Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process1[OPEN] Po-Kai Huang, Po-Ting Chan 2, Pai-Hsiang Su 3, Lih-Jen Chen, and Hsou-min Li* Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan ORCID IDs: 0000-0002-0400-0556 (P.-H.S.); 0000-0002-0211-7339 (H.-m.L.).

Three stromal chaperone ATPases, cpHsc70, Hsp90C, and Hsp93, are present in the chloroplast translocon, but none has been shown to directly bind preproteins in vivo during import, so it remains unclear whether any function as a preproteintranslocating motor and whether they have different functions during the import process. Here, using protein crosslinking followed by ionic detergent solubilization, we show that Hsp93 directly binds to the transit peptides of various preproteins undergoing active import into chloroplasts. Hsp93 also binds to the mature region of a preprotein. A time course study of import, followed by coimmunoprecipitation experiments, confirmed that Hsp93 is present in the same complexes as preproteins at an early stage when preproteins are being processed to the mature size. In contrast, cpHsc70 is present in the same complexes as preproteins at both the early stage and a later stage after the transit peptide has been removed, suggesting that cpHsc70, but not Hsp93, is important in translocating processed mature proteins across the envelope.

Most chloroplast proteins are encoded by the nuclear genome as higher M r preproteins that are fully synthesized in the cytosol before being imported into the chloroplast. The import process is initiated by binding of the N-terminal transit peptide of the preprotein to the translocon at the outer envelope membrane of chloroplasts (TOC) complex, in which Toc159 and Toc34 function as receptors and Toc75 is the outer membrane channel. This step is followed by binding of the transit peptide to the translocon at the inner envelope membrane of chloroplasts (TIC) machinery, the central components of which include the Tic20/Tic56/ Tic100/Tic214 channel complex and Tic110. Tic110 functions as the stromal receptor for transit peptides and also as a scaffold for tethering other translocon components (for reviews, see Li and Chiu, 2010; Shi and Theg, 2013; Paila et al., 2015). The actual translocation of the bound preproteins across the envelope is powered 1

This work was supported by the Ministry of Science and Technology, Taiwan (grant nos. MOST 103-2321-B-001-053 and MOST 104-2321-B-001-021 to H.-m.L.) and the Academia Sinica of Taiwan (to H.-m.L.). 2 Present address: AbbVie Biopharmaceuticals GmbH, Taiwan Branch, Taipei 10478, Taiwan. 3 Present address: Academia Sinica Biotechnology Center in Southern Taiwan, Tainan 741, Taiwan. * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hsou-min Li ([email protected]). H.-m.L. and P.-H.S. designed the study; P.-K.H., P.-T.C., P.-H.S., and L.-J.C. performed experiments; H.-m.L., P.-K.H., P.-T.C., P.-H.S., and L.-J.C. analyzed the data; H.-m.L. and P.-K.H. wrote the article. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.15.01830

by hydrolysis of ATP in the stroma (Pain and Blobel, 1987; Theg et al., 1989), and it is therefore assumed that some stromal ATPase motor proteins bind the preproteins as they emerge from the inner membrane and use the energy of ATP hydrolysis to translocate the preproteins across the envelope into the stroma. Three stromal ATPases have been identified in the translocon complex: cpHsc70 (chloroplast heat shock cognate protein 70 kD), Hsp90C (chloroplast heat shock protein 90), and Hsp93/ClpC (93-kD heat shock protein). Hsp93, the first to be identified, belongs to the Hsp100 subfamily of AAA+ proteins (ATPases associated with various cellular activities) and was detected in coimmunoprecipitation experiments in complexes containing other translocon components and preproteins undergoing import (Akita et al., 1997; Nielsen et al., 1997; Chou et al., 2003; Rosano et al., 2011). In Arabidopsis (Arabidopsis thaliana), Hsp93 exists as two isoforms encoded by the genes HSP93III and HSP93V. Removal of the more abundant Hsp93V results in protein import defects, while double knockout of the two genes causes lethality (Constan et al., 2004; Kovacheva et al., 2007; Chu and Li, 2012; Lee et al., 2015). Purified recombinant Hsp93III can bind to the transit peptide of pea (Pisum sativum) ferredoxin-NADP+ reductase in vitro (Rosano et al., 2011). In addition, the N-terminal domain of Hsp93 is critical both for its in vivo functions and its association with chloroplast membranes and Tic110, suggesting that one of the major functions of Hsp93 requires it to be localized at the envelope with Tic110 (Chu and Li, 2012). However, because many prokaryotic Hsp100 family proteins function as the regulatory components of the Clp proteases (Kress et al., 2009; Nishimura and van Wijk, 2015), and, in Arabidopsis, some Clp proteolytic core components have also been found at the envelope fraction, it has

