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Article |
Reconstitution of protein targeting to the inner envelope membrane of chloroplasts
Correspondence to Danny J. Schnell: dschnell{at}biochem.umass.edu
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The chloroplast envelope plays critical roles in the synthesis and regulated transport of key metabolites, including intermediates in photosynthesis and lipid metabolism. Despite this importance, the biogenesis of the envelope membranes has not been investigated in detail. To identify the determinants of protein targeting to the inner envelope membrane (IM), we investigated the targeting of the nucleus-encoded integral IM protein, atTic40. We found that pre-atTic40 is imported into chloroplasts and processed to an intermediate size (int-atTic40) before insertion into the IM. Int-atTic40 is soluble and inserts into the IM from the internal stromal compartment. We also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. Collectively, our experiments are consistent with a "postimport" mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.
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Introduction |
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Except for the translocon component, Toc75, outer envelope membrane proteins are targeted directly from the cytoplasm to the membrane via targeting signals contained within and adjacent to their transmembrane helices (Kessler and Schnell, 2006). The majority of thylakoid membrane proteins contain cleavable N-terminal transit peptides that target them across the envelope. Import is mediated by the same translocon complexes within the outer (Toc) and inner (Tic) envelope membranes that mediate the import of soluble proteins (Schnell, 1998; Jarvis and Robinson, 2004). Upon import, thylakoid membrane proteins are released into the soluble stroma and processed to remove their transit peptides, and intrinsic secondary targeting signals direct them to the thylakoid (Jarvis and Robinson, 2004). The protein translocons at the thylakoid are homologous to protein export translocons found in prokaryotic cytoplasmic membranes (e.g., Sec, SRP, and TAT pathways), indicating conservation in these targeting systems from the original bacterial endosymbiont (Keegstra and Cline, 1999; Jarvis and Robinson, 2004).
The limited studies on targeting to the chloroplast inner envelope membrane (IM) leave open the question of whether nucleus-encoded proteins insert into the membrane during the import process via a stop-transfer mechanism or target to the membrane after the completion of import by inserting from the stromal side of the membrane. Two classes of nucleus-encoded integral IM proteins are known to exist. The first class contains proteins that lack cleavable transit peptides (Miras et al., 2002; Nada and Soll, 2004). Although the targeting determinants for these proteins have not been completely defined, they appear not to use the Toc–Tic translocons for import.
The second, larger class consists of IM proteins, is synthesized with cleavable transit peptides, and initially engages the Toc–Tic import machinery. The abundant inner membrane protein, Tic110, is a member of this class (Lubeck et al., 1996, 1997). The analysis of deletion mutants and fusion proteins indicates that the N-terminal region of Tic110, including its two transmembrane segments, is required for targeting to the IM (Lubeck et al., 1997). Interestingly, a fusion protein containing the N-terminal targeting determinants of Tic110 transiently accumulated as a soluble intermediate in the stroma before inserting into the IM (Lubeck et al., 1997). In a subsequent study, dominant-negative mutants of Tic110 that disrupt Tic complex formation resulted in the accumulation of normal Tic110 in the stroma in vivo (Inaba et al., 2005). Consistent with the fusion protein studies, chloroplasts isolated from these mutants transiently accumulated a soluble, mature form of Tic110 in in vitro import assays (Inaba et al., 2005). Collectively, the two studies suggested that Tic110 inserts into the membrane from the stroma after import.
In contrast to the studies with Tic110, in vitro import studies with several chloroplast polytopic membrane transporters led to the conclusion that IM proteins do not use stromal intermediates en route to the membranes (Li et al., 1992; Brink et al., 1995; Knight and Gray, 1995). In one case, soluble forms of fusion proteins to the envelope phosphate translocator were observed but were not shown to represent targeting intermediates (Knight and Gray, 1995). These results suggested that chloroplast IM proteins use a stop-transfer mechanism of targeting that results in direct insertion of the proteins into the inner membrane during import through the Toc–Tic system.
In this report, we wished to examine the process of targeting to the chloroplast IM by studying the import and insertion of a simple IM protein. To this end, we investigated the targeting of pre-atTic40, a nucleus-encoded chloroplast inner membrane protein with a single transmembrane helix (Stahl et al., 1999; Chou et al., 2003). We show that the import and membrane insertion of native pre-atTic40 involves a size intermediate that inserts into the inner membrane after import from the cytoplasm through the Toc–Tic machinery. Furthermore, we demonstrate that atTic40 and atTic110 can insert directly and selectively into isolated IM vesicles. These data are consistent with a pathway for the targeting of IM proteins that is independent of their import from the cytoplasm.
