Journal of Experimental Botany, Vol. 65, No. 18, pp. 5257–5265, 2014
doi:10.1093/jxb/eru290 Advance Access publication 10 July, 2014
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
v
Research Paper
Targeting of a polytopic membrane protein to the inner
envelope membrane of chloroplasts in vivo involves multiple
transmembrane segments
Kumiko Okawa1, Hitoshi Inoue2, Fumi Adachi1, Katsuhiro Nakayama1, Yasuko Ito-Inaba3, Danny J. Schnell2,
Susumu Uehara1 and Takehito Inaba1,*
1 Department of Agricultural and Environmental Sciences, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, 01003 MA, USA
3 Organization for Promotion of Tenure Track, University of Miyazaki, Miyazaki 889-2192, Japan
2 * To whom correspondence should be addressed. E-mail: [email protected]
Received 12 March 2014; Revised 21 May 2014; Accepted 5 June 2014
Abstract
The inner envelope membrane (IEM) of the chloroplast plays crucial roles in forming an osmotic barrier and controlling metabolite exchange between the organelle and the cytosol. The IEM therefore harbours a number of membrane
proteins and requires the import and integration of these nuclear-encoded proteins for its biogenesis. Recent studies
have demonstrated that the transmembrane segment of single-spanning IEM proteins plays key roles in determining their IEM localization. However, few studies have focused on the molecular mechanisms by which polytopic
membrane proteins are targeted to the IEM. In this study, we investigated the targeting mechanism of polytopic IEM
proteins using the protein Cor413im1 as a model substrate. Cor413im1 does not utilize a soluble intermediate for its
targeting to the IEM. Furthermore, we show that the putative fifth transmembrane segment of Cor413im1 is necessary for its targeting to the IEM. The C-terminal portion containing this transmembrane segment is also able to deliver
Cor413im1 protein to the IEM. However, the fifth transmembrane segment of Cor413im1 itself is insufficient to target
a fusion protein to the IEM. These data suggest that the targeting of polytopic membrane proteins to the chloroplast
IEM in vivo involves multiple transmembrane segments and that chloroplasts have evolved a unique mechanism for
the integration of polytopic proteins to the IEM.
Key words: Arabidopsis, chloroplast, Cor413im1, inner envelope membrane, polytopic proteins, protein targeting.
Introduction
Plastid biogenesis is dependent on the import of several
thousand nuclear-encoded proteins into plastids (Inaba and
Schnell, 2008; Jarvis, 2008; Li and Chiu, 2010). The archetypal plastid is the chloroplast found in photosynthetic cells.
Chloroplasts consist of six distinct sub-compartments: the
outer envelope membrane (OEM), the inner envelope membrane (IEM), stroma, intermembrane space (IMS), thylakoid
membrane, and the thylakoid lumen. Each subcompartment
plays roles in synthesizing, storing, catabolizing, and transporting metabolites. Hence, the biogenesis and function of
each compartment rely on the import and proper suborganelle targeting of nuclear-encoded proteins (Kessler and
Schnell, 2009).
Most nuclear-encoded chloroplast proteins are encoded as
precursors and harbour an N-terminal transit peptide that
is cleaved off after import into the chloroplast (Inaba and
Abbreviations: IMS, intermembrane space of the chloroplast envelope; TOC, translocon at the outer membrane of chloroplasts; TIC, translocon at the inner membrane of chloroplasts; IEM, inner envelope membrane; OEM, outer envelope membrane; RFP, red fluorescent protein; PCR, polymerase chain reaction; SDS-PAGE,
sodium dodecyl sulfate polyacrylamide gel electrophoresis.
© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
5258 | Okawa et al.
Schnell, 2008; Jarvis, 2008; Li and Chiu, 2010). The transit
peptide is first recognized by the receptor components of
the translocon at the OEM of chloroplasts (TOC) (Andres
et al., 2010). Subsequently, the unfolded precursor protein is
inserted into the channels of the TOC and the translocon at
the IEM (TIC). Translocation of the precursor protein across
the TOC-TIC channel is achieved by chaperone activities in
the stroma (Flores-Perez and Jarvis, 2013). At the same time,
the transit peptide is cleaved by stromal processing peptidase
during the translocation. This series of reactions allows precursor proteins to be imported into the chloroplast stroma.
