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

Yih-Lin Chen, Lih-Jen Chen, Hsou-min Li

Published September 2016. DOI: https://doi.org/10.1104/pp.16.00919

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.

Most chloroplast proteins are encoded by the nuclear genome and synthesized in the cytosol as higher Mr preproteins with an N-terminal transit peptide. Transit peptides direct preprotein import into chloroplasts through the translocons at the outer and inner envelope membranes of chloroplasts (the TOC and TIC complexes). The TOC core complex consists of two GTPase receptors, Toc159 and Toc34, and one protein translocation channel, Toc75. The principle components of the TIC machinery include the Tic20-Tic56-Tic100-Tic214 channel complex and Tic110, with the latter functioning as the stroma-side receptor for transit peptides and a scaffold for the attachment of stromal translocon proteins such as the Hsp93 ATPase motor (for review, see Li and Chiu, 2010; Shi and Theg, 2013; Paila et al., 2015). Tic22 is a translocon component located in the intermembrane space (IMS) of the two envelope membranes and has been proposed to function as a link between the TIC and TOC complexes (Kasmati et al., 2013; Rudolf et al., 2013).

Toc75 is a member of the Omp85/TpsB superfamily. Proteins in this family all have a domain structure consisting of a variable number of polypeptide transport-associated (POTRA) domains at the N terminus, followed by a C-terminal β-barrel domain. Structure analyses of BamA, the canonical Omp85 family member in bacteria, shows a 16-strand β-barrel forming a channel spanning the outer membrane and five POTRA domains located in the periplasm. POTRA domains are hydrophilic globular domains and function as protein-protein interaction and polypeptide-binding domains. In bacteria, all Omp85 homologs analyzed have their POTRA domains protruding from the β-barrel into the periplasm, where the POTRA domains interact with other partner proteins to accomplish protein secretion and outer membrane protein assembly (Sánchez-Pulido et al., 2003; Knowles et al., 2009; Hagan et al., 2011; Noinaj et al., 2013, 2015; Bakelar et al., 2016; Gu et al., 2016). Sam50, an Omp85 family member in yeast mitochondria, also has its single POTRA domain located in the IMS of the mitochondrial envelope (Habib et al., 2007).

In chloroplasts, the membrane topology of Toc75 is still debated (Inoue and Potter, 2004; Sommer et al., 2011; Paila et al., 2015). Protease protection analyses have shown that, in isolated intact chloroplasts, Toc75 is resistant to thermolysin but sensitive to trypsin treatments (Schnell et al., 1994; Jackson et al., 1998; Inoue and Potter, 2004; Chiu et al., 2010; Paila et al., 2016). Since thermolysin cannot penetrate the outer membrane, whereas trypsin can penetrate the outer but not the inner membrane, the protease treatment results suggest that Toc75 has no cytosolically exposed domain but has trypsin-sensitive domains located in the IMS. Recently, the POTRA domains of Arabidopsis (Arabidopsis thaliana) Toc75 have been shown to interact with Tic22 in vitro (Paila et al., 2016), further suggesting that the POTRA domains are located in the IMS. OEP80 (also named atToc75-V, At5g19620) is another Omp85 family member in the chloroplast outer membrane. Transgenic Arabidopsis plants expressing a C-terminal T7-tagged OEP80 have been produced (Hsu et al., 2012). Treatments of isolated intact chloroplasts from these transgenic plants with thermolysin showed that the C-terminal T7 tag was resistant to thermolysin digestion, suggesting that the C terminus of OEP80 is located in the IMS (Hsu et al., 2012). Since Omp85 family members usually have their N and C termini facing the same side of a membrane because their β-barrels usually have an even number of β-strands (Noinaj et al., 2013, 2015), the result suggests that the N-terminal POTRA domains of OEP80 are also located in the IMS. However, the opposite conclusion has been reached when the topology of Toc75 and OEP80 was analyzed using the self-assembling split-GFP assay in a protoplast transient expression system (Sommer et al., 2011). GFP is composed of 11 β-strands. In the split-GFP system, engineered GFP is separated into a large N-terminal fragment containing the first 10 β-strands and a small C-terminal fragment of the 11th β-strand. When located in the same subcellular compartment, the two fragments associate and fluoresce (Cabantous et al., 2005). For the analysis of Toc75 POTRA domain orientation, the Toc75-11N construct, consisting of the 11th β-strand of GFP inserted in-between the Arabidopsis Toc75 transit peptide and the N terminus of the POTRA domains, was transiently coexpressed with the first 10 β-strands of GFP localized either in the cytosol or IMS (abbreviated as 1-10CYT or 1-10IMS, respectively). Green fluorescence signals resulting from reconstitution of all 11 β-strands of GFP were detected in the periphery of chloroplasts only when Toc75-11N was coexpressed with 1-10CYT but not with 1-10IMS (Sommer et al., 2011). Similar results were obtained when the analysis was performed with OEP80, suggesting that POTRA domains of both Toc75 and OEP80 are located on the cytosolic side of the outer membrane. It was therefore concluded that chloroplast Omp85 family members had changed their orientation during evolution (Sommer et al., 2011).

