The chloroplast twin arginine transport (Tat) component, Tha4, undergoes conformational changes leading to Tat protein transport

Abstract

Twin arginine transport (Tat) systems transport folded proteins using proton-motive force as sole energy source. The thylakoid Tat system comprises three membrane components. A complex composed of cpTatC and Hcf106 is the twin arginine signal peptide receptor. Signal peptide binding triggers assembly of Tha4 for the translocation step. Tha4 is thought to serve as the protein-conducting element, and the topology it adopts during transport produces the transmembrane passageway. We analyzed Tha4 topology and conformation in actively transporting translocases and compared that with Tha4 in nontransporting membranes. Using cysteine accessibility labeling techniques and diagnostic protease protection assays, we confirm an overall N(OUT)-C(IN) topology for Tha4 that is maintained under transport conditions. Significantly, the amphipathic helix (APH) and C-tail exhibited substantial changes in accessibility when actively engaged in protein transport. Compared with resting state, cysteines within the APH became less accessible to stromally applied modifying reagent. The APH proximal C-tail, although still accessible to Cys-directed reagents, was much less accessible to protease. We attribute these changes in accessibility to indicate the Tha4 conformation that is adopted in the translocase primed for translocation. We propose that in the primed translocase, the APH partitions more extensively and uniformly into the membrane interface and the C-tails pack closer together in a mesh-like network. Implications for the mode by which the substrate protein crosses the bilayer are discussed.

FIGURE 1.

Testing Cys labeling approaches using cpTatC. Two topology-labeling strategies were employed: biocytin/streptavidin (SA) labeling in intact or detergent-solubilized thylakoid membranes (B) and direct Br-PEG labeling (C). A, topology of the control protein cpTatC (25). Amino acid residues substituted with cysteine are indicated in black with the residue number. B, cpTatC biocytin/SA labeling: demonstration of SA accessibility patterns for cysteines in the stromal region (K72C), the transmembrane domain (L92C), and the lumen region (L126C) of cpTatC in thylakoid in the presence (+) or absence of digitonin, where accessibility is indicated by the appearance of a higher molecular weight cpTatC:SA adduct (arrow). C, cpTatC branched-PEG labeling: demonstrated accessibility of Br-PEG (+) where accessibility is indicated by the appearance of the cpTatC:Br-PEG adduct (arrowhead). Samples were analyzed by SDS-PAGE and fluorography. Gels are representative of at least two experiments.

FIGURE 2.

Tha4 topology labeling shows a N_OUT-C_IN conformation. A, Tha4 labeling: survey of SA accessibility to cysteines placed in the predicted lumen region (F3C, F4C), the transmembrane domain (V13C), the near hinge region (V21C), the APH domain (F48C), and the C-tail (T78C) as indicated by Tha4:SA adduct bands (arrow). All samples were treated with biocytin followed by SA either in the absence or presence (+) of digitonin solubilization of the membrane. B, Tha4 labeling: survey of Br-PEG accessibility to cysteine for the same residues tested in B. The Tha4:Br-PEG adduct is marked with an arrowhead. Samples were analyzed by SDS-PAGE and fluorography. Gels are representative of at least two experiments. C, amino acid sequence of Tha4 as modeled from B. subtilis TatA_d (29). Amino acid residues substituted with cysteine are indicated in black with the residue number.

FIGURE 3.

Tha4 emerges from the hydrophobic membrane core at the N-proximal APH. A, biocytin/SA labeling: accessibility of cysteines placed in the hinge region of Tha4 to added SA with intact thylakoids or after (+) digitonin solubilization. B, branched-PEG labeling: accessibility of cysteines placed in the hinge region of Tha4 to Br-PEG. Assays were as described in the legend to Fig. 1 and under ”Experimental Procedures.“ Samples were analyzed by SDS-PAGE and fluorography. Gels are representative of at least two experiments.

FIGURE 4.