Because the transit peptide only serves as the signal for
translocation across envelope membranes, proteins destined
to the IEM and thylakoid membranes contain a second,
discrete signal that is necessary for their targeting to these
locations. Several targeting pathways have been shown to be
involved in the targeting of these proteins within the interior of chloroplasts (Celedon and Cline, 2013). Recent studies have demonstrated that IEM proteins utilize two distinct
pathways for their targeting to the IEM (Lubeck et al., 1997;
Inaba et al., 2005; Li and Schnell, 2006; Tripp et al., 2007;
Chiu and Li, 2008; Firlej-Kwoka et al., 2008; Viana et al.,
2010; Froehlich and Keegstra, 2011). One is the stop-transfer
pathway, and the other is the post-import or conservative
pathway. In the stop-transfer pathway, substrate proteins are
integrated into the IEM during protein translocation such
that they do not utilize soluble intermediates. Substrates of
this pathway include ARC6 (Froehlich and Keegstra, 2011),
APG1 (Viana et al., 2010), and several other proteins (Chiu
and Li, 2008; Firlej-Kwoka et al., 2008). In contrast, substrates of the post-import pathway, such as Tic40, are first targeted to the stroma through the TOC-TIC complex and then
re-inserted into the IEM (Li and Schnell, 2006; Tripp et al.,
2007). Hence, chloroplasts accumulate a soluble intermediate
in the stroma during targeting of the Tic40 protein. The presence of these two pathways has been analyzed in detail using
proteins carrying a single transmembrane domain. However,
much less is known about the insertion of polytopic IEM
proteins. A few studies have suggested that a portion of the
polytopic protein may act to provide targeting information to
the IEM (Knight and Gray, 1995; Teng et al., 2006; Sattasuk
et al., 2011). According to a transient expression assay, the
N-terminal half of Tic21 fused to red fluorescent protein
(RFP) was delivered to the IEM (Teng et al., 2006). Likewise,
the N-terminal portion of a putative amino acid transporter
in Arabidopsis was sufficient for delivery to the IEM in vitro
(Sattasuk et al., 2011). Although there is additional evidence
that a small portion of polytopic IEM proteins may play a
role in their targeting (Knight and Gray, 1995), little is known
about the targeting mechanism of polytopic membrane proteins to IEM.
In this work, we investigated the mechanism by which a
polytopic membrane protein is targeted to the IEM of chloroplasts. To this end, we chose the Cor413im1 protein as
the model substrate. Cor413im1 is a cold-regulated protein
in plants (Okawa et al., 2008). It is predicted to have several transmembrane helices and has been shown to localize
to the IEM of chloroplasts (Okawa et al., 2008). We show
that Cor413im1 does not utilize a soluble intermediate for
its targeting to the IEM. Furthermore, we demonstrate that
targeting of Cor413im1 to the inner envelope membrane of
chloroplasts involves multiple transmembrane segments.
Materials and methods
Construction of vectors and Arabidopsis transformation
For the construction of the in vitro import assay, the pre-COR413IM1
gene was amplified from Arabidopsis cDNA by polymerase chain
reaction (PCR) using primers that introduced a 5ʹ NcoI site and a
3ʹ XhoI site. The cDNA was inserted into the NcoI and XhoI sites
of pET21d. The pre-APG1 and pre-AtTic40 constructs have been
described elsewhere (Li and Schnell, 2006; Viana et al., 2010).
For the construction of TP-pA-Cor413im1 and TP-pA, the
transit peptide plus the first five residues of the mature portions
of Cor413im1 was amplified by PCR and subcloned into the NcoI
and EcoRI sites of pET21d-TEV-pA to generate pET21d-TP-pA.
The mature portion of Cor413im1 (amino acids 77–225) was then
inserted into the C-terminus of staphylococcal protein A (pA) to
create pET-TP-pA-Cor413im1. These plasmids were then digested
with NcoI and XbaI and the inserts subcloned into the NcoI and
XbaI sites of pCAMBIA3300.
For the construction of a C-terminal deletion series (K30, K50,
E73, R95, and K124), a portion of the pre-COR413IM1 gene was
first subcloned into the NcoI and EcoRI sites of pET21d-TEV-pA.
The predicted transmembrane helices of Cor413im1 protein and
positions of each deletion site are shown in Supplementary Figure
S1 (available at JXB online). The resulting pET vectors were then
digested with NcoI and XbaI and the insert subcloned into the NcoI
and XbaI sites of pCAMBIA3300. The pre-Cor413im1-pA construct has been described elsewhere (Okawa et al., 2008).
For the construction of K50-C and R95-C, a portion of the
COR413IM1 gene was subcloned into the C-terminus of pA in
pET21d-TP-pA. The resulting pET vectors were then digested with
NcoI and XbaI and the insert subcloned into the NcoI and XbaI
sites of pCAMBIA3300.
For the construction of TM5-C and K124-C, a portion of the
COR413IM1 gene was subcloned into the C-terminus of pA in
pET21d-TP-pA using In-Fusion HD Cloning Kit (Takara). The
resulting insert, TP-pA-TM5 and TP-pA-K124, was further amplified by PCR, and the amplified fragment was subcloned into the
NcoI and XbaI sites of pCAMBIA3300 using In-Fusion HD
Cloning Kit.
All pCAMBIA constructs were introduced into Arabidopsis thaliana (accession Columbia) via Agrobacterium tumefaciens-mediated
transformation by the floral dip method (Clough and Bent, 1998).
Arabidopsis chloroplast isolation and membrane fractionation
For chloroplast isolation, Arabidopsis plants were grown on 0.5x
MS plates supplemented with 1% sucrose at 23°C for 2 weeks.
Chloroplasts were isolated from 14- to 18-day-old transgenic plants
expressing each chimeric protein as described previously (Smith
et al., 2002).
For the preparation of total membrane and soluble proteins, isolated chloroplasts were diluted in either 0.2 M Na2CO3 (pH 12.0),
or 1% Triton X-100, and incubated for 10 min on ice. The samples
were then separated into soluble and membrane fractions by ultracentrifugation at 100 000 g for 15 min.
