranylgeranylation) in HEK293T cells labeled with [³H]mevalonate. Cleavage was assessed by loss of the ³H-geranylgeranyl group from RhoA (Upper). Expression of RhoA was detected by anti-RhoA Western blot (Lower). (B) YopT cleavage assay with recombinant RhoA that was geranylgeranylated in vitro (no aaX proteolysis and methylation). Recombinant GST-RhoA was geranylgeranyl-modified in vitro using geranylgeranyl transferase and ³H-geranylgeranyl pyrophosphate as described in the text. The resulting labeled GST-RhoA (RhoA-CLVL) was immobilized onto glutathione beads and incubated with recombinant YopT. Fully modified GST-RhoA produced from HEK293T cells was used as a positive control (RhoA-CoMe). (C) In vivo YopT cleavage assay with coexpressed YopT and RhoA V192Y, a RhoA mutant that blocks the aaX proteolysis as well as subsequent methylation of the geranylgeranylated cysteine. (D) GST-pulldown assay of the RhoA V192Y mutant. The experiment was performed as described in the text. The binding affinity between the mutant RhoA and YopT C139S was assessed by anti-Flag Western blot of the GST pulldowns.