A toc159 import receptor mutant, defective in hydrolysis of GTP, supports preprotein import into chloroplasts

Abstract

The heterotrimeric Toc core complex of the chloroplast protein import apparatus contains two GTPases, Toc159 and Toc34, together with the protein-conducting channel Toc75. Toc159 and Toc34 are exposed at the chloroplast surface and function in preprotein recognition. Together, they have been shown to facilitate the import of photosynthetic proteins into chloroplasts in Arabidopsis. Consequently, the ppi2 mutant lacking atToc159 has a non-photosynthetic albino phenotype. Previous mutations in the conserved G1 and G3 GTPase motifs abolished the function of Toc159 in vivo by disrupting targeting of the receptor to chloroplasts. Here, we demonstrate that a mutant in a conserved G1 lysine (atToc159 K868R) defective in GTP binding and hydrolysis can target and assemble into Toc complexes. We show that atToc159 K868R can support protein import into isolated chloroplasts, albeit at lower preprotein binding and import efficiencies compared with the wild-type receptor. Considering the absence of measurable GTPase activity in the K868R mutant, we conclude that GTP hydrolysis at atToc159 is not strictly required for preprotein translocation. The data also indicate that preprotein import requires at least one additional GTPase other than Toc159.

FIGURE 1.

Complementation of the ppi2 mutant by GTPase deficient Toc159GM K868R. A, phenotypes of homozygous ppi2 plants transformed with TAP-Toc159GM and TAP-Toc159GM K868R and untransformed A. thaliana plants. In the upper panel, untransformed plants are seedlings descended from a heterozygous ppi2 plant; in the lower panel an untransformed homozygous wild-type plant ecotype Wassilewskija is shown. B, chlorophyll levels in leaves of untransformed wild-type plants and homozygous ppi2 plants complemented by TAP-Toc159GM or TAP-Toc159GM K868R at 19 days after germination. C, confirmation of genotypes and the presence of the K868R single point mutation. PCR analysis of genomic DNA from plants shown above with primer sets specific for the ppi2 allele, the non-disrupted TOC159 gene (WT) and the transgene. Digestion of the transgene-specific PCR product by AseI is indicative of the presence of the K868R mutation that was introduced along with an AseI cleavage site. D, expression of the TAP-tagged proteins in the absence of endogenous Toc159. 50 μg of total protein extracts each were used for Western blotting with rabbit IgG for the detection of TAP-tagged protein and anti-Toc159 A-domain for the detection of endogenous full-length atToc159. The numbers in brackets represent the numbers of the transgenic plant lines (see supplemental Table S1).

FIGURE 2.

Expression of wild-type or mutated TAP-Toc159GM remedies the deficiency of ppi2 for Rubisco and chlorophyll a/b-binding protein. Western blot analysis using antibodies as indicated of 10, 20, and 40 μg of total protein of wild-type A. thaliana (ecotype Wassilewskija), ppi2 (toc159) mutant plants as well as ppi2 plants transformed with TAP-Toc159GM WT or K868R. For TAP-Toc159GM K868R:ppi2 plants of two different transgenic lines (B16) and (B20) were analyzed. Western blotting against actin was used to monitor gel loading.

FIGURE 3.

Membrane association and integration of TAP-Toc159GM K868R into the Toc complex. A, Western blot analysis of protein distribution in 100,000 × g supernatant (S) and pellet (P) fractions of plant protein extracts (L). The pellet fraction was treated in parallel with extraction buffer, 2 m NaCl or Na₂CO₃, and re-centrifuged. 50 μg of soluble or pelletable protein were analyzed by immunoblotting with antibodies as indicated. B, as control the same experiment as described in A but with Na₂CO₃ extraction only was performed with untransformed wild-type plants. C, co-isolation of Toc core complex components with TAP-Toc159GM WT or K868R. Western blot of the fractions obtained from affinity purification of TAP-tagged proteins on IgG-Sepharose. L, load; UB, unbound; E, eluate; PRK, phosphoribulokinase; BIP, luminal-binding protein.

FIGURE 4.

The effects of the GTPase mutations K868R and mGTP (A864R, K868N, S869R) on the in vitro membrane insertion of Toc159GM into chloroplasts. A, in vitro translated, [³⁵S]methionine-labeled Toc159 lacking the A-domain (Toc159GM) with or without the K868R or mGTP mutation were incubated with isolated Arabidopsis chloroplasts. Chloroplasts were reisolated and incubated in the absence (-) or presence (+) of 50 μg/ml of thermolysin (TL) for 30 min on ice. Samples were analyzed by SDS-PAGE and Coomassie Blue staining followed by PhosphorImager visualization and quantification. A section of the Coomassie Blue-stained gel is shown as a loading control. B, quantitative analysis of data from three replicate experiments using the Quantity One® software (BioRad). The experiments were calibrated to the amount of in vitro translated radioactive protein added to the chloroplasts (is 100%). For the quantification of insertion of Toc159GM, the data were normalized based on the methionine content of Toc159GM (15) and Toc159M (8).

FIGURE 5.

Complementation of the ppi2 import deficiency in a transient expression system using protoplasts. Protoplasts derived from leaf cells of homozygous ppi2 plants were transformed with a plasmid encoding the N-terminal transit peptide of the small subunit of Rubisco fused to GFP (RbcS-nt:GFP) together with the empty vector (1) or together with different Toc159 constructs as indicated (2-7). Total protein extracts were prepared from protoplasts 12 h after transformation and subjected to Western blot analysis with anti-GFP and anti-Toc159 antibodies (for the upper panel, anti-atTOC159 serum was used, for the lower panel, affinity-purified anti-atTOC159 A-domain). ^*, proteolytic product of the mature form (26).

FIGURE 6.

Import deficiency in TAP-Toc159GM K868R chloroplasts. A, in vitro translated, [³⁵S]methionine-labeled preprotein of the small subunit of Rubisco (pSSu) was incubated with isolated chloroplasts in the absence or presence of 0.1 mm ATP and GTP. The low concentrations of ATP and GTP allow for formation of the early import intermediate but not for complete translocation. Chloroplasts were reisolated and analyzed by SDS-PAGE and Coomassie Blue staining followed by PhosphorImager visualization and quantification. A section of the Coomassie Blue-stained gel is shown as a loading control (Coomassie). The graph shows quantitative data of three experiments. The pSSu preprotein bound to TAP-Toc159GM WT chloroplasts in the presence of ATP and GTP was set to 100%. B, chloroplasts were isolated from plants of the indicated genotypes, and used in protein import assays with the radioactive pSSu. Import was allowed to proceed for 2, 5, 10, and 15 min. Samples were analyzed by SDS-PAGE and Coomassie Blue staining followed by PhosphorImager visualization and quantification. The bands corresponding to mature SSu were quantified from triplicate experiments. The amount of imported SSu after 15 min of import into TAP-Toc159GM WT chloroplasts, was set to 100%. A section of the Coomassie Blue-stained gel is shown as a loading control (Coomassie). C, protein import into isolated chloroplasts was allowed to proceed for 5 and 15 min in the absence (-) or presence (+) of 10 mm of the non-hydrolyzable GTP analog GMP-PNP. The graph shows the quantification of mature SSu from three independent experiments. The amount of SSu after 15 min of import into TAP-Toc159GM WT chloroplasts in the absence of GMP-PNP was set to 100%. ^*, pSSu modified in the course of the import reactions.