Analysis of the localization of truncated proteins within
chloroplasts
To determine the localization of truncated proteins within chloroplasts, isolated chloroplasts were fractionated into stroma, envelope,
and thylakoid membranes as described previously (Smith et al.,
Targeting of polytopic protein to chloroplast inner envelope | 5259
samples was performed with a Typhoon FLA7000 PhosphorImager
and ImageQuant TL software (GE Healthcare Life Sciences).
2002). After the quantification of proteins in each fraction, total
chloroplast (3 μg), stroma (3 μg), envelope (1 μg), and thylakoid
(1.5 μg) fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting
with the antisera indicated in the figures. Although we sometimes
loaded different amount of proteins for the analysis, the protein
ratio of total chloroplast:stroma:envelope:thylakoid was always
3:3:1:1.5. The trypsin sensitivity of Cor413im1-pA was examined
using intact chloroplasts, as described previously (Jackson et al.,
1998; Li and Schnell, 2006).
Antibodies against Tic110 (Inaba et al., 2005) and ACCase
(Kozaki et al., 2000) have already been described. Rabbit polyclonal
antibody against Toc75 was produced using Toc75(141–397)-His as the
antigen based on previous literature (Hiltbrunner et al., 2001). The
anti-protein A IgG was purchased from Sigma-Aldrich.
The fold enrichment of each truncated protein in the envelope
fraction was estimated as follows. The signal intensity of each chimeric protein band in total chloroplast and envelope fractions was
first measured by densitometric software (CS analyzer, ATTO) and
the signal intensity per microgram protein was calculated based on
the amount of protein loaded. Then, the value for the envelope fraction was divided by the value for the total chloroplast fraction to
estimate the fold enrichment in the envelope fraction compared to
the total chloroplast. As the positive control, we also estimated fold
enrichment of Tic110 protein in the envelope fraction.
Results
Cor413im1 does not utilize a soluble intermediate for
its integration into the chloroplast inner envelope
We first examined the pathway Cor413im utilizes for its integration into the chloroplast inner envelope. To this end, we
conducted a time-course import experiment using in vitrotranslated 35S-labelled pre-Cor413im1. According to previous
studies, substrates for the stop-transfer pathway do not utilize
soluble intermediates (Chiu and Li, 2008; Firlej-Kwoka et al.,
2008; Viana et al., 2010; Froehlich and Keegstra, 2011). In
contrast, precursors that use the post-import pathway, such as
Tic40, have been shown to accumulate soluble intermediates
during the early time points in the import assay (int-atTic40 in
Supplementary Figure S2, available at JXB online). We took
this approach to discriminate between these two pathways.
Aliquots were taken from the import reaction mixture at each
time point and added into excess ice-cold HEPES-sorbitol
buffer to stop the import reaction. The samples were then
fractionated into the membrane versus soluble fractions. As
shown in Fig. 1A, B, mature Cor413im1 accumulated in the
membrane fraction in a time-dependent manner. In contrast,
we did not observe a detectable amount of soluble Cor413im1
(Fig. 1A, B). This observation was further confirmed by the
in vitro import assay using protein A-tagged pre-Cor413im1
In vitro translation and protein import assay
[35S]Met-labelled wild-type and mutant preCor413im1 proteins
were generated in a coupled transcription-translation system containing reticulocyte lysate according to the manufacturer’s instructions (Promega, Madison, WI, USA). Time-course import assays
were performed using intact pea chloroplasts as described previously (Viana et al., 2010). The quantitative analysis of radiolabeled
A
IVT
2 min.
5 min.
30 min.
S
S
S
P
P
P
pre-Cor413im1
Cor413im1
pre-Cor413im1-pA
Cor413im1-pA
1
2
3
4
5
6
7
3
2
Cor413im1
S
P
1
0
2 min.
5 min.
Cor413im1-pA
8
% of added protein
% of added protein
B
30 min
S
P
6
4
2
0
2 min.
35
5 min.
30 min
Fig. 1. Cor413im1 does not utilize a soluble intermediate during targeting to the IEM. (A) [ S]pre-Cor413im1 (upper panel) or [35S]pre-Cor413im1-pA
(lower panel) was imported into chloroplasts for the times indicated in the presence of 5 mM ATP. The reactions were stopped by the addition of an
excess of HEPES-sorbitol buffer. The chloroplasts were then lysed and separated into soluble (S) and membrane (P) fractions. IVT, in vitro translation
product. (B) The graphs represent the quantification of lanes 2–7 for mature Cor413im1 or mature Cor413im1-pA.
