Department of Biology I, Botany, University of Munich, Menzinger Str. 67, Munich 80638, Germany
* Author for correspondence (e-mail: soll{at}uni-muenchen.de)
Accepted 13 April 2004
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Summary |
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Key words: Protein import, Targeting signal, Chloroplast, Envelope membranes
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Introduction |
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Several reports indicate that specialised import routes into chloroplasts might exist. A nuclear encoded heat-shock-induced protein of Chlamydomonas thylakoids did not contain a cleavable pre-sequence as deduced from sequence comparison. However no in vitro imports were conducted (Grimm et al., 1989). From in vivo studies using a GFP-fusion of the chloroplast inner envelope localized quinone oxido reductase (QORH) it was shown that internal sequence information was required for correct targeting and that neither N- or C-terminal transit peptides were required (Miras et al., 2002
). Tic22 takes a different route again. Whereas the preprotein contains a cleavable pre-sequence and requires protease-sensitive receptors, its import needed only low concentrations of ATP, consistent with the idea that stromal chaperones are not involved in Tic22 import (Kouranov et al., 1999
).
In this paper we provide evidence for a distinct import pathway into the inner envelope of chloroplasts. The inner envelope protein IEP32, also named HP32, is targeted to chloroplasts independent of a cleavable pre-sequence and any protease-sensitive surface-exposed receptor protein. Import of IEP32 does not seem to require the Toc75 import channel as deduced from inhibitor studies. The involvement of stromal chaperones is also unlikely because ATP concentrations below 20 µM are sufficient for import.
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Materials and Methods |
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Chloroplast isolation and protein import
Chloroplasts were isolated from leaves of 10- to 12-day-old pea plants (Pisum sativum, var. Golf) and purified through Percoll density gradients as described (Waegemann and Soll, 1991). A standard import reaction contained chloroplasts equivalent to 15 µg chlorophyll in 100 µl import buffer (10 mM methionine, 10 mM cysteine, 20 mM potassium gluconate, 10 mM NaHCO3, 330 mM sorbitol, 50 mM HEPES-KOH, pH 7.6, 5 mM MgCl2) and 1-5% in vitro translation product. Import reactions were initiated by the addition of translation product and carried out for 15 minutes at 25°C unless indicated otherwise. Reactions were terminated by separation of chloroplasts from the reaction mixture by centrifugation through a 40% (v/v) Percoll cushion in import buffer. Chloroplasts were washed once and import products separated by SDS-PAGE and radiolabelled proteins analysed by a phosphor-imager.
Chloroplasts were treated with the protease thermolysin either before or after import at a protease concentration of 20 µg/ml for 20 minutes on ice (Waegemann and Soll, 1995). Chloroplasts were repurified through Percoll density gradients before further use (Waegemann and Soll, 1995
). Inhibitors like CuCl2 (1 mM) (Seedorf and Soll, 1995
) or spermine (5 mM) (Hinnah et al., 2002
) were incubated with chloroplasts 20 minutes prior to import.
In some cases chloroplasts were treated with 6 M urea after import to separate bound pre-proteins from membrane-integrated polypeptides. Urea (6 M) treatment in 50 mM HEPES-KOH, pH 7.6 was carried out for 15 minutes at 25°C. Insoluble proteins were collected by centrifugation at 250,000 g for 10 minutes.
Chemical crosslinking was performed after separation of chloroplasts from the import mixture by centrifugation as above in the presence of 0.5 mM dithiobis-succinimidyl-proprionate (DSP) for 30 minutes at 4°C. The reaction was stopped by the addition of lysine (125 mM) and further incubation for 15 minutes. Chloroplasts were washed twice in 330 mM sorbitol, 50 HEPES-KOH, pH 7.6, 3 mM MgCl2 and finally lysed in hypotonic buffer 20 mM HEPES-KOH, pH 7.0, 5 mM EDTA. A total-membrane fraction was recovered by centrifugation at 125,000 g for 30 minutes. Membranes were solubilized in 1% SDS (w/v), 25 mM HEPES-KOH pH 7.6, 150 mM NaCl for 10 minutes at 25°C, diluted tenfold in the above buffer in the absence of SDS and used for immunoprecipitation experiments with antisera to Tic110, Toc75 V, Toc75III, Tic22 and OEP21. Antisera incubations were continued for 1 hour followed by purification by Protein A-Sepharose. The affinity matrix was washed with 50 bed-volumes of the above buffer before elution with Laemmli sample buffer in the presence of mercaptoethanol to split the crosslink products.
