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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 Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC.A. GST–HrcU255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU255–357 and GST–HrcU255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown.B. HrcUY318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcUY318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.
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fig08: The Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC.A. GST–HrcU255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU255–357 and GST–HrcU255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown.B. HrcUY318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcUY318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.

Mentions: As HrcUY318D presumably mimics a protein conformation that is permissive for the secretion of late substrates, we investigated a possible influence of the Y318D mutation on the interaction of HrcUC with HrpB2 and HpaC. For this, GST, GST–HrcU255–357 and GST–HrcU255–357/Y318D were immobilized on glutathione sepharose and incubated with HrpB2-c-Myc and HpaC-c-Myc respectively. Figure 8A shows that HrpB2-c-Myc and HpaC-c-Myc co-eluted with GST–HrcU255–357 as expected but were not detectable in the eluate of GST–HrcU255–357/Y318D, suggesting that the Y318D mutation prevents the stable binding of both HrpB2 and HpaC to HrcUC. Given the finding that HrpB2 is oversecreted by strain 85*hrcUY318DΔhpaC, it is conceivable that the interaction of HrcUC and HrpB2 is not required for efficient HrpB2 secretion after the substrate specificity switch.


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 Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC.A. GST–HrcU255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU255–357 and GST–HrcU255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown.B. HrcUY318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcUY318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040844&req=5

fig08: The Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC.A. GST–HrcU255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU255–357 and GST–HrcU255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown.B. HrcUY318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcUY318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.
Mentions: As HrcUY318D presumably mimics a protein conformation that is permissive for the secretion of late substrates, we investigated a possible influence of the Y318D mutation on the interaction of HrcUC with HrpB2 and HpaC. For this, GST, GST–HrcU255–357 and GST–HrcU255–357/Y318D were immobilized on glutathione sepharose and incubated with HrpB2-c-Myc and HpaC-c-Myc respectively. Figure 8A shows that HrpB2-c-Myc and HpaC-c-Myc co-eluted with GST–HrcU255–357 as expected but were not detectable in the eluate of GST–HrcU255–357/Y318D, suggesting that the Y318D mutation prevents the stable binding of both HrpB2 and HpaC to HrcUC. Given the finding that HrpB2 is oversecreted by strain 85*hrcUY318DΔhpaC, it is conceivable that the interaction of HrcUC and HrpB2 is not required for efficient HrpB2 secretion after the substrate specificity switch.

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