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Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.

Aldridge C, Ma X, Gerard F, Cline K - J. Cell Biol. (2014)

Bottom Line: Substrate binding triggers assembly of Tha4 onto the interior site.We provide evidence that the substrate signal peptide inserts between cpTatC subunits arranged in a manner that conceivably forms an enclosed chamber.The location of the inserted signal peptide and the Tha4-cpTatC contact data suggest a model for signal peptide-gated Tha4 entry into the chamber to form the translocase.

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Affiliation: Horticultural Sciences Department and Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611.

ABSTRACT
The twin-arginine translocase (Tat) transports folded proteins across tightly sealed membranes. cpTatC is the core component of the thylakoid translocase and coordinates transport through interactions with the substrate signal peptide and other Tat components, notably the Tha4 pore-forming component. Here, Cys-Cys matching mapped Tha4 contact sites on cpTatC and assessed the role of signal peptide binding on Tha4 assembly with the cpTatC-Hcf106 receptor complex. Tha4 made contact with a peripheral cpTatC site in nonstimulated membranes. In the translocase, Tha4 made an additional contact within the cup-shaped cavity of cpTatC that likely seeds Tha4 polymerization to form the pore. Substrate binding triggers assembly of Tha4 onto the interior site. We provide evidence that the substrate signal peptide inserts between cpTatC subunits arranged in a manner that conceivably forms an enclosed chamber. The location of the inserted signal peptide and the Tha4-cpTatC contact data suggest a model for signal peptide-gated Tha4 entry into the chamber to form the translocase.

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Contact between the Tha4 TM and cpTatC TM4 in the translocase. (A) Radiolabeled pre-cpTatC L231C was imported into chloroplasts. Recovered thylakoids were incubated with in vitro–translated unlabeled Tha4 single Cys variants (XnC) followed by in vitro–translated SpF16 Tat substrate (see Materials and methods). Samples at 15°C were illuminated to assemble the translocase and CuP was added to promote disulfide formation (Materials and methods). Analysis was by SDS-PAGE/fluorography under nonreducing or reducing (+ β-mercaptoethanol) conditions. (B) Assays received SpF16 or mock translation extract before cross-linking. (C) Assays received either the full-size substrate tOE17-20F or the inactive twin lysine variant (KK-tOE17-20F). (D) Models of Tha4 and cpTatC with the positions of Cys substitutions marked with stars. Tha4 F4C E10Q is a nonfunctional Tha4 variant.
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fig1: Contact between the Tha4 TM and cpTatC TM4 in the translocase. (A) Radiolabeled pre-cpTatC L231C was imported into chloroplasts. Recovered thylakoids were incubated with in vitro–translated unlabeled Tha4 single Cys variants (XnC) followed by in vitro–translated SpF16 Tat substrate (see Materials and methods). Samples at 15°C were illuminated to assemble the translocase and CuP was added to promote disulfide formation (Materials and methods). Analysis was by SDS-PAGE/fluorography under nonreducing or reducing (+ β-mercaptoethanol) conditions. (B) Assays received SpF16 or mock translation extract before cross-linking. (C) Assays received either the full-size substrate tOE17-20F or the inactive twin lysine variant (KK-tOE17-20F). (D) Models of Tha4 and cpTatC with the positions of Cys substitutions marked with stars. Tha4 F4C E10Q is a nonfunctional Tha4 variant.

Mentions: Rollauer et al. (2012) postulated that TM4 residue Glu170 of E. coli TatC, which is on the concave surface of TatC, hydrogen-bonds to TM Gln8 of E. coli TatA during translocase assembly. We used Cys–Cys cross-linking to investigate such an interaction between comparable residues cpTatC Q234 and Tha4 E10. Single Cys substitutions were engineered into cpTatC and Tha4 and the resulting variants were shown to be functional in substrate binding and transport assays (Fig. S2 and Fig. S4; Aldridge et al., 2012; Ma and Cline, 2013). In Fig. 1, cpTatC with a Cys substitution of L231, which is on the same TM4 face and one helical turn removed from Q234, was paired with Tha4 with Cys in different domains (Fig. 1 D). Radiolabeled cpTatC L231C was imported into chloroplasts, and thylakoids obtained from the chloroplasts were incubated with in vitro–translated unlabeled Tha4-Cys and in vitro–translated substrate peptide (see Materials and methods). Reactions were illuminated to generate the proton gradient and assemble the translocase (Celedon and Cline, 2012), copper phenanthroline (CuP) was added to promote disulfide formation, and samples were analyzed by nonreducing SDS-PAGE and fluorography. In this assay, imported cpTatC assembles with a free pool of Hcf106 to form new receptor complexes, is fully functional, and is distinguishable from endogenous cpTatC (Ma and Cline, 2013). Added Tha4 is fully functional and present at ∼10 times the amount of endogenous Tha4 (Dabney-Smith et al., 2003; Celedon and Cline, 2012). We cannot presently incorporate significant amounts of exogenous Hcf106 into the receptor complex, but Hcf106 contains no Cys residues and cannot participate in disulfide cross-linking reactions.