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been proposed that Hsp93 is involved in degradation of misfolded or damaged proteins at the envelope (Sjögren et al., 2014). However, whether the Clp proteolytic core can form a stable complex with Hsp93 in higher plant chloroplasts remains to be shown. In mitochondria and the endoplasmic reticulum, protein import is driven by the Hsp70 family of proteins. In chloroplasts, accumulating evidence also supports that Hsp70 is important for chloroplast protein import. Purified recombinant Hsp70 can bind in vitro to the transit peptide of the small subunit of RuBP carboxylase preprotein (prRBCS; Ivey et al., 2000). Stromal Hsp70 can be coimmunoprecipitated with preproteins undergoing import and with other translocon components, and mutations resulting in reduced or altered stromal Hsp70 activity cause protein import defects (Shi and Theg, 2010; Su and Li, 2010). Recently, it has been shown, in moss, that increasing the Km for Hsp70 ATP hydrolysis results in an increased Km for ATP usage in chloroplast protein import, indicating that stromal Hsp70 is indeed one of the proteins supplying ATP-derived energy to power import (Liu et al., 2014). Finally, stromal Hsp90C has been shown to be part of active translocon complexes in coimmunoprecipitation experiments (Inoue et al., 2013). As further evidence that Hsp90 is important for protein import into chloroplasts, the Hsp90 ATPase activity inhibitor radicicol reversibly inhibits the import of preproteins into chloroplasts (Inoue et al., 2013). Presence of the three ATPases in the translocon was demonstrated by coimmunoprecipitation after solubilization of chloroplast membranes under conditions that preserve the large membrane protein complexes, either by solubilization with nonionic detergents or by treating chloroplasts with crosslinkers that link all proteins in a complex together (Akita et al., 1997; Nielsen et al., 1997; Shi and Theg, 2010; Su and Li, 2010; Inoue et al., 2013). These complexes contain translocon components that directly bind to preproteins, and also other proteins that are associated with these translocon components but have no direct contacts with the preproteins. For example, Nielsen et al. (1997) demonstrated the presence of Hsp93 in the translocon by binding of prRBCS to isolated pea chloroplasts and then solubilization of chloroplast membranes with the nonionic detergent decylmaltoside. Under these conditions, an anti-Hsp93 antibody specifically immunoprecipitated Hsp93 together with Toc159, Toc75, Toc34, Tic110, and prRBCS (Nielsen et al., 1997). The result showed that Hsp93 is in the same complexes with these proteins but did not provide information whether Hsp93 directly binds to them. It is possible that Hsp93 only has direct contacts with, for example, Tic110, which then binds to prRBCS. Direct binding, in particular to the transit peptide region, would provide strong evidence that an ATPase functions as a protein translocating motor, rather than in assisting the assembly of other translocon components or in the folding or degradation of imported proteins. Furthermore, if all three ATPases were found to be involved in preprotein translocation, it would be 858