POTRA domains are essential for Toc75 function. Deletion of even the first POTRA domain abolishes the function of Toc75 (Paila et al., 2016). Knowing the orientation of the POTRA domains is critical for understanding their functions in the protein import process. For example, if the POTRA domains are located on the cytosolic side of the outer membrane, they may be important for docking of cytosolic factors or may participate in the initial recognition of transit peptides. If the POTRA domains are located in the IMS, they may be important for binding transit peptides in the IMS and chaperoning preproteins during their translocation across the IMS as suggested by Paila et al. (2016), or they may even function in linking the TOC and TIC machineries.

In this study, we used bimolecular fluorescence complementation (BiFC; also called split-YFP assays), protease treatment, and immunogold electron microscopy to investigate the membrane topology of the Toc75 POTRA domains. The BiFC approach takes advantage of the ability of the yellow fluorescent protein (YFP) to fluoresce after being reconstituted from its N- and C-terminal halves (YN and YC; Waadt et al., 2008), similar to the split-GFP system. In Agrobacterium-infiltrated tobacco (Nicotiana benthamiana) leaves, BiFC YFP signals were detected when YN-Toc75 was coexpressed with YC-Toc33 in the cytosol and also with Tic22-YC in the IMS, indicating that there were two populations of YN-Toc75 with different orientations in the transient expression system. However, in stably transformed Arabidopsis plants, BiFC YFP signals were only detected when YN-Toc75 was coexpressed with Tic22-YC in the IMS, but not with YC-Toc33 or YC in the cytosol. Finally, immunogold labeling of endogenous Toc75 POTRA domains in both isolated pea (Pisum sativum) and Arabidopsis chloroplasts showed that POTRA domains are located in the IMS.

RESULTS

BiFC Analyses by Transient Expression in Tobacco Epidermal Cells

For BiFC analyses, we used the pVYNE series binary vectors (Waadt et al., 2008), which can be used both for Agrobacterium-mediated transient expression and for generating stably transformed plants. The fluorescent protein used in the vectors is Venus, a variant of enhanced YFP exhibiting significantly brighter fluorescence than enhanced YFP (Nagai et al., 2002). The N- and C-terminal halves, i.e. YN and YC, consist of amino acids 1 to 173 and amino acids 155 to 239 of Venus, respectively (Waadt et al., 2008). The BiFC system was originally designed to assay protein-protein interactions because the YN and YC fragments normally only assemble if brought together through interactions of proteins fused to each fragment. However, when overexpressed, for example, through the strong constitutive 35S promoter, YN and YC often assemble spontaneously when they are located in the same subcellular compartment (Kudla and Bock, 2016), similar to the self-assembling split-GFP system. In this study, we only used the BiFC system to assay if the two expressed fusion proteins were located in the same subcellular compartment.

