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Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.

Lallemand M, Login FH, Guschinskaya N, Pineau C, Effantin G, Robert X, Shevchik VE - PLoS ONE (2013)

Bottom Line: We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane.Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS.These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

View Article: PubMed Central - PubMed

Affiliation: INSA-Lyon, Villeurbanne, France ; CNRS, UMR5240, Microbiologie Adaptation et Pathogénie, Lyon, France.

ABSTRACT
The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

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Related in: MedlinePlus

Dissection of the interacting regions of OutC, OutL and OutM in pull-down assays.The GST-fused derivatives of OutM (A), OutL (B) or OutC (C) (indicated at the top) were immobilized on Glutathione Sepharose beads, to constitute the affinity matrices (upper panels). Next, the indicated proteins of interest were incubated with these matrices for 1 h and unbound proteins were washed away. Bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE and either stained (upper panels), or (lower panels) probed with the indicated antibodies, or revealed by autoradiography (35S). GST-fused degradation products are indicated by asterisks. Schematic representation of the used derivatives is shown in Figure S1.
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pone-0079562-g001: Dissection of the interacting regions of OutC, OutL and OutM in pull-down assays.The GST-fused derivatives of OutM (A), OutL (B) or OutC (C) (indicated at the top) were immobilized on Glutathione Sepharose beads, to constitute the affinity matrices (upper panels). Next, the indicated proteins of interest were incubated with these matrices for 1 h and unbound proteins were washed away. Bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE and either stained (upper panels), or (lower panels) probed with the indicated antibodies, or revealed by autoradiography (35S). GST-fused degradation products are indicated by asterisks. Schematic representation of the used derivatives is shown in Figure S1.

Mentions: First, a GST pull-down assay was used to search for potential bi-partner interactions between OutC, OutL and OutM. The full-length proteins, carrying a GST-tag, were bound onto Glutathione Sepharose and used as baits, whereas those carrying a His-tag were solubilized with Triton X-100 and used as prey in the liquid phase. These assays revealed that each of the three proteins interacts in vitro with itself and with the two other proteins (Figure 1A-C, compare lanes 1 and 2).


Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.

Lallemand M, Login FH, Guschinskaya N, Pineau C, Effantin G, Robert X, Shevchik VE - PLoS ONE (2013)

Dissection of the interacting regions of OutC, OutL and OutM in pull-down assays.The GST-fused derivatives of OutM (A), OutL (B) or OutC (C) (indicated at the top) were immobilized on Glutathione Sepharose beads, to constitute the affinity matrices (upper panels). Next, the indicated proteins of interest were incubated with these matrices for 1 h and unbound proteins were washed away. Bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE and either stained (upper panels), or (lower panels) probed with the indicated antibodies, or revealed by autoradiography (35S). GST-fused degradation products are indicated by asterisks. Schematic representation of the used derivatives is shown in Figure S1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815138&req=5

pone-0079562-g001: Dissection of the interacting regions of OutC, OutL and OutM in pull-down assays.The GST-fused derivatives of OutM (A), OutL (B) or OutC (C) (indicated at the top) were immobilized on Glutathione Sepharose beads, to constitute the affinity matrices (upper panels). Next, the indicated proteins of interest were incubated with these matrices for 1 h and unbound proteins were washed away. Bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE and either stained (upper panels), or (lower panels) probed with the indicated antibodies, or revealed by autoradiography (35S). GST-fused degradation products are indicated by asterisks. Schematic representation of the used derivatives is shown in Figure S1.
Mentions: First, a GST pull-down assay was used to search for potential bi-partner interactions between OutC, OutL and OutM. The full-length proteins, carrying a GST-tag, were bound onto Glutathione Sepharose and used as baits, whereas those carrying a His-tag were solubilized with Triton X-100 and used as prey in the liquid phase. These assays revealed that each of the three proteins interacts in vitro with itself and with the two other proteins (Figure 1A-C, compare lanes 1 and 2).

Bottom Line: We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane.Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS.These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

View Article: PubMed Central - PubMed

Affiliation: INSA-Lyon, Villeurbanne, France ; CNRS, UMR5240, Microbiologie Adaptation et Pathogénie, Lyon, France.

ABSTRACT
The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

Show MeSH
Related in: MedlinePlus