The N terminus of the APH is accessible to protease cleavage. A, schematic of Factor Xa protease cleavage site at the APH N terminus of Tha4. B, Factor Xa cleavage of Tha4 over time (min) (left panel). Disruption of the cryptic site with an R32K substitution confirms the location of the cleavage site (right panel). Assays are as described under ”Experimental Procedures.“ Samples were analyzed by SDS-PAGE and fluorography. Gels are representative of three experiments.

FIGURE 5.

A gradient of accessibility of residues in the APH. A, total biocytin labeling of Tha4 single cysteine residues placed along the entire APH as determined by SA binding with digitonin-solubilized thylakoids calculated from scintillation counting of radiolabeled proteins extracted from gel bands as described (17). Biocytin labeling is expressed as a percentage of total integrated Tha4 for each Tha4 Cys variant. B, percent stromal accessibility of biocytin-labeled APH residues as determined by SA binding to intact thylakoid membranes. Percent stromal accessibility is expressed as a percentage of the total amount of biocytin-labeled Tha4. Percent of biocytin labeling was assigned relative to the totally accessible A65C control. Includes data from at least two separate trials for each residue. C, Br-PEG labeling: accessibility of Br-PEG to select hydrophobic and hydrophilic substitutions within the APH of Tha4 measured with intact thylakoids. Samples were analyzed by SDS-PAGE and fluorography. Gels are representative of at least two experiments.

FIGURE 6.

Differential accessibility of APH and C-tail residues to Br-PEG with or without transport conditions. A, Br-PEG labeling (PEG adduct arrowhead) was compared in the absence or presence (+) of transport of the OE17V-20F precursor to determine whether cysteine accessibility is increased (upward arrow), decreased (downward arrow), or unchanged (sideways arrow). Solid arrows indicate a more significant accessibility change. Some residues were not labeled under either condition (U). Labeling was for 2.5 min during the period of maximum precursor transport as described under ”Experimental Procedures.“ Precursor (p) and mature (m) tOE17V-20F, and Tha4 monomers are indicated. Samples were analyzed by SDS-PAGE and fluorography. Gels are representative of at least two separate trials for each residue tested.

FIGURE 7.

Differential accessibility of APH residue T37C is independent of precursor size. Precursor proteins of different molecular sizes were tested for their ability to induce the differential Br-PEG accessibility of Tha4T37C. The mature domain of SufI is 49 kDa. tOE17V-20F has a 17-kDa mature domain, SpF16, SpF12, and SpF8 are truncated versions of tOE17V-20F with only 16, 12, and 8 mature domain residues, respectively. Tha4F41C is labeled to the same extent with or without transport and is used as a control. Samples were analyzed by SDS-PAGE and fluorography. Gels are representative of three similar experiments.

FIGURE 8.

Reduced accessibility of the APH proximal C-tail to protease during protein transport. Effect of transport on Tha4 C-tail accessibility was studied by insertion of two different thrombin cleavage sites; one placed at the extreme C terminus followed by a His₆ tag; the other near the APH after Tha4 residue 62. Thrombin was added during the period of maximum precursor transport and Tha4 cleavage was monitored over time as described under ”Experimental Procedures.“ A, Tha4CT-Throm: thrombin cleavage of a site inserted at the C terminus of Tha4 in the light or in the light with precursors tOE17V-20F, SpF16, or SpF8. Samples were analyzed by SDS-PAGE followed by immunoblotting with anti-His antibody. Cleavage was indicated by disappearance of the His tag signal and quantified through densitometry using Quantity One (Bio-Rad). Data shown are representative of two experiments. B, Tha4I62-Throm: thrombin cleavage of a site inserted in the C-tail of Tha4 closer to the APH (Ile⁶²) was monitored in the presence of light ± precursors tOE17V-20F, SpF16, or SpF8. Samples were analyzed by SDS-PAGE and fluorography. Cleavage was calculated from scintillation counting of radiolabeled proteins extracted from gel bands as described (17). Error bars represent mean ± S.E. of 3 experiments (light, light + tOE17V-20F) or mean ± range of two experiments (light + SpF16). SpF8 represents a single experiment. Nonlinear regression analysis was performed using GraphPad Prism, version 5.0d for Mac (GraphPad Software, San Diego, CA).