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Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome.

Diepold A, Kudryashev M, Delalez NJ, Berry RM, Armitage JP - PLoS Biol. (2015)

Bottom Line: Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells.Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion.These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells. While the structure of the distal "needle complex" is well documented, the composition and role of the functionally important cytosolic complex remain less well understood. Using functional fluorescent fusions, we found that the C-ring, an essential and conserved cytosolic component of the system, is composed of ~22 copies of SctQ (YscQ in Yersinia enterocolitica), which require the presence of YscQC, the product of an internal translation initiation site in yscQ, for their cooperative assembly. Photoactivated localization microscopy (PALM) reveals that in vivo, YscQ is present in both a free-moving cytosolic and a stable injectisome-bound state. Notably, fluorescence recovery after photobleaching (FRAP) shows that YscQ exchanges between the injectisome and the cytosol, with a t½ of 68 ± 8 seconds when injectisomes are secreting. In contrast, the secretin SctC (YscC) and the major export apparatus component SctV (YscV) display minimal exchange. Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion. The stabilization of the C-ring depends on the presence of the functional ATPase SctN (YscN). These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion.

No MeSH data available.


Related in: MedlinePlus

The C-ring subunit YscQ is a dynamic element of the injectisome.Fluorescent foci formed by EGFP-YscQ, but not by YscC-mCherry or YscV-mCherry show exchange with a cytosolic pool. (A) Position of the three studied components in the injectisome. Secretin YscC, blue; IM export apparatus component YscV, green; C-ring YscQ, red. Conformation and localization of the dashed components have not been experimentally determined. (B) Activity of strains expressing the indicated fusion proteins in an export kinetics assay based on the translocation of an artificial export substrate, YopH1–17-β-lactamase (Yop-bla). Wild-type control, set to 100%, black; negative controls (bla fused to the non-T3SS substrate glutathione-S-transferase GST and T3SS defective strain ΔYscN), grey. Error bars represent standard errors of the mean of n = 5 measurements (n = 4 for ΔYscN) in technical triplicates. (C) Micrographs showing representative images before and after photobleaching of a single fluorescent spot (marked by red circle) of YscC-mCherry, YscV-mCherry, and EGFP-YscQ. (D) Fluorescence recovery over time. Circles indicate the relative spot intensity in single frames for the micrographs shown in (C). (E) Localization of single PAmCherry-YscQ molecules in PALM. (F) Cellular distribution of motile (blue) and stationary (red) PAmCherry-YscQ molecules. (G) Single step diffusion coefficients for PAmCherry-YscQ and YscV-PAmCherry. See Materials and Methods for details. Scale bars, 1 μm. Numerical values and raw data (B, D, G) can be found in S1 Data.
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pbio.1002039.g003: The C-ring subunit YscQ is a dynamic element of the injectisome.Fluorescent foci formed by EGFP-YscQ, but not by YscC-mCherry or YscV-mCherry show exchange with a cytosolic pool. (A) Position of the three studied components in the injectisome. Secretin YscC, blue; IM export apparatus component YscV, green; C-ring YscQ, red. Conformation and localization of the dashed components have not been experimentally determined. (B) Activity of strains expressing the indicated fusion proteins in an export kinetics assay based on the translocation of an artificial export substrate, YopH1–17-β-lactamase (Yop-bla). Wild-type control, set to 100%, black; negative controls (bla fused to the non-T3SS substrate glutathione-S-transferase GST and T3SS defective strain ΔYscN), grey. Error bars represent standard errors of the mean of n = 5 measurements (n = 4 for ΔYscN) in technical triplicates. (C) Micrographs showing representative images before and after photobleaching of a single fluorescent spot (marked by red circle) of YscC-mCherry, YscV-mCherry, and EGFP-YscQ. (D) Fluorescence recovery over time. Circles indicate the relative spot intensity in single frames for the micrographs shown in (C). (E) Localization of single PAmCherry-YscQ molecules in PALM. (F) Cellular distribution of motile (blue) and stationary (red) PAmCherry-YscQ molecules. (G) Single step diffusion coefficients for PAmCherry-YscQ and YscV-PAmCherry. See Materials and Methods for details. Scale bars, 1 μm. Numerical values and raw data (B, D, G) can be found in S1 Data.

Mentions: Protein complexes can be dynamic assemblies, adapting their structure and function to changing environmental conditions. Given the inconsistent data on the cytosolic versus injectisome-bound localization of the C-ring, we wanted to test whether the C-ring exchanges subunits in the assembled structure. To this end, we followed the ability of injectisome components to exchange with cellular pools. Besides the C-ring subunit YscQ, we analyzed the secretin YscC and the major export apparatus component YscV, as these proteins cover the major functional subcomplexes of the injectisome (cytosolic components, membrane rings, and export apparatus, respectively) (Fig. 3A). All fusion proteins were stable and fully functional for secretion (S2 and S6 Figs.). To ensure that the fluorescent tags have only minimal influence on the kinetics of effector export, we devised a sensitive assay based on the quantification of export of an engineered T3SS substrate, β-lactamase fused to the YopH secretion signal, YopH1–17-bla [50–52]. In this assay, the strains expressing YscC-mCherry, YscV-mCherry, and EGFP-YscQ showed export rates of over 75% of the wild-type (WT) strain (Fig. 3B). This shows that the fluorescent tags have very little influence on protein functionality, suggesting that the exchange rates of labeled proteins resemble those of unlabeled subunits.


Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome.

Diepold A, Kudryashev M, Delalez NJ, Berry RM, Armitage JP - PLoS Biol. (2015)

The C-ring subunit YscQ is a dynamic element of the injectisome.Fluorescent foci formed by EGFP-YscQ, but not by YscC-mCherry or YscV-mCherry show exchange with a cytosolic pool. (A) Position of the three studied components in the injectisome. Secretin YscC, blue; IM export apparatus component YscV, green; C-ring YscQ, red. Conformation and localization of the dashed components have not been experimentally determined. (B) Activity of strains expressing the indicated fusion proteins in an export kinetics assay based on the translocation of an artificial export substrate, YopH1–17-β-lactamase (Yop-bla). Wild-type control, set to 100%, black; negative controls (bla fused to the non-T3SS substrate glutathione-S-transferase GST and T3SS defective strain ΔYscN), grey. Error bars represent standard errors of the mean of n = 5 measurements (n = 4 for ΔYscN) in technical triplicates. (C) Micrographs showing representative images before and after photobleaching of a single fluorescent spot (marked by red circle) of YscC-mCherry, YscV-mCherry, and EGFP-YscQ. (D) Fluorescence recovery over time. Circles indicate the relative spot intensity in single frames for the micrographs shown in (C). (E) Localization of single PAmCherry-YscQ molecules in PALM. (F) Cellular distribution of motile (blue) and stationary (red) PAmCherry-YscQ molecules. (G) Single step diffusion coefficients for PAmCherry-YscQ and YscV-PAmCherry. See Materials and Methods for details. Scale bars, 1 μm. Numerical values and raw data (B, D, G) can be found in S1 Data.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4295842&req=5

pbio.1002039.g003: The C-ring subunit YscQ is a dynamic element of the injectisome.Fluorescent foci formed by EGFP-YscQ, but not by YscC-mCherry or YscV-mCherry show exchange with a cytosolic pool. (A) Position of the three studied components in the injectisome. Secretin YscC, blue; IM export apparatus component YscV, green; C-ring YscQ, red. Conformation and localization of the dashed components have not been experimentally determined. (B) Activity of strains expressing the indicated fusion proteins in an export kinetics assay based on the translocation of an artificial export substrate, YopH1–17-β-lactamase (Yop-bla). Wild-type control, set to 100%, black; negative controls (bla fused to the non-T3SS substrate glutathione-S-transferase GST and T3SS defective strain ΔYscN), grey. Error bars represent standard errors of the mean of n = 5 measurements (n = 4 for ΔYscN) in technical triplicates. (C) Micrographs showing representative images before and after photobleaching of a single fluorescent spot (marked by red circle) of YscC-mCherry, YscV-mCherry, and EGFP-YscQ. (D) Fluorescence recovery over time. Circles indicate the relative spot intensity in single frames for the micrographs shown in (C). (E) Localization of single PAmCherry-YscQ molecules in PALM. (F) Cellular distribution of motile (blue) and stationary (red) PAmCherry-YscQ molecules. (G) Single step diffusion coefficients for PAmCherry-YscQ and YscV-PAmCherry. See Materials and Methods for details. Scale bars, 1 μm. Numerical values and raw data (B, D, G) can be found in S1 Data.
Mentions: Protein complexes can be dynamic assemblies, adapting their structure and function to changing environmental conditions. Given the inconsistent data on the cytosolic versus injectisome-bound localization of the C-ring, we wanted to test whether the C-ring exchanges subunits in the assembled structure. To this end, we followed the ability of injectisome components to exchange with cellular pools. Besides the C-ring subunit YscQ, we analyzed the secretin YscC and the major export apparatus component YscV, as these proteins cover the major functional subcomplexes of the injectisome (cytosolic components, membrane rings, and export apparatus, respectively) (Fig. 3A). All fusion proteins were stable and fully functional for secretion (S2 and S6 Figs.). To ensure that the fluorescent tags have only minimal influence on the kinetics of effector export, we devised a sensitive assay based on the quantification of export of an engineered T3SS substrate, β-lactamase fused to the YopH secretion signal, YopH1–17-bla [50–52]. In this assay, the strains expressing YscC-mCherry, YscV-mCherry, and EGFP-YscQ showed export rates of over 75% of the wild-type (WT) strain (Fig. 3B). This shows that the fluorescent tags have very little influence on protein functionality, suggesting that the exchange rates of labeled proteins resemble those of unlabeled subunits.

Bottom Line: Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells.Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion.These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells. While the structure of the distal "needle complex" is well documented, the composition and role of the functionally important cytosolic complex remain less well understood. Using functional fluorescent fusions, we found that the C-ring, an essential and conserved cytosolic component of the system, is composed of ~22 copies of SctQ (YscQ in Yersinia enterocolitica), which require the presence of YscQC, the product of an internal translation initiation site in yscQ, for their cooperative assembly. Photoactivated localization microscopy (PALM) reveals that in vivo, YscQ is present in both a free-moving cytosolic and a stable injectisome-bound state. Notably, fluorescence recovery after photobleaching (FRAP) shows that YscQ exchanges between the injectisome and the cytosol, with a t½ of 68 ± 8 seconds when injectisomes are secreting. In contrast, the secretin SctC (YscC) and the major export apparatus component SctV (YscV) display minimal exchange. Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion. The stabilization of the C-ring depends on the presence of the functional ATPase SctN (YscN). These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion.

No MeSH data available.


Related in: MedlinePlus