5260 | Okawa et al.
mature portion of Cor413im1 to the C-terminus of TP-pA
(Fig. 2A, TP-pA-Cor413im1). We previously demonstrated
that the pre-Cor413im1-pA (Fig. 2A) was properly localized
to the IEM (Okawa et al., 2008). These three constructs were
transformed into Arabidopsis and the expressed proteins were
detected by immunoblotting (Fig. 2B). When chloroplasts
isolated from Arabidopsis expressing TP-pA-Cor413im1 were
further fractionated into stroma, envelope, and thylakoid
fractions, pA-Cor413im1 signals were enriched in the envelope fraction (Fig. 2C). The pA-Cor413im1 was resistant to
trypsin treatment of intact chloroplasts (Fig. 2D), indicating
that the expressed proteins were targeted to the interior of
the IEM. It was also shown that pA-Cor413im1 was resistant to Na2CO3 extraction but can be solubilized by Triton
X-100 (Fig. 2E, lanes 10–13). This localization was similar
to Cor413im1-pA (Okawa et al., 2008) as well as the inner
envelope protein Tic110 (Fig. 2C, 2E). We also confirmed
that the peripherally associated IEM protein, acetyl CoA
carboxylase (ACCase) (Sasaki et al., 1993; Shorrosh et al.,
1996; Nakayama et al., 2007), could be extracted by Na2CO3
treatment (Fig. 2E), indicating that pA-Cor413im1 is an integral membrane protein of the IEM. In contrast, signals from
TP-pA plants were observed in the stroma before (Fig. 2C,
pA) and after (Fig. 2E, pA) Na2CO3 extraction. These results
indicate that the transit peptide portion of Cor413im1 acts as
the targeting signal into the stroma. Furthermore, the mature
The mature portion of Cor413im1 contains information
necessary for its targeting to the IEM
It has been shown that the mature portion plays a key role in
the integration of IEM proteins into the membrane, regardless of the pathways they use. This proposal is based on the
analysis of IEM proteins that have either one or two transmembrane domains (Li and Schnell, 2006; Tripp et al., 2007;
Viana et al., 2010; Froehlich and Keegstra, 2011). However,
much less is known about polytopic transmembrane proteins.
Therefore, we next investigated the role of the transit peptide
and the mature portions in the integration of Cor413im1 into
the IEM. We generated constructs in which the transit peptide of Cor413im1 was fused to the amino terminus of protein A (Fig. 2A, TP-pA). In one construct, we also fused the
B
pre-Cor413im1
TP
Protein A
TP
Protein A
Protein A
C
A
or
4
pA 13i
-C m1
pA or4 -pA
13
im
1
(pre-Cor413im1-pA in Fig. 1). Both Cor413im1 and
Cor413im1-pA proteins were also resistant to thermolysin
treatment in intact chloroplasts (Supplementary Figure S2,
available at JXB online), indicating that they were imported
into the chloroplast interior. The distribution of Cor413im1
and Cor413im1-pA is quite similar to that of APG1, a known
substrate of the stop-transfer pathway (Supplementary
Figure S2, available at JXB online). These results indicate that
Cor413im1 does not utilize a soluble intermediate for its integration into the chloroplast inner envelope.
pre-Cor413im1-pA
mCor413im1 TP-pA-Cor413im1
TP-pA
kDa
29
24
20
14
C
E
D
pA-Cor413im1
-
+
pA-Cor413im1
Tic110
5
6
7
TX-100
M
S
M
S
10
11
12
13
pA-Cor413im1
ACCase
Toc75
4
3
Tic110
pA
pA
2
Na2CO3
Trypsin
Chl Str Env Thy
1
8
9
pA
Fig. 2. The mature portion of Cor413im1 contains the information necessary for targeting to the IEM. (A) Schematic diagram of chimeric Cor413im1
constructs used in these studies. The pre-Cor413im1-pA was previously shown to correctly target to the chloroplast IEM. (B) Expression analysis of
chimeric proteins in transgenic Arabidopsis. Total protein extracts from true leaves were resolved by SDS-PAGE and immunoblotted using an antibody
to protein A. (C) Localization of pA-Cor413im1 and pA in chloroplasts. Isolated chloroplasts (Chl) were fractionated into stroma (Str), envelope (Env),
and thylakoid (Thy) fractions. The protein ratio of Chl:Str:Env:Thy used in the analysis was 3:3:1:1.5. Each fraction was resolved by SDS-PAGE and
immunoblotted with antibodies against protein A (pA-Cor413im1 and pA) or Tic110. (D) Trypsin sensitivity of chimeric Cor413im1 proteins in intact
chloroplasts. Chloroplasts equivalent to 25 µg chlorophyll were treated with trypsin on ice for 30 min. The trypsin was inactivated and intact chloroplasts
were re-isolated, resolved by SDS-PAGE, and immunoblotted with antibody against protein A. The protease sensitivity of the OEM protein, Toc75, was
included as a control for trypsin activity. (E) Localization of pA-Cor413im1 and pA in the soluble and membrane fractions of chloroplasts. Chloroplasts were
lysed in the buffer containing 0.2 M Na2CO3, pH 12 (Na2CO3) or 1% Triton X-100 (TX-100) and separated into membrane (M) and soluble (S) fractions. The
extracts were resolved by SDS-PAGE and immunoblotted with antibodies against protein A (pA-Cor413im1 and pA), ACCase CTα subunit, or Tic110.
Targeting of polytopic protein to chloroplast inner envelope | 5261
portion of Cor413im1 contains the information necessary for
its targeting to the IEM.
Identification of a specific transmembrane segment
necessary for the efficient integration of Cor413im1
into the IEM
Cor413im1 is predicted to contain six potential transmembrane segments (Breton et al., 2003; Okawa et al., 2008).