important to understand how they work together; for example, whether they preferentially bind different preproteins, bind to different regions of a preprotein, or act at different stages of the import process. Here, we examined whether Hsp93 can directly bind to preproteins undergoing import into chloroplasts, and compared the timing of the binding of Hsp93 and cpHsc70 to the preproteins. We used isolated pea chloroplasts, rather than isolated Arabidopsis chloroplasts, because pea chloroplasts exhibit more robust import ability (Fitzpatrick and Keegstra, 2001). Various crosslinkers that react with cysteines were then used to achieve more specific crosslinkings, followed by solubilization with the ionic detergent lithium dodecyl sulfate (LDS) to thoroughly solubilize chloroplast membranes and to disrupt noncovalent protein-protein interactions. Our results show that Hsp93 directly binds to preproteins undergoing import. Import time course experiments further revealed that Hsp93 functions primarily during the early stage of import, whereas cpHsc70 associates with substrates being imported at both the early stage and a later stage after transit peptide removal. RESULTS Hsp93 Binds to Preproteins Undergoing Import

To determine whether Hsp93 directly binds to preproteins undergoing import, we incubated the [35S]Met-labeled prRBCS (Fig. 1A, top) with isolated pea chloroplasts under low ATP conditions to allow binding, reisolated intact chloroplasts, and performed a chase for 2 to 4 min with 3 mM ATP to induce translocation into the stroma, then treated the chloroplasts with various crosslinkers and solubilized the membranes using the ionic detergent LDS. A similar approach has been taken by Akita et al. (1997). After binding of prRBCS to chloroplasts and crosslinking, they also solubilized chloroplasts with LDS, but they used homobifunctional crosslinkers that react with primary amines. Due to the high abundance of Lys residues on protein surfaces, these crosslinkers are expected to covalently crosslink all proteins in a complex together. Indeed, under these conditions, the anti-Toc75 antibody specifically precipitated a 600-kD complex containing prRBCS, Toc75, Toc159, Toc34, Tic110, and Hsp93 (Akita et al., 1997). This result demonstrated that Hsp93 is a member of this 600-kD complex but did not provide information on whether Hsp93 directly binds to prRBCS. To achieve a more specific crosslinking, we first tested the crosslinker succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), which is membrane permeable and heterobifunctional, with one end reactive with cysteines and the other with primary amines (Fig. 1B). After crosslinking, the chloroplasts were lysed and the thylakoid and envelope membrane fractions collected and solubilized with 1% LDS to disrupt noncovalent protein-protein interactions, then, after clarification by centrifugation, the supernatant Plant Physiol. Vol. 170, 2016

Hsp93 Binds Transit Peptides During Import

Figure 1. A, Schematic representation of the constructs used. The red, blue, and green lines represent, respectively, the transit peptide, the mature region, and the 3xFLAG-His6 tag. The numbers above each construct indicate the positions of Cys residues, with the first residue of the mature protein designated +1 and residues in the transit peptide designated with negative numbers. All constructs are drawn to scale. B, Summary of the crosslinkers used in this study.

(Fig. 2A, lane 1) was used for immunoprecipitation. As a positive control, we first performed immunoprecipitation using antiserum against Toc75, the proteintranslocating channel of the outer membrane, which precipitated two of the major crosslinked products with approximate molecular masses of 95 kD and 135 kD (Fig. 2A, lane 2, black circles), suggesting that the former is an adduct of one Toc75 molecule (75 kD) with one prRBCS molecule (20 kD), whereas the 135-kD complex could potentially contain either one Toc75, one prRBCS, and another protein with a molecular mass of about 40 kD, or one Toc75 and several prRBCS molecules. When the same solubilized membranes were Plant Physiol. Vol. 170, 2016

