Results As a starting point to investigate the import character-istics of the intermembrane space proteins Tic22 and MGD1, in vitro import experiments into isolated chloroplasts were per
Trang 1intermembrane space
Lea Vojta, Ju¨rgen Soll and Bettina Bo¨lter
University of Munich, Department of Botany, Munich, Germany
Protein import into organelles not only requires
trans-location into the organelle, but also sorting to the
various subcompartments Chloroplasts are highly
structured organelles that contain three distinct
brane systems (i.e the outer and inner envelope
mem-brane and the photosynthetic thylakoid memmem-branes) as
well as three soluble subcompartments, the thylakoid
lumen, the stroma and the intermembrane space The
large majority of chloroplast proteins are encoded by
nuclear genes, synthesized in the cytosol and
post-translationally imported into the organelle [1,2] In the
cytosol, preproteins associate with different molecular
chaperones (e.g Hsp70 or Hsp90) This interaction
determines the primary receptor at the chloroplast
surface that is chosen by the preprotein chaperone
complex [3]
Toc64 recognizes Hsp90-associated preproteins,
which are released from Hsp90 and transferred to
Toc34 in an ATP dependent process [3] Toc34
func-tions as a primary receptor of Hsp70-associated as well
as monomeric precursor proteins Toc34 receptor
func-tion is regulated by GTP binding and hydrolysis [4–7]
Toc34GDP interacts with the second G-protein in the
Toc complex, Toc159, and simultaneously transfers the preprotein to Toc159 Toc159 action facilitates pre-protein translocation through the Toc75 channel [8] Translocation of stromal or thylakoid preproteins occurs simultaneously through the Toc and Tic trans-locon Translocation across the inner envelope mem-brane requires ATP, probably for the action of stromal molecular chaperones [9,10]
Beside this standard import pathway, which is taken
by the majority of chloroplast preproteins, several spe-cialized translocation pathways have been described These can be distinguished in general by the differ-ences in ATP-, Toc-receptor or presequence require-ment For example, Tic32 and QORH, two proteins of the chloroplasts inner envelope, do not contain an N-terminal targeting sequence, but Tic32 and QORH are targeted to chloroplasts by internal sequence infor-mation present at the N- or C-terminus, respectively [11,12] In addition, Tic32 translocation requires
< 20 lm ATP in contrast to stromal preproteins or the precursor of Tic110, which require > 20 lm ATP This indicates that translocation of Tic32 does not involve the action of stromal chaperones Insertion of
Keywords
intermembrane space; MGD1; Tic; Tic22;
Toc
Correspondence
J Soll, University of Munich, Botany,
Menzinger Strasse 67, 80638 Munich,
Germany
Fax: +49 89 17861185
Tel: +49 89 17861245
E-mail: soll@lmu.de
(Received 29 May 2007, revised 31 July
2007, accepted 1 August 2007)
doi:10.1111/j.1742-4658.2007.06023.x
The import of proteins destined for the intermembrane space of chloro-plasts has not been investigated in detail up to now By investigating energy requirements and time courses, as well as performing competition experiments, we show that the two intermembrane space components Tic22 and MGD1 (E.C 2.4.1.46) both engage the Toc machinery for crossing the outer envelope, whereas their pathways diverge thereafter Although MGD1 appears to at least partly cross the inner envelope, Tic22 very likely reaches its mature form in the intermembrane space without involving stromal components Thus, different pathways for intermembrane space targeting probably exist in chloroplasts
Abbreviation
LSU, large subunit of RubisCO.
