Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system

Abstract

The chloroplast Twin arginine translocation (Tat) pathway uses three membrane proteins and the proton gradient to transport folded proteins across sealed membranes. Precursor proteins bind to the cpTatC-Hcf106 receptor complex, triggering Tha4 assembly and protein translocation. Tha4 is required only for the translocation step and is thought to be the protein-conducting component. The organization of Tha4 oligomers was examined by substituting pairs of cysteine residues into Tha4 and inducing disulfide cross-links under varying stages of protein translocation. Tha4 formed tetramers via its transmembrane domain in unstimulated membranes and octamers in membranes stimulated by precursor and the proton gradient. Tha4 formed larger oligomers of at least 16 protomers via its carboxy tail, but such C-tail clustering only occurred in stimulated membranes. Mutational studies showed that transmembrane domain directed octamers as well as C-tail clusters require Tha4's transmembrane glutamate residue and its amphipathic helix, both of which are necessary for Tha4 function. A novel double cross-linking strategy demonstrated that both transmembrane domain directed- and C-tail directed oligomerization occur in the translocase. These results support a model in which Tha4 oligomers dock with a precursor-receptor complex and undergo a conformational switch that results in activation for protein transport. This possibly involves accretion of additional Tha4 into a larger transport-active homo-oligomer.

Figure 1.

Double cysteine substitutions in Tha4. (A) Predicted structure of Tha4 with primary cysteines shaded black and secondary cysteines shaded gray. (B) Complementation efficiency of recombinant Tha4. Complementation efficiency (the relative amount of precursor transported) was calculated as the amount of DT23 transported in anti-Tha4–treated thylakoids normalized by the relative amount of recombinant Tha4 integrated into thylakoids. Complementation by wild-type (WT) in each experiment was arbitrarily set at 100%, and the amount of DT23 transported in the no-integration control, generally <2%, was subtracted from the test samples and set at 0%. Complementation efficiencies of Cys-substituted Tha4 were calculated relative to that of wild-type Tha4 in the same experiment.

Figure 2.

Tha4 C-tail oligomerizes in a transport-dependent manner. (A) Thylakoids were treated with 2.5 or 5.0 mM NEM or mock treated (0 NEM) before integration of the indicated in vitro-translated, radiolabeled Tha4. The oligomerization assays were done under transport conditions (precursor and the ΔpH) as described in Materials and Methods. Cys-Cys disulfide bond formation was initiated with 0.25 mM CuP and terminated after 5 min with 50 mM NEM and 12.5 mM EDTA as described in Materials and Methods. Recovered thylakoids were analyzed by 4–15% acrylamide Tris-tricine SDS-PAGE and fluorography under nonreducing conditions. Molecular weight markers are to the left and Tha4 oligomers are to the right. (B) Image taken from lane 9 in A and enlarged to show detail. Average M_r (relative molecular mass) of oligomers (in kilodaltons) was determined from relative mobilities of bands fit to a curve from the molecular mass standards. (C) Transport efficiency of thylakoids from A with or without recombinant Tha4 integrated as indicated below the chart. Efficiencies were calculated as the amount of DT23 transported in NEM or mock (no NEM)-pretreated thylakoids relative to that of an untreated control, which was arbitrarily set at 100%. (D) Cross-linking of Tha4 induced with different amounts of CuP. Assay conditions of dark (no ΔpH), light (ΔpH), and light + RR (ΔpH and precursor) are shown above the panel and described in Materials and Methods. Thylakoids were pretreated with 2.5 mM NEM before Tha4 integration. Disulfide cross-linking was as described in A.

Figure 3.

Tha4 TMDs oligomerize in stimulated thylakoids. Disulfide cross-linking of Tha4 directed by double Cys in the TMD was conducted in the presence of precursor and the ΔpH by the procedure described in Figure 2A. (A) Oligomerization via the TMD on the lumenal proximal side. An arrowhead indicates the anomalous G7CP9C cross-linking product. The gel in the left panel was a 12.5% Tris-glycine SDS-polyacrylamide gel and those in the right hand panel were 4–15% gradient Tris-tricine SDS-polyacrylamide gels. (B) Oligomerization via the TMD on the stromal proximal side. Samples were analyzed by 4–15% acrylamide Tris-tricine SDS-PAGE. Molecular weight standards are indicated to the left and the Tha4 oligomers to the right. Average M_r of oligomers is indicated in parentheses (in kilodaltons).

Figure 4.

