Essential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane

Abstract

Two homologous GTP-binding proteins, atToc33 and atToc159, control access of cytosolic precursor proteins to the chloroplast. atToc33 is a constitutive outer chloroplast membrane protein, whereas the precursor receptor atToc159 also exists in a soluble, cytosolic form. This suggests that atToc159 may be able to switch between a soluble and an integral membrane form. By transient expression of GFP fusion proteins, mutant analysis, and biochemical experimentation, we demonstrate that the GTP-binding domain regulates the targeting of cytosolic atToc159 to the chloroplast and mediates the switch between cytosolic and integral membrane forms. Mutant atToc159, unable to bind GTP, does not reinstate a green phenotype in an albino mutant (ppi2) lacking endogenous atToc159, remaining trapped in the cytosol. Thus, the function of atToc159 in chloroplast biogenesis is dependent on an intrinsic GTP-regulated switch that controls localization of the receptor to the chloroplast envelope.

Figure 1.

Transient expression of atToc159 domains fused to GFP. GFP fusion constructs were transformed into isolated Arabidopsis protoplasts using the PEG method. Expression of GFP fusion proteins was monitored by Western blotting to confirm functionality of a GFP fusion to the NH₂ terminus of the G- and M-domains of atToc159 (Fig. 1 A; GFP-Toc159GM) or confocal laser scanning microscopy using a Leica DM IRBE microscope and a Leica TCS SP laser (Fig. 1, B–F). (A) Western blotting of the carbonate-extracted total membrane fraction (P) and soluble fraction (S) of protoplasts expressing either GFP or GFP-Toc159GM. Proteins were detected using either antibodies against GFP (α-GFP) or the A-domain of atToc159 (α-Toc159) to detect full-length endogenous atToc159. (B) GFP-Toc159GM. (C) GFP-Toc159G, NH₂-terminal GFP fusion to the G-domain. (D) GFP-Toc159M, NH₂-terminal GFP fusion to the M-domain. (E) Toc159A-GFP, COOH-terminal GFP fusion to the A-domain. (F) GFP control. Lane 1, GFP fluorescence; lane 2, autofluorescence; lane 3, merge of GFP and autofluorescence.

Figure 1.

Transient expression of atToc159 domains fused to GFP. GFP fusion constructs were transformed into isolated Arabidopsis protoplasts using the PEG method. Expression of GFP fusion proteins was monitored by Western blotting to confirm functionality of a GFP fusion to the NH₂ terminus of the G- and M-domains of atToc159 (Fig. 1 A; GFP-Toc159GM) or confocal laser scanning microscopy using a Leica DM IRBE microscope and a Leica TCS SP laser (Fig. 1, B–F). (A) Western blotting of the carbonate-extracted total membrane fraction (P) and soluble fraction (S) of protoplasts expressing either GFP or GFP-Toc159GM. Proteins were detected using either antibodies against GFP (α-GFP) or the A-domain of atToc159 (α-Toc159) to detect full-length endogenous atToc159. (B) GFP-Toc159GM. (C) GFP-Toc159G, NH₂-terminal GFP fusion to the G-domain. (D) GFP-Toc159M, NH₂-terminal GFP fusion to the M-domain. (E) Toc159A-GFP, COOH-terminal GFP fusion to the A-domain. (F) GFP control. Lane 1, GFP fluorescence; lane 2, autofluorescence; lane 3, merge of GFP and autofluorescence.

Figure 2.

Alignment of the GTP-binding domains of hsRas, atToc33, and atToc159 encompassing the G1 and G3 motifs. (A) Triple-point mutations in the G1 motif of atToc159mGTP are indicated in red. GenBank/EMBL/DDBJ accession nos.: hsRas, P01112; atToc159, AF069298; atToc33, U89959. (B) GTP binding to wild-type and mutant atToc159 G-domains (159G). Purified 159G and 159G-mGTP were bound to nitrocellulose and incubated with 50 nM α-[³²P]GTP (3,000 Ci/mmol) in the presence of 1 μM ATP. Bound α-[³²P]GTP was quantitated using a phosphorimager. Error bars indicate SD. (C) GTP hydrolysis by wild-type and mutant atToc159 G-domains. 1 μM α-[³²P]GTP (150 mCi/μmol) was incubated with 0.5 μM 159G or 159G-mGTP for 60 min at 25°C. Radiolabeled GTP and GDP were resolved by TLC on PEI-cellulose F plates using 1 M LiCl as the solvent, and radioactivity was quantitated using a phosphorimager. Error bars indicate SD. N.D., not detectable above background.

Figure 3.