Trang 2preproteins into the outer envelope is less well
charac-terized and no components have yet been identified,
except for the import of the precursor of Toc75 Toc75
is made with a cleavable N-terminal presequence and
uses the Toc and Tic translocon in a specialized import
pathway [13] The Toc75 preprotein contains a
bipar-tite targeting signal The N-terminal domain is
respon-sible for chloroplast targeting and translocation
initiation Translocation is halted when this domain is
translocated across the Tic complex only to become
processed by the stromal processing peptidase The
protein subsequently retracts from the Tic translocon
and is redirected to the outer envelope [13] Toc75
itself could play a role in the insertion of the outer
envelope protein OEP14 [14]
In the present study, we describe the import
charac-teristics of two proteins, namely Tic22 and the MGDG
synthase (MGD1, E.C 2.4.1.46), localized in the
inter-membrane space between the outer and the inner
enve-lope Tic22 is a subunit of a soluble intermembrane
space complex, which facilitates the transfer of
prepro-teins from the Toc to the Tic translocon [15] The
import behaviour of pea Tic22 has previously been
described by Kuoranov et al [16]; thus, we used this
protein as a reference for intermembrane space
target-ing However, in the course of our studies, we obtained
contrasting results to those reported by Kouranov
et al [16], which led us to a refined model for the
import of pTic22 MGDG synthases are proposed to
be associated with either the inner or the outer
enve-lope, dependent on the plant species studied [17–19]
Two different enzyme types are found in chloroplasts;
type B enzymes (MDG2⁄ 3) appear to be in the outer
envelope membrane [20], whereas further studies in
Arabidopsis strongly indicated that type A MGDG
synthase, represented by MGD1, is an intermembrane
space component and bound to the intermembrane
face of the inner envelope, although this has not been
demonstrated unequivocally [20] Our results show that
both pTic22 and pMGD1 use the Toc translocon, but
they differ in their ATP-requirement for translocation,
indicating clear differences in the final translocation
steps Furthermore, pMGD1 import is greatly
stimu-lated by the addition of potassium phosphate in the
import reaction These data suggest that chloroplasts
have established a number of specialized translocation
pathways
Results
As a starting point to investigate the import
character-istics of the intermembrane space proteins Tic22
and MGD1, in vitro import experiments into isolated
chloroplasts were performed using 35S-labelled precur-sor proteins Both pTic22 and pMGD1 were imported and processed to a smaller mature form in the presence
of ATP (Fig 1) Upon protease treatment after com-pletion of the import assay, the organellar surface bound pMGD1 was completely removed as expected, although a significant amount of pTic22 was protease resistant (Fig 1A) This phenomenon was consistently observed, indicating that the rate of translocation exceeds the rate of processing for pTic22 The pTic22 translation product is completely degraded by the pro-tease thermolysin, indicating that the precursor is not protease resistant per se (data not shown) Recognition and translocation of pTic22 is completely dependent
on the N-terminal cleavable presequence An N-termi-nal truncation of 60 amino acids, most likely repre-senting the entire targeting signal, was neither bound, nor translocated into isolated chloroplasts (Fig 1A, lanes 4–6) The import of QORH into the inner enve-lope of chloroplasts was shown to depend on targeting information present in the C-terminus of the pre-protein [11] Therefore, we constructed a pTic22DC mutant in which the carboxy-terminal amino acids were deleted but still contained the N-terminal target-ing signal PTic22DC imported with an efficiency simi-lar to the wild-type protein (Fig 1A, lanes 7–9), indicating that the N-terminal presequence is both nec-essary and sufficient for recognition and translocation [16]
The import yield for pMGD1 was consistently low and the running behaviour of the processed mature form was partially distorted by the large subunit of RubisCO (LSU) at 54 kDa, resulting in a sharp band
in front of LSU, and a smear of radioactively-labelled protein mixed with LSU (Fig 1B, lanes 3–6, indicated
by an asterisk) We have demonstrated earlier that the presence of KPi buffer could greatly stimulate the import yield of the inner envelope protein IEP96 by an unknown mechanism [21] When we used 80 mm KPi
in the import reaction of pMGD1, both binding and translocation were stimulated by several-fold (Fig 1C, lanes 3–5), whereas the import of pTic22 was not influ-enced (data not shown) These initial data already indi-cate subtle, but clearly distinguishable differences in the import characteristics for these two intermembrane space localized preproteins
First, we wanted to verify the localization of MGD1
to the inter envelope space in chloroplasts An immu-noblot was carried out using purified inner and outer envelope membranes from pea chloroplasts and anti-sera against Tic110 and Toc75 as a marker for the localization (Fig 1D) MGD1 was found to be in almost equal distribution in both membrane fractions,
Trang 3whereas the marker proteins Toc75 and Tic110 were
largely confined to their respective localization,
indicat-ing that MGD1 spans the intermembrane space and is
in contact with both envelope membranes Inner and
outer envelopes are separated by treatment of intact
chloroplasts with hypertonic buffer during the
fraction-ation procedure; this may result in the observed
distri-bution behaviour However, the MGD1 population in
the inner envelope membrane behaves differently from
that in the outer envelope upon treatment with 6 m
urea or 0.1 m Na2CO3 Although MGD1 present in
the inner envelope is recovered almost exclusively in
the urea or Na2CO3 insoluble membrane fraction,
MGD1 in the outer envelope is partly or largely
recov-ered in the Na2CO3 or urea soluble fraction,
respec-tively MGD1 contains no obvious hydrophobic
transmembrane a-helices Therefore, we propose that
MGD1 binds to the outer leaflet of the inner envelope
by hydrophobic interactions, which is in accordance with its behaviour with respect to urea and Na2CO3 extraction The protein might span the intermembrane space and simultaneously interact with the inner leaflet
of the outer envelope (but less strongly than with the inner membrane), which could explain the different behaviour upon treatment with high salt concentra-tions and basic pH
To determine these differences more clearly, import experiments were carried out into isolated chloroplasts that contained two different radioactively-labelled preproteins simultaneously, either pTic22 or pMGD1 together with pSSU, a stromal marker, respectively, or
a mixture of the two intermembrane space proteins This was performed for better comparison of the import behaviour Under these conditions, we can exclude any differences in the treatment of the sam-ples No difference in the import efficiency was observed with respect to the number of precursor pro-teins present in one sample (data not shown) The results from these experiments were quantified and a representative example of each is shown as a gel image
A
B
D
C
Fig 1 (A) AtTic22 is imported into pea chloroplasts In vitro
syn-thesized [35S]pTic22 (lanes 1–3), [35S]mTic22 (lanes 4–6) and
[ 35 S]Tic22DC (lanes 7–9) were incubated with isolated intact
chlo-roplasts at 25 C for 20 min, in a standard import reaction
contain-ing 3 m M ATP After import, samples were reisolated on a Percoll
cushion and treated with thermolysin (Th) (lanes 3, 6 and 9) The
results were analyzed by SDS ⁄ PAGE Lanes 1, 4 and 7 represent
10% of the translation product used for the import reactions The
positions of pTic22, mTic22 and Tic22DC are indicated by arrows.