We placed YN in-between the transit peptide and POTRA domains of Arabidopsis Toc75 (At3g46740), creating the construct YN-Toc75 (Fig. 1). We placed YC at the cytosolically localized N terminus of Toc33 (At1g02280). The outer membrane targeting and insertion signal of Toc33 family proteins is located at their C-terminal transmembrane domain (Chen and Schnell, 1997; Li and Chen, 1997). Therefore, the N-terminal YC tag does not interfere with proper membrane insertion of Toc33. For an IMS marker, we fused YN or YC to the C terminus of Arabidopsis Tic22-IV (At4g33350). All YN and YC fusion constructs were placed under the control of the 35S promoter (Fig. 1) in the pVYNE, pVYCE, or pVYCE(R) binary vectors. Different combinations of YN and YC constructs were then transiently coexpressed in tobacco leaf epidermal cells by Agrobacterium infiltration. When YN-Toc75 was coexpressed with Tic22-YC, BiFC YFP signals were detected in the periphery of chloroplasts and also in stromules (Fig. 2), suggesting that the N terminus of Toc75 was located in the IMS like Tic22. However, when YN-Toc75 was coexpressed with YC-Toc33, BiFC signals were also detected at the periphery of chloroplasts, suggesting that there were also some YN-Toc75 molecules with their YN portion exposed to the cytosol. BiFC signals were also detected at the periphery of chloroplasts when YC-Toc33 was coexpressed with cytosolic YN but not when YC-Toc33 was coexpressed with Tic22-YN, confirming that the Toc33 N terminus was exposed to the cytosol and Tic22 was localized in the IMS opposite to YC-Toc33. However, some punctuated YFP signals were observed when Tic22-YC was coexpressed with cytosolic YN, possibly caused by aggregation of some Tic22-YC on the chloroplast surface. These data suggest that the transiently overexpressed YN-Toc75 molecules had mixed orientations, possibly due to incomplete translocation or aggregation of some of the expressed proteins.

Figure 1.
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.
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.

BiFC Analyses in Stably Transformed Arabidopsis Plants

We next generated stably transformed plants expressing individual BiFC constructs to see whether we could circumvent the mixed-orientation problem encountered in the transient overexpression system. Arabidopsis plants were transformed with the YN-Toc75 construct. T2 plants with confirmed expression of the YN-Toc75 fusion protein were crossed with Arabidopsis plants with stably transformed Tic22-YC, YC-Toc33, or cytosolic YC. F1 or F2 plants confirmed to contain the two fusion proteins under testing were examined for BiFC signals. When plants expressing both YN-Toc75 and Tic22-YC were examined, BiFC signals were detected in the periphery of chloroplasts (Fig. 3). No BiFC signal was detected when YN-Toc75 was coexpressed with YC-Toc33 or cytosolic YC. BiFC signals were detected at the periphery of chloroplasts when cytosolic YN was coexpressed with YC-Toc33 but not when cytosolic YN was coexpressed with Tic22-YC, confirming that the YC portions of YC-Toc33 and Tic22-YC were on the cytosolic and IMS sides of the outer membrane, respectively. These data show that the N terminus of stably expressed YN-Toc75 was located in the IMS.

Figure 3.
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.

