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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.

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

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Model of the molecular mechanisms underlying the HpaC-HrcUC-mediated substrate specificity switch in X. campestris pv. vesicatoria.A. HpaC controls secretion of early and late T3S substrates. The T3S system of X. campestris pv. vesicatoria consists of approximately 20 components, eleven of which (abbreviated with single letters) are designated Hrc (Hrp conserved) and presumably constitute the core components of the membrane-spanning secretion apparatus. Cytoplasmic components of the T3S apparatus are shown in light blue, the C-terminal domain of HrcU in green. During the initial step of T3S the early T3S substrate HrpB2 (abbreviated B2), which is required for pilus assembly, interacts with the C-terminal cytoplasmic domain of HrcU and is secreted. The efficient secretion of HrpB2 is inhibited upon binding of the T3S4 protein HpaC to HrpB2 and/or to HrcUC. The cleavage of HrcU at the conserved NPTH motif and a conformational change in HrcUC lead to the release of HrcUC-bound HpaC and HrpB2 and activate the secretion of late substrates including translocon and effector proteins. Dashed lines refer to reduced secretion of HrpB2, the arrow next to HrcUC to the predicted conformational change.B. The Y318D mutation in HrcUC activates secretion of late substrates in the absence of HpaC. The Y318D mutation presumably leads to a conformational change in HrcUC, which allows the efficient secretion of late substrates but leads to reduced cleavage of HrcU and also abolishes the interaction between HrcUC and HrpB2. Secretion of HrpB2 is not affected by the Y318D exchange in HrcU.IM, inner membrane; OM, outer membrane; PM, plasma membrane of the host cell.
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fig09: Model of the molecular mechanisms underlying the HpaC-HrcUC-mediated substrate specificity switch in X. campestris pv. vesicatoria.A. HpaC controls secretion of early and late T3S substrates. The T3S system of X. campestris pv. vesicatoria consists of approximately 20 components, eleven of which (abbreviated with single letters) are designated Hrc (Hrp conserved) and presumably constitute the core components of the membrane-spanning secretion apparatus. Cytoplasmic components of the T3S apparatus are shown in light blue, the C-terminal domain of HrcU in green. During the initial step of T3S the early T3S substrate HrpB2 (abbreviated B2), which is required for pilus assembly, interacts with the C-terminal cytoplasmic domain of HrcU and is secreted. The efficient secretion of HrpB2 is inhibited upon binding of the T3S4 protein HpaC to HrpB2 and/or to HrcUC. The cleavage of HrcU at the conserved NPTH motif and a conformational change in HrcUC lead to the release of HrcUC-bound HpaC and HrpB2 and activate the secretion of late substrates including translocon and effector proteins. Dashed lines refer to reduced secretion of HrpB2, the arrow next to HrcUC to the predicted conformational change.B. The Y318D mutation in HrcUC activates secretion of late substrates in the absence of HpaC. The Y318D mutation presumably leads to a conformational change in HrcUC, which allows the efficient secretion of late substrates but leads to reduced cleavage of HrcU and also abolishes the interaction between HrcUC and HrpB2. Secretion of HrpB2 is not affected by the Y318D exchange in HrcU.IM, inner membrane; OM, outer membrane; PM, plasma membrane of the host cell.

Mentions: In contrast to translocon and effector proteins, HrpB2 was efficiently secreted by HrcU cleavage mutants carrying alanine substitutions within the NPTH motif (Fig. 2). For yet unknown reasons, ectopic expression of hrcU under control of the lac promoter in a hrcU deletion mutant background led to increased HrpB2 secretion that was independent of HrcU cleavage. This implies that HrpB2 secretion is controlled by the amounts of HrcU and occurs prior to HrcU cleavage, which is in agreement with the notion that HrpB2 is an early substrate of the T3S system (Fig. 9). We previously reported that HrpB2 interacts with the C-terminal domain of HrcU (Lorenz et al., 2008b). Here, we show that HrpB2 does not stably interact with GST–HrcU deletion derivatives lacking the NPTH motif or carrying a P265G mutation (shown in the context of both GST–HrcU255–357 and GST–HrcU; Fig. 6). In contrast, N264A and P265A mutations in GST–HrcU255–357 did not significantly affect the interaction between HrcUC and HrpB2. It is conceivable that binding of HrpB2 depends on a certain conformation of HrcUC in or around the NPTH motif that is altered in P265G but not in N264A or P265A HrcU mutant derivatives. However, the P265G mutation presumably did not lead to a complete misfolding of HrcU because the interaction with the putative ATPase regulator HrcL and the general T3S chaperone HpaB was not affected. Notably, HrcUP265G did not promote secretion of HrpB2, which is in contrast to the mutant derivatives HrcUN264A and HrcUP265A (Fig. 2). It is therefore possible that the interaction between HrpB2 and HrcUC is required for the efficient secretion of HrpB2 during the early stage of the T3S process, i.e. prior to HrcU cleavage (Fig. 9).