immunoprecipitated with anti-Hsp93 antiserum, a band slightly larger than 110 kD was observed (Fig. 2A, lane 4); precipitation was shown to be specific as no band was seen using preimmune serum from the rabbit used to raise the anti-Hsp93 antiserum (Fig. 2A, lane 3). Its size corresponded to approximately one Hsp93 crosslinked to one prRBCS, suggesting that a Lys or Cys residue in Hsp93 was in close proximity to prRBCS during translocation. The crosslinker SMCC is noncleavable, so the crosslinked substrates cannot be separated from Hsp93. Due to the small size difference between prRBCS and mature RBCS, it was difficult to know whether the crosslinked products consisted of only Hsp93-prRBCS or were mixtures of Hsp93-prRBCS and Hsp93-RBCS. To investigate whether Hsp93 was still directly associated with the mature form of the preprotein after transit peptide cleavage, we repeated the crosslinking assays using another membrane permeable crosslinker, succinimidyl 6-[3(2-pyridyldithio)propionamido]hexanoate (LC-SPDP), which, like SMCC, is heterobifunctional and crosslinks Cys and primary amine residues (Fig. 1B), but can be cleaved by a reducing agent, such as dithiothreitol (DTT). When binding-chase reaction mixtures containing prRBCS were treated with LC-SPDP, the membranes solubilized with LDS, and the immunoprecipitates analyzed by SDS-PAGE using nonreducing sample buffer, the major crosslinked complexes (Fig. 2B, lane 1) were indeed similar to those produced using SMCC (Fig. 2A, lane 1). The anti-Toc75 antiserum again precipitated two of the major complexes (Fig. 2B, lane 3), although their molecular masses appeared slightly smaller due to compression by intact IgG molecules migrating immediately above them (Fig. 2B, asterisks). Cleaving the immunoprecipitates by including DTT in the SDS-PAGE sample buffer revealed that Toc75 was only crosslinked to prRBCS (lane 4). When the solubilized membranes were precipitated with anti-Hsp93 antiserum, again a 110-kD product similar to that seen with SMCC was precipitated when nonreducing SDS buffer was used (lane 6), but, when the Hsp93-containing immunoprecipitates were cleaved using DTT-containing SDS-PAGE sample buffer, both prRBCS and mature RBCS were observed (lane 7); again, the preimmune serum did not precipitate any protein (lane 5). This result shows that Hsp93 was crosslinked to both prRBCS and processed RBCS during import, suggesting that Hsp93 binds to prRBCS when prRBCS is being processed to mature RBCS. This result with the cleavable crosslinker LC-SPDP also confirmed that the high molecular weight crosslinked adduct indeed contained the import substrate prRBCS. To narrow down the crosslinked region, we used the membrane-permeable homobifunctional Cys-Cys crosslinker 1,6-bis-maleimidohexane (BMH; Fig. 1B) and also created two prRBCS variants as substrates. Our initial prRBCS construct had a Cys as the last residue of the transit peptide, i.e. at 21, and two other cysteines at residues 41 and 112 in the mature region (Fig. 1A). We first replaced the Cys in the transit peptide 859

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Figure 2. Crosslinking of Hsp93 to prRBCS during import. A, Chloroplasts were incubated with [35S]Met-labeled prRBCS in the presence of 50 mM ATP to allow binding, then bound prRBCS was chased into the chloroplasts with 3 mM ATP for 4 min. The chloroplasts were then reisolated and treated with the crosslinker SMCC, then the total membrane fraction was isolated and solubilized with 1% LDS. The clarified supernatant (Input) was then immunoprecipitated with the antiserum against Toc75 or Hsp93 or the Hsp93 preimmune serum and protein A beads, and the immunoprecipitates eluted using reducing SDS sample buffer and analyzed by SDS-PAGE and fluorography. B, Binding-chase experiments were performed as described in A, except LC-SPDP, a DTT cleavable crosslinker, was used. The plus and minus signs (+ and 2) indicate whether DTT was included in the sample buffer. The asterisks indicate the position of intact IgG molecules. C and D, The experiments were performed as in A, but using BMH as crosslinker and prRBCS(21C/A) or prRBCS(252S/C) as import substrate in C or D, respectively, and with a chase of 2 min.