Transport-related conditions required for Tha4 oligomerization via the TMD and via the C-tail. (A) Tha4V8CP9C was integrated into isolated, NEM-pretreated thylakoids and oxidative cross-linking was performed in the absence (lane 1) or presence of light (lane 2), light plus methylviologen (lanes 3–7) and either urea (lanes 1–3), 1.5 μM nonfunctional precursor (KK; lane 4), 1.5 μM functional precursor (RR; lane 5), or 1.5 μM synthetic signal peptide (tpOE17; lanes 6 and 7). Nigericin (0.5 μM) and valinomycin (1.0 μM) were added to completely dissipate the protonmotive force before cross-linking (lane 7). (B) A densitometry scan of lanes 3 and 5 of the film shown in A. This film was overexposed to detect the pentamer through octamer bands in the unstimulated membrane lanes. The film image was acquired with a scanner by using transmitted light and the peak profile produced by ImageJ (National Institutes of Health, Bethesda, MD). (C) As in A except Tha4A65CT78C was integrated into isolated, NEM-pretreated thylakoids. (D) A densitometry scan of lanes 5 and 6 of the film shown in C to demonstrate the similar oligomer patterns triggered by the signal peptide (SP, tpOE17) versus the full RR precursor DT23. Cross-linking was as described in Materials and Methods and recovered thylakoids were analyzed by 4–15% acrylamide Tris-tricine SDS-PAGE under nonreducing conditions.

Figure 5.

Tha4 structural requirements for oligomerization. Integration of different variants of Tha4 and cross-linking are as described in Materials and Methods. (A) Cross-linking in the TMD of Tha4. Tha4V8CP9C (WT), Tha4V8CP9CE10A (E10A), Tha4V8CP9CΔAPH (ΔAPH), Tha4V8CP9CΔ29 (ΔC-tail), and Tha4V8CP9CΔ54 (ΔC-tail/ΔAPH) were integrated into isolated thylakoids and assayed in the presence of ΔpH alone (−) or ΔpH and either nonfunctional (KK) or functional (RR) precursor as described in Figure 2 except that 1 mM CuP was used for the cross-linking. (B) Cross-linking in the C-tail. Thylakoids were pretreated with NEM. Tha4A65CT78C (WT), Tha4E10AA65CT78C (E10A), or Tha4A65CT78CΔAPH (ΔAPH) were integrated and assayed as described in A. (C) Cartoon diagram depicting the domain organization of Tha4. Deletion of these domains in the Tha4 used in A and B is denoted as Δ.

Figure 6.

Tha4 oligomers via the TMD are in the translocase. Tha4V8CP9C was integrated into isolated thylakoids and subjected to cross-linking in the presence of light and the absence or presence of functional precursor (RR) as indicated across the top of the panels. The sulfhydryl reactive cross-linker BMOE and the amine reactive cross-linker DSP were added simultaneously at a final concentration of 1 mM. DSP cross-linking was for 5 min and terminated with 100 mM glycine, whereas BMOE cross-linking continued for a total of 10 min as described in Materials and Methods. After cross-linking, samples were washed and subjected to coimmunoprecipitation by IgG-linked protein A-Sepharose beads. (A) Aliquots of samples before immunoprecipitation (pre-IP) were taken and used for SDS-PAGE under nonreducing (top) or reducing conditions (bottom). Each lane contains the equivalent of 0.9 μg of chlorophyll, and the gels were exposed to film for 9 d. (B) The remaining samples were subjected to coimmunoprecipitation with antiHcf106 (αHcf106 IP, top) to detect the presence of Tha4 in the translocase and with antipsAlb3 (αAlb3 IP, bottom) as a control for nonspecific interactions. Samples were eluted from the beads and =analyzed by SDS-PAGE under reducing conditions. Each immunoprecipitation lane contains the equivalent of 3.6 μg of chlorophyll of starting material, and the gels were exposed to film for 16 d.

Figure 7.

Tha4 oligomers via the C-tail are in the translocase. Tha4A65CT78C was integrated into isolated thylakoids and subjected to cross-linking in the presence of light and the absence or presence of functional precursor (RR). The sulfhydryl reactive cross-linker BM(PEO)₃ and the amine reactive cross-linker DSP were added simultaneously to 1 mM. Cross-linking and analysis was as described in Figure 6. (A) SDS-PAGE under nonreducing (top) or reducing conditions (bottom) before immunoprecipitation (pre-IP). Each lane contains the equivalent of 0.9 μg of chlorophyll. (B) The remaining samples were subjected to coimmunoprecipitation with antiHcf106 (αHcf106 IP, top) and with antipsAlb3 (αAlb3 IP, bottom). Samples were eluted from the beads and analyzed by SDS-PAGE under reducing conditions. Each lane contains the equivalent of 3.6 μg of chlorophyll. Gels in A and B were exposed to film for 16 d.