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35-kD soluble region extending into the stroma (Stahl et al., 1999; Chou et al., 2003). As a first step in examining the targeting of pre-atTic40, we performed a time course of import of in vitro–translated [35S]-labeled pre-atTic40 into isolated pea chloroplasts (Fig. 1 B, lanes 2–4). In addition to pre-atTic40, two other major forms of atTic40 are apparent in the import assays. The polypeptide with the highest mobility was confirmed to be mature atTic40 by comparing its mobility with that of endogenous atTic40 as detected by immunoblotting (unpublished data). In addition to mature atTic40, a major intermediate-sized form of the protein (int-atTic40) with a mobility between pre-atTic40 and atTic40 is observed in the import assays.
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To ensure that the lack of atTic40 and int-atTic40 proteolysis was not a result of incomplete digestion, samples from the 5-min time point in import were treated with a range of thermolysin and trypsin concentrations (Fig. 1 C). Increasing the protease concentrations had no effect on the proportions of protease-insensitive atTic40 or int-atTic40. Furthermore, the addition of Triton X-100 to the samples before protease treatment resulted in complete degradation of int-atTic40 and atTic40 (Fig. 1 E), indicating that the proteins are not intrinsically protease resistant. These data confirm that both forms are localized inside the inner membrane.
Although atTic40 and int-atTic40 accumulate inside the chloroplast with time, a portion of each population is sensitive to both proteases. The highest levels of sensitive forms are observed at early time points in import (Fig. 1 D). Their sensitivity to both proteases indicates that they are exposed at the surface of the chloroplast and therefore appear to be in the process of import into the organelle. These forms are similar to import intermediates observed with stromal preproteins that are captured within the Toc–Tic machinery (Waegemann and Soll, 1991; Olsen and Keegstra, 1992; Schnell and Blobel, 1993).
To obtain additional evidence that int-atTic40 accessed the stroma, we tested whether the stromal processing peptidase (SPP) that cleaves the transit peptides of soluble proteins (Lamppa and Abad, 1987; Abad et al., 1989) was involved in converting pre-atTic40 to the intermediate form. Analysis of the pre-atTic40 sequence identifies a potential SPP processing site after amino acid 42 of the transit peptide (Emanuelsson et al., 1999). Incubation of in vitro–translated pre-atTic40 with a stromal extract results in the conversion of 35% of the precursor to an intermediate-size form coincident in size with int-atTic40 (Fig. 1 F). The processing by SPP is likely to occur at the predicted cleavage site (residue 42) because the mobility of a truncated form of pre-atTic40 lacking the first 42 residues is identical to int-atTic40 (Fig. S1 B). Furthermore, the truncated form is not cleaved by the stromal extract (Fig. 1 F), eliminating the possibility of processing at another site. As a control, we also tested cleavage of the precursor to the triose-phosphate translocator (pre-TPT; Knight and Gray, 1995). As expected, 50% of pre-TPT was converted to its mature form in the assay (Fig. 1 F). These data provide additional evidence that int-atTic40 is exposed to the stroma before IM insertion and processing to mature atTic40.
To determine if fully imported int-atTic40 and mature atTic40 had reached the IM, we fractionated chloroplasts from 2-, 5-, and 30-min import reactions. The samples were treated with trypsin to remove external or partially imported atTic40, osmotically lysed and separated into membrane and soluble fractions. As expected, the majority of mature atTic40 (75%) partitioned with the membrane fraction at the 5-min and 30-min time points (Fig. 2 B, left).
In contrast, 88–94% of int-atTic40 was not associated with the membrane at the three time points (Fig. 2 B, right). Collectively, the data in Figs. 2 and 3 suggest that the protease-insensitive form of int-Tic40 is soluble and located in the chloroplast stroma.
