Chloroplast Preproteins Bind to the Dimer Interface of the Toc159 Receptor during Import

Abstract

Most chloroplast proteins are synthesized in the cytosol as higher molecular weight preproteins and imported via the translocons in the outer (TOC) and inner (TIC) envelope membranes of chloroplasts. Toc159 functions as a primary receptor and directly binds preproteins through its dimeric GTPase domain. As a first step toward a molecular understanding of how Toc159 mediates preprotein import, we mapped the preprotein-binding regions on the Toc159 GTPase domain (Toc159G) of pea (Pisum sativum) using cleavage by bound preproteins conjugated with the artificial protease FeBABE and cysteine-cysteine cross-linking. Our results show that residues at the dimer interface and the switch II region of Toc159G are in close proximity to preproteins. The mature portion of preproteins was observed preferentially at the dimer interface, whereas the transit peptide was found at both regions equally. Chloroplasts from transgenic plants expressing engineered Toc159 with a cysteine placed at the dimer interface showed increased cross-linking to bound preproteins. Our data suggest that, during preprotein import, the Toc159G dimer disengages and the dimer interface contacts translocating preproteins, which is consistent with a model in which conformational changes induced by dimer-monomer conversion in Toc159 play a direct role in facilitating preprotein import.

Figure 1.

The prRBCS transit peptide directs the specific binding and import of prRBCS-GST into chloroplasts. A, Schematic representation of constructs used in this study. The red rectangles represent the transit peptides, and the blue rectangles represent the mature protein regions or the passenger proteins. The numbers above each construct indicate the positions of Cys residues, with the first residue of the mature protein designated as +1 and residues in the transit peptide designated with negative numbers. The AtToc159G recombinant protein construct, composed of AtToc159 residues 727 to 1,092 with a C-terminal His₆ tag, also is depicted. All constructs are drawn to scale. a.a., Amino acids. B, Binding and import of prRBCS-GST into isolated chloroplasts. GST (lane 1) and prRBCS-GST (lane 2) were purified from the soluble fraction of E. coli. Two different amounts were incubated with ATP-depleted chloroplasts in the presence of 0.1 and 3 mm ATP for the binding and import reactions, respectively. Samples from the import reaction were further treated with 0.2 mg mL⁻¹ thermolysin. Samples were analyzed by SDS-PAGE and immunoblotting with anti-GST antibody.

Figure 2.

Recombinant prRBCS-GST binds to AtToc159G. A, AtToc159G was expressed and purified from E. coli. Protein integrity was analyzed by SDS-PAGE, and the gel was stained with Coomassie Blue. B, Purified AtToc159G was incubated with GST or prRBCS-GST. Proteins were pulled down by GSH resin and analyzed by SDS-PAGE. The bound proteins were visualized by immunoblotting using the anti-GST or anti-AtToc159G antibody as indicated. The anti-AtToc159G antibody was raised in rats against the purified AtToc159G protein as shown in A.

Figure 3.

Cleavage of AtToc159G by FeBABE-conjugated preproteins. A, Schematic representation of the FeBABE cleavage mechanism. FeBABE was conjugated onto the thiol group of a Cys residue in the preprotein represented by the thick green line. In the presence of ascorbate and hydrogen peroxide (H₂O₂), the hydroxyl group of FeBABE becomes nucleophilic and attacks a peptide bond within a distance of 12 Å. B, prRBCS-GST with or without FeBABE conjugation was incubated with AtToc159G. Protein complexes were purified by GSH resin, and FeBABE cleavage was activated. The reactions were analyzed by SDS-PAGE and immunoblotting using the anti-AtToc159G or anti-His₆ antibody. S1_C and S2_C, AtToc159G C-terminal fragments generated by FeBABE cleavage; S1_N and S2_N, AtToc159G N-terminal fragments generated by FeBABE cleavage. C, The experiment was performed as described in B, except that prFd-protA_His or FeBABE-conjugated prFd-protA_His was used and IgG Sepharose was used to pull down prFd-protA_His and bound AtToc159G.

Figure 4.

AtToc159G structure model and sequence alignment. A, The two deduced sites cleaved by FeBABE-conjugated preproteins are colored in red. Additional residues mutated to Cys are indicated and colored in green. The N- and C-terminal ends, α-helix 2, and the central loop in switch II also are indicated. At left, one of the monomers is rotated 90° horizontally to show the dimer interface. B, Sequence alignment of the G domain region of Toc159 homologs from representative higher plant species and the pea Toc34 G domain. Secondary structure elements are marked above the sequence, and G1 to G3 motifs are marked below the sequence, all of which are labeled according to the pea Toc34 crystal structure (Protein Data Bank no. 1h65). The predicted switch II is boxed. Deduced residues cleaved by FeBABE and residues that generated significant cross-linking to preproteins when mutated to Cys are indicated with red and black numbers, respectively. Accession numbers for the sequences shown are as follows: At4g02510 (Arabidopsis Toc159), AAF75761 (pea Toc159), XP_009769991 (tobacco [Nicotiana tabacum] Toc159), NP_001147969 (maize [Zea mays] Toc159), XP_015639221 (rice [Oryza sativa] Toc159), XP_004152365 (cucumber [Cucumis sativus] Toc159), XP_002312975 (Populus spp. Toc159), and Q41009 (pea Toc34).

Figure 5.