To confirm that the stably expressed YN-Toc75 has the same topology as endogenous Toc75, we performed protease protection analyses. Intact chloroplasts were isolated from the YN-Toc75 transgenic plants, treated with thermolysin or trypsin, analyzed by SDS-PAGE and immunoblotting, and probed with anti-Toc75 antibodies. As controls, the same samples were also probed with anti-Toc159 and anti-Gln synthase 2 (GS2) antibodies. Toc159, with its large cytosolically exposed domain, was sensitive to both thermolysin and trypsin, while the stromally localized GS2 was protected from both proteases in intact chloroplasts (Fig. 4). As shown previously (Schnell et al., 1994; Jackson et al., 1998; Inoue and Potter, 2004; Chiu et al., 2010; Paila et al., 2016), endogenous Toc75 was thermolysin-resistant and trypsin-sensitive in intact chloroplasts. YN-Toc75 was also thermolysin-resistant, just like the endogenous Toc75. A large fraction of YN-Toc75 was trypsin-sensitive, suggesting that most of the YN-Toc75 molecules had the same topology as endogenous Toc75. However, a small fraction of YN-Toc75 was trypsin-resistant in intact chloroplasts. These YN-Toc75 molecules were degraded when trypsin treatment was performed in the presence of 0.5% Triton X-100 to allow access of the protease to the interior of chloroplasts, suggesting that these YN-Toc75 molecules might have been mistargeted to a location inside the inner membrane. Under the same conditions with 0.5% Triton X-100, GS2 was degraded by trypsin, indicating that trypsin had indeed gained access to the stroma.

Figure 4.
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.

Immunogold Labeling of Endogenous Toc75 POTRA Domains

We next used immunogold electron microscopy to determine the membrane topology of endogenous Toc75 POTRA domains. For better visualization of the outer membranes, isolated pea chloroplasts were first incubated in 0.6 m Suc to increase the distance between the two envelope membranes (Keegstra and Yousif, 1986) before being processed by high-pressure freezing and freeze-substitution fixation and embedding. Ultrathin sections were immunolabeled with an antibody raised against the first POTRA domain of pea Toc75 (pea Toc75POTRA-1, Agrisera AS08 345), then with colloidal gold-conjugated secondary antibodies, and examined under an electron microscope. Gold particles were predominantly found along the periphery of chloroplasts close to the outer membrane (Fig. 5A). We observed 544 gold particles in total around the outer membrane; 78.5% of these particles were located on the IMS side of the outer membrane, 12.7% overlapped with the outer membrane, and 8.8% were on the cytosolic side of the outer membrane (Fig. 5F). As a control, we performed immunogold labeling of Toc34. The antibody used was raised against the cytosolically localized GTPase domain of pea Toc34 (Toc34G; Tu et al., 2004) and further affinity-purified using purified recombinant Toc34G. Gold particles labeling Toc34G appeared to be located even closer to the outer membrane than the gold particles labeling Toc75 POTRA domains, mostly adhered to the surfaces of the outer membrane (Fig. 5B). Of the 705 gold particles labeling Toc34G, 41.7% were on the cytosolic side of the outer membrane, 26.7% overlapped with the outer membrane, and 31.6% were on the IMS side of the outer membrane (Fig. 5F). No gold particles were detected when purified nonimmune rabbit IgG was used for the same immunogold labeling experiments (Fig. 5C).

Figure 5.
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.

We further verified the immunogold labeling results using Arabidopsis chloroplasts. Because our available anti-Toc75 antibodies were raised against pea Toc75 and do not recognize Arabidopsis well, we overexpressed the N-terminal region (residue 141–468) of Arabidopsis Toc75 encompassing the three POTRA domains. The overexpressed recombinant protein, termed atToc75POTRA-His6, was used to raise antibodies (Supplemental Fig. S1). The antibodies were further affinity-purified using atToc75POTRA-His6 before being used for the immunogold labeling experiments. As shown in Figure 5D, on ultrathin sections of isolated Arabidopsis chloroplasts, gold particles labeling Arabidopsis Toc75 POTRA domains were found along the periphery of chloroplasts and mostly on the IMS side of the outer membrane (Fig. 5D). We observed a total of 514 gold particles around the outer membrane; 81.5% of these particles were on the IMS side of the outer membrane, 12.5% overlapped with the outer membrane, and 6% were on the cytosolic side of the outer membrane (Fig. 5F). No gold particles were detected when purified IgG from the preimmune serum of the anti-atToc75POTRA-His6 antiserum was used for the same labeling experiment (Fig. 5E). The immunogold electron microscopy results using both pea and Arabidopsis chloroplasts support that the POTRA domains of Toc75 are located on the IMS side of the outer membrane.