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To test whether int-atTic40 was the direct precursor to atTic40, we performed a variation of the pulse-chase experiment. For this assay, isolated chloroplasts were incubated with pre-atTic40 for 5 min under import conditions to accumulate soluble int-atTic40 (Fig. 4 A). The chloroplasts were chilled to stop the import reaction and were treated with trypsin to remove pre- atTic40 and int-atTic40 that had not been completely imported into the organelle (Fig. 4 A). The treated chloroplasts were isolated to remove the protease and incubated again under import conditions for 5 or 60 min (Fig. 4 A). The soluble int-atTic40 that accumulated after 5 min of import could be quantitatively converted to membrane-integrated mature atTic40 by the additional incubation (Fig. 4 B). On the basis of these results, we conclude that the targeting of atTic40 involves a soluble intermediate that targets to the IM after import into the stroma.
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We generated a series of increasingly larger deletion mutants in the intermediate sequence (residues 43–76; Fig. 1 A) and examined their import and insertion properties. All deletions disrupt the putative processing site that gives rise to mature atTic40. Elimination of the four C-terminal residues of the transit peptide does not significantly affect import efficiency or processing to an intermediate size (Fig. 5 A). However, larger deletions dramatically reduce import efficiency in addition to inhibiting processing to the mature size (Fig. 5 A).
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58-76 appeared to target and insert into the IM, albeit with somewhat lower efficiencies than pre-atTic40 (Fig. 5 B). The effects of deletions larger than 58-76 on IM targeting could not be determined because of the combination of poor import efficiency and aberrant cleavage (Fig. 5 A), presumably because of disruption of the SPP processing site. To further ensure that the soluble forms of the deletions were productive intermediates in the targeting pathway, we performed a chase experiment similar to that shown in Fig. 4 using the 58-76 mutant (Fig. 5 C). The 58-76 soluble intermediate can be chased to the membrane with a 30-min incubation, similar to authentic int-atTic40. Collectively, these results indicate that the intermediate sequence of the transit peptide participates in import but is not essential for membrane insertion.
To further explore the determinants for pre-atTic40 targeting, we replaced its transit peptide with that of the small subunit of rubisco (psTP-Tic40). The import efficiency of this construct was comparable to pre-atTic40, but the protein was converted to mature atTic40 directly with no apparent intermediate processing (Fig. 6 A).
Remarkably, the chimera targeted to the IM, albeit with approximately one half the efficiency of pre-atTic40 (Fig. 6 B). These results are consistent with the conclusion that the int-atTic40 intermediate sequence is not required for membrane integration but might facilitate the process. Finally, we tested whether the transmembrane helix of atTic40 was required for IM targeting. As expected, deletion of the transmembrane region (TM-atTic40) had little effect on import but completely eliminated membrane integration (Fig. 6, A and B).
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95 kD) extends into the stroma (Kessler and Blobel, 1996; Jackson et al., 1998), whereas psTi22 is bound to the outer face of the IM in the intermembrane space (Kouranov et al., 1999). The majority of psTic110 is degraded by either trypsin or thermolysin, whereas psTic22 is resistant to both proteases, indicating that the isolated vesicles are predominantly inside out (Fig. 7 A i). Disruption of the vesicles with Triton X-100 resulted in complete degradation of psTic110 and psTic22 (Fig. 7 A i). To quantify the proportion of inside- out vesicles in the population, we imported radiolabeled atTic110 into chloroplasts and treated isolated IM vesicles from these chloroplasts with thermolysin (Fig. 7 A ii). Greater than 90% of the radiolabeled atTic110 was degraded within 30 min, suggesting that at least 90% of the vesicle population was in the inside-out orientation.
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As a control for vesicle targeting specificity, we tested the binding and insertion of int-atTic40 to canine pancreatic microsomal membranes. Binding to the heterologous membranes was insignificant when compared with control reactions lacking membranes (Fig. 8 A). As an additional control, we tested the protein dependence of int-atTic40 insertion into IM vesicles by treating the IM vesicles with varying concentrations of thermolysin before the insertion reaction (Fig. 8 B). The protease treatments dramatically reduced the binding and processing of int-atTic40 in IM vesicles, demonstrating the requirement of proteinaceous components at the IM for insertion.
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Finally, we examined the role of stromal factors in int-atTic40 targeting by testing whether in vitro–translated forms of atTic40 could target directly to vesicles. We compared the targeting of in vitro–translated int-atTic40, atTic40, or the TM-atTic40 mutant to stromal int-atTic40 (Fig. 9).
