PMC

Ankyrin region of cpSRP43 and Alb3-Cterm coprecipitate. A, equimolar concentrations of GST or GST-43 construct were incubated with His-S_tag-Alb3-Cterm and then recovered using glutathione-Sepharose resin and eluted with SDS buffer. Eluates were analyzed by SDS-PAGE and Coomassie Blue staining. B, equimolar concentrations of His-S_tag-Alb3-Cterm were incubated with cpSRP43, His-Ank1-CD2, ΔCD2/CD3, His-Ank1–4, or His-CD2 and then recovered using S-protein-agarose resin and eluted with SDS buffer. Lanes show proteins precipitated by His-S_tag-Alb3-Cterm (+) compared with background binding to resin alone (−). Lanes labeled RC (recombinant control) show appropriate migration distance of each cpSRP43 construct into the gel. Eluates were analyzed as in A.

Interaction of cpSRP43 and Alb3-Cterm destabilizes transit complex. A, in vitro translated transit complex components (pLHCP, cpSRP43, and cpSRP54) were incubated in the presence of increasing concentrations (0–83.3 μm and 0–5000 pmol) of His-S_tag-Alb3-Cterm as indicated. Transit complex formation was examined using native PAGE and phosphorimaging for the radiolabeled component as indicated (*). TC indicates transit complex band. B, recombinant cpSRP43 and cpSRP54, in combination with in vitro translated and radiolabeled pLHCP, were used to form transit complex, which was monitored as in A after the addition of increasing concentrations (0–33.3 μm and 0–2000 pmol) of His-S_tag-Alb3-Cterm, GST, or CD3 as indicated. TC indicates transit complex band.

Current cpSRP43-dependent targeting model. Interactions with thylakoid membranes prime cpFtsY for binding cpSRP54 and GTP. Interactions with cpSRP43/LHCP prime cpSRP54 for binding GTP. The GTP-bound cpSRP43·LHCP·cpSRP54 complex associates with GTP-bound cpFtsY on thylakoid membranes. The membrane-associated complex is directed to Alb3 via an interaction between the Ank1–4 region of cpSRP43 and the C terminus of Alb3. cpSRP43 binding to the C terminus of Alb3 initiates LHCP release from cpSRP. LHCP, which acts as a negative regulator of hydrolysis, is released from cpSRP for insertion into thylakoids. In the absence of LHCP, interactions with thylakoid membranes, cpSRP43, and Alb3 trigger reciprocal stimulation of GTP hydrolysis by cpSRP54 and cpFtsY. GTP hydrolysis leads to dissociation of cpSRP43/54 and cpFtsY components from Alb3 and the thylakoid membrane. cpSRP43 may remain associated with Alb3 following departure of the GTPases from the membrane.

cpSRP43 is the predominant interacting partner with the translocase Alb3 in thylakoids. A, SW thylakoids (75 μg of Chl) were incubated with 10 μg of His-tagged constructs as indicated. Membranes were solubilized and used for purification with Talon Superflow metal affinity resin. Western blots of copurified proteins are shown probed for proteins indicated to the right. Protein Loading Control lanes contain thylakoid membranes or 50 ng of His-tagged construct for comparing relative amounts precipitated. aa, amino acids. B, graph depicts the amount of Alb3 copurified with His-tagged constructs. Total precipitated Alb3 was calculated from the relative signal of total thylakoid lane and eluate lanes in A.

Alb3-Cterm binding to cpSRP43 stimulates GTP hydrolysis by the cpSRP GTPases. The effect of Alb3-Cterm on the GTP hydrolysis activity of cpSRP54 and cpFtsY was examined in the presence or absence of cpSRP43. Assays contained 150 pmol (1 μm final concentration) of cpSRP43, cpSRP54, and cpFtsY and 0–6000 pmol (0–40 μm final concentration) of His-S_tag-Alb3-Cterm as indicated with 2 mm GTP as described under “Experimental Procedures.” GTPase activity resulting in the release of P_i was determined according to González-Romo et al. () using known phosphate standards. The average and S.D. were calculated from three separate experiments.

Ankyrin region of cpSRP43 is the interacting domain with the C terminus of Alb3. A, C, D, and E, ITC curves showing data characterizing interactions between His-FLAG-Alb3-Cterm with cpSRP43 constructs as indicated. All experiments were done at 25 °C. The insets and larger panels show the raw and integrated data, respectively, of the titration of cpSRP43 construct with Alb3-Cterm as indicated. The solid line in the larger panels represents the best fit curve of the data (Microcal Origin). B, depiction of the domain organization of cpSRP43, with triangles representing chromodomains and rounded rectangles representing ankyrins. Domains are listed in the N to C termini order across the top. Protein constructs described in this study are shown as listed on the left.

Ankyrin region of cpSRP43 and chromodomain 2 are necessary for Alb3-Cterm stimulation of GTP hydrolysis by the cpSRP GTPases. The effect of Alb3-Cterm on the GTP hydrolysis activity of cpSRP54 and cpFtsY was examined in the presence or absence of cpSRP43, His-Ank1–4, ΔCD1, ΔCD2, ΔCD3, His-Ank1-CD2, ΔCD2/CD3, His-CD1, and His-CD2. Assays contained 150 pmol (1 μm final concentration) of cpSRP43 construct, cpSRP54, and cpFtsY and 4000 pmol (27 μm final) of His-S_tag-Alb3-Cterm as indicated with 2 mm GTP. GTPase activity resulting in the release of P_i was determined according to González-Romo et al. () using known phosphate standards. The average and S.D. were calculated from three separate experiments.