(B) Import of atMGD1 into pea chloroplasts In vitro synthesized
[ 35 S]pMGD1 was incubated with isolated intact pea chloroplasts at
25 C for 20 min, in a standard import reaction Lane 1 represents
10% of the translation product used for the import In lane 2,
trans-lation product was treated with thermolysin Import was performed
in the absence (lanes 3 and 4) or presence (lanes 5 and 6) of 3 m M
ATP After import, chloroplasts were reisolated on a Percoll cushion
and subjected to the treatment with 0.5 lg thermolysin (Th) per lg
chlorophyll (lanes 4 and 6) Untreated samples are shown in lanes
3 and 5 The results were analyzed by SDS ⁄ PAGE (C) Import of
pMGD1 performed in the presence of 80 m M KPi Precursor protein
(pMGD1), mature form (mMGD1) and typical thermolysin
degrada-tion pattern (Th) are indicated Lane 1 represents 10% of the
trans-lation product Import was performed in the absence (lanes 2 and
3) or presence (lanes 4 and 5) of 3 m M ATP After import
chloro-plasts were reisolated on a Percoll cushion and subjected to the
treatment with 0.5 lg thermolysin (Th) per lg chlorophyll (lanes 3
and 5) Untreated samples are shown in lanes 2 and 4 The results
were analyzed by SDS⁄ PAGE The mature form of MGD1, which is
compressed by LSU without addition of KPi, is marked with an
asterisk (D) Extraction of MGD1 from inner and outer envelope
vesicles from pea by 0.1 M Na 2 CO 3 , 6 M urea or 1 M NaCl
Chloro-plast envelopes were pelleted at 256 000 g for 10 min at 4 C using
a Himac CS150GX centrifuge and S150AT rotor (Hitachi, Tokyo,
Japan) and resuspended in either 0.1 M Na 2 CO 3 (pH 11.5) (lanes 3,
4, 9 and 10), 6 M urea (lanes 1, 2, 7 and 8) or 1 M NaCl (lanes 5, 6,
11 and 12) for 20 min at room temperature, followed by
centrifuga-tion at 256 000 g for 10 min at 4 C The pellet and the supernatant
were analyzed by SDS ⁄ PAGE and immunoblotting a-MGD1,
a-Tic22, a-Tic110 and a-Toc75 were used for immunodecoration.
Trang 4(Fig 2) Initially, we tested ATP dependence and the
kinetics of the import reaction using radiolabelled
pre-cursor proteins depleted of ATP by gel-filtration as
well as chloroplasts stored in the dark to deplete
intra-organellar ATP (Fig 2A) Subsequently, import
reac-tions were carried out in the dark under dim green
safelight, which does not support photosynthetic ATP production We consistently observed that pTic22 was imported and processed even in the absence of exoge-nous ATP The yield of pTic22 import in the absence
of ATP varied between 20–50% of that in the presence
of ATP The import of pMGD1 was efficient only at
C
Fig 2 Comparison of ATP- and time-demands for import of Tic22, MGD1 and SSU Import into intact pea chloroplasts was performed under standard conditions,
by incubating in vitro synthesized [ 35 S]pTic22 and [ 35 S]pMGD1 with chloro-plasts corresponding to 20 lg chlorophyll
at 25 C Parallel imports combining [ 35 S]pMGD1 and [ 35 S]pSSU, [ 35 S]pTic22 and [ 35 S]pSSU and [ 35 S]pTic22 and [ 35 S]pMGD1
in the same reaction were performed After import, chloroplasts were reisolated on a Percoll cushion and all samples were trea-ted with thermolysin The results were analyzed by SDS ⁄ PAGE The respective precursor and mature forms are indicated
by arrow heads (A) ATP scale import into intact pea chloroplasts was performed using increasing concentrations of ATP from 0 to
3000 l M for 15 min at 25 C The top three panels represent gel images; the bottom panel depicts the quantification graph For quantification, import at 3 m M ATP was taken as maximal import rate (B) Time-scale import into intact pea chloroplasts was per-formed using increasing times as indicated and 3 m M ATP at 25 C ATP- and time-dependent import reactions from five inde-pendent experiments were quantified and the results presented graphically The top three panels represent gel images; the bot-tom panel depicts the quantification graph Import after 20 min was calculated as maxi-mal import rate (C) Stroma was isolated from pea chloroplasts and incubated with radioactively-labelled translation products [ 35 S]pTic22, [ 35 S]pMGD1 and [ 35 S]pSSU for
90 min at 26 C Reactions were stopped by addition of Laemmli buffer and samples were analyzed by SDS ⁄ PAGE Lanes 1, 3 and 5 represent 2 lL of the corresponding translation products, and lanes 2, 4 and 6 represent 2 lL of the translation product which was incubated with isolated stromal fraction, respectively Precursor and mature forms of MGD1 and SSU, appearing after processing by a stromal processing assay, are indicated by arrow heads.