DISCUSSION

We used four different approaches to investigate the orientation of the Toc75 POTRA domains relative to the outer membrane. BiFC analyses in tobacco epidermis showed that, when transiently overexpressed, at least two populations of YN-Toc75 were observed: one with the YN portion located in the IMS and the other with the YN portion exposed to the cytosol. It was not clear which one of the two populations resulted from mislocalization. Toc75 preproteins have a bipartite transit peptide. The first part of the transit peptide is cleaved by the stromal processing peptidase (Tranel and Keegstra, 1996), while the second part is cleaved by the Plsp1 signal peptidase in the IMS (Inoue et al., 2005), indicating that the N terminus of mature Toc75 is exposed to the IMS during the import of Toc75 preproteins. If the final location of the POTRA domains is in the IMS, as has been suggested (Hsu et al., 2012; Paila et al., 2016), then the cytosolically localized YN-Toc75 molecules most likely resulted from failure in initial translocation across the outer membrane. If the final location of POTRA domains is in the cytosol, as otherwise suggested (Sommer et al., 2011), then the IMS-localized YN-Toc75 may represent transport intermediates that had not yet flipped back across the outer membrane. To circumvent the potential problems of protein aggregation or incomplete translocation with the transient overexpression system (Dixit et al., 2006; Tanz et al., 2013), we created Arabidopsis transgenic lines stably expressing the YN and YC fusion proteins. Stably expressed YN-Toc75 only produced BiFC signals with Tic22-YC (Fig. 3). Protease protection assays further showed that most of the stably expressed YN-Toc75 had the same topology as endogenous Toc75 (Fig. 4). Finally, immunogold electron microscopy in intact chloroplasts isolated from both pea and Arabidopsis confirmed that the POTRA domains are located on the IMS side of the outer membrane (Fig. 5). Therefore, the cytosolically localized YN-Toc75 molecules in the tobacco transient expression system were most likely the result of failure in initial translocation across the outer membrane. In this case, it would be expected that these YN-Toc75 would be incompletely processed. Indeed, when analyzed by immunoblots, stably expressed YN-Toc75 in Arabidopsis chloroplasts comigrated with 110-kD marker (Supplemental Fig. S2, lane 3), while transiently overexpressed YN-Toc75 in tobacco leaves was larger in size and migrated slower than the 110-kD marker (Supplemental Fig. S2, lanes 1 and 2). This result suggests that most of the transiently overexpressed YN-Toc75 molecules in tobacco leaves were incompletely processed.

Using the split-GFP reporter and a transient expression system in Arabidopsis leaf mesophyll protoplasts, reconstituted GFP signals were observed in the periphery of chloroplasts when Toc75-11N was transiently coexpressed with 1-10CYT but not with 1-10IMS (Sommer et al., 2011). We partially confirmed this result by showing that, in a transient expression system using tobacco epidermis, some YN-Toc75 remained on the cytosolic side of the outer membrane. Both translocation and assembly of Toc75 preproteins seem to be very slow, and in vitro import of Toc75 preproteins into chloroplasts is always very inefficient (Tranel et al., 1995; Inoue and Keegstra, 2003). Even for endogenous Toc75 in plants, incompletely processed Toc75 intermediates can be detected in young leaves of pea seedlings, and complete processing is not observed until the leaves mature and open (Tranel et al., 1995). Transient expression in a protoplast system may not allow sufficient time for Toc75 to attain its final conformation. The overexpressed Toc75-11N fusion protein may also be prone to aggregation. The topology analysis of OEP80 by Sommer et al. (2011) may have additional complications. The 11th β-strand of GFP was placed at the N terminus of an OEP80 construct that contained a 5′-untranslated region and the potential transit peptide (Hsu et al., 2012; Day et al., 2014), resulting in more than 50 amino acids in front of the transit peptide. This fusion protein may not be able to attain the final topology of OEP80.