Therefore, we generated a series of deletion constructs carrying one to five transmembrane segments fused to protein
A and investigated their localization within the chloroplast
(Fig. 3A and Supplementary Figure S1, available at JXB
online). These constructs were transformed into Arabidopsis
and the protein expression was confirmed by immunoblotting
(Fig. 3B). When intact chloroplasts isolated from these plants
were treated with trypsin, all of the expressed proteins were
resistant to trypsin digestion (Fig. 3C), indicating that the
expressed proteins were targeted to the interior of the IEM.
The control outer envelope protein, Toc75, was digested by
trypsin, indicating that the protease was active (Fig. 3C).
We further fractionated chloroplasts into stroma, envelope, and thylakoid fractions and investigated the localization of the truncated proteins (Fig. 4A). To assess the IEM
localization of each chimeric protein, we quantified the signal
intensity per microgram of protein of each band in the total
chloroplast and envelope fractions and estimated the fold
enrichment of each protein in the envelope fraction. When
we used the 12.5% acrylamide resolving gel for this analysis, the average fold enrichment of Tic110, a marker protein
of the IEM, was ~15 (Fig. 4B). Consistent with the previous
observation, the full-length chimeric protein, Cor413im1-pA,
was also enriched more than 10 times (Fig. 4B). When the
fold enrichment of each expressed protein was estimated, the
A
TP
Protein A
pre-Cor413im1-pA
I
K30
I II
K50
I II III
E73
I II III IV
R95
I II III IV V
K124
kDa
29
24
E7
3
R
95
K1
24
I II III IV V VI
K3
0
K5
0
B
mCor413im1
20
1
C
2
3
4
Trypsin
-
K30
5
Trypsin
-
+
+
R95
K124
K50
E73
Toc75
6
7
8
9
Fig. 3. Expression of C-terminal truncated Cor413im1 proteins
in transgenic Arabidopsis. (A) Schematic diagram of Cor413im1
C-terminal deletion constructs. Roman numerals indicate the predicted
positions of each transmembrane segment. (B) Expression analysis of
C-terminal truncated Cor413im1 proteins in transgenic Arabidopsis.
Total proteins were extracted from true leaves, resolved by SDS-PAGE
and immunoblotted with antibodies against protein A to detect chimeric
proteins. (C) Trypsin sensitivity of C-terminal truncated Cor413im1
proteins in intact chloroplasts. Chloroplasts equivalent to 25 µg chlorophyll
were treated with trypsin on ice for 30 min. The trypsin was inactivated
and intact chloroplasts were re-isolated, resolved by SDS-PAGE, and
immunoblotted with antibody against protein A. The protease sensitivity of
the OEM protein, Toc75, was included as a control for trypsin activity.
Fig. 4. Identification of the transmembrane segment necessary for the
targeting of Cor413im1 to the IEM. (A) Localization of C-terminal truncated
Cor413im1 chimeric proteins within chloroplasts. Isolated chloroplasts
(Chl) were fractionated into stroma (Str), envelope (Env), and thylakoid (Thy)
fractions. Each fraction was resolved by SDS-PAGE, and immunoblotted
with antibodies against protein A or Tic110. The protein ratio of
Chl:Str:Env:Thy used in these analyses was always 3:3:1:1.5. (B) Fold
enrichment of each truncated protein in the envelope fraction. The signal
intensity of each band (lanes 1 and 3) was first measured by densitometry
and the signal intensity per microgram protein was calculated based on
the amount of protein loaded. Then, the value for the envelope fraction
was divided by the value for the total chloroplast fraction to estimate the
fold enrichment in the envelope fraction compared to the total chloroplast.
As a control, average fold enrichment of Tic110 protein in the envelope
fraction was also calculated. For Tic110, lanes 5 and 7 were used to
measure signal intensity. Because we used 12.5% resolving gel for SDSPAGE in Fig. 4, the fold enrichment of Tic110 and other envelope-localized
proteins is higher than those found in Fig. 7.
5262 | Okawa et al.
TP
Protein A
III IV V VI K50-C
V VI R95-C
VI K124-C
TM5-C
B
kDa
29
24
20
kDa
29
24
24
TM -C
5C
V
20
1
C
2
3
Trypsin
-
+
TM5-C
R95-C
Toc75
5
4
Trypsin
K50-C
K124-C
Fig. 5. Membrane association of C-terminal truncated Cor413im1
proteins. Localization of C-terminal truncated Cor413im1 proteins in
soluble and membrane fractions of chloroplasts. Chloroplasts were lysed
in buffer containing 0.2 M Na2CO3, pH 12 (Na2CO3), or 1% Triton X-100
(TX-100) and separated into membrane (M) and soluble (S) fractions. The
extracts were resolved by SDS-PAGE and immunoblotted with antibodies
against protein A.
mCor413im1
I II III IV V VI TP-pA-Cor413im1
K1
Given our results indicating that the deletion of the C-terminus
resulted in the inefficient targeting of Cor413im1 proteins to
the envelope membrane, we wished to demonstrate whether
the C-terminal portion is sufficient to deliver chimeric proteins to the IEM. To this end, we generated amino-terminal
deletion constructs of Cor413im1 and fused them to the
TP-pA construct (Fig. 6A). These chimeric constructs contain the transit peptide and the C-terminal portion of mature
Cor413im1, but lack the N-terminal portion (Fig. 6A).
