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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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Proteolytic cleavage of HrcU depends on the NPTH motif.A. Schematic representation of HrcU. HrcU contains four transmembrane helices and a C-terminal cytoplasmic region that is proteolytically cleaved. Cleavage presumably occurs at the NPTH motif and results in a conformational change of the PTH loop as was shown for HrcU homologues from animal pathogenic bacteria. Numbers refer to amino acid positions. IM, inner membrane.B. Proteolytic cleavage of HrcU and point mutant derivatives. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU (ΔhrcU) encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A) and HrcUH267A-c-Myc (H267A), respectively, from corresponding expression constructs were analysed by immunoblotting using a c-Myc epitope-specific antibody.C. HrcUP265A-c-Myc is partially cleaved. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A) and HrcUP265G-c-Myc (P265G), respectively, from corresponding expression constructs were analysed as described in (B). For the better visualization of the HrcU cleavage product, the blot was overexposed.D. Mutations in the NPTH motif of HrcU206–357-c-Myc affect proteolytic cleavage. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU265–357 (ΔhrcU265–357) encoding HrcU206–357-c-Myc (wt), HrcU206–357/N264A-c-Myc (N264A), HrcU206–357/P265A-c-Myc (P265A), HrcU206–357/T266A-c-Myc (T266A) and HrcU206–357/H267A-c-Myc (H267A), respectively, from corresponding expression constructs were analysed as described in (B).
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fig01: Proteolytic cleavage of HrcU depends on the NPTH motif.A. Schematic representation of HrcU. HrcU contains four transmembrane helices and a C-terminal cytoplasmic region that is proteolytically cleaved. Cleavage presumably occurs at the NPTH motif and results in a conformational change of the PTH loop as was shown for HrcU homologues from animal pathogenic bacteria. Numbers refer to amino acid positions. IM, inner membrane.B. Proteolytic cleavage of HrcU and point mutant derivatives. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU (ΔhrcU) encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A) and HrcUH267A-c-Myc (H267A), respectively, from corresponding expression constructs were analysed by immunoblotting using a c-Myc epitope-specific antibody.C. HrcUP265A-c-Myc is partially cleaved. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A) and HrcUP265G-c-Myc (P265G), respectively, from corresponding expression constructs were analysed as described in (B). For the better visualization of the HrcU cleavage product, the blot was overexposed.D. Mutations in the NPTH motif of HrcU206–357-c-Myc affect proteolytic cleavage. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU265–357 (ΔhrcU265–357) encoding HrcU206–357-c-Myc (wt), HrcU206–357/N264A-c-Myc (N264A), HrcU206–357/P265A-c-Myc (P265A), HrcU206–357/T266A-c-Myc (T266A) and HrcU206–357/H267A-c-Myc (H267A), respectively, from corresponding expression constructs were analysed as described in (B).

Mentions: The FlhB/YscU homologue HrcU from X. campestris pv. vesicatoria strain 85-10 contains four transmembrane helices and a C-terminal cytoplasmic region that is proteolytically cleaved in both E. coli and X. campestris pv. vesicatoria (Fig. 1A; Lorenz et al., 2008b; Berger et al., 2010). Cleavage of HrcU presumably occurs at the conserved NPTH motif (amino acids 264–267) as was described for HrcU homologues from animal pathogenic bacteria. To study the contribution of the NPTH motif of HrcU to protein cleavage and function, we introduced point mutations that led to an exchange of each amino acid residue of the NPTH motif by alanine respectively. The resulting HrcU mutant derivatives were analysed as C-terminally c-Myc epitope-tagged proteins in E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU by immunoblotting. Using a c-Myc epitope-specific antibody, we detected the full-length HrcU-c-Myc, HrcUT266A-c-Myc and HrcUH267A-c-Myc proteins and/or corresponding cleavage products (Fig. 1B). As full-length HrcU-c-Myc was only detectable in E. coli but not in X. campestris pv. vesicatoria, we assume that the proteolytic cleavage of HrcU-c-Myc in X. campestris pv. vesicatoria was nearly complete (Fig. 1B). We detected increased levels of uncleaved HrcUT266A-c-Myc and HrcUH267A-c-Myc when compared with HrcU-c-Myc, suggesting that mutations of amino acids T266 and H267 of HrcU affect the efficiency of the proteolytic cleavage. The C-terminal HrcU cleavage product was not observed for HrcUN264A-c-Myc and only in significantly reduced amounts for HrcUP265A-c-Myc (upon overexposure of the blot; Fig. 1B and C). We also introduced an additional mutation into HrcU that led to an exchange of the proline residue at position 265 by a glycine. Notably, the P265G exchange resulted in a complete loss of detectable HrcU cleavage (Fig. 1C).