with an Ala, creating the construct prRBCS(21C/A), which only contained cysteines in the mature region (Fig. 1A). When this mutant was used in binding-chase studies, followed by crosslinking with BMH, solubilization with LDS, and immunoprecipitation, a 95-kD crosslinked product was precipitated by the anti-Toc75 antiserum and an approximately 110-kD crosslinked product was precipitated by the anti-Hsp93 antiserum (Fig. 2C, lanes 2 and 4). Since prRBCS(21C/A) only contained Cys residues in the mature region, this shows that both Toc75 and Hsp93 contact the mature region of prRBCS during import. To determine whether Hsp93 also binds to the transit peptide of preproteins undergoing import, we performed the same BMH crosslinking experiment using another construct, prRBCS(252S/C), in which all three endogenous cysteines were replaced with alanines and the Ser at residue 252, close to the N terminus of the transit peptide, was mutated to a Cys (Fig. 1A). Interestingly, the BMH crosslinking pattern of prRBCS(252S/C) was very different from that of 860

prRBCS(21C/A) (compare lanes 1 in Fig. 2, C and D), suggesting that residue 252 and the mature region of prRBCS form contacts with different proteins during the initial translocation stage. When the anti-Toc75 antiserum was used for immunoprecipitation, very little crosslinked product was precipitated (Fig. 2D, lane 2), suggesting that, in most of the bound prRBCS (252S/C) molecules, residue 252 was either already outside the Toc75 channel or not in close proximity to any Cys in Toc75. When the anti-Hsp93 antiserum was used, a complex with a size slightly larger than 110 kD was again precipitated (Fig. 2D, lane 4), indicating that Hsp93 was in close proximity to the N terminus of the transit peptide during preprotein translocation. Hsp93 Directly Binds to the Transit Peptide During Preprotein Import

In the experiments described above, we used molecular mass as evidence to infer that the 110-kD Plant Physiol. Vol. 170, 2016

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crosslinked product contained one Hsp93 molecule and one prRBCS molecule. However, we could not exclude the possibility that the 110-kD complex was actually one Hsp93 crosslinked to a small protein, which itself was crosslinked to prRBCS, i.e. Hsp93 had no direct contact with prRBCS. We therefore used another crosslinker, 4-(N-maleimido)benzophenone (MBP; Fig. 1B), which can be preconjugated to the Cys residues of the preprotein, then crosslinking to any residue in close proximity can be induced by exposure to UV light. As import substrate, we used a variant of the 58-kD recombinant preprotein pre-atTic40-3xFLAG (Inoue et al., 2013), which is composed of Arabidopsis Tic40 preprotein, with three cysteines in the transit peptide (residues 268, 257, and 234) and one in the mature region (residue 415), followed by three FLAG tags and one His6 tag. This recombinant preprotein has high import efficiency (Li and Schnell, 2006; Inoue et al., 2010, 2013), and the presence of three cysteines in the transit peptide should also increase the probability of detecting proteins that bind to the transit peptide. We mutated the Cys in the mature region to Ser and created the construct pre-atTic40-3xFLAG(415C/S) (Fig. 1A). The nonradiolabeled recombinant protein was purified from Escherichia coli, conjugated to MBP, and used in import experiments. Because MBP is preconjugated to pre-atTic40-3xFLAG(415C/S), only proteins directly within reach of the Cys-MBP arms in the transit peptide can be crosslinked to it. To confirm the feasibility of this approach, we first tested whether the MBP-conjugated pre-atTic40-3xFLAG(415C/S) could be crosslinked to Toc75 and Toc159. MBP-conjugated pre-atTic403xFLAG(415C/S) was incubated with chloroplasts under import conditions for 1 min, then the crosslinking reaction was activated by exposure to UV light, total membranes were solubilized with LDS, immunoprecipitation was performed with anti-FLAG antibody or anti-Toc75 or Toc159 antiserum, and pre-atTic403xFLAG(415C/S) in the immunoprecipitates was detected by immunoblotting with anti-FLAG antibody. As shown in Figure 3A, when chloroplasts were exposed to UV light during the import of pre-atTic403xFLAG(415C/S), several high Mr complexes recognized by anti-FLAG antibody were observed in the solubilized membrane preparation (lane 2) and were immunoprecipitated by anti-FLAG antibody (lane 4). The anti-Toc75 antiserum precipitated two crosslinked complexes in the 130- to 160-kD region and one in the 200-kD region (lane 6, black circles). Based on the molecular masses, we suggest that the former contains one Toc75 molecule and one pre-atTic40-3xFLAG(415C/S) molecule and that the different sizes may represent different conformations resulting from crosslinking of pre-atTic40-3xFLAG(415C/S) to different regions of Toc75, while the 200-kD band might contain one Toc75 molecule and two pre-atTic40-3xFLAG(415C/S) molecules. The anti-Toc159 antiserum precipitated a single FLAG-containing complex with a molecular mass of about 160 kD (lane 8), slightly larger than the two 130- to 160-kD Toc75 complexes, which are probably Plant Physiol. Vol. 170, 2016