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)

Model of the molecular mechanisms underlying the HpaC-HrcUC-mediated substrate specificity switch in X. campestris pv. vesicatoria.A. HpaC controls secretion of early and late T3S substrates. The T3S system of X. campestris pv. vesicatoria consists of approximately 20 components, eleven of which (abbreviated with single letters) are designated Hrc (Hrp conserved) and presumably constitute the core components of the membrane-spanning secretion apparatus. Cytoplasmic components of the T3S apparatus are shown in light blue, the C-terminal domain of HrcU in green. During the initial step of T3S the early T3S substrate HrpB2 (abbreviated B2), which is required for pilus assembly, interacts with the C-terminal cytoplasmic domain of HrcU and is secreted. The efficient secretion of HrpB2 is inhibited upon binding of the T3S4 protein HpaC to HrpB2 and/or to HrcUC. The cleavage of HrcU at the conserved NPTH motif and a conformational change in HrcUC lead to the release of HrcUC-bound HpaC and HrpB2 and activate the secretion of late substrates including translocon and effector proteins. Dashed lines refer to reduced secretion of HrpB2, the arrow next to HrcUC to the predicted conformational change.B. The Y318D mutation in HrcUC activates secretion of late substrates in the absence of HpaC. The Y318D mutation presumably leads to a conformational change in HrcUC, which allows the efficient secretion of late substrates but leads to reduced cleavage of HrcU and also abolishes the interaction between HrcUC and HrpB2. Secretion of HrpB2 is not affected by the Y318D exchange in HrcU.IM, inner membrane; OM, outer membrane; PM, plasma membrane of the host cell.
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fig09: Model of the molecular mechanisms underlying the HpaC-HrcUC-mediated substrate specificity switch in X. campestris pv. vesicatoria.A. HpaC controls secretion of early and late T3S substrates. The T3S system of X. campestris pv. vesicatoria consists of approximately 20 components, eleven of which (abbreviated with single letters) are designated Hrc (Hrp conserved) and presumably constitute the core components of the membrane-spanning secretion apparatus. Cytoplasmic components of the T3S apparatus are shown in light blue, the C-terminal domain of HrcU in green. During the initial step of T3S the early T3S substrate HrpB2 (abbreviated B2), which is required for pilus assembly, interacts with the C-terminal cytoplasmic domain of HrcU and is secreted. The efficient secretion of HrpB2 is inhibited upon binding of the T3S4 protein HpaC to HrpB2 and/or to HrcUC. The cleavage of HrcU at the conserved NPTH motif and a conformational change in HrcUC lead to the release of HrcUC-bound HpaC and HrpB2 and activate the secretion of late substrates including translocon and effector proteins. Dashed lines refer to reduced secretion of HrpB2, the arrow next to HrcUC to the predicted conformational change.B. The Y318D mutation in HrcUC activates secretion of late substrates in the absence of HpaC. The Y318D mutation presumably leads to a conformational change in HrcUC, which allows the efficient secretion of late substrates but leads to reduced cleavage of HrcU and also abolishes the interaction between HrcUC and HrpB2. Secretion of HrpB2 is not affected by the Y318D exchange in HrcU.IM, inner membrane; OM, outer membrane; PM, plasma membrane of the host cell.
Mentions: In contrast to translocon and effector proteins, HrpB2 was efficiently secreted by HrcU cleavage mutants carrying alanine substitutions within the NPTH motif (Fig. 2). For yet unknown reasons, ectopic expression of hrcU under control of the lac promoter in a hrcU deletion mutant background led to increased HrpB2 secretion that was independent of HrcU cleavage. This implies that HrpB2 secretion is controlled by the amounts of HrcU and occurs prior to HrcU cleavage, which is in agreement with the notion that HrpB2 is an early substrate of the T3S system (Fig. 9). We previously reported that HrpB2 interacts with the C-terminal domain of HrcU (Lorenz et al., 2008b). Here, we show that HrpB2 does not stably interact with GST–HrcU deletion derivatives lacking the NPTH motif or carrying a P265G mutation (shown in the context of both GST–HrcU255–357 and GST–HrcU; Fig. 6). In contrast, N264A and P265A mutations in GST–HrcU255–357 did not significantly affect the interaction between HrcUC and HrpB2. It is conceivable that binding of HrpB2 depends on a certain conformation of HrcUC in or around the NPTH motif that is altered in P265G but not in N264A or P265A HrcU mutant derivatives. However, the P265G mutation presumably did not lead to a complete misfolding of HrcU because the interaction with the putative ATPase regulator HrcL and the general T3S chaperone HpaB was not affected. Notably, HrcUP265G did not promote secretion of HrpB2, which is in contrast to the mutant derivatives HrcUN264A and HrcUP265A (Fig. 2). It is therefore possible that the interaction between HrpB2 and HrcUC is required for the efficient secretion of HrpB2 during the early stage of the T3S process, i.e. prior to HrcU cleavage (Fig. 9).

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