The three chimeric proteins, K50-C, R95-C and K124-C,
were stably expressed in transgenic Arabidopsis (Fig. 6B).
When chloroplasts isolated from transgenic plants were treated
with trypsin, all the proteins examined were resistant to trypsin
treatment (Fig. 6C, left panel), indicating that they were
imported into the interior of the IEM. We further fractionated
chloroplasts into the stroma, envelope, and thylakoid fractions.
A
0R C
95
-C
Multiple transmembrane segments containing the fifth
transmembrane segment are indispensable for the
targeting of Cor413im1 to the IEM
When each fraction was resolved by 5–20% acrylamide gradient gel in SDS-PAGE and immunoblotting, K50-C and R95-C
proteins were enriched in the envelope fraction and their distribution was similar to those of Tic110 and pA-Cor413im1 (Fig.
7A, B). These proteins were also resistant to Na2CO3 extraction
(Fig. 7C). In contrast, K124-C protein failed to be targeted to
the envelope membrane efficiently (Fig. 7A, B). Furthermore,
K124-C was extractable from the membrane with Na2CO3,
indicating that the protein was not properly integrated into the
membrane. Again, these data indicate that the fifth transmembrane segment is necessary for the targeting of Cor413im1 to
the IEM in vivo.
We next investigated if the fifth transmembrane segment is
sufficient for the targeting to the IEM. To this end, we constructed a chimeric gene consisting of the fifth transmembrane segment and TP-pA (Fig 6A, TM5-C). The TM5-C
protein was expressed in transgenic Arabidopsis (Fig 6B, lane
4). Furthermore, TM5-C was imported into the interior of the
K5
enrichment of K124 was as good as those of Cor413im1-pA
and Tic110 (Fig. 4B). In contrast, other chimeric proteins,
K30, K50, E73, and R95, were not enriched in the envelope
fraction efficiently (Fig. 4B).
To investigate whether the chimeric proteins were integrated
into the membrane or associated with the membrane peripherally, chloroplasts were lysed with Na2CO3 and fractionated
into the membrane and soluble fractions. As shown in Fig. 5,
the K30 protein was partitioned into both the soluble and
membrane fractions. However, most of the other truncated
proteins were exclusively localized in the membrane fraction
and could be solubilized by Triton X-100 (Fig. 5).
Collectively, these data suggest that the fifth transmembrane segment play crucial roles in the efficient targeting of
Cor413im1 to the IEM. Furthermore, it seems that a portion
of K30, K50, E73 and R95 was mistargeted and integrated at
the thylakoid membranes instead of the IEM.
-
+
7
8
6
Fig. 6. Expression of N-terminal truncated Cor413im1 proteins in
transgenic Arabidopsis. (A) Schematic diagram of N-terminal truncated
Cor413im1 constructs. Roman numerals indicate the predicted positions
of each transmembrane segment. (B) Accumulation of N-terminal
truncated Cor413im1 protein in Arabidopsis. Total proteins were extracted
from true leaves, resolved by SDS-PAGE, and immunoblotted using
antibodies against protein A to detect chimeric proteins. (C) Trypsin
sensitivity of the N-terminal truncated Cor413im1 proteins in intact
chloroplasts. Chloroplasts equivalent to 25 µg of chlorophyll were treated
with trypsin on ice for 30 min. Intact chloroplasts were re-isolated, resolved
by SDS-PAGE, and immunoblotted with antibody against protein A. The
protease sensitivity of the OEM protein, Toc75, was included as a control
for trypsin activity.
Targeting of polytopic protein to chloroplast inner envelope | 5263
As shown in Fig. 7A, B, TM5-C was no longer enriched in
the envelope fraction. Instead, we observed strong signal in
the thylakoid fraction. The protein was extracted by Na2CO3
(Fig. 7C), indicating that TM5-C was not properly integrated
into the chloroplast membrane.
Taken together, we conclude that the fifth transmembrane
segment is necessary for the targeting of Cor413im1 to the
IEM. However, the fifth transmembrane segment itself is
insufficient to target fusion proteins to the IEM. Instead,
multiple transmembrane segments containing the fifth transmembrane segment are indispensable for the efficient targeting of Cor413im1 to the IEM.
Discussion
Fig. 7. Targeting of Cor413im1 to the chloroplast IEM involves multiple
transmembrane segments containing the fifth transmembrane segment.
(A) Localization of the N-terminal truncated Cor413im1 proteins within
chloroplasts. Isolated chloroplasts (Chl) were fractionated into stroma (Str),
envelope (Env), and thylakoid (Thy) fractions. Each fraction was resolved
by SDS-PAGE, and immunoblotted with the antibodies against protein
A or Tic110. The protein ratio of Chl:Str:Env:Thy used in these analyses
was always 3:3:1:1.5. (B) Fold enrichment of each truncated protein in
the envelope fraction. The signal intensity of each band [lanes 1 and 3 in
panel (A)] was first measured by densitometry and the signal intensity per
microgram protein was calculated based on the amount of protein loaded.
