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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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Substrate protein signal peptides insert between cpTatC subunits. Thylakoid membranes containing radiolabeled or unlabeled cpTatC E73C (domain S1, shown below the panels) were incubated in a binding reaction with radiolabeled or unlabeled single and double Cys variants of the full-length tOE17-20F substrate as shown above the panels. Disulfide cross-linking was conducted as described in the Materials and methods. The identification of B3 as substrate × cpTatC is inferred from the migration of the cross-linking product between cpTatC E73C and substrate −25C (lanes 6 and 11). The identification of band B2 as substrate dimer is inferred from the migration of the bands from the reaction containing radiolabeled substrate −3C (lane 12). Identification of the B4 band as substrate2 × cpTatC and the B5 band as substrate2 × cpTatC2 was based on their Mr and the substrate/cpTatC abundance in the band. The ratio of abundance was determined in an experiment in which substrate was labeled with 3H-leucine and cpTatC was labeled with 35S-methionine (lane 13). The ratio of tritium to 35S for the B3 band was set as 1:1 (asterisk) and the ratios of B4 and B5 calculated normalized to that value. Ratios are the average and standard deviation obtained from six individual cross-linking assays.
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fig7: Substrate protein signal peptides insert between cpTatC subunits. Thylakoid membranes containing radiolabeled or unlabeled cpTatC E73C (domain S1, shown below the panels) were incubated in a binding reaction with radiolabeled or unlabeled single and double Cys variants of the full-length tOE17-20F substrate as shown above the panels. Disulfide cross-linking was conducted as described in the Materials and methods. The identification of B3 as substrate × cpTatC is inferred from the migration of the cross-linking product between cpTatC E73C and substrate −25C (lanes 6 and 11). The identification of band B2 as substrate dimer is inferred from the migration of the bands from the reaction containing radiolabeled substrate −3C (lane 12). Identification of the B4 band as substrate2 × cpTatC and the B5 band as substrate2 × cpTatC2 was based on their Mr and the substrate/cpTatC abundance in the band. The ratio of abundance was determined in an experiment in which substrate was labeled with 3H-leucine and cpTatC was labeled with 35S-methionine (lane 13). The ratio of tritium to 35S for the B3 band was set as 1:1 (asterisk) and the ratios of B4 and B5 calculated normalized to that value. Ratios are the average and standard deviation obtained from six individual cross-linking assays.

Mentions: To investigate this possibility we conducted an experiment to determine if a pair of substrates cross-linked through their dimerization region could simultaneously be cross-linked from their RR-proximal regions to S1 domains of two different cpTatCs. The tOE17-20F substrates contained a Cys substitution at the RR-proximal −25 position and a second Cys substitution in the substrate dimerization region in the signal peptide (−3) or early mature domain (+3 or +17). Substrates were bound to membranes containing cpTatC E73C (S1 domain) and were subjected to disulfide cross-linking with CuP. As shown in Fig. 7, either the substrate or the cpTatC are radiolabeled and the other unlabeled, as designated above and below the panels. All three double Cys-substituted substrates produced five substrate-labeled bands when paired with cpTatC E73C (designated B1 through B5; lanes 1, 2, and 10), with the clearest banding pattern occurring with the −25C −3C substrate (lanes 9–13). B1 is the substrate monomer; B2 is the substrate dimer as shown by comparison to the banding for the −3C substrate, which forms dimers but does not cross-link to cpTatC E73C (lane 12). B3 is a 1:1 substrate/cpTatC adduct as shown in lanes 6 and 11, and as reported previously (Ma and Cline, 2013), that the −25C substrate cross-links to cpTatC E73C but does not form substrate dimers. The largest cross-linking product that could be obtained from the combination of −25C −3C substrate and cpTatC E73C would result from two cpTatC subunits individually cross-linked to the −25C positions of a substrate dimer linked through the −3 Cys. The B5 band migrated at the molecular weight expected for such an adduct. B4 migrated as expected for an adduct of two substrates linked to one cpTatC. This assignment was confirmed by an experiment in which cpTatC was labeled with 35S-methionine and substrate with 3H-leucine (lane 13). Quantification of 35S and 3H in bands yielded the expected 1:1 substrate/cpTatC ratio for B5 and 2:1 substrate/cpTatC ratio for B4. Because the −3 position is only 4 residues from the −7 position that contacts TM5 V270C on the concave side of cpTatC (Fig. 6), this result suggests that the signal peptide H-domains of the substrate dimer are inserted between concave faces of at least two cpTatC subunits (Fig. 8 B). A chase experiment (transport of bound precursor) verified that substrate moieties of B5 and B4 undergo transport when the membrane is energized (Fig. S5).