Trang 5ATP concentrations above 100 lm The import of the
stromal marker pSSU, which imports constantly with
high yield, clearly requires also exogenous ATP
Prom-inent amounts of mSSU accumulated at 25 lm ATP,
which we consistently observed for this highly import
competent preprotein Import increased almost linearly
up to 0.5–1 mm ATP
Likewise, the import kinetics clearly differ between
the three preproteins Although pSSU imports
extre-mely rapidly, and mSSU is detectable already after a
few seconds of import (zero time point) and continues
linearly only for up to 5 min, the import of both
pTic22 and pMGD1 is much slower Import becomes
detectable only after 1–2 min and then continues
line-arly for up to 20 min (Fig 2B) Another indication for
the different import pathways of pTic22 and pMGD1,
respectively, is precursor cleavage by the stromal
pro-cessing peptidase Whereas the control protein pSSU
as well as pMGD1 are processed by the SPP in a
stro-mal processing assay, pTic22 remains intact (Fig 2C)
As already indicated by the results presented in
Fig 1 and corroborated by those shown in Fig 2, clear
differences in the import behaviour can be determined
not only between pTic22 and pMGD1, respectively,
but also between each of the two and pSSU Another
requirement for the import competence of chloroplasts
for preproteins such as pSSU is the presence of protease sensitive translocon components in the outer chloroplasts envelope In an attempt to determine the involvement of such protease sensitive components in the import pathway of pTic22 and pMGD1, we treated isolated chloroplasts with the protease thermolysin, which removes exposed parts of translocon components such as Toc159, Toc64 and Toc34 (Fig 3) The import yield of pSSU into protease treated chloroplasts dropped to approximately 20–30%, which corresponds well to the results described earlier The import effi-ciency of pTic22 and pMGD1 were consistently less susceptible to protease pretreatment of organelles, and residual imports rates vary between 20–45% for pTic22 and 40–60% for MGD1, respectively (Fig 3, gel images are presented on the left side, quantification is depicted on the right hand side) These data suggest that import of all preproteins tested is mediated by proteinaceous components of the outer membrane Although differences were observed in the import behaviour between the intermembrane space proteins pTic22 and pMGD1 in comparison with the stromal precursor pSSU, the similarities that were observed raised the possibility that targeting to the intermem-brane space may involve subunits of the general import pathway In an initial attempt to test this
Fig 3 Import of pTic22 and pMGD1 is
reduced by thermolysin pretreatment of
chloroplasts Gel images are depicted on
the left side Precursor and mature forms
are indicated by arrow heads A graphical
presentation is shown of the influence of
thermolysin pretreatment of chloroplasts on
the import of pTic22, pMGD1 and pSSU in
the presence of ATP, derived by 2D
densi-tometry evaluation (AIDA image analyser) of
five independently performed experiments
for each protein For import, intact
chloro-plasts were used that were either
pretreat-ed or not treatpretreat-ed with 1 mg of thermolysin
per 1 mg of chlorophyll Import of
[ 35 S]pTic22, [ 35 S]pMGD1 and [ 35 S]pSSU into
intact pea chloroplasts corresponding to
15 lg of chlorophyll was performed for
15 min at 25 C for pTic22 and pMGD1, and
5 min for pSSU After import, chloroplasts
were either subjected to thermolysin
post-treatment (+Th) or not (–Th) Import without
pre- and post-treatment was considered to
be the maximal import rate.
