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. 2004 Aug;186(16):5366-75.
doi: 10.1128/JB.186.16.5366-5375.2004.

Role of the pilot protein YscW in the biogenesis of the YscC secretin in Yersinia enterocolitica

Peter Burghout  1 , Frank Beckers, Emmie de Wit, Ria van Boxtel, Guy R Cornelis, Jan Tommassen, Margot Koster
Affiliations

Role of the pilot protein YscW in the biogenesis of the YscC secretin in Yersinia enterocolitica

Peter Burghout et al. J Bacteriol. 2004 Aug.

Abstract

The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that approximately 30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.

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Figures

FIG. 1.
FIG. 1.
Analysis of the cell envelope proteins of the pYV-cured strain CE1525 with either plasmid pRS6 (yscW) (lane 1), plasmid pSM3km (yscC) (lane 2), or both plasmids (lane 3). (A) Cell envelopes were analyzed by SDS-PAGE on an 11% polyacrylamide gel, and YscC was detected by immunoblotting. The positions of the molecular mass standard proteins (in kilodaltons) and the boundary between the stacking and running gel (st) are indicated at the left. (B) Cell envelopes were analyzed by SDS-PAGE on a 3 to 9% polyacrylamide gradient gel, and proteins were stained with silver. Only the relevant part of the gel is shown. Strains were grown at 37°C in BHI-OX with IPTG to induce yscC expression. Cell envelopes from an equal amount of cells were loaded in each lane.
FIG. 2.
FIG. 2.
Subcellular localization of YscC in the presence or absence of YscW and of YscW itself. Membrane preparations of French press lysates of the Y. enterocolitica wild-type strain KNG22703 (A), the yscW mutant KNG22703(pRS227) (B), and the pYV-cured strain CE1525 with either plasmid pSM3km (yscC) (C), plasmids pSM3km (yscC) and pRS6 (yscW) (D), or plasmid pRS6 (yscW) (E) were applied onto 30 to 55% sucrose gradients, which were centrifuged and fractionated. Aliquots of the fractions were analyzed by SDS-PAGE on 3 to 9% polyacrylamide gradient gels (A to D) or on a 14% polyacrylamide gel (E). For the detection of YscC oligomers, protein bands were stained with silver (A to D), and the YscW protein was immunodetected on a Western blot (E). The fractions that represent the inner (dotted line) and outer membranes (continuous line) were identified based on NADH-oxidase activity and the presence of the porins, respectively. The strains were grown at 37°C in BHI-OX with IPTG to induce yscC expression.
FIG. 3.
FIG. 3.
Stability and localization of YscCΔC-term. (A) Cell envelopes of the pYV-cured Y. enterocolitica strain CE1525 with either plasmid pSM3km (yscC) (lane 1), plasmid pSM3kmΔ7 (yscCΔC-term) (lane 2), or plasmids pSM3kmΔ7 (yscCΔC-term) and pRS6 (yscW) (lane 3) were analyzed by SDS-PAGE on a 3 to 9% polyacrylamide gradient gel, and YscC was detected by silver staining. (B) Cell envelopes of the pYV-cured Y. enterocolitica strain CE1525 with either plasmid pSM3kmΔ7 (yscCΔC-term) (lane 1) or plasmids pSM3kmΔ7 (yscCΔC-term) and pRS6 (yscW) (lane 2) were analyzed by SDS-PAGE on an 11% polyacrylamide gel, and YscC was detected by immunoblotting. The positions of the molecular mass standard proteins (in kilodaltons) and the boundary between the stacking and running gel (st) are indicated on the left. Cell envelopes from an equal amount of cells were loaded in each lane. (C and D) The cell envelope fraction of the French press lysates of the pYV-cured strain CE1525 with either plasmid pSM3kmΔ7 (yscCΔC-term) (C) or plasmid pSM3kmΔ7 and plasmid pRS6 (yscW) (D) were applied on a 30 to 55% sucrose gradient, which was centrifuged and fractionated. The samples were analyzed by SDS-PAGE on 3 to 9% polyacrylamide gradient gels, and YscC oligomers were detected by silver staining. The fractions that represent the inner (dotted line) and outer membranes (continuous line) were identified based on NADH-oxidase activity and the presence of the porins, respectively. The strains were grown at 37°C in BHI-OX with IPTG to induce yscC expression.
FIG. 4.
FIG. 4.
Complementation of an yscC mutation by YscCΔC-term. Secreted protein patterns are shown of the Y. enterocolitica yscC mutant KNG22703(pAA203) (lane 1) with pSM3 (yscC) (lane 2) or pSM3Δ7 (yscCΔC-term) (lane 3), which were grown at 37°C in BHI-OX. The expression of yscC or yscCΔC-term was induced by the addition of IPTG to a final concentration of 0.1 mM. Proteins from the extracellular medium were analyzed by SDS-PAGE on an 11% polyacrylamide gel and stained with Coomassie brilliant blue. The positions of the most abundant Yop proteins are indicated on the right.