Remarkably, in vitro–translated int-atTic40 and atTic40 both associated with vesicles and inserted into the membranes with efficiencies only slightly lower than that observed for stromal int-atTic40 (Fig. 9, graph). Furthermore, in vitro–translated int-atTic40 was partially processed to mature atTic40. As expected, in vitro–translated mature atTic40 was not processed (Fig. 9). These data confirm that int-atTic40 processing to mature atTic40 occurs upon association with the IM. The TM-atTic40 mutant showed no significant association or insertion into the membrane vesicles (Fig. 9), confirming the selectivity of the insertion assay. Collectively, these results indicate that in vitro–translated int-atTic40 is capable of inserting into isolated IM vesicles, suggesting that specific stromal factors are not required for the targeting reaction.
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50% of the bound protein is integrated into the membrane.
As controls, we tested the binding and insertion of atTic110 lacking one (atTic110-TM1 or atTic110-TM2) or both of its transmembrane segments (atTic110Sol; Fig. 10 A). Consistent with previous in vitro import experiments, the binding and insertion of the deletion mutants was dramatically lower than for full-length atTic110. atTic110 showed considerably lower binding and no appreciable insertion into pancreatic microsomal membranes (Fig. 10 B), demonstrating the membrane specificity of the reaction. Overall, the data with atTic40 and atTic110 demonstrate that these proteins can insert directly into the IM from a soluble state independent of protein import from the cytoplasm.
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Discussion |
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Both preTic40 and preTic110 appear to initially use the general Toc–Tic pathway for import into the organelle (Lubeck et al., 1996, 1997; Stahl et al., 1999) and are processed by SPP, generating soluble, stromal intermediates. The intermediates insert into the IM from the stroma side of the membrane. Insertion is dependent on information within the transmembrane helices of the proteins (Figs. 6, 9, and 10) and requires proteinaceous components of the inner membrane (Fig. 8). Our initial studies suggest that insertion occurs independent of nucleoside triphosphate hydrolysis (Fig. 8) or the need for stromal factors (Fig. 9).
The precise role of the two-step processing of the pre-atTic40 transit peptide in targeting, if any, remains to be determined. Our data indicate that the intermediate region, spanning amino acids 43–76 of the transit peptide, participates in import but is not required for IM insertion (Fig. 5). Furthermore, the intermediate processing site does not appear to be conserved because the pea orthologue of Tic40 appears to be initially processed at a site much closer to the C-terminal end of the transit peptide (Stahl et al., 1999; unpublished data). The initial cleavage to generate int-atTic40 during import is mediated by SPP (Fig. 1). The data in Figs. 7, 8, and 9 indicate that the final processing step requires association with the inner membrane. Therefore, the processing could be performed by a specific envelope protease. Although the protease responsible for processing int-atTic40 to mature atTic40 remains to be identified, two potential processing peptidases have been identified at the envelope (Inoue et al., 2005; Bolter et al., 2006).
Although our data demonstrate that import and IM targeting are independent processes, one observation suggests that the two reactions can be coupled under some circumstances. The protease protection studies of pre-atTic40 import demonstrate that both int-atTic40 and mature atTic40 are largely exposed to the chloroplast surface at the earliest time points in import and, therefore, have not completed the import process (Fig. 1). Protease-resistant forms corresponding to fully imported int-atTic40 and mature atTic40 accumulate with time in the standard import assay (Fig. 1) and in our import pulse-chase experiment (Fig. 3), reaching their maximum levels at later time points. Collectively, these observations suggest that IM targeting can initiate while int-atTic40 is in the process of import. The fact that the intermediate accumulates in the stroma with time suggests that the rate of import of pre-atTic40 from the cytoplasm exceeds the rate of insertion into the inner membrane, resulting in the inability of the insertion reaction to keep pace with import.
