Figure 7.

Reconstitution into Thylakoid Lipid Vesicles Recovers the Activity of Prereduced and Redox-Inactive Plsp1.

(A) T7-Plsp1 (wild type or C286A) was purified in 0.25% Triton X-100 (micelles, final pH∼7.5) and reconstituted into liposomes to yield proteoliposomes (PLs) and were then divided into soluble (S) and pellet (P) fractions by centrifugation as described in Methods. The soluble fraction was treated with 0.1 M Na₂CO₃ for 5 min on ice and then subjected to ultracentrifugation to yield soluble and pellet fractions; nr = aliquot run in non-reducing sample buffer. Samples were analyzed by SDS-PAGE and immunoblotting.

(B) PLs at pH 8.0 were subjected to treatment with thermolysin (10 μg/mL with 0.5 mM CaCl₂) with or without 2% Triton X-100 (TX) at 25°C for 40 min. Samples were analyzed by SDS-PAGE and immunoblotting. The arrowhead indicates the T7-Plsp1 doublet, and “dp” indicates degradation products observed after thermolysin treatment.

(C) PLs at pH 8.0 were pretreated with or without 50 mM DTT and/or 2% Triton X-100 (TX) for 30 min on ice followed by incubation with ³⁵S-prPsbP for 30 min at 25°C. As a control, 60 nM purified T7-Plsp1 pretreated with or without 50 mM DTT was included. Samples were analyzed by SDS-PAGE and autoradiography. Δ = Boiled for 10 min before adding substrate; TP = translation product.

(D) Quantification of mature PsbP bands in C relative to the TP on the same gel. Shown are the means ± sd from three independent experiments. WT =wild type. Asterisks indicate undetectable activity.

(E) T7-Plsp1 in 1% octyl glucoside was treated with 50 mM DTT followed by reconstitution into liposomes made from thylakoid lipids as described in Methods except that 50 mM DTT was present throughout the reconstitution procedure. The recovered PLs were subjected to thermolysin treatment as in (B). S = soluble fraction after resuspending pelleted liposomes; P = pellet fraction after resuspending pelleted liposomes.

(F) T7-Plsp1 in 1% octyl glucoside was treated with or without DTT followed by TCA precipitation and incubation in the presence of 10 mM AMS. An aliquot of the DTT-treated reconstituted (recons.) T7-Plsp1 was also precipitated and treated with 10 mM AMS. 2S-AMS = reduced and AMS-labeled form of Plsp1; S-S = oxidized form of Plsp1.

(G) PLs described in E were incubated with ³⁵S-prPsbP as in (C), and samples were analyzed by SDS-PAGE and autoradiography.

(H) Quantification of mature PsbP bands in G relative to the untreated control. Shown are the means ± sd from two independent reconstitution experiments.