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Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU.

Lorenz C, Büttner D - Mol. Microbiol. (2010)

Bottom Line: T3S substrate specificity is controlled by HpaC, which promotes secretion of translocon and effector proteins but prevents efficient secretion of the early substrate HrpB2.The results of mutant studies showed that cleavage of HrcU contributes to pathogenicity and secretion of late substrates but is dispensable for secretion of HrpB2, which is presumably secreted prior to HrcU cleavage.As HrcU(Y318D) did not interact with HrpB2 and HpaC, we propose that the substrate specificity switch leads to the release of HrcU(C) -bound HrpB2 and HpaC.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Department of Genetics, Martin-Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany.

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The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC.A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU255–357, GST–HrcU265–357 and GST–HrcU268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.B. Mutations within the NPTH motif abolish the interaction between HrcUC and HpaC. GST, GST–HrcU255–357, GST–HrcU255–357/N264A, GST–HrcU255–357/P265A and GST–HrcU255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU255–357 and thus to enhanced amounts of the full-length fusion proteins.C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU255–357, GST–HrcUN264A, GST–HrcUP265A and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.
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fig05: The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC.A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU255–357, GST–HrcU265–357 and GST–HrcU268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.B. Mutations within the NPTH motif abolish the interaction between HrcUC and HpaC. GST, GST–HrcU255–357, GST–HrcU255–357/N264A, GST–HrcU255–357/P265A and GST–HrcU255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU255–357 and thus to enhanced amounts of the full-length fusion proteins.C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU255–357, GST–HrcUN264A, GST–HrcUP265A and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

Mentions: We have previously shown that the T3S4 protein HpaC interacts with a GST–HrcU255–357 fusion protein (Lorenz et al., 2008b). To investigate whether the interaction depends on the NPTH motif (amino acids 264–268) of HrcU, we generated additional expression constructs encoding GST–HrcU265–357, which lacks the conserved asparagine residue, and GST–HrcU268–357, which is deprived of the complete NPTH motif. For protein–protein interaction studies, GST, GST–HrcU255–357, GST–HrcU265–357 and GST–HrcU268–357 were synthesized in E. coli, immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. Eluted proteins were analysed by immunoblotting using a c-Myc epitope-specific antibody. Figure 5A shows that HpaC-c-Myc was detected in the eluate of GST–HrcU255–357 as expected but not of GST, GST–HrcU265–357 and GST–HrcU268–357. We also performed interaction studies with GST–HrcU255–357 derivatives carrying single amino acid substitutions of the conserved asparagine and proline residues (N264A, P265A and P265G) of the NPTH motif. When GST–HrcU255–357/N264A, GST–HrcU255–357/P265A and GST–HrcU255–357/P265G were immobilized on glutathione sepharose and incubated with HpaC-c-Myc, HpaC-c-Myc was not detected in the eluates, suggesting that mutations of N264 and P265 abolish the efficient binding of HpaC to HrcUC (Fig. 5B).


Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU.

Lorenz C, Büttner D - Mol. Microbiol. (2010)

The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC.A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU255–357, GST–HrcU265–357 and GST–HrcU268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.B. Mutations within the NPTH motif abolish the interaction between HrcUC and HpaC. GST, GST–HrcU255–357, GST–HrcU255–357/N264A, GST–HrcU255–357/P265A and GST–HrcU255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU255–357 and thus to enhanced amounts of the full-length fusion proteins.C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU255–357, GST–HrcUN264A, GST–HrcUP265A and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.
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fig05: The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC.A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU255–357, GST–HrcU265–357 and GST–HrcU268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.B. Mutations within the NPTH motif abolish the interaction between HrcUC and HpaC. GST, GST–HrcU255–357, GST–HrcU255–357/N264A, GST–HrcU255–357/P265A and GST–HrcU255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU255–357 and thus to enhanced amounts of the full-length fusion proteins.C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU255–357, GST–HrcUN264A, GST–HrcUP265A and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.
Mentions: We have previously shown that the T3S4 protein HpaC interacts with a GST–HrcU255–357 fusion protein (Lorenz et al., 2008b). To investigate whether the interaction depends on the NPTH motif (amino acids 264–268) of HrcU, we generated additional expression constructs encoding GST–HrcU265–357, which lacks the conserved asparagine residue, and GST–HrcU268–357, which is deprived of the complete NPTH motif. For protein–protein interaction studies, GST, GST–HrcU255–357, GST–HrcU265–357 and GST–HrcU268–357 were synthesized in E. coli, immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. Eluted proteins were analysed by immunoblotting using a c-Myc epitope-specific antibody. Figure 5A shows that HpaC-c-Myc was detected in the eluate of GST–HrcU255–357 as expected but not of GST, GST–HrcU265–357 and GST–HrcU268–357. We also performed interaction studies with GST–HrcU255–357 derivatives carrying single amino acid substitutions of the conserved asparagine and proline residues (N264A, P265A and P265G) of the NPTH motif. When GST–HrcU255–357/N264A, GST–HrcU255–357/P265A and GST–HrcU255–357/P265G were immobilized on glutathione sepharose and incubated with HpaC-c-Myc, HpaC-c-Myc was not detected in the eluates, suggesting that mutations of N264 and P265 abolish the efficient binding of HpaC to HrcUC (Fig. 5B).

Bottom Line: T3S substrate specificity is controlled by HpaC, which promotes secretion of translocon and effector proteins but prevents efficient secretion of the early substrate HrpB2.The results of mutant studies showed that cleavage of HrcU contributes to pathogenicity and secretion of late substrates but is dispensable for secretion of HrpB2, which is presumably secreted prior to HrcU cleavage.As HrcU(Y318D) did not interact with HrpB2 and HpaC, we propose that the substrate specificity switch leads to the release of HrcU(C) -bound HrpB2 and HpaC.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Department of Genetics, Martin-Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany.

Show MeSH
Related in: MedlinePlus