Polypeptide Transport-Associated Domains of the Toc75
Channel Protein Are Located in the Intermembrane
Space of Chloroplasts1[OPEN]
Yih-Lin Chen, Lih-Jen Chen, and Hsou-min Li*
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan
ORCID IDs: 0000-0003-1151-1706 (Y.-L.C.); 0000-0002-0211-7339 (H.-m.L.).
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 b-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).
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.
* Address correspondence to [email protected].
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 ([email protected]).
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.
[OPEN]
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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 b-barrel domain.
Structure analyses of BamA, the canonical Omp85
family member in bacteria, shows a 16-strand b-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 polypeptidebinding domains. In bacteria, all Omp85 homologs
analyzed have their POTRA domains protruding from
the b-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
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235
Chen et al.
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
b-barrels usually have an even number of b-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 b-strands. In the split-GFP
system, engineered GFP is separated into a large
N-terminal fragment containing the first 10 b-strands
and a small C-terminal fragment of the 11th b-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 b-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 b-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 b-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 Nand 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
Figure 1. Schematic representation of the BiFC constructs used. The
three POTRA domains and the b-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.
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Toc75 POTRA Domains Reside in the Intermembrane Space
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
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 mm.
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Chen et al.
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.
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 YNToc75 construct. T2 plants with confirmed expression of
the YN-Toc75 fusion protein were crossed with Arabidopsis plants with stably transformed Tic22-YC, YCToc33, 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
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 mm.
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Toc75 POTRA Domains Reside in the Intermembrane Space
outer membrane, respectively. These data show that the
N terminus of stably expressed YN-Toc75 was located
in the IMS.
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 trypsinsensitive, suggesting that most of the YN-Toc75 molecules had the same topology as endogenous Toc75.
However, a small fraction of YN-Toc75 was trypsinresistant 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
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 SDSPAGE and immunoblotting. For samples without Triton, 10 mg of proteins were loaded in each lane. For samples with Triton, 0.75 mg
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.
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.
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).
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-His 6, 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
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Chen et al.
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 goldconjugated 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.
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
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Toc75 POTRA Domains Reside in the Intermembrane Space
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 b-strand of GFP was placed at the N terminus
of an OEP80 construct that contained a 59-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 transitpeptide 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 mg/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).
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Chen et al.
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 mg of chlorophyll each) of intact chloroplasts
isolated from wild-type and YN-Toc75 transgenic Arabidopsis plants were
treated with 0, 100, or 200 mg/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 icecold import buffer containing trypsin inhibitor or EDTA. For treatments in the
presence of 0.5% Triton X-100, an equal volume of 23 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.
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.
Supplemental Figure S1. Antibody against Arabidopsis Toc75 POTRA
domains.
Supplemental Figure S2. YN-Toc75 transiently expressed in tobacco
leaves was slightly larger than YN-Toc75 in Arabidopsis transgenic
plant chloroplasts.
Supplemental Table S1. List of primers used in the study.
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 290°C for 72 h,
gradually warmed to 260°C, and kept for another 24 h. The temperature was
then gradually increased to 220°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 220°C for 24 h. (3) The sample was
infiltrated with London Resin Gold (Electron Microscopy Science) at 220°C
over 3 d. (4) Polymerization was conducted with UV light at 220°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 b-barrel (Arnold et al., 2010). Therefore, for a gold particle
labeling Toc75 POTRA, its maximum distance from the outer membrane would
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.
Received June 16, 2016; accepted July 5, 2016; published July 7, 2016.
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