Sommer et al. (2011) also used electron cryotomography to investigate the orientation of the Toc75 POTRA domains. However, rather than employing intact chloroplasts, isolated outer membrane vesicles were used. The vesicles were inferred to be “right-side out” based on protease protection assays. However, while Toc159 is thermolysin-sensitive and Toc75 is thermolysin-resistant in intact chloroplasts, some Toc159 in their outer membrane vesicle preparation were thermolysin-resistant while some Toc75 became thermolysin-sensitive, indicating that their vesicles most likely had mixed orientations. Therefore, it would be difficult to conclude from the subsequent electron cryotomography whether an observed vesicle was right-side out or inside out.

POTRA domains are essential for the functions of Toc75 (Paila et al., 2016). Our data showing their IMS localization, together with the in vitro data showing direct binding of POTRA domains to transit peptides, suggest that one major function of the POTRA domains is to serve as the trans-side receptor for transit peptides as preproteins are translocated across the outer membrane. In addition, Toc75 POTRA domains have also been shown to directly bind Tic22 in vitro (Paila et al., 2016). Transformation of POTRA-deletion constructs into wild-type plants caused a dominant-negative effect in growth and triggered a massive increase in Tic22 protein levels (Paila et al., 2016). These data suggest that, in chloroplasts, Toc75 POTRA domains and Tic22 functionally interact. Interestingly, in the cyanobacterium Anabaena sp. PCC 7120, a Tic22 homolog has also been shown to directly interact with the POTRA domains of a Toc75 homolog (Tripp et al., 2012). Therefore, not only has the orientation of Toc75 POTRA domains been preserved during evolution, as we show here, but the functional and physical interaction of POTRA domains with Tic22 in the IMS has also been preserved. It would be interesting to analyze how their function has been adapted to facilitate protein traffic in opposite directions, i.e. toward the outer membrane in cyanobacteria and toward the inner membrane in chloroplasts.

MATERIALS AND METHODS

Constructs for BiFC

All desired coding regions were amplified by PCR and cloned into the binary vectors pVYNE, pVYCE, and pVYCE(R) (Waadt et al., 2008). Primers used are listed in Supplemental Table S1. To construct YN-Toc75, the transit-peptide coding region of Arabidopsis (Arabidopsis thaliana) Toc75 (At3g46740) was inserted into the XbaI site at the N terminus of YN in pVYNE. The region encoding the entire mature Toc75 without the transit peptide was then amplified and inserted into the SpeI/XhoI site at the C terminus of YN. To construct Tic22-YC or Tic22-YN, the cDNA encoding Arabidopsis Tic22-IV preprotein (At4g33350) was inserted into the SpeI/XhoI site at the N terminus of YC in pVYCE or into the SpeI/XhoI site at the N terminus of YN in pVYNE. To construct YC-Toc33, the cDNA encoding full-length Arabidopsis Toc33 (At1g02280) was inserted into the SpeI/XhoI site of pVYCE(R) at the C terminus of YC. All plasmids were verified by sequencing and transformed into Agrobacterium tumefaciens strain GV3101.

BiFC Analyses

For transient expression in tobacco epidermal cells, Agrobacterium strains carrying the BiFC constructs were infiltrated into leaves of 5- to 6-week-old Nicotiana benthamiana plants as described (Llave et al., 2000). The BiFC YFP signals were examined by confocal microscopy in leaf epidermis 2 d after the infiltration.

For analyses in transgenic Arabidopsis, Arabidopsis plants (Columbia ecotype) were transformed by the BiFC Agrobacterium strains using the floral spray method (Chung et al., 2000). Transgenic plants harboring the introduced DNA fragment encoding YN-Toc75, Tic22-YC, YC-Toc33, YC, or YN were screened on Murashige and Skoog medium containing 50 µg/mL hygromycin or kanamycin, and further confirmed by PCR and immunoblotting. BiFC YFP signals were examined in leaves of transgenic plants carrying different combinations of YN and YC constructs generated by crossing plants carrying individual constructs.