FIG. 5.
FIG. 5.
Time course of the formation of stable oligomers of the YscC secretin. Cells of the strain CE1525 with pSM3km (yscC) and pRS6 (yscW) were induced for the production of YscC and pulse-labeled for 1 min with [35S]methionine. (A) Samples were taken after the chase periods indicated, and YscC was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. The exposure time and the positions of the monomer and the oligomer are indicated on the left. The data are representative of three independent experiments. (B) Samples obtained after 2 and 120 min chase were treated with TCA and TFA to dissociate YscC oligomers, and YscC was immunoprecipitated and analyzed by SDS-PAGE and autoradiography (left). For quantification of the YscC levels, the autoradiogram was analyzed on a PhosphorImager (right), and the value obtained for the 2-min chase time was set at 100%.
FIG. 6.
FIG. 6.
Time course of YscC oligomerization in the absence of YscW. Cells of strain CE1525 with pSM3km (yscC) were induced for the production of YscC and pulse-labeled for 1 min with [35S]methionine. (A) Samples were taken after the chase periods indicated, and YscC was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. Different exposure times were used for the detection of the monomer (lower panel) and the oligomer (upper panel), as indicated in parentheses on the left. The data are representative of three independent experiments. (B) Samples obtained after 2 and 120 min of chase were treated with TCA and TFA to dissociate YscC oligomers, and YscC was immunoprecipitated and analyzed by SDS-PAGE and autoradiography (left). For quantification of the YscC levels, the gel was exposed in a PhosphorImager (right), and the value obtained for the 2-min chase time was set at 100%.
FIG. 7.
FIG. 7.
Localization of unlipidated YscW and its dominant-negative effect on Yop secretion. (A) Cell envelopes (CE) and soluble fractions (S) of the Y. enterocolitica pYV-cured strain CE1525 with either pRS6 (yscW) or pEW4 (yscWΔlip) were analyzed by SDS-PAGE on a 14% polyacrylamide gel. YscW was detected by immunoblotting. (B) The Y. enterocolitica yscW mutant KNG22703(pRS227) with either pRS6 (yscW) (lane 1) or pEW4 (yscWΔlip) (lane 2), and the wild-type strain KNG22703 (lane 3) with either pRS6 (yscW) (lane 4) or pEW4 (yscWΔlip) (lanes 5 to 7) were grown at 37°C in BHI-OX. The expression of YscWΔlip was induced by the addition 0.1 mM (lanes 2 and 5), 0.5 mM (lane 6), or 1.0 mM (lane 7) IPTG. Yops from the extracellular medium were precipitated and analyzed by SDS-PAGE on an 11% polyacrylamide gel. Proteins were stained with Coomassie brilliant blue. The positions of the most abundant Yop proteins are indicated on the right.
FIG. 8.
FIG. 8.
Stability and localization of YscC in the presence of YscWΔlip. (A) Cell envelope proteins of the pYV-cured Y. enterocolitica strain CE1525 with either plasmid pSM3km (yscC) (lane 1), plasmids pSM3km (yscC) and pRS6 (yscW) (lane 2), or plasmids pSM3km (yscC) and pEW4 (yscWΔlip) (lane 3) were separated by SDS-PAGE on a 3 to 9% polyacrylamide gradient gel. YscC oligomers were detected with Coomassie brilliant blue. Cell envelopes from an equal amount of cells were loaded in each lane. (B and C) The cell envelope fractions of French press lysates of the pYV-cured strain CE1525 with pEW4 (yscWΔlip) and pSM3km (yscC) (B) and that of the wild-type strain KNG22703 with pEW4 (yscWΔlip) (C) were applied onto 30 to 55% sucrose gradients, which were centrifuged and fractionated. Samples from all fractions were analyzed by SDS-PAGE on 3 to 9% polyacrylamide gradient gels. YscC oligomers were detected by silver staining. The fractions that represent the inner (dotted line) and outer membranes (continuous line) were identified based on NADH-oxidase activity and the presence of the porins, respectively. (D) Proteins from whole cells of the pYV-cured strain CE1525 bearing either plasmid pRS6 (yscW) (lane 1), plasmids pRS6 (yscW) and pSM3km (yscC) (lane 2), plasmid pEW4 (yscWΔlip) (lane 3), or plasmids pEW4 (yscWΔlip) and pSM3km (yscC) (lane 4) and the Y. enterocolitica yscW mutant KNG22703(pRS227) with either plasmid pRS6 (yscW) (lane 5) or plasmid pEW4 (yscWΔlip) (lane 6) were separated on a 14% polyacrylamide gel, and YscW was immunodetected on Western blots. The same amount of total cell extracts was loaded in each lane. The strains were grown at 37°C in BHI-OX with IPTG to induce yscC and/or yscWΔlip expression.
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