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Results |
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In the presence of <20 µM ATP, which is carried into the import reaction from the translation mixture, binding of pSSU still occurred but in accordance with published data (Olsen et al., 1989; Theg et al., 1989
) import was greatly reduced (Fig. 1A, lanes 4,5). Surprisingly the import of IEP32 in the presence of <20 µM ATP was as efficient as in the presence of 3 mM ATP (Fig. 1A, compare lanes 3 and 5). In order to examine if IEP32 import required ATP the translation products were treated with the ATP hydrolysing enzyme apyrase to remove exogenous ATP and the subsequent import reaction was carried out in the dark. In the absence of ATP the import of IEP32 is largely diminished. In general a 4- to 5-fold stimulation in the presence of ATP can be seen. Residual IEP32 bound to the chloroplast surface might be caused by partial import or aggregation of the protein at the chloroplast surface. PSSU neither bound nor imported in the absence of ATP. We conclude that the import of IEP32 is dependent on ATP but the ATP concentration required is much lower than for proteins that enter the stroma like pSSU (Fig. 1D, lanes 4,5).
In order to verify that IEP32 had actually reached the inner envelope, chloroplasts were fractionated into the soluble stroma, which contained processed mSSU as expected. Most IEP32 was recovered with the inner envelope, whereas only a little IEP32 co-fractionated with the outer envelope membrane (Fig. 1E). As outer envelope preparations always contain some inner envelope membrane contamination, we conclude that IEP32 has successfully reached its target membrane. The results from urea extraction, ATP dependence and chloroplast fractionation indicate that most IEP32 is imported into the inner envelope and that only about 20% of chloroplast-recovered IEP32 is on a non-productive pathway.
The data indicated that IEP32 could be imported without a cleavable pre-sequence, we therefore wanted to know how IEP32 is targeted to chloroplasts. Amino-terminal deletion mutants of IEP32 were constructed, in which we progressively removed the first 10-40 amino acids (Fig. 2A). Wild-type IEP32 and the deletion mutants were synthesized in vitro and used for protein import studies. The mutated pre-proteins showed a strong reduction in the extent of chloroplast binding (Fig. 2B, lanes 2,5,8,11,14). With the exception of the wild-type IEP32 all mutant proteins remained protease accessible demonstrating that no productive interaction with chloroplasts had occurred. Deletion of the first ten amino acids already abolished import (Fig. 2B, lanes 5,6), indicating that this part constitutes an indispensable part of the signal.
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The ability of typical precursor proteins such as pSSU to interact with chloroplasts is susceptible to protease treatment of the organelle, owing to the removal of receptor polypeptides at the outer envelope. We questioned whether IEP32 is dependent on protease-sensitive chloroplast surface components. Therefore chloroplasts were treated with the protease thermolysin prior to conducting the import reaction. Under appropriate conditions thermolysin removes surface-exposed epitopes of outer envelope proteins, but leaves inner envelope and deeply embedded outer envelope proteins intact (Joyard et al., 1983; Cline et al., 1984
). The thermolysin pretreatment was assessed by immunoblotting (Fig. 3A) and showed that surface exposed domains of the receptor proteins Toc159 and Toc34 as well as Toc64 were removed. The Toc75III import channel, its homologue Toc75V (Eckart et al., 2002
) as well as the inner envelope protein Tic110 remained intact (Fig. 3A). Chloroplasts from the same batch as used for the immunoblot analysis were used for the import assays. As shown in Fig. 3B (lanes 4,5) pre-treatment of chloroplasts with thermolysin resulted in a loss of pSSU binding and import, demonstrating the receptor dependence of recognition and subsequent translocation. Binding of IEP32 to chloroplasts was however not affected by protease pre-treatment (Fig. 3B, lanes 3,5). Furthermore IEP32 was in a protease-resistant environment as indicated by thermolysin treatment after completion of translocation (Fig. 3B, lane 5). We conclude that IEP32 is recognized by factors other than Toc34 or Toc159.
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Translocation across the outer envelope occurs through the import channel Toc75III (Hinnah et al., 2002). In order to investigate if Toc75 is involved in IEP32 translocation we used two different approaches. First, import reactions were carried out in the absence or presence of chemical amounts of the precursor of the 33-kD oxygen-evolving-complex subunit of pOE33. In the presence of the competitor pOE33 the import of pSSU was largely inhibited and binding to the chloroplast surface was also reduced (Fig. 4A, lanes 4-7). In contrast, binding and import of IEP32 was not influenced in the presence of the competitor pOE33 (Fig. 4A, lanes 2-5). An increase of the competitor concentrations up to 600 µg/ml still did not effect the import of IEP32 into chloroplasts (Fig. 4B, lanes 2-5).