Our studies provide compelling evidence that the import and membrane insertion of atTic40 and atTic110 are independent processes. However, we cannot rule out the possibility that other IM proteins (e.g., polytopic transporters) use a stop-transfer mechanism. In mitochondria, both processes appear to operate for targeting of proteins to the IM (Herrmann, 2003; Herrmann and Neupert, 2003; Herrmann and Bonnefoy, 2004; Preuss et al., 2005). Nucleus-encoded mitochondrial IM proteins appear to initially engage the general import machinery of the outer mitochondrial membrane (TOM complex; Rehling et al., 2004). A small number of nucleus-encoded mitochondrial IM proteins are inserted into the membrane from the matrix after they complete import (Herrmann, 2003; Herrmann and Neupert, 2003; Herrmann and Bonnefoy, 2004). The Oxa1 pathway mediates insertion of several of these proteins (Herrmann et al., 1997; Baumann et al., 2002; Herrmann and Bonnefoy, 2004). This pathway is a member of the Oxa1/Alb3/YidC family of protein export pathways that are conserved from prokaryotes. Oxa1 and a second pathway also mediate the insertion of mitochondria-encoded IM proteins (Herrmann, 2003; Frazier et al., 2006). However, for the majority of mitochondrial IM proteins, complete translocation into the matrix is interrupted by stop-transfer sequences, resulting in the lateral insertion of the proteins into the lipid bilayer via the Tim22 or Tim23 complexes (Herrmann and Neupert, 2003; Rehling et al., 2004). Additional studies with a wider array of chloroplast IM proteins should demonstrate whether stop-transfer mechanisms also exist in chloroplasts.
Although the pathway of Tic40 and Tic110 targeting in chloroplasts and the Oxa1 pathway in mitochondria are analogous at first examination, it is unclear whether the chloroplast proteins use a conservative targeting pathway. Genomic and proteomic studies fail to identify proteins homologous to bacterial export pathways in the chloroplast IM (Ferro et al., 2002, 2003; Froehlich et al., 2003; Jarvis, 2004; Gerdes et al., 2006). Furthermore, the conserved protein export pathways, including the Oxa1/YidC homologue, Alb3, are all present at the thylakoid membrane (Jarvis and Robinson, 2004). Therefore, the relationship of the chloroplast IM targeting translocon to the conserved translocons at the molecular level remains to be determined. Identification of the factors involved in IM targeting in the chloroplast will provide more definitive evidence for the molecular mechanism of this novel membrane protein targeting system.
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Materials and methods |
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The atTic40 deletion mutants were generated by overlap extension PCR of the atTic40 cDNA to eliminate the specific residues indicated in the figures. The psTP-Tic40 construct (pET21d-pssuTP-matTic40) corresponds to the 57 amino acids of the complete transit peptide of the pea small subunit of rubisco (Pain and Blobel, 1987), fused to mature atTic40. The recombinant int-atTic40 (pET21d-int-atTic40) and mature atTic40 (pET21d-atTic40) constructs for in vitro translation lack the N-terminal 42 and 76 amino acids of pre-atTic40, respectively. The int-atTic40 construct contains an additional Gly codon following the initiation codon to introduce a 5' Nco1 restriction site. The atTic110TM1 (pET21d-mature atTic110-TM1-His) and atTic110TM2 (pET21d-mature atTic110-TM2-His) constructs contained deletions of amino acids 43–62 and 70–89 of mature atTic110, respectively. The atTic110 and atTic110Sol constructs were previously described (Inaba et al., 2005).
Chloroplast isolation and in vitro import assays
Import substrates were generated using a coupled in vitro transcription–translation system derived from reticulocyte lysate (Promega). Chloroplast isolation from pea and A. thaliana and the in vitro import experiments were performed as previously described (Smith et al., 2002). For the time course of in vitro import, reactions were stopped at the indicated time points by rapid dilution with three volumes of ice-cold 50 mM Hepes-KOH, pH 7.5, and 330 mM Sorbitol (HS buffer). Protease treatments were performed by dividing the import reactions into three equal parts and diluting each with threefold excess ice-cold HS buffer alone or buffer containing trypsin (50 µg/ml final concentration) or thermolysin (100 µg/ml final concentration). The reactions were incubated on ice for 30 min, and proteolysis was stopped with 1 mM PMSF, 0.05 mg/ml TLCK, 0.1 mg/ml soybean trypsin inhibitor, and 2 µg/ml aprotinin (trypsin inhibitor; Jackson et al., 1998) or 10 mM EDTA (thermolysin inhibitor). The chloroplasts were isolated through 40% Percoll silica gel containing the corresponding protease inhibitor, washed once with HS buffer, and processed for SDS-PAGE analysis. All quantitative analysis of radiolabeled samples was performed with a FLA-5000 phosphorimager and Multi Gauge v. 3.0 software (Fujifilm).