Figure 3. Hsp93 directly binds to transit peptides of preproteins undergoing import. A, MBP-conjugated pre-atTic40-3xFLAG(415C/S) was imported into isolated chloroplasts for 1 min, then the chloroplasts were reisolated and half of them were exposed to UV to activate MBP crosslinking while the other half were kept in the dark. The total membrane fraction from each was then isolated and solubilized with 1% LDS. The clarified supernatant of the solubilized membranes (Input) was immunoprecipitated with antibody against the FLAG tag or antiToc75 or anti-Toc159 antiserum and the immunoprecipitates eluted using reducing SDS sample buffer and analyzed by SDS-PAGE followed by immunoblotting with the anti-FLAG antibody. B, The same MBP crosslinking experiment was performed, but using antiserum against cpHsc70 or Hsp93 for immunoprecipitation or the preimmune serum from the rabbit used to raise the anti-Hsp93 antiserum as a control. The immunoprecipitates were eluted using reducing SDS sample buffer and analyzed by SDS-PAGE followed by immunoblotting with the antiFLAG antibody.

composed of one pre-atTic40-3xFLAG(415C/S) molecule and one molecule of Toc86, the major degraded form of Toc159 (Bölter et al., 1998). We then performed the same experiment using anti-cpHsc70 or anti-Hsp93 antiserum and the preimmune serum from the rabbit used to raise the anti-Hsp93 antiserum. As shown in Figure 3B, no crosslinked products were precipitated by the anti-cpHsc70 antiserum (lane 4), but two crosslinked products were precipitated by the Hsp93 antiserum and recognized by anti-FLAG antibody (lane 8), whereas no bands were seen when the preimmune serum was used (lane 6). The sizes of the Hsp93 antiserum 861

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precipitated products were slightly larger than the adduct between pre-atTic40-3xFLAG(415C/S) and Toc86, suggesting that they comprise one pre-atTic40-3xFLAG (415C/S) molecule and one Hsp93 molecule. The two different sizes may represent different conformations resulting from crosslinking of pre-atTic40-3xFLAG (415C/S) to different regions of Hsp93, similar to the result observed for crosslinking to Toc75 (Fig. 3A, lane 6). These results suggest that Hsp93 directly binds to the transit peptide of pre-atTic40-3xFLAG(415C/S) during import. Hsp93 and cpHsc70 Associate With Preproteins at Overlapping, But Distinct, Stages of Import

We next performed import time course experiments followed by coimmunoprecipitation to investigate the stages at which Hsp93 and cpHsc70 associate with the preproteins being imported. Chloroplasts with bound [35S]Met-labeled prRBCS produced under low ATP conditions were chased with 3 mM ATP for 0 to 24 min, treated with 1 mM dithiobis(succinimidylpropionate) (DSP), which crosslinks primary amines with primary amines (Fig. 1B), to stabilize protein complexes, and lysed. Total membranes were then solubilized with 1% decylmaltoside, a nonionic detergent that preserves translocon complexes (Nielsen et al., 1997; Su and Li, 2010), and immunoprecipitated with antiserum against Toc75, Tic110, Hsp93, or cpHsc70. As shown in Figure 4A, bound preproteins were mostly in the precursor form from 0 to 2 min, were gradually converted to the mature form between 4 to 8 min, and were all in the mature form after 8 min (Fig. 4A, Input). Toc75 coimmunoprecipitated with preproteins at a very early stage (0 to 4 min) and almost only with the precursor form. Tic110 and Hsp93 coimmunoprecipitated with both the precursor and mature forms from 0 to 4 min, and their association with the substrates being imported tapered off after 8 min. This result agrees with the result of LC-SPDP crosslinking (Fig. 2B) showing that Hsp93 is associated with preproteins when preproteins are being processed to the mature size. In contrast, although