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In a second approach we used the inhibitors spermine and CuCl2. CuCl2 inhibits import because it catalyses the formation of disulfide bonds between Toc subunits and therefore inactivates its translocation activity (Seedorf and Soll, 1995). The positively charged spermine binds to Toc75 and blocks the import channel (Hinnah et al., 2002
). In the presence of 2 mM spermine import of pSSU was strongly reduced (Fig. 4C, compare lanes 3 and 5), whereas binding is slightly increased. The data corroborate earlier findings that the effect of spermine on import is at a step after recognition but before translocation (Hinnah et al., 2002
). In the presence of CuCl2, translocation of pSSU is again completely inhibited and binding is reduced (Fig. 4B, lanes 6,7). IEP32 import was however influenced neither by spermine nor by CuCl2 (Fig. 4B, lanes 2-7). We conclude from the competitor and inhibitor experiments that Toc75III is not involved in translocation of IEP32, but that IEP32 uses a so far unidentified pathway across the outer envelope. Diethylpyrocarbonate (DEPC) has been shown to inhibit import of pSSU at the level of the inner envelope (Caliebe et al., 1997
). The import of pSSU into chloroplasts in the presence of DEPC yields a typical translocation intermediate, indicating that the Toc complex is still functional (Caliebe et al., 1997
). IEP32 import was hardly affected in the presence of 1 mM DEPC, whereas pSSU import was drastically reduced (Fig. 4D). More pSSU remained in the precursor form, which could be converted into a lower molecular weight translocation intermediate upon thermolysin treatment (Fig. 4D, lane 5/TIM). These data indicate that IEP32 might insert into the inner envelope independent of the standard Tic pathway.
Chemical crosslinking can be used to identify proteins that are in close proximity to each other. In order to identify potential factors that are involved in the translocation of IEP32, we used a crosslinking approach under different import conditions, which should result in the formation of distinct translocation intermediates (Waegemann and Soll, 1991; Ma et al., 1996
). Chloroplasts were incubated in the presence of the pSSU and IEP32 pre-proteins in the presence of the crosslinker DSP. Each pre-protein gave rise to a distinct set of labelled crosslinked products in the presence of chloroplasts (Fig. 5A). Though IEP32 gave rise to fewer crosslinked products than pSSU (Fig. 5A, lanes 3,4) they were clearly visible and distinct from the crosslinking profile of pSSU. In order to identify at least some of the crosslinked products they were coimmunoprecipitated by antibodies against the receptor polypeptides Toc159, Toc64 and Toc34 (Fig. 5B) as well as the import channel Toc75III, its isoforms Toc75V, the intermembrane space subunit Tic22 and Tic110. As a control we used an antibody raised against the outer envelope solute channel OEP21. Binding to the chloroplast receptors Toc159 and Toc34 is GTP dependent (Kessler et al., 1994
; Schleiff et al., 2003
). We therefore investigated the role of these two GTP receptors for IEP32 recognition in the presence of the non-hydrolyzable GTP analog GMP-PNP, which leads to an accumulation of pre-proteins at this very early import step. The pre-protein pSSU was crosslinked efficiently to Toc34, Toc64 and Toc159 (Fig. 5B), whereas IEP32 shows no detectable interaction. These results corroborate the data shown in Fig. 3, which also indicate that protease-sensitive Toc subunits are not involved in the import of IEP32. Under conditions that allow binding as well as partial translocation but not complete import, i.e. incubation for 5 minutes at <20 µM ATP and 4°C, crosslinking of pSSU was observed predominantly to Toc75III, Tic22 and to a lesser extent Tic110. No crosslinked products of pSSU could be observed to Toc75V and OEP21 (Fig. 5C). IEP32 did not yield any significant crosslink products under these conditions. When import reactions were carried out at 25°C for 5 minutes in the presence of 3 mM ATP further crosslinked products were formed. Although the pSSU-Toc75III, -Tic22, -Tic110 crosslinked products were still prominent, crosslinking of Tic22 and Tic110 to processed mature mSSU started to appear, supporting their role in the later stages of translocation (Kessler and Blobel, 1996
; Kouranov and Schnell, 1997
). In addition, low but reproducible amounts of IEP32 were recovered together with Tic110 and Tic22, but not with Toc75III or Toc75V (Fig. 5D), corroborating our earlier finding that none of the well-documented Toc subunits is involved in IEP32 translocation (compare Figs 3 and 4).