For confocal microscopy, leaf epidermis of tobacco or Arabidopsis was excited with a 514-nm argon laser; the emission bandwidth was 524 to 559 nm for YFP detection and 675 to 765 nm for chlorophyll autofluorescence. Images were acquired using a laser scanning confocal microscope (LSM780; Zeiss) equipped with a Plan-Apochromat 40X NA 0.95 objective lens (Zeiss) or LD C-Apochromat 40X NA 1.1 water objective lens (Zeiss), and driven by Zen acquisition and analysis software (Zeiss).

Chloroplast Isolation, Protease Treatments, Antibody Production, and Immunoblotting

Plant growth conditions and chloroplast isolations were performed as described previously (Chiu and Li, 2008). Isolated chloroplasts were adjusted to 1 mg chlorophyll/mL in import buffer (330 mm sorbitol, 50 mm HEPES-KOH, pH 8.0) and subjected to trypsin and thermolysin treatments as described (Jackson et al., 1998). Aliquots (75 µg of chlorophyll each) of intact chloroplasts isolated from wild-type and YN-Toc75 transgenic Arabidopsis plants were treated with 0, 100, or 200 µg/mL trypsin in import buffer with or without 0.5% Triton X-100 at room temperature for 1 h, or with thermolysin in import buffer supplemented with 1 mm CaCl2 on ice for 30 min. After protease treatments, trypsin and thermolysin were quenched by 2 mg/mL (w/v) trypsin inhibitor and 5 mm EDTA, respectively. For treatments in the absence of Triton X-100, intact chloroplasts were reisolated through 40% Percoll and washed with ice-cold import buffer containing trypsin inhibitor or EDTA. For treatments in the presence of 0.5% Triton X-100, an equal volume of 2× SDS-PAGE sample buffer was added directly after quenching the trypsin and the samples were boiled immediately.

Protease-treated chloroplasts were analyzed by SDS-PAGE and transferred to Immobilon-P PVDF membranes (Merck). Antibodies against Toc159 (dilution 1:1000; Tu et al., 2004), Arabidopsis Toc75 POTRAs (dilution 1:1000; see below), and GS2 (1:5000; Agrisera AS08 296) were used for immunoblotting. For immunoblotting, horseradish peroxidase-conjugated secondary antibodies were used for chemiluminescence detection as described (Chu and Li, 2012).

To overexpress the three POTRA domains of Arabidopsis Toc75, the coding region for residues 141 to 468 was amplified by PCR using primers that added a NdeI site to the N terminus and a XhoI site to the C terminus of the PCR fragment. The PCR fragment was cloned into the NdeI/XhoI site of pET-22b (Merck), and the resulting recombinant protein produced was named atToc75POTRA-His6. Recombinant atToc75POTRA-His6 was purified from Escherichia coli and used to raise antibodies in rabbits.

Immunogold Electron Microscopy

Intact chloroplasts isolated from leaves of 9-d-old pea (Pisum sativum) seedlings or 14-d-old Arabidopsis seedlings were incubated in import buffer containing 0.6 m Suc for 10 min on ice. The chloroplasts were pelleted down and transferred to 3-mm metal carriers for immediate high-pressure freezing in Leica EM HPM100. Freeze substitution was conducted in Leica EM ASF as follows. (1) Samples in metal carriers were incubated in anhydrous ethanol containing 0.2% glutaraldehyde and 0.1% uranyl acetate at −90°C for 72 h, gradually warmed to −60°C, and kept for another 24 h. The temperature was then gradually increased to −20°C. (2) Prior to packing the sample into the plastic capsule, the solution was substituted with anhydrous ethanol, and the capsule was kept in anhydrous ethanol at −20°C for 24 h. (3) The sample was infiltrated with London Resin Gold (Electron Microscopy Science) at −20°C over 3 d. (4) Polymerization was conducted with UV light at −20°C for 24 h and then at room temperature for 48 h. Ultrathin sections were put on nickel grids. The grids were first blocked with 4% (w/v) bovine serum albumin at room temperature for 30 min, and then incubated with primary antibodies in TBST (20 mm Tris-HCl, pH 7.4, 500 mm NaCl, and 0.05% Tween 20) at 4°C overnight. The rabbit anti-pea Toc75 POTRA-1 antibodies (dilution 1:800; Agrisera AS08 345), affinity-purified anti-Toc34G antibody (dilution 1:1), and purified nonimmune rabbit IgG (dilution 1:600) were used as the primary antibodies for labeling pea chloroplasts. Affinity-purified antibodies against Arabidopsis Toc75 POTRA domains (dilution 1:4) and the preimmune serum IgG of the same rabbit (dilution 1:600) were used for labeling Arabidopsis chloroplasts. The grids were then washed in TBST, incubated with 12-nm gold-conjugated goat anti-rabbit IgG (dilution 1:30; Jackson Immunoresearch), and washed with TBST and then with distilled water. The sections were further stained with uranyl acetate. Images were obtained by a transmission electron microscope (Tecnai G2 Spirit TWIN; FEI Company) equipped with a Gatan CCD camera (794.10.BP2 MultiScan) and the acquisition software Digital Micrograph (Gatan).

