Protein transport in chloroplasts ) targeting to the intermembrane 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 mem- brane systems (i.e. the outer and inner envelope mem- brane and the photosynthetic thylakoid membranes) 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]. Toc34 GDP 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. FEBS Journal 274 (2007) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS 5043 preproteins 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 35 S-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, Intermembrane space targeting in chloroplasts L. Vojta et al. 5044 FEBS Journal 274 (2007) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS whereas 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 Na 2 CO 3 . Although MGD1 present in the inner envelope is recovered almost exclusively in the urea or Na 2 CO 3 insoluble membrane fraction, MGD1 in the outer envelope is partly or largely recov- ered in the Na 2 CO 3 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 Na 2 CO 3 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 [ 35 S]pTic22 (lanes 1–3), [ 35 S]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 ,6M 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. L. Vojta et al. Intermembrane space targeting in chloroplasts FEBS Journal 274 (2007) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS 5045 (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 AB 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. Intermembrane space targeting in chloroplasts L. Vojta et al. 5046 FEBS Journal 274 (2007) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS ATP 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 treated 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. L. Vojta et al. Intermembrane space targeting in chloroplasts FEBS Journal 274 (2007) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS 5047 possibility, 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 35 S-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 tested (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 4 °C. 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 [ 35 S]pTic22, [ 35 S]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. Intermembrane space targeting in chloroplasts L. Vojta et al. 5048 FEBS Journal 274 (2007) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS indicated 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 translocation (i.e. in the presence of 3 mm ATP at 4 °C). 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 AB Fig. 5. Import of pTic22 is inhibited by the soluble domain of the receptor protein Toc34. (A) Increasing concentrations (0–10 lM) of overex- pressed soluble receptor Toc34DTM, 3 m M ATP and 3 mM GTP, were added to the import mix prior to import of [ 35 S]pTic22, [ 35 S]pMGD1 and [ 35 S]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. L. Vojta et al. Intermembrane space targeting in chloroplasts FEBS Journal 274 (2007) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS 5049 Toc75 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 lM pOE33 in the import mixture. Intermembrane space targeting in chloroplasts L. Vojta et al. 5050 FEBS Journal 274 (2007) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS (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 L. Vojta et al. Intermembrane space targeting in chloroplasts FEBS Journal 274 (2007) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS 5051 forms 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 [ 35 S]methionine ⁄ cysteine mix- ture (MGD1) or [ 35 S]cysteine (Tic22) for radioactive label- ling. After translation, the reaction mixture was centrifuged at 50 000 g for 20 min at 4 °C 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 25 °C 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 MgSO 4, 10 mm methionine, 10 mm cysteine, 20 mm K-gluconate, 10 mm NaHCO 3 , 2% BSA (w ⁄ v)], up to 3 mm ATP and 35 S-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 25 °C, 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 25 °C. 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 lgof chlorophyll were added and the reaction was incubated for 10–12 min at 25 °C 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 1mm MgSO 4 and 0.4% glucose for mOE33) to an D 600 nm of 0.6. Expression was induced by 1 mm isopropyl thio-b- d-galactoside and cells were grown for 3 h at 37 °C. 