Trang 6possibility, we conducted import experiments in the
presence or absence of an excess of unlabelled
hetero-logously expressed soluble chloroplast precursor
protein, the 33 kDa subunit of the oxygen evolving
complex pOE33 (Fig 4) Unlabelled pOE33, but not
its mature form mOE33, efficiently competed for the
import of 35S-labelled pSSU; the maximum inhibition
of approximately 90% was reached at a competitor
concentration of 2 lm (Fig 4, middle panel) The
import efficiency of pTic22 also clearly decreased in
the presence of pOE33 However, at 2 lm competitor,
we still observed a 50% import yield and, at the
high-est competitor concentration thigh-ested (10 lm), import
yield was still approximately 30% (Fig 4, upper
panel) This result is clearly distinct from those
previ-ously obtained [16], which indicate that pTic22 import
is not competed for by standard chloroplasts
prepro-teins such as pSSU (see below) The import of
pMGD1 was reduced to only approximately 50% at
the highest competitor concentration tested and
inhibi-tion was barely detectable at 2 lm pOE33 (Fig 4,
lower panel) However, in every case, the reduction of
import yield depended on the precursor from of OE33,
as can be deduced from the gel images and the
quanti-fication data In no case did we observe any significant effect of the mature form of OE33 on the import of any of the three preproteins A slight decrease of import efficiency was observed at 10 lm mOE33, but this effect was much weaker than at the same concen-trations of the precursor form
In an effort to address the involvement of known Toc subunits more directly, we expressed the soluble domain of one of the receptor proteins, Toc34, and used this peptide as a competitor for import (Fig 5) Toc34 or the deletion Toc34DTM, which does not con-tain the transmembrane anchor and can therefore serve
as a soluble receptor, interact with preproteins but not their mature forms in solution [5] To this end, purified Toc34DTM was preloaded with 3 mm GTP in the import mix for 10 min at 4C Subsequently, 35 S-labelled preproteins pTic22, pMGD1 and pSSU were added to the mixture and incubation continued for
10 min The import reaction was initiated by addition
of chloroplasts and carried out as described above Soluble Toc34DTM competed for the import of all three preproteins tested (Fig 5A) Import inhibition
by Toc34DTM was approxiately 60% for pMGD1 and approxiately 50% for both pTic22 and pSSU, as
Fig 4 pTic22 and pMGD1 compete with pOE33 for import into chloroplasts Increas-ing concentrations of overexpressed protein pOE33 or its mature form mOE33, as indi-cated, were added into the import mix prior
to import of [35S]pTic22, [35S]pMGD1 and [ 35 S]pSSU into intact pea chloroplasts corre-sponding to 15 lg chlorophyll Import reac-tion was performed for 12 min at 25 C for pTic22 and pMGD1 and 5 min for the pSSU control After import, chloroplasts were sub-jected to thermolysin post-treatment Repre-sentative gel images are depicted on the left-hand side, and quantifications on the basis of five independently performed com-petition experiments are shown on the right Import without competitor was considered
to be the maximal import rate.
Trang 7indicated in the autoradiographs as well as in the
quantification data (Fig 5A) Furthermore, we could
demonstrate that Toc34DTM interacts directly with
each of the three preproteins (Fig 5B) To do so,
over-expressed purified Toc34DTM was coupled to Ni-NTA
matrix (see Experimental procedures) and preloaded
with 1 mm GTP Subsequently, radioactively-labelled
translation products of pTic22, pMGD1 and pSSU
were incubated with the matrix for 45 min Unbound
preproteins were washed off and bound preproteins
eluted with 250 mm imidazole containing buffer In
every case, precursor protein was detected in the
imid-azole eluate, indicating a direct interaction with
Toc34DTM This interaction was not observed when
we used the mature form mTic22 or mSSU (data not
shown) or the empty Ni-NTA matrix incubated solely
with radioactively-labelled preproteins Taken together,
these results indicate that the two intermembrane space proteins, pTic22 and pMGD1, are deduced to share common import components with pSSU
In a further attempt to test this idea, we used a chemical cross-link approach (Fig 6) Chloroplasts were incubated with radioactively-labelled precursor proteins under conditions that allow binding and inser-tion into the translocon but not complete translocainser-tion (i.e in the presence of 3 mm ATP at 4C) After preincubation, preproteins were cross-linked using 0.5 mm dithiobis-succinimdyl-proprionate Chloroplasts were then solubilized with 1% SDS and coimmunopre-cipitation was performed using antisera against the translocon subunits Toc34, Toc75, Tic110 and the outer envelope protein OEP16 as a control (Fig 6A) The intermembrane space preproteins pTic22 and pMGD1 appears to interact strongly with Toc34 and
Fig 5 Import of pTic22 is inhibited by the soluble domain of the receptor protein Toc34 (A) Increasing concentrations (0–10 l M ) of overex-pressed soluble receptor Toc34DTM, 3 m M ATP and 3 m M GTP, were added to the import mix prior to import of [ 35 S]pTic22, [ 35 S]pMGD1 and [35S]pSSU into intact pea chloroplasts corresponding to 15 lg of chlorophyll The import reaction was performed for 12 min at 25 C for pTic22 and pMGD1 and 5 min for the pSSU control After import, chloroplasts were subjected to thermolysin post-treatment (+Th) or not ( )Th) After import of MGD1, all samples were post-treated with thermolysin Representative gel images are shown on the upper left-hand side The results were quantified on the basis of five independently performed competition experiments for each preprotein The quanifica-tion graph is depicted at the bottom Import without competitor was considered to be the maximal import rate (B) pTic22 and pMGD1 inter-act with the soluble domain of the receptor protein Toc34 For each separate experiment, 300 lg of overexpressed soluble receptor Toc34DTM was coupled to 10 lL of Ni-NTA matrix and preloaded with 1 m M GTP Ni-NTA matrix without bound Toc34DTM was used as negative control [ 35 S]pTic22, [ 35 S]pMGD1 and [ 35 S]pSSU were added to the column in binding buffer and incubated for 45 min The flow through after incubation (Ft), the third wash of the matrix (W) and the elution with 300 m M imidazole (E) were analyzed by SDS ⁄ PAGE Tp represents 10% of the translation product used in each experiment.