To quantify the distribution of gold particles around the outer membrane, we considered the sizes of the molecules involved. Each IgG molecule is about 12 nm long. The crystal structure of the Toc75 homolog in the cyanobacterium Thermosynechococcus elongatus shows that the three POTRA domains extend about 10 nm from the β-barrel (Arnold et al., 2010). Therefore, for a gold particle labeling Toc75 POTRA, its maximum distance from the outer membrane would be approximately 34 nm (primary and secondary antibodies plus the three POTRA domains). Although almost all of the gold particles we observed were in close vicinity to the outer membrane, only particles that were on the cytosolic side and within 34 nm of the cytosolic surface of the outer membrane were counted as being on the “cytosolic side” in Figure 5F. Gold particles that were on the IMS side and within 34 nm of the IMS surface of the outer membrane were counted as being on the “intermembrane space side.” For Toc34, the crystal structure of the pea Toc34 GTPase domain shows that the GTPase domain extends 5.6 nm into cytosol (Sun et al., 2002). Therefore, gold particles that were on the cytosolic side and within 29.6 nm (primary and secondary antibodies plus one Toc34 GTPase domain) of the cytosolic surface of the outer membrane were counted as being on the cytosolic side. Particles that were on the IMS side and within 24 nm (primary and secondary antibodies) of the cytosolic surface of the outer membrane were counted as being on the intermembrane space side.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL/TAIR data libraries under accession numbers At3g46740 (Arabidopsis Toc75), At4g33350 (Arabidopsis Tic22), and At1g02280 (Arabidopsis Toc33).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Dr. Jörg Kudla for the binary vectors used for the BiFC analyses. We thank the IMB Imaging Core for assistance with confocal and electron microscopy, and the English Editing Core for English editing.

Footnotes

  • www.plantphysiol.org/cgi/doi/10.1104/pp.16.00919

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hsou-min Li (mbhmli{at}gate.sinica.edu.tw).

  • Y.-L.C. designed the experiments, performed most of the experiments, and analyzed the data; H.-m.L. and L.-J.C. provided technical assistance; H.-m.L. conceived the project and designed the experiments; H.-m.L. and Y.-L.C. wrote the article.

  • ↵1 This work was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 104-2321-B-001-021), and Academia Sinica of Taiwan to H.-m.L.

  • ↵[OPEN] Articles can be viewed without a subscription.

Glossary

IMS
intermembrane space
BiFC
bimolecular fluorescence complementation
  • Received June 16, 2016.
  • Accepted July 5, 2016.
  • Published July 7, 2016.

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Polypeptide Transport-Associated Domains of the Toc75 Channel Protein Are Located in the Intermembrane Space of Chloroplasts
Yih-Lin Chen, Lih-Jen Chen, Hsou-min Li
Plant Physiology Sep 2016, 172 (1) 235-243; DOI: 10.1104/pp.16.00919
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