Then, the value for the envelope fraction was divided by the value for the total
chloroplast fraction to estimate the fold enrichment in the envelope fraction.
As a control, average fold enrichment of Tic110 protein in the envelope
fraction was also calculated. For Tic110, lanes 5 and 7 in panel (A) were used
to measure signal intensity. Because we used 5–20% acrylamide gradient gel
for SDS-PAGE in Fig. 7, the fold enrichment of Tic110 and other envelopelocalized proteins is lower than those found in Fig. 4. (C) Localization of
N-terminal truncated Cor413im1 proteins in the soluble and membrane
fractions of chloroplasts. Chloroplasts were lysed in the buffer containing
0.2 M Na2CO3, pH 12 (Na2CO3), or 1% Triton X-100 (TX-100) and separated
into membrane (M) and soluble (S) fractions. The extracts were resolved by
SDS-PAGE and immunoblotted with antibodies against protein A.
IEM in vivo (Fig. 6C, right panel) as it was resistant to trypsin
treatment of intact chloroplasts. We next fractionated chloroplasts into the stroma, envelope, and thylakoid fractions.
The plastid IEM plays key roles in synthesizing, catabolizing, and transporting metabolites. However, the biogenesis of
IEM proteins, especially polytopic IEM proteins, remains to
be characterized in detail. In this study, we aimed to investigate how polytopic proteins are targeted to the IEM. We
chose Cor413im1 as the model substrate and showed that
Cor413im1 does not utilize a soluble intermediate for its targeting to the IEM. This suggests that Cor413im1 most likely
utilizes the stop-transfer pathway for its integration to the
IEM. Furthermore, we also provide evidence that the small
portion of Cor413im1 plays crucial roles in determining IEM
localization in vivo.
We observed that C-terminal truncated Cor413im1 proteins that lack the fifth transmembrane segment were not efficiently enriched into the IEM (Fig. 4A, B), but were resistant
to Na2CO3 extraction (Fig. 5). This suggests that a portion of
truncated Cor413im1 was mislocalized to the thylakoid membrane. Indeed, TM5-C protein carrying insufficient targeting
information to the IEM was also mislocalized to the thylakoid
membrane (Fig. 7A). This implies that hydrophobic proteins
lacking the IEM targeting signal are targeted to thylakoid
membranes once they are imported into chloroplasts. We
speculate that the thylakoid membrane is the default destination for hydrophobic membrane proteins within chloroplasts
if they do not have the IEM insertion signal. Another possible explanation is that the truncated forms of Cor413im1
lacking the IEM targeting signal cannot discriminate the
IEM from the thylakoid membrane. The thylakoid membrane
is far more abundant than the IEM within the chloroplast.
Hence, unless membrane proteins have an additional signal
to direct them to the IEM, they predominantly localize to
the thylakoid membrane. The second hypothesis is consistent
with the observation that some truncated proteins lacking the
IEM targeting signal were also found in the envelope fraction
(e.g. E73 and R95 in Fig. 4; and K124-C in Fig.7). Our results
reemphasize that the IEM targeting signal is indispensable for
the biogenesis of polytopic IEM proteins.
Both C-terminal (K124 in Fig. 4) and N-terminal (K50-C
and R95-C in Fig. 7) truncated Cor413im1 containing the
fifth transmembrane segment were integrated into the IEM.
However, the fifth transmembrane segment itself is insufficient for efficient targeting to the IEM (TM5-C in Fig. 7). This
5264 | Okawa et al.
result implies that the presence of multiple transmembrane
segments is indispensable for the integration of polytopic
membrane proteins to the IEM. A similar observation was
made for targeting of the polytopic phosphate translocator
to the chloroplast IEM (Knight and Gray, 1995). When the
putative first transmembrane segment of the phosphate translocator (Willey et al., 1991) was fused to the small subunit of
ribulose bisphosphate carboxylase/oxygenase, the majority of
chimeric proteins were detected in the soluble fraction (Knight
and Gray, 1995). Our results also raise the possibility that the
targeting pathway of polytopic IEM proteins is distinct from
that of monotopic IEM proteins. This hypothesis is supported
by the fact that the transmembrane segment derived from
monotopic protein, such as APG1 and ARC6, is sufficient to
integrate chimeric proteins to IEM in in vitro import assays
(Viana et al., 2010; Froehlich and Keegstra, 2011).
In mitochondria, the targeting mechanism of polytopic
inner membrane proteins is distinct from that of presequencecarrying precursors. The TOM complex harbours distinct
receptors for each class of proteins (Becker et al., 2012).
Furthermore, they also have the specialized TIM complex,
the TIM22 complex, for the integration of polytopic proteins from the intermembrane space to the inner membrane
(Jensen and Dunn, 2002). In chloroplasts, it appears that
discrimination between abundant photosynthetic proteins
and less-abundant non-photosynthetic proteins are mediated by multiple TOC receptors (Inaba and Schnell, 2008;
Jarvis, 2008; Kessler and Schnell, 2009; Li and Chiu, 2010).
Although it remains unclear whether the specialized pathway
for polytopic membrane proteins exists, the hypothesis that
chloroplasts contain specialized translocation machinery for
polytopic proteins is consistent with our observations.
