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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 contributes to the interaction between HrcUC and HrpB2.A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU255–357, GST–HrcU265–357 and GST–HrcU268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-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. The P265G exchange abolishes the interaction between HrcUC and HrpB2. 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 HrpB2-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. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown.D. GST–HrcUP265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-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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fig06: The NPTH motif of HrcU contributes to the interaction between HrcUC and HrpB2.A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU255–357, GST–HrcU265–357 and GST–HrcU268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-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. The P265G exchange abolishes the interaction between HrcUC and HrpB2. 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 HrpB2-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. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown.D. GST–HrcUP265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-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: In addition to HpaC, GST–HrcU255–357 also interacts with a C-terminally c-Myc epitope-tagged derivative of the early T3S substrate HrpB2 (Lorenz et al., 2008b). To investigate whether the NPTH motif of HrcU contributes to the interaction between HrcUC and HrpB2, we performed additional pull-down assays with GST or GST–HrcU derivatives as described above. When GST–HrcU fusion proteins were immobilized on glutathione sepharose and incubated with HrpB2-c-Myc, we detected HrpB2-c-Myc in the eluate of GST–HrcU255–357 as expected but not in the eluates of GST–HrcU265–357 and GST–HrcU268–357 (Fig. 6A; Lorenz et al., 2008b). The presence of N264A and P265A point mutations in GST–HrcU255–357, respectively, did not significantly affect the binding of HrpB2-c-Myc (Fig. 6B). In contrast, HrpB2-c-Myc was not detected in the eluate of GST–HrcU255–357/P265G indicating that the P265G exchange abolishes the stable interaction between GST–HrcU255–357 and HrpB2 (Fig. 6B).


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 contributes to the interaction between HrcUC and HrpB2.A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU255–357, GST–HrcU265–357 and GST–HrcU268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-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. The P265G exchange abolishes the interaction between HrcUC and HrpB2. 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 HrpB2-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. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown.D. GST–HrcUP265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-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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fig06: The NPTH motif of HrcU contributes to the interaction between HrcUC and HrpB2.A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU255–357, GST–HrcU265–357 and GST–HrcU268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-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. The P265G exchange abolishes the interaction between HrcUC and HrpB2. 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 HrpB2-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. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown.D. GST–HrcUP265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcUP265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-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: In addition to HpaC, GST–HrcU255–357 also interacts with a C-terminally c-Myc epitope-tagged derivative of the early T3S substrate HrpB2 (Lorenz et al., 2008b). To investigate whether the NPTH motif of HrcU contributes to the interaction between HrcUC and HrpB2, we performed additional pull-down assays with GST or GST–HrcU derivatives as described above. When GST–HrcU fusion proteins were immobilized on glutathione sepharose and incubated with HrpB2-c-Myc, we detected HrpB2-c-Myc in the eluate of GST–HrcU255–357 as expected but not in the eluates of GST–HrcU265–357 and GST–HrcU268–357 (Fig. 6A; Lorenz et al., 2008b). The presence of N264A and P265A point mutations in GST–HrcU255–357, respectively, did not significantly affect the binding of HrpB2-c-Myc (Fig. 6B). In contrast, HrpB2-c-Myc was not detected in the eluate of GST–HrcU255–357/P265G indicating that the P265G exchange abolishes the stable interaction between GST–HrcU255–357 and HrpB2 (Fig. 6B).

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