Chloroplast lysis and fractionation
Chloroplasts were lysed by suspension in HS buffer to a concentration of 0.5–1 mg chlorophyll/ml and diluted with five volumes of 2 mM EDTA. The lysate was mixed vigorously and incubated on ice for 10 min. The samples were adjusted to 0.2 M NaCl, and the membrane fraction was collected by centrifugation at 18,000 g for 30 min at 4°C (Smith et al., 2002). For alkaline extraction, the membrane pellet was resuspended with a small volume of HS buffer and diluted with 20 volumes of 0.2 M Na2CO3, pH 12. The samples were homogenized with a Teflon homogenizer (Kontes Glass Co.) and incubated at room temperature for 10 min, and the membrane fraction was collected by centrifugation at 100,000 g for 15 min. The soluble fractions were removed and concentrated by precipitation in 20% trichloroacetic acid.
SPP assay
The stromal extract was prepared as previously described (Abad et al., 1989; Tranel and Keegstra, 1996). For the SPP processing assays, 1 µl of in vitro–translation product was incubated with 20 µl stromal extract at 26°C for 90 min. As a control, 1 µl of in vitro–translation product was mock treated with 20 µl of 5 mM Hepes-KOH, pH 8.0.
Pulse-chase experiments
For the pre-atTic40 binding and chase experiments in Fig. 3, isolated chloroplasts were depleted of internal ATP by incubation in the dark at room temperature for 15 min. In vitro–translated pre-atTic40 was depleted of nucleotides by gel filtration. The early import intermediate was generated by incubating chloroplasts with pre-atTic40 in the presence of 100 µM ATP under import conditions (Olsen et al., 1989; Schnell and Blobel, 1993). The chloroplasts were reisolated through 40% Percoll silica gel, washed once with ice-cold HS buffer, and resuspended in ice-cold HS buffer containing 50 mM KOAc and 4 mM MgOAc (import buffer) without ATP. A fraction was removed as the 0-min sample, and the remaining chloroplasts were diluted into 10 volumes of import buffer containing 2 mM ATP at 26°C. Equal fractions of the import reaction were removed at 2, 4, 7.5, and 30 min, reisolated, and separated into membrane and soluble fractions.
For the experiment in Fig. 4 to test int-atTic40 conversion to atTic40, chloroplasts (400 µg chlorophyll) were incubated with pre-atTic40 for 5 min under standard import conditions. The reaction was stopped, and chloroplasts were treated in the absence or presence of trypsin as described in Chloroplast isolation and in vitro import assays. After recovery through Percoll silica gel, the chloroplasts were suspended to 2 ml in prewarmed (26°C) import buffer containing 2 mM ATP. Equal fractions of the reaction mixture were collected at 5 and 60 min, reisolated, and separated into membrane and soluble fractions.
In vitro insertion of int40 into isolated IM vesicles
The chloroplast IM vesicles were isolated as described previously (Keegstra and Yousif, 1986). The membranes were recovered by differential centrifugation and stored in HS buffer containing 1 mM dithiothreitol at –80°C.
The stromal extract containing int-atTic40 was isolated using chloroplasts from a 5-min pre-atTic40 import reaction as described in SPP assay. The chloroplasts (1–2 mg chlorophyll/ml final concentration) were lysed hypertonically with 10 mM Hepes-KOH, pH 8.0, and 10 mM MgCl2 (Yuan et al., 1991) on ice for 10 min. The stromal fraction was separated from the membrane fraction by centrifugation at 40,000 g for 30 min, adjusted to 50 mM Hepes-KOH, pH 7.5, 50 mM KOAc, and 4 mM MgCl2, and stored at –80°C.
For a typical in vitro insertion assay, 15 µl of isolated stromal extract (50 µg protein) or 1 µl in vitro–translated atTic40 or atTic110 protein was incubated with 30 µg inner membrane vesicles under conditions identical to those used for standard chloroplast import assays. The vesicle insertion was performed at 26°C for 1 or 2.5 h. Then, three volumes of ice-cold HS buffer were added and the vesicles were reisolated by spinning at 100,000 g for 20 min. The vesicles were washed once with HS buffer and resuspended in 30 µl HS buffer before further treatments, such as carbonate extraction.
Online supplemental material
Fig. S1 shows that the int-atTic40 targeting intermediate is observed during import into both pea and A. thaliana chloroplasts. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200605126/DC1.
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Acknowledgments |
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This work was supported by National Science Foundation grant MCB-0090727 and National Institutes of Health grant GM-61893 to D.J. Schnell.
Submitted: 25 May 2006
Accepted: 20 September 2006
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References |
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