Polypeptide Transport-Associated Domains of the Toc75 Channel Protein Are Located in the Intermembrane Space of Chloroplasts

Abstract

Toc75 is the channel for protein translocation across the chloroplast outer envelope membrane. Toc75 belongs to the Omp85 protein family and consists of three N-terminal polypeptide transport-associated (POTRA) domains that are essential for the functions of Toc75, followed by a membrane-spanning β-barrel domain. In bacteria, POTRA domains of Omp85 family members are located in the periplasm, where they interact with other partner proteins to accomplish protein secretion and outer membrane protein assembly. However, the orientation and therefore the molecular function of chloroplast Toc75 POTRA domains remain a matter of debate. We investigated the topology of Toc75 using bimolecular fluorescence complementation and immunogold electron microscopy. Bimolecular fluorescence complementation analyses showed that in stably transformed plants, Toc75 N terminus is located on the intermembrane space side, not the cytosolic side, of the outer membrane. Immunogold labeling of endogenous Toc75 POTRA domains in pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana) confirmed that POTRA domains are located in the intermembrane space of the chloroplast envelope.

Figure 1.

Schematic representation of the BiFC constructs used. The three POTRA domains and the β-barrel domain of Arabidopsis Toc75 (At3g46740) as well as the transmembrane domain of Arabidopsis Toc33 (At1g02280) are marked. The Arabidopsis Tic22 used is Tic22-IV (At4g33350). Transit peptides are shown in purple, and mature regions are shown in various shades of green. YN and YC are the N- and C-terminal fragments of YFP, respectively.

Figure 2.

BiFC analyses by transient expression in tobacco epidermal cells. Combinations of Agrobacterium strains containing the constructs indicated on the left were infiltrated into tobacco leaves. BiFC YFP signals, colored in green, and chlorophyll autofluorescence, colored in red, were examined in epidermal cells using a confocal microscope. Differential interference contrast (DIC) images of the same cells are shown on the right. Scale bars = 5 μm.

Figure 3.

BiFC analyses in stably transformed Arabidopsis plants. Combinations of YN and YC fusion proteins, as indicated at left, were coexpressed in Arabidopsis plants by stable transformation. BiFC YFP signals, colored in green, and chlorophyll autofluorescence, colored in red, were examined in guard cells on leaf epidermis using a confocal microscope. Differential interference contrast (DIC) images of the same cells are shown on the right. Scale bars = 5 μm.

Figure 4.

Endogenous Toc75 and most transgenic YN-Toc75 are thermolysin-resistant and trypsin-sensitive in intact chloroplasts. Intact chloroplasts isolated from wild-type (Columbia [Col]) or YN-Toc75 transgenic Arabidopsis plants were treated with different concentrations of thermolysin, trypsin, or trypsin supplemented with 0.5% Triton X-100 (+Triton). The protease-treated chloroplasts were analyzed by SDS-PAGE and immunoblotting. For samples without Triton, 10 µg of proteins were loaded in each lane. For samples with Triton, 0.75 µg chlorophyll equivalent of chloroplasts were loaded in each lane. The anti-Toc75 antibody used was raised against Arabidopsis Toc75 POTRA domains as described in “Materials and Methods” and Supplemental Figure S1.

Figure 5.

Gold particles labeling Toc75 POTRA domains were mostly located on the IMS side of the outer membrane in both pea and Arabidopsis chloroplasts. A to E, Ultrathin sections of pea (A–C) and Arabidopsis (D and E) chloroplasts were hybridized with antibodies against pea Toc75 POTRA-1 (A), pea Toc34G (B), and Arabidopsis Toc75 POTRA domains (D). C was hybridized with nonimmune rabbit IgG, and E was hybridized with IgG purified from the preimmune serum of the anti-Arabidopsis Toc75 POTRA domains antiserum. The sections were then hybridized with gold-conjugated secondary antibodies and observed under an electron microscope. Gold particles are indicated with red arrowheads. Total numbers of particles observed are 544 (A), 705 (B), and 514 (D). F, Percentages of gold particles observed on each side of the outer membrane or that overlap with the outer membrane (OM). See “Materials and Methods” for criteria to determine the positions of particles. Scale bars = 100 nm.