Substrate-gated docking of pore subunit Tha4 in the TatC cavity initiates Tat translocase assembly.

Aldridge C, Ma X, Gerard F, Cline K - J. Cell Biol. (2014)

Contact between the Tha4 TM and cpTatC TM4 in the translocase. (A) Radiolabeled pre-cpTatC L231C was imported into chloroplasts. Recovered thylakoids were incubated with in vitro–translated unlabeled Tha4 single Cys variants (XnC) followed by in vitro–translated SpF16 Tat substrate (see Materials and methods). Samples at 15°C were illuminated to assemble the translocase and CuP was added to promote disulfide formation (Materials and methods). Analysis was by SDS-PAGE/fluorography under nonreducing or reducing (+ β-mercaptoethanol) conditions. (B) Assays received SpF16 or mock translation extract before cross-linking. (C) Assays received either the full-size substrate tOE17-20F or the inactive twin lysine variant (KK-tOE17-20F). (D) Models of Tha4 and cpTatC with the positions of Cys substitutions marked with stars. Tha4 F4C E10Q is a nonfunctional Tha4 variant.
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fig1: Contact between the Tha4 TM and cpTatC TM4 in the translocase. (A) Radiolabeled pre-cpTatC L231C was imported into chloroplasts. Recovered thylakoids were incubated with in vitro–translated unlabeled Tha4 single Cys variants (XnC) followed by in vitro–translated SpF16 Tat substrate (see Materials and methods). Samples at 15°C were illuminated to assemble the translocase and CuP was added to promote disulfide formation (Materials and methods). Analysis was by SDS-PAGE/fluorography under nonreducing or reducing (+ β-mercaptoethanol) conditions. (B) Assays received SpF16 or mock translation extract before cross-linking. (C) Assays received either the full-size substrate tOE17-20F or the inactive twin lysine variant (KK-tOE17-20F). (D) Models of Tha4 and cpTatC with the positions of Cys substitutions marked with stars. Tha4 F4C E10Q is a nonfunctional Tha4 variant.
Mentions: Rollauer et al. (2012) postulated that TM4 residue Glu170 of E. coli TatC, which is on the concave surface of TatC, hydrogen-bonds to TM Gln8 of E. coli TatA during translocase assembly. We used Cys–Cys cross-linking to investigate such an interaction between comparable residues cpTatC Q234 and Tha4 E10. Single Cys substitutions were engineered into cpTatC and Tha4 and the resulting variants were shown to be functional in substrate binding and transport assays (Fig. S2 and Fig. S4; Aldridge et al., 2012; Ma and Cline, 2013). In Fig. 1, cpTatC with a Cys substitution of L231, which is on the same TM4 face and one helical turn removed from Q234, was paired with Tha4 with Cys in different domains (Fig. 1 D). Radiolabeled cpTatC L231C was imported into chloroplasts, and thylakoids obtained from the chloroplasts were incubated with in vitro–translated unlabeled Tha4-Cys and in vitro–translated substrate peptide (see Materials and methods). Reactions were illuminated to generate the proton gradient and assemble the translocase (Celedon and Cline, 2012), copper phenanthroline (CuP) was added to promote disulfide formation, and samples were analyzed by nonreducing SDS-PAGE and fluorography. In this assay, imported cpTatC assembles with a free pool of Hcf106 to form new receptor complexes, is fully functional, and is distinguishable from endogenous cpTatC (Ma and Cline, 2013). Added Tha4 is fully functional and present at ∼10 times the amount of endogenous Tha4 (Dabney-Smith et al., 2003; Celedon and Cline, 2012). We cannot presently incorporate significant amounts of exogenous Hcf106 into the receptor complex, but Hcf106 contains no Cys residues and cannot participate in disulfide cross-linking reactions.

Bottom Line: Substrate binding triggers assembly of Tha4 onto the interior site.We provide evidence that the substrate signal peptide inserts between cpTatC subunits arranged in a manner that conceivably forms an enclosed chamber.The location of the inserted signal peptide and the Tha4-cpTatC contact data suggest a model for signal peptide-gated Tha4 entry into the chamber to form the translocase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Horticultural Sciences Department and Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611.

ABSTRACT
The twin-arginine translocase (Tat) transports folded proteins across tightly sealed membranes. cpTatC is the core component of the thylakoid translocase and coordinates transport through interactions with the substrate signal peptide and other Tat components, notably the Tha4 pore-forming component. Here, Cys-Cys matching mapped Tha4 contact sites on cpTatC and assessed the role of signal peptide binding on Tha4 assembly with the cpTatC-Hcf106 receptor complex. Tha4 made contact with a peripheral cpTatC site in nonstimulated membranes. In the translocase, Tha4 made an additional contact within the cup-shaped cavity of cpTatC that likely seeds Tha4 polymerization to form the pore. Substrate binding triggers assembly of Tha4 onto the interior site. We provide evidence that the substrate signal peptide inserts between cpTatC subunits arranged in a manner that conceivably forms an enclosed chamber. The location of the inserted signal peptide and the Tha4-cpTatC contact data suggest a model for signal peptide-gated Tha4 entry into the chamber to form the translocase.

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