Trang 8Toc75 in the outer membrane but not with OEP16.
This result is identical to the data obtained for the
control precursor pSSU, which is clearly established as
a substrate for the general import pathway The
stro-mal precursor pSSU also interacted strongly with the
inner envelope translocon subunit Tic110, whereas
pTic22 interaction was weak and that of pMGD1
barely detectable The interaction of pTic22 and
Tic110 might be explained by Tic22 being a
compo-nent of the inner envelope translocon, although a
direct interaction with Tic110 has not yet been shown
[15] Therefore, this weak interaction might not
neces-sarily indicate a role of Tic110 in the translocation of
pTic22
Besides the translocation pore, the outer chloroplast
envelope contains Toc75-III, commonly called Toc75,
which constitutes the import channel for the general
import pathway, an evolutionary more ancient isoform
named Toc75-V [22] The function of Toc75-V, which
constitutes approximately 10% of the total Toc75-like
proteins present in chloroplasts, is not yet clear There-fore, we considered whether pTic22 and pMGD1 might also use this channel protein An identical cross-link approach to that described above was used (Fig 6B) Although we could again detect a cross-link product between Toc75-III and pTic22, pMGD1 and pSSU, no interaction of Toc75-V could be found with any of the three preproteins Because the coimmuno-precipitation using Toc75-III and Toc75-V antisera were carried out from identical samples, we conclude that Toc75-V plays no role in the translocation of pTic22 and pMGD1
The cross-linking of pTic22 and pMGD1 to Toc34 and Toc75 might be nonspecific because these two translocon subunits are very abundant polypeptides in the chloroplast outer envelope Therefore, we repeated the cross-linking experiments in the presence of an excess of the soluble chloroplast preprotein pOE33 to compete for specific binding, whereas the control experiment contained an equal amount of mOE33
A
C
B
Fig 6 Chemical cross-linking and immunoprecipitation of pTic22 and pMGD1 to the major components of the translocation machinery (A) [ 35 S]pTic22, [ 35 S]pMGD1 and [ 35 S]pSSU were incubated with intact pea chloroplasts corresponding to 20 lg of chlorophyll for 8 min on ice After reisolation on a Percoll cushion and subsequent washing, chloroplasts with bound precursor proteins were subjected to cross-linking using 0.5 m M dithiobis-succinimdyl-proprionate Immunoprecipitation was performed after lysis of chloroplasts, centrifugation and solubiliza-tion of the membranes Antibodies raised against Toc34, Toc75, Tic110, and OEP16 were used and incubated for 1 h at room temperature Antibodies were bound to protein A-sepharose Ten percent of the flow through after incubation with protein A-sepharose (Ft), 10% of the third wash (W), and the elution with Laemmli sample buffer (E) were analyzed by SDS ⁄ PAGE TL indicates 10% of the translation product used for each experiment (B) Cross-linking and immunoprecipitation were performed under the same conditions, using antibodies against two Arabidopsis Toc75-isoforms: atToc75(III) and atToc75(V) (C) Cross-linking and immunoprecipitation were performed in the presence of
10 l M mOE33 or 10 l M pOE33 in the import mixture.