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)

Substrate protein signal peptides insert between cpTatC subunits. Thylakoid membranes containing radiolabeled or unlabeled cpTatC E73C (domain S1, shown below the panels) were incubated in a binding reaction with radiolabeled or unlabeled single and double Cys variants of the full-length tOE17-20F substrate as shown above the panels. Disulfide cross-linking was conducted as described in the Materials and methods. The identification of B3 as substrate × cpTatC is inferred from the migration of the cross-linking product between cpTatC E73C and substrate −25C (lanes 6 and 11). The identification of band B2 as substrate dimer is inferred from the migration of the bands from the reaction containing radiolabeled substrate −3C (lane 12). Identification of the B4 band as substrate2 × cpTatC and the B5 band as substrate2 × cpTatC2 was based on their Mr and the substrate/cpTatC abundance in the band. The ratio of abundance was determined in an experiment in which substrate was labeled with 3H-leucine and cpTatC was labeled with 35S-methionine (lane 13). The ratio of tritium to 35S for the B3 band was set as 1:1 (asterisk) and the ratios of B4 and B5 calculated normalized to that value. Ratios are the average and standard deviation obtained from six individual cross-linking assays.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig7: Substrate protein signal peptides insert between cpTatC subunits. Thylakoid membranes containing radiolabeled or unlabeled cpTatC E73C (domain S1, shown below the panels) were incubated in a binding reaction with radiolabeled or unlabeled single and double Cys variants of the full-length tOE17-20F substrate as shown above the panels. Disulfide cross-linking was conducted as described in the Materials and methods. The identification of B3 as substrate × cpTatC is inferred from the migration of the cross-linking product between cpTatC E73C and substrate −25C (lanes 6 and 11). The identification of band B2 as substrate dimer is inferred from the migration of the bands from the reaction containing radiolabeled substrate −3C (lane 12). Identification of the B4 band as substrate2 × cpTatC and the B5 band as substrate2 × cpTatC2 was based on their Mr and the substrate/cpTatC abundance in the band. The ratio of abundance was determined in an experiment in which substrate was labeled with 3H-leucine and cpTatC was labeled with 35S-methionine (lane 13). The ratio of tritium to 35S for the B3 band was set as 1:1 (asterisk) and the ratios of B4 and B5 calculated normalized to that value. Ratios are the average and standard deviation obtained from six individual cross-linking assays.
Mentions: To investigate this possibility we conducted an experiment to determine if a pair of substrates cross-linked through their dimerization region could simultaneously be cross-linked from their RR-proximal regions to S1 domains of two different cpTatCs. The tOE17-20F substrates contained a Cys substitution at the RR-proximal −25 position and a second Cys substitution in the substrate dimerization region in the signal peptide (−3) or early mature domain (+3 or +17). Substrates were bound to membranes containing cpTatC E73C (S1 domain) and were subjected to disulfide cross-linking with CuP. As shown in Fig. 7, either the substrate or the cpTatC are radiolabeled and the other unlabeled, as designated above and below the panels. All three double Cys-substituted substrates produced five substrate-labeled bands when paired with cpTatC E73C (designated B1 through B5; lanes 1, 2, and 10), with the clearest banding pattern occurring with the −25C −3C substrate (lanes 9–13). B1 is the substrate monomer; B2 is the substrate dimer as shown by comparison to the banding for the −3C substrate, which forms dimers but does not cross-link to cpTatC E73C (lane 12). B3 is a 1:1 substrate/cpTatC adduct as shown in lanes 6 and 11, and as reported previously (Ma and Cline, 2013), that the −25C substrate cross-links to cpTatC E73C but does not form substrate dimers. The largest cross-linking product that could be obtained from the combination of −25C −3C substrate and cpTatC E73C would result from two cpTatC subunits individually cross-linked to the −25C positions of a substrate dimer linked through the −3 Cys. The B5 band migrated at the molecular weight expected for such an adduct. B4 migrated as expected for an adduct of two substrates linked to one cpTatC. This assignment was confirmed by an experiment in which cpTatC was labeled with 35S-methionine and substrate with 3H-leucine (lane 13). Quantification of 35S and 3H in bands yielded the expected 1:1 substrate/cpTatC ratio for B5 and 2:1 substrate/cpTatC ratio for B4. Because the −3 position is only 4 residues from the −7 position that contacts TM5 V270C on the concave side of cpTatC (Fig. 6), this result suggests that the signal peptide H-domains of the substrate dimer are inserted between concave faces of at least two cpTatC subunits (Fig. 8 B). A chase experiment (transport of bound precursor) verified that substrate moieties of B5 and B4 undergo transport when the membrane is energized (Fig. S5).

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.

Show MeSH