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 Toc34 DTM 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 Na i PO 4 , 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 MgCl 2 ,20mm 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. Intermembrane space targeting in chloroplasts L. Vojta et al. 5052 FEBS Journal 274 (2007) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... v), 25 mm Hepes ⁄ KOH (pH 7. 6), 150 mm NaCl, diluted ten-fold in the above buffer in the absence of SDS, centrifuged for 2 min at 20 000 g using an Eppendorf (Hamburg, Germany) 5417R centrifuge and EL033 rotor and the supernatant used for immunoprecipitation with antisera against Toc75(III), Toc75(V), Toc34, Tic110 and OEP16 All antibodies are directed against the corresponding proteins from pea The. .. Dabney-Smith C, Subramanian C & Bruce BD (200 2) Structural and guanosine triphosphate ⁄ diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34 Biochemistry 41, 1934–1946 Intermembrane space targeting in chloroplasts 6 Schnell DJ & Hebert DN (200 3) Protein translocons: multifunctional mediators of protein translocation across membranes... buffer in the presence of b-mercaptoethanol to split the cross-link products Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie and the excellence cluster CIPSM References 1 Gutensohn M, Fan E, Frielingsdorf S, Hanner P, Hou B, Hust B, Klosgen RB (200 6) Toc, Tic, Tat-et al.: structure and function of protein transport machineries in chloroplasts. .. Soll J & Schleiff E (200 0) Toc34 is a preprotein receptor regulated by GTP and phosphorylation Proc Natl Acad Sci USA 97, 4973–4978 8 Schleiff E, Jelic M & Soll J (200 3) A GTP-driven motor moves proteins across the outer envelope of chloroplasts Proc Natl Acad Sci USA 100, 4604–4609 9 Kessler F & Blobel G (199 6) Interaction of the protein import and folding machineries in the chloroplast Proc Natl... Soll J & Schleiff E (200 4) Protein import into chloroplasts Nat Rev Mol Cell Biol 5, 198–208 3 Qbadou S, Becker T, Mirus O, Tews I, Soll J & Schleiff E (200 6) The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64 EMBO J 25, 1836–1847 4 Becker T, Jelic M, Vojta A, Radunz A, Soll J & Schleiff E (200 4) Preprotein recognition by the Toc complex EMBO J 23, 520–530... cross-linking and immunoprecipitation After import, chloroplasts were reisolated on a Percoll cushion, washed, and chemical cross-linking was performed by incubation of chloroplasts with 0.5 mm dithiobissuccinimdyl-proprionate (Pierce, Munchen, Germany) in ¨ 330 mm sorbit, 50 mm Hepes ⁄ KOH (pH 7. 6) and 0.5 mm CaCl2, for 15 min at 4 °C The reaction was stopped by the addition of 125 mm glycin and further... chloroplast interior proteins and the outer-membrane protein OEP14 converge at Toc75 Plant Cell 16, 2078–2088 15 Kouranov A, Chen X, Fuks B & Schnell DJ (199 8) Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane J Cell Biol 143, 991–1002 16 Kouranov A, Wang H & Schnell DJ (199 9) Tic22 is targeted to the intermembrane space of chloroplasts by a novel pathway... Joyard J (199 9) Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope FEBS Journal 274 (200 7) 5043–5054 ª 2007 The Authors Journal compilation ª 2007 FEBS 5053 Intermembrane space targeting in chloroplasts L Vojta et al enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG Eur J Biochem 265, 990–1001 20 Benning C & Ohta H (200 5) Three enzyme... & Joyard J (200 1) Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana Proc Natl Acad Sci USA 97, 10960–10965 18 Jarvis P, Dormann P, Peto CA, Lutes J, Benning C & Chory J (200 0) Galactolipid deficiency and abnormal chloroplast development in the Arabidopsis MGD... envelope protein 32 is imported into chloroplasts by a novel pathway J Cell Sci 117, 3975–3982 13 Inoue K, Baldwin AJ, Shipman RL, Matsui K, Theg SM & Ohme-Takagi M (200 5) Complete maturation of the plastid protein translocation channel requires a type I signal peptidase J Cell Biol 171, 425–430 14 Tu SL, Chen LJ, Smith MD, Su YS, Schnell DJ & Li HM (200 4) Import pathways of chloroplast interior proteins . starting point to investigate the import character- istics of the intermembrane space proteins Tic22 and MGD1, in vitro import experiments into isolated chloroplasts. precursor proteins. Toc34 receptor func- tion is regulated by GTP binding and hydrolysis [4–7]. Toc34 GDP interacts with the second G -protein in the Toc complex,