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)

Proteolytic cleavage of HrcU depends on the NPTH motif.A. Schematic representation of HrcU. HrcU contains four transmembrane helices and a C-terminal cytoplasmic region that is proteolytically cleaved. Cleavage presumably occurs at the NPTH motif and results in a conformational change of the PTH loop as was shown for HrcU homologues from animal pathogenic bacteria. Numbers refer to amino acid positions. IM, inner membrane.B. Proteolytic cleavage of HrcU and point mutant derivatives. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU (ΔhrcU) encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A) and HrcUH267A-c-Myc (H267A), respectively, from corresponding expression constructs were analysed by immunoblotting using a c-Myc epitope-specific antibody.C. HrcUP265A-c-Myc is partially cleaved. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A) and HrcUP265G-c-Myc (P265G), respectively, from corresponding expression constructs were analysed as described in (B). For the better visualization of the HrcU cleavage product, the blot was overexposed.D. Mutations in the NPTH motif of HrcU206–357-c-Myc affect proteolytic cleavage. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU265–357 (ΔhrcU265–357) encoding HrcU206–357-c-Myc (wt), HrcU206–357/N264A-c-Myc (N264A), HrcU206–357/P265A-c-Myc (P265A), HrcU206–357/T266A-c-Myc (T266A) and HrcU206–357/H267A-c-Myc (H267A), respectively, from corresponding expression constructs were analysed as described in (B).
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fig01: Proteolytic cleavage of HrcU depends on the NPTH motif.A. Schematic representation of HrcU. HrcU contains four transmembrane helices and a C-terminal cytoplasmic region that is proteolytically cleaved. Cleavage presumably occurs at the NPTH motif and results in a conformational change of the PTH loop as was shown for HrcU homologues from animal pathogenic bacteria. Numbers refer to amino acid positions. IM, inner membrane.B. Proteolytic cleavage of HrcU and point mutant derivatives. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU (ΔhrcU) encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A) and HrcUH267A-c-Myc (H267A), respectively, from corresponding expression constructs were analysed by immunoblotting using a c-Myc epitope-specific antibody.C. HrcUP265A-c-Myc is partially cleaved. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A) and HrcUP265G-c-Myc (P265G), respectively, from corresponding expression constructs were analysed as described in (B). For the better visualization of the HrcU cleavage product, the blot was overexposed.D. Mutations in the NPTH motif of HrcU206–357-c-Myc affect proteolytic cleavage. Equal amounts of total-cell extracts from E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU265–357 (ΔhrcU265–357) encoding HrcU206–357-c-Myc (wt), HrcU206–357/N264A-c-Myc (N264A), HrcU206–357/P265A-c-Myc (P265A), HrcU206–357/T266A-c-Myc (T266A) and HrcU206–357/H267A-c-Myc (H267A), respectively, from corresponding expression constructs were analysed as described in (B).
Mentions: The FlhB/YscU homologue HrcU from X. campestris pv. vesicatoria strain 85-10 contains four transmembrane helices and a C-terminal cytoplasmic region that is proteolytically cleaved in both E. coli and X. campestris pv. vesicatoria (Fig. 1A; Lorenz et al., 2008b; Berger et al., 2010). Cleavage of HrcU presumably occurs at the conserved NPTH motif (amino acids 264–267) as was described for HrcU homologues from animal pathogenic bacteria. To study the contribution of the NPTH motif of HrcU to protein cleavage and function, we introduced point mutations that led to an exchange of each amino acid residue of the NPTH motif by alanine respectively. The resulting HrcU mutant derivatives were analysed as C-terminally c-Myc epitope-tagged proteins in E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU by immunoblotting. Using a c-Myc epitope-specific antibody, we detected the full-length HrcU-c-Myc, HrcUT266A-c-Myc and HrcUH267A-c-Myc proteins and/or corresponding cleavage products (Fig. 1B). As full-length HrcU-c-Myc was only detectable in E. coli but not in X. campestris pv. vesicatoria, we assume that the proteolytic cleavage of HrcU-c-Myc in X. campestris pv. vesicatoria was nearly complete (Fig. 1B). We detected increased levels of uncleaved HrcUT266A-c-Myc and HrcUH267A-c-Myc when compared with HrcU-c-Myc, suggesting that mutations of amino acids T266 and H267 of HrcU affect the efficiency of the proteolytic cleavage. The C-terminal HrcU cleavage product was not observed for HrcUN264A-c-Myc and only in significantly reduced amounts for HrcUP265A-c-Myc (upon overexposure of the blot; Fig. 1B and C). We also introduced an additional mutation into HrcU that led to an exchange of the proline residue at position 265 by a glycine. Notably, the P265G exchange resulted in a complete loss of detectable HrcU cleavage (Fig. 1C).

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