Trang 9(Fig 6C) The interaction of both pTic22 and pMGD1
was largely abolished in the presence of the competitor
pOE33 but not in the presence of mOE33 Taken
together, we conclude that the intermembrane space
constituents pTic22 and pMGD1 are bona fide
sub-strates of the Toc translocon in chloroplasts
Discussion
The data presented in the present study suggest that
proteins located in the intermembrane space of
chloro-plasts use components of the general import pathway
in the outer envelope membrane, but nevertheless
clearly show distinctive import behaviour compared to
stromal precursors as well as to each other We
investi-gated the import characteristics of two intermembrane
space localized proteins, Tic22 and MGDG synthase
(MGD1) The first noticable difference was the
concen-tration of exogenously added ATP that was required
for import Whereas stromal proteins such as pSSU
need > 0.1 mm ATP for complete translocation, most
likely for the action of stromal chaperones, pTic22 was
already imported at a concentration of less than 20 lm
ATP, indicating that no stromal components are
involved in this process By contrast, the import of
pMGD1 required more than 100 lm ATP to be
effi-cient, suggesting that this precursor might reach the
stroma before being released to its final destination in
the intermembrane space This was corroborated by
stromal processing assays in which pMGD1 was
pro-cessed by the SPP, whereas pTic22 was not (Fig 2C)
With respect to the Toc75 import and processing
features, it is feasible that MGD1 also is partly
trans-located to the stroma, where the transit peptide is
cleaved off by the stromal processing peptidase, and is
then released to its final localization in the
intermem-brane space
Another difference between pTic22, pMGD1 and
pSSU is their import rate The stromal precursor
reaches its destination within seconds, whereas the
intermembrane space proteins require 1–2 min Again,
pTic22 and pMGD1 show distinctive features: the
pro-cessing of pTic22 appears to be very slow compared to
translocation (i.e because the precursor becomes
resis-tant to externally added protease), whereas pMGD1
import and processing occur at similar rates In
addi-tion to the differences in ATP requirements and the
stromal processing assay, the import kinetics definitely
indicate that the two preproteins not only use different
pathways, but also are processed by different
prote-ases The protease responsible for maturation of
pTic22 appears to be located in the intermembrane
space but this requires further investigation
Competition experiments using pOE33 and Toc34DTM, as well as cross-linking and immunopre-cipitation assays, clearly show that both pTic22 and pMGD1 engage the Toc complex We could demon-strate interaction with the receptor Toc34 and the gen-eral import pore Toc75 This contradicts previously published studies [16] that showed no competition of a stromal precursor (i.e the authors used overexpressed pSSU as competitor, which should not make a differ-ence because both pSSU and pOE33 engage the Toc translocon) with pTic22 In the previous study [16], however, import rates of pTic22 were generally very low (approximately 5%) so that the competition effect clearly demonstrated in the present study might not have been detectable Import kinetics have to be estab-lished for each precursor individually to determine biochemically relevant data in further experiments Therefore, import experiments to elucidate the effect
of competitors were conducted only for 12 min, which
is within the linear time course of pTic22 and pMGD1 import (cf Fig 2), whereas Kouranov et al [16] incu-bated the import reaction for much longer This might
be one reason to explain their negative competition data Even under our optimal conditions, the import
of pTic22 and pMGD1 is much slower than that of pSSU, which makes the competition of pSSU import more visible due to the greater difference of the import rate with and without competitor, respectively Fur-thermore, it is possible that not all components involved in pSSU translocation play a role in the import of pTic22 and pMGD1, and therefore the com-petition by pOE33 is not as pronounced as it is for pSSU Nevertheless, the effect of pOE33 on import of pTic22 and pMGD1 is clearly apparent
Taken together, our experiments indicate that pTic22 and pMGD1 use the general import pathway
to traverse the outer envelope and diverge at the level
of the intermembrane space⁄ inner envelope The results of the present study clearly indicate distinct import pathways not only for proteins destined for stromal or membrane compartments, but also for the intermembrane space of chloroplasts
Experimental procedures
In vitro transcription and translation The coding region for Tic22 from Arabidopsis thaliana (At4g33350) was cloned into the vector pSP65 (Promega, Madison, WI, USA) under the control of the SP6 promoter The coding region for MGD1 from A thaliana (At4g38170) was cloned into the vector pET21d (Merck, Darmstadt, Germany) under the control of the T7 promoter Mature
Trang 10forms of these proteins were produced by the removal of the
sequences encoding the transit peptides (177 bp for Tic22