Binding of preproteins reduces AtToc159G dimerization. A, Purified GST or prRBCS-GST was incubated with in vitro-translated [³⁵S]Met-labeled AtToc159G. BMH was added to cross-link protein complexes. Reactions were analyzed by SDS-PAGE and fluorography. The cross-linked AtToc159G dimer is indicated by arrowheads. The cross-linked adduct of AtToc159G-prRBCS-GST is indicated by the arrow. B, Binding and cross-linking experiments were performed as in A, except that purified prFd-protA_His was used.

Figure 6.

prRBCS-GST was cross-linked to Cys residues at the AtToc159G dimer interface and switch II. prRBCS-GST or GST was incubated with [³⁵S]Met-labeled single-Cys AtToc159G. After BMH cross-linking, reactions were analyzed by SDS-PAGE and fluorography. The locations of the single Cys residues are indicated above the lanes. A nonspecific cross-linked product from a protein in the reticulocyte lysate is indicated by the asterisk.

Figure 7.

The mature region of prFd-protA_His preferentially contacts the dimer interface of AtToc159G. A, prFd-protA_His was incubated with various [³⁵S]Met-labeled single-Cys AtToc159G mutants. After BMH cross-linking, reactions were analyzed by SDS-PAGE and fluorography. The locations of the single Cys residues are indicated above the lanes. Cys-864 and Cys-951 are located around FeBABE cleavage site 1 on the dimer interface, whereas Cys-917, Cys-920, and Cys-924 are located around cleavage site 2 in the switch II region. The cross-linked adduct of AtToc159G-prFd-protA_His is indicated by an arrow. A nonspecific cross-linked product from a protein in the reticulocyte lysate is indicated by the asterisk. B, Binding and cross-linking experiments were performed as described in A, except that prFd-protA_His (-37S/C) was used. Quantification of the cross-linked products is shown at right for each preprotein. The value for each single-Cys AtToc159G preprotein cross-linked product was normalized to the amount of cross-linking observed for AtToc159G Cys-864 to the same preprotein. Values plotted are means ± sd of three independent experiments.

Figure 8.

Genotypic and phenotypic analyses of transgenic plants. A, Total genomic DNA was isolated from the ppi2 mutant, the corresponding wild-type plant (WT), and three transgenic lines containing the transgene encoding the different AtToc159GM variants transformed into the ppi2 mutant. DNA was amplified by PCR using primer pairs specific for the endogenous AtToc159 gene, the ppi2 T-DNA insertion, or the transgene encoding the introduced AtToc159GM. Primer positions are depicted at right with red arrows. The attB1 and attB2 sites resulting from the LR recombination reaction are shown in blue, and the FLAG tag from the pEarleyGate 202 vector is shown in green. B, Phenotypes of 12-d-old wild-type, AtToc159GM transgenic, and ppi2 mutant plants grown on Murashige and Skoog (MS) medium. C, AtToc159 protein expression. Protein extracts from 12-d-old plants as shown in B were analyzed by SDS-PAGE and immunoblotting using anti-AtToc159 antibody.

Figure 9.

Cys placed at the dimer interface results in increased cross-linking of AtToc159 to preproteins bound on chloroplasts. A, prRBCS(-1C/A) exhibited more cross-linking to AtToc159 than prRBCS did. Chloroplasts were isolated from wild-type plants (WT) or AtToc159GM transgenic plants. [³⁵S]Met-labeled prRBCS or prRBCS(-1C/A) was bound to the chloroplasts under 100 µm ATP and cross-linked to the chloroplasts with BMH. Samples were analyzed by SDS-PAGE (lanes 1, 2, 5, and 6). Part of the samples from the AtToc159GM transgenic plant chloroplasts was solubilized with 1% lithium dodecyl sulfate (LDS) and subjected to immunoprecipitation (IP) using anti-AtToc75 or anti-AtToc159 antibody. The immunoprecipitates were analyzed by SDS-PAGE (lanes 3, 4, 7, and 8). B, AtToc159GM(A864C) showed more cross-linking to preproteins. Chloroplasts were isolated from AtToc159GM, AtToc159GM(A864C), and AtToc159GM(R894C) transgenic plants. [³⁵S]Met-labeled prRBCS(-1C/A) was bound to the chloroplasts under 100 µm ATP. Intact chloroplasts were reisolated. Part of the samples was analyzed directly to obtain the values of total prRBCS(-1C/A) bound on chloroplasts (lanes 1–3). Another part of the samples was used to perform BMH cross-linking assays (lanes 4–6). Samples were analyzed by SDS-PAGE and fluorography. C, Levels of AtToc159GM in transgenic plant chloroplasts. Chloroplasts from binding and cross-linking experiments as shown in B were serially diluted and analyzed by SDS-PAGE and immunoblotting using anti-AtToc159 antibody. The samples were analyzed on the same blotting membrane and developed for the same amount of time with intervening lanes removed. D, Quantification of the amount of cross-linking between AtToc159GM and prRBCS(-1C/A). Binding and cross-linking experiments as shown in B were performed, and the amount of AtToc159GM-prRBCS(-1C/A) cross-linked adduct (blue circle in B) was quantified by a phosphorimager. The value obtained was first normalized to the amount of prRBCS(-1C/A) bound to the chloroplasts of each transgenic line without BMH added (lanes 1–3 in B) and then further normalized to the amount of AtToc159GM in each transgenic plant as shown in C. Values plotted are means ± sd of three independent experiments.