Segregation analysis of ppi2 plants expressing atToc159wt (159wt) or atToc159mGTP (159mGTP). (A) Complementation of the ppi2 mutant. Three Arabidopsis lines each, expressing either of the constructs, were analyzed. Note that lines B0101, B0201, B1105, B0505, and B0502 are heterozygous for ppi2 (ppi2/TOC159), whereas line B0205 expressing atToc159wt is homozygous for ppi2 (ppi2/ppi2). PPT, phosphinothricine resistance gene; 35S, cauliflower mosaic virus 35S promoter; nos, nopaline synthase terminator; LB, left border of T-DNA; RB, right border of T-DNA. (B) Phenotypes of complemented plants. ppi2 plants expressing atToc159wt (top panel; line B0205, homozygous for ppi2) or atToc159mGTP (bottom panel; line B0502, heterozygous for ppi2). Plants were grown on selection media containing kanamycine and phosphinothricine.

Figure 4.

Genetic background of complementation lines. (A) PCR analysis of heterozygous (ppi2/TOC159) and homozygous (ppi2/ppi2) ppi2 plants expressing atToc159mGTP (159mGTP; line B0502) and homozygous ppi2 plants expressing full-length wild-type atToc159 (159wt, line B0205). The first reaction indicates the presence of undisrupted TOC159, the second the presence of the respective transgene, and the third indicates the presence of the 3′ end of TOC159 (control reaction). (B) Expression analysis of atToc159 (159wt) and atToc159mGTP (159mGTP) in a homozygous ppi2 background using RT-PCR. (C) Control PCR on total RNA after DNase treatment, but without reverse transcription.

Figure 5.

AtToc159mGTP (159mGTP) and atToc159wt (159wt) protein expression. (A) Total protein extracts were prepared from homozygous (ppi2/ppi2, lane 1) and heterozygous (ppi2/TOC159, lane 2) ppi2 plants expressing atToc159mGTP as well as homozygous (ppi2/ppi2) ppi2 plants expressing atToc159wt (lane 3). 50 μg of protein each were separated by SDS-PAGE, blotted onto nitrocellulose and stained with amido black (bottom). The blot was probed with affinity-purified antibodies raised against the A-domain of Toc159 (top). RbcL, large subunit of Rubisco. (B) Distribution of atToc159mGTP (159mGTP) and atToc159wt (159wt) between an alkaline-extracted membrane fraction (M, lanes 1 and 3) and a soluble fraction (S, lanes 2 and 4) prepared from homozygous ppi2 plants (ppi2/ppi2) expressing atToc159mGTP (lanes 1 and 2) or atToc159wt (lanes 3 and 4), respectively. 20 μg of protein each were separated by SDS-PAGE, blotted onto nitrocellulose, and stained with amido black (bottom). The blot was probed with affinity-purified antibodies raised against the A-domain of Toc159 (top). RbcL, large subunit of Rubisco.

Figure 6.

Association of atToc159 with isolated chloroplasts and integration into the outer chloroplast membrane. (A) In vitro synthesized [³⁵S]atToc159wt and [³⁵S]atToc159mGTP, respectively, were incubated with isolated Arabidopsis chloroplasts. Chloroplasts were subsequently reisolated and either directly analyzed by SDS-PAGE followed by autoradiography (lanes 2 and 6) or subjected to protease treatment with thermolysin at the concentrations indicated (T-lysin, lanes 3, 4, 7, and 8) before analysis. (B) Phosphorimager quantitation of the chloroplast association experiment. The relative amounts of [³⁵S]atToc159wt and [³⁵S]atToc159mGTP, respectively, associated with isolated chloroplasts (Chloroplast Association) as well as the 52-kD proteolytic fragments formed on treatment with thermolysin (52-kD Fragment) are indicated.

Figure 7.

Binding of [³⁵S]atToc159wt, [³⁵S]atToc159mGTP, and [³⁵S]atToc159_1–731 to atToc33_1–265-H₆. (A) Increasing concentrations of atToc33_1–265-H₆ were incubated with in vitro synthesized [³⁵S]atToc159wt, [³⁵S]atToc159mGTP, and [³⁵S]atToc159_1–731, respectively, reisolated using Ni-NTA chromatography and eluted with imidazole. The imidazole eluates were analyzed by SDS-PAGE followed by autoradiography. (B) Phosphorimager quantitation of the binding experiment. Binding of [³⁵S]atToc159wt (159wt), [³⁵S]atToc159mGTP (159mGTP), and [³⁵S]atToc159_1-731 (159A) is given in arbitrary units. Binding in the absence of atToc33_1-265-H₆ was adjusted to zero.