Froehlich and Keegstra suggested that the transmembrane
segments of monotopic IEM proteins are rich in aromatic
amino acids and lack proline residues relative to thylakoid
proteins (Froehlich and Keegstra, 2011). However, the
requirement of multiple transmembrane segments for IEM
targeting of polytopic membrane proteins (Figs 4 and 7) will
make definition of the physical characteristics of any IEM
targeting signal difficult to define. In mitochondria, polytopic proteins are inserted into the inner membrane from
intermembrane space by the TIM22 complex (Jensen and
Dunn, 2002). During their targeting, a module consisting of
two helices connected by a matrix-facing loop serves as a signal for integration into inner membrane (Davis et al., 1998).
Therefore, requirement of multiple transmembrane segments
is reminiscent of polytopic protein targeting in mitochondrial
inner membrane. However, this mechanism also requires the
membrane potential across inner mitochondrial membrane
(Ferramosca and Zara, 2013). Unlike inner mitochondrial
membrane, the chloroplast IEM lacks a significant membrane potential. Furthermore, polytopic IEM proteins also
possess a cleavable transit peptide that is not usually found
in mitochondrial carrier proteins (Inaba and Schnell, 2008;
Ferramosca and Zara, 2013). Hence, polytopic chloroplast
IEM proteins are unlikely to utilize the same mechanism as
mitochondrial inner membrane proteins. Rather, we hypothesize that additional IEM components participate in the
integration of polytopic IEM proteins. A recent study identified a second type of Sec machinery, cpSec2, which is predominantly localized in the IEM (Skalitzky et al., 2011). The
level of cpSec2 is co-regulated with the levels of Tic40 and
Tic110 (Celedon and Cline, 2013). Hence, it is intriguing to
hypothesize that cpSec2 and TIC machinery cooperate to act
as a membrane integrase for polytopic IEM proteins that is
targeted by the stop-transfer mechanism (Celedon and Cline,
2013). If this is the case, it is possible that the separate transmembrane segments are distinctively recognized by Tic and
Sec machineries such that multiple transmembrane segments
are required for efficient targeting of polytopic proteins to the
IEM. The cpSec might also help the flanking transmembrane
segments at the stromal side (e.g. TM1-TM4 of translocating
Cor413im1) avoid aggregation during translocation through
the TIC channel.
According to our analysis, the protein A portion of K124
is resistant to trypsin treatment (Fig. 3C). Because K124 lacks
the sixth transmembrane segment, the topology of K124
seems to be reverted compared to Cor413im1-pA (Okawa
et al., 2008). These data suggest that the IEM-targeting signal of polytopic proteins plays key roles in precise targeting to
the IEM but does not determine the transmembrane topology.
A previous study suggested that IEP37 is flipped at the IEM
(Viana et al., 2010), suggesting the presence of a topologyflipping mechanism at the IEM. Therefore, it is plausible to
speculate that some of the truncated proteins, such as K124,
are subjected to this regulation. Furthermore, in contrast to
the transgenic study (Fig. 7C), we found that in vitro imported
K124-C and TM5-C proteins were fractionated into the
membrane fraction after Na2CO3 extraction (Supplementary
Figure S3, available at JXB online). This suggests that the in
vitro import assay using isolated chloroplasts could not completely reconstitute the targeting of truncated Cor413im1
proteins observed in vivo. Chloroplasts appear to regulate the
biogenesis of polytopic IEM proteins at multiple levels.
It appears that chloroplasts have evolved stop-transfer
and post-import/conservative IEM protein targeting systems
that are distinct from those in mitochondria. In mitochondria, Oxa1p, a homologue of bacterial YidC in mitochondria, participates in the insertion of membrane proteins from
mitochondrial matrix via a conservative pathway (Wang and
Dalbey, 2011). However, in chloroplasts, the YidC homologue, Alb3, is relocated to the thylakoid membrane (Moore
et al., 2000), and there is no evidence that YidC/Oxa1/Alb3
homologues exist on the IEM. Identification of the components mediating the chloroplast IEM-targeting pathways
will shed additional light on the unique mechanisms of inner
membrane biogenesis in this organelle.
Supplementary material
Supplementary data can be found at JXB online.
Supplementary Figure S1. A putative secondary structural
model of Arabidopsis Cor413im1 protein.
Supplementary Figure S2. Distribution of Cor413im1,
Cor413im1-pA, APG1, int-atTic40, and atTic40 after thermolysin treatment of intact chloroplasts.
Targeting of polytopic protein to chloroplast inner envelope | 5265
Supplementary Figure S3. In vitro protein import assay of
truncated Cor413im1 proteins.
Funding
This work was supported by the Programme to Disseminate
Tenure Tracking System from the Japanese Ministry of
Education, Culture, Sports, Science, and Technology, a
grant for Scientific Research on Priority Areas from the
University of Miyazaki (to TI and YII), Naito Foundation,
Strategic Young Researcher Overseas Visits Programme for
Accelerating Brain Circulation, Grant-in-Aid for Young
Scientists (B, no. 25850073) to TI, and a National Institutes
of Health Grant 2RO1-GM061893 (to DJS). HI was a recipient of a Japan Society for the Promotion of Science postdoctoral fellowship for research abroad.
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