and 321 bp for MGD1) in the same vectors, using standard
PCR protocols Tic22DC was produced by removal of the
coding sequence corresponding to 225 C-terminal amini
acids of the preprotein Transcription of linearized plasmids
was carried out in the presence of SP6 (for Tic22) or T7
polymerases (for MGD1) using chemicals obtained from
MBI Fermentas (St Leon-Rot, Germany) Translation was
carried out using the Flexi Rabbit Reticulocyte Lysate
System or the TNT Coupled Reticulocyte Lysate System
(Promega) in the presence of [35S]methionine⁄ cysteine
mix-ture (MGD1) or [35S]cysteine (Tic22) for radioactive
label-ling After translation, the reaction mixture was centrifuged
at 50 000 g for 20 min at 4C using a Himac CS150GX
centrifuge (Hitachi, Tokyo, Japan) and S150AT rotor
and the postribosomal supernatant was used for import
experiments
Chloroplast isolation and protein import
Chloroplasts were isolated from leaves of 9–11-day-old pea
seedlings (Pisum sativum var Arvica) as described
previ-ously [23] Prior to import, ATP was depleted from
chlo-roplasts and the in vitro translation product Intact
chloroplasts were left on ice in the dark for 30 min
Trans-lation products were treated with 0.5 U apyrase per 10 lL
of translation product at 25C for 15 min, or passed
through Micro Bio-Spin Chromatography Columns
(Bio-Rad, Hercules, CA, USA) A standard import reaction
con-tained chloroplasts equivalent to 15–20 lg of chlorophyll in
100 lL of import buffer [330 mm sorbit, 50 mm Hepes⁄
KOH (pH 7.6), 3 mm MgSO4, 10 mm methionine, 10 mm
cysteine, 20 mm K-gluconate, 10 mm NaHCO3, 2% BSA
(w⁄ v)], up to 3 mm ATP and35S-labelled translation
prod-ucts in the maximal amount of 10% (v⁄ v) The import
reactions were initiated by the addition of translation
prod-uct and carried out for 20 min at 25C, unless indicated
otherwise Reactions were terminated by separation of
chloroplasts from the reaction mixture by centrifugation
through 40% (v⁄ v) Percoll cushion Chloroplasts were
washed once, and import products were separated by
SDS⁄ PAGE Radiolabelled proteins were analyzed by a
phosphoimager or by exposure on X-ray films
Chloroplasts were treated before or after import with the
protease thermolysin For pretreatment, 1 mg thermolysin
per mg chlorophyll was applied for 30 min on ice The
reaction was terminated by reisolation on a Percoll density
gradient in the presence of 5 mm EDTA [24] For
post-treatment, 0.5 lg of thermolysin per lg chlorophyll was
applied for 20 min on ice The reaction was stopped by the
addition of 5 mm EDTA, sedimenting the chloroplasts and
resuspending them in Laemmli buffer [50 mm Tris pH 6.8,
100 mm b-MeOH, 2% (w/v) SDS, 0.1% bromophenol blue
(w/v), 10% glycerol (v/v)]
Import competition experiments were performed by the addition of up to 10 lm of purified competitor protein pOE33, as well as its mature form mOE33, into the import mixture prior to import Fifteen micrograms of chlorophyll per reaction were used and the import reaction lasted 5 (pSSU) to 12 min (Tic22, MGD1) at 25C Competition for import by the cytosolic domain of Toc34 receptor was performed in the presence of 3 mm GTP and up to 10 lm Toc34DTM in the standard import mixture First, Toc34DTM was preincubated with GTP in the import mix-ture for 10 min on ice Subsequently, radioactively-labelled translation product was added and incubated for another
10 min to allow the interaction of preprotein with Toc34DTM Finally, chloroplasts equivalent to 15 lg of chlorophyll were added and the reaction was incubated for 10–12 min at 25C for Tic22 and MGD1 and 5 min for pSSU
Overexpression and purification of pOE33-His6, mOE33-His6 and Toc34DTM-His6 for competition experiments
Transformed Escherichia coli BL21(DE3) cells were grown
in LB medium containing 100 lgÆmL)1 of ampicilin (and
1 mm MgSO4and 0.4% glucose for mOE33) to an D600 nm
of 0.6 Expression was induced by 1 mm isopropyl
thio-b-d-galactoside and cells were grown for 3 h at 37C pOE33 and mOE33 were purified from inclusion bodies under denaturing conditions via Ni-affinity chromatogra-phy and eluted by decreasing the pH Refolding of the pro-teins was accomplished using stepwise dialysis against 6, 4,
2 and 0 m urea (in 50 mm Tris, pH 8.0), respectively Toc34DTM was expressed in a soluble form and purified under native conditions and elution by 250 mm imidazole The protein was always used fresh and diluted so that the final imidazole concentration in the import reaction did not exceed 30 mm
Binding of Toc34DTM to precursor proteins Three hundred micrograms of purified Toc34DTM were coupled to 10 lL of Ni-NTA matrix in binding buffer (50 mm NaCl, 50 mm NaiPO4, 0.5% BSA, pH 7.9) for
45 min, rotating at room temperature The prepared matrix was preincubated with 1 mm GTP, and subsequently 10–12 lL of a radioactively-labelled translation product were applied in the reaction containing 1 mm GTP, 2 mm MgCl2, 20 mm Tris⁄ HCl (pH 7.6), 50 mm NaCl and 0.5% BSA The incubation lasted 45–50 min The matrix was subsequently washed three times with wash buffer (50 mm NaCl, 50 mm NaPi, 30 mm imidazole, pH 7.9) and eluted with 50 lL of elution buffer (50 mm NaCl, 50 mm NaPi,
300 mm imidazole, pH 7.9) The obtained fractions were analysed by SDS⁄ PAGE