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When crosslinking was carried out after 15 minutes' import at 25°C in the presence of 3 mM ATP the detectable products changed quite significantly. In comparison to the 5-minute time point much less pSSU was crosslinked to Toc75, Tic22 or Tic110 (Fig. 5E). Instead mSSU was the most prominent crosslinked product associated with Tic110 and Tic22 indicating that the import reaction was near completion. Under these conditions IEP32 was still crosslinked only to Tic22 and Tic110, indicating that these two proteins could be involved in IEP32 import. The association of IEP32 with Tic110 might however also represent the endpoint of IEP32 import, because it is closely associated with Tic110 in situ (F. Hörmann and J.S., unpublished).
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Discussion |
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Tic22 is localized to the intermembrane space between the inner and the outer envelope (Kouranov et al., 1998). Its import characteristics are also different from pre-proteins that normally traverse both envelope membranes and import into the stroma (Kouranov et al., 1999
). Tic22 is made in the cytosol with a cleavable pre-sequence, binding and import is stimulated by protease-sensitive chloroplast surface components, however import is not competed for by excess of a `typical' pre-protein like pSSU and it requires ATP concentrations below 100 µM for maximum import yields (Kouranov et al., 1999
). These characteristics again differ in some respect from IEP32 import as IEP32 is not synthesized with a cleavable pre-sequence and does not require protease-sensitive receptors. Similarities do exist however; for example import of both pre-proteins requires only low ATP concentration and is not competed for by an excess of precursor protein. Together the data indicate that alternative import routes into chloroplasts exist, which can be distinguished from the general route into chloroplasts (Chen and Schnell, 1999
; Keegstra and Froehlich, 1999
; Bauer et al., 2001
; Soll, 2002
) by the following criteria: (i) energy requirement, (ii) presence of a cleavable pre-sequence, (iii) involvement of protease-sensitive surface components; (iv) competition by stroma-targeted pre-proteins; (v) usage of a different import channel than Toc75III. It should be noted however that the import characteristics found here and in other published studies all describe pathway(s) for polypeptides that are affiliated with the inner envelope membrane (Kouranov et al., 1999
; Miras et al., 2002
). These proteins most likely do not traverse the inner envelope completely, but are sorted to their final destination by as yet unidentified signals. This is also supported by the missing effect of DEPC on IEP32 translocation. This system could resemble a stop-transfer mechanism described for mitochondrial inner membrane protein import (Pfanner and Geissler, 2001
).
Chemical crosslinking studies as well as competition experiments indicate that IEP32 does not use a Toc75 import channel, but that the outer envelope must contain further channel proteins that allow the passage of polypeptides. The outer envelope is known to contain several solute channels (Bölter et al., 1999), however none of them seem to be involved in IEP32 import. CuCl2 treatment inhibits not only the Toc translocon but also the amino acid-selective ion channel OEP16 (Pohlmeyer et al., 1998
). Furthermore, no crosslinks between the anion-selective channels OEP21 and OEP24 could be observed (Fig. 5 and not shown). Although this is negative evidence it supports our idea that additional ion channels have to be present in the outer envelope membrane.
IEP32 behaves as an integral membrane protein. How these hydrophobic proteins cross the aqueous intermembrane space is unknown. Tic22, which is localized in the envelope space might be a cofactor in this process and might function in a way similar to the small Tim proteins (Pfanner and Geissler, 2001) in the space between the outer and the inner mitochondrial membrane. The small Tim proteins are required for the translocation of hydrophobic carrier proteins into the inner mitochondrial membrane (Pfanner and Geissler, 2001
). We obtained a clear crosslink product between Tic22 and IEP32 indicating that Tic22 is involved in the import of IEP32. The other crosslinked product was found to be Tic110. Whether Tic110 is involved in the actual import process of IEP32 cannot be resolved, because IEP32 and Tic110 are closely associated with each other in situ (F. Hörmann and J.S., unpublished). Therefore the observed crosslink could represent only the end point of import and indicate that IEP32 assembled correctly.
We conclude that IEP32 follows a distinct import pathway into chloroplasts, which shows many special features not observed by precursor proteins that are translocated across both envelope membranes. The major challenge for the future will be to identify the outer envelope components involved in recognition and translocation of IEP32.
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Acknowledgments |
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References |
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