Figure 4. Hsp93 and cpHsc70 associate with preproteins at overlapping, but distinct, stages during import. A, [35S]Met-labeled prRBCS was bound to chloroplasts in the presence of 0.1 mM ATP on ice for 5 min, then intact chloroplasts were reisolated and chased with 3 mM ATP 862

for the indicated time, when a portion of the chase reaction mixture was removed and import terminated. Reisolated chloroplasts were treated with 1 mM DSP to stabilize protein complexes and lysed by freeze-thaw cycles. The total membrane fraction was isolated and solubilized with 1% decylmatoside (Input) and immunoprecipitated with antiserum against Toc75, Tic110, Hsp93, or cpHsc70, and the immunoprecipitates eluted using reducing SDS sample buffer and analyzed by SDSPAGE and fluorography. p, Precursor form; m, mature form. B, Import time course assays were performed as described above, except that [35S] Met-labeled prOE33 was used as the import substrate. p, Precursor form; i, intermediate form; m, mature form. C, Quantification of the 4 and 8 min results shown above for each antiserum. The ratios of mature to precursor (for prRBCS shown in A) or intermediate to precursor (for prOE33 in B) are shown. Result of anti-Toc75 in prOE33 import was not quantified because no intermediate was observed. Plant Physiol. Vol. 170, 2016

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cpHsc70 coimmunoprecipitated with a similar ratio of precursor to mature proteins as Tic110 and Hsp93 at 2 min after the chase, by 4 min it was mainly associated with mature RBCS and this continued to the end of the study (24 min after chase), with the highest association being observed at about 12 min. Quantification of the mature-to-precursor ratio at 4 and 8 min for immunoprecipitates from each antiserum (Fig. 4C, left) shows that cpHsc70 already associated with a higher ratio of mature RBCS than the other three translocon components at 4 min. By 8 min, a further and sharp increase in the ratio was observed for cpHsc70. These results suggest that, in the later stage of import, mature RBCS is preferentially associated with cpHsc70. It was possible that the association of cpHsc70 with mature RBCS at later time points was not related to the actual translocation step but to its assisting the folding of mature RBCS after import, or that the anti-cpHsc70 antiserum nonspecifically precipitated mature RBCS from the total solubilized membranes. We therefore repeated the experiments with another substrate to verify the specificity and also chose a substrate that should not be folded in the stroma, that of the 33-kD subunit of the oxygen-evolving complex (OE33). Preprotein of OE33 (prOE33) is processed to an intermediate size in the stroma, and processing to its mature size and folding occurs in the thylakoid lumen (Robinson et al., 2001). After the same binding-chase experiments, the solubilized membranes were immunoprecipitated with anti-Toc75, anti-Hsp93, or anticpHsc70 antiserum. As shown in Figure 4B, Toc75 was again associated with the precursor form of prOE33. Hsp93 and cpHsc70 were associated with both the precursor form and the intermediate form (p and i forms, respectively, in Fig. 4B), but cpHsc70 had a greater preference for the intermediate form than Hsp93. Quantification of the intermediate-to-precursor

ratio at 4 and 8 min for immunoprecipitates from the anti-Hsp93 and anti-cpHsc70 antisera (Fig. 4C, right) shows that at 4 min cpHsc70 and Hsp93 associated with a similar ratio of intermediate to precursor, but by 8 min the ratio for cpHsc70 was 2-fold of that for Hsp93. These results again support the idea that cpHsc70 plays an important role during the later stage of import after the removal of the transit peptide. Moreover, while mature OE33 was the major component in the total solubilized membranes after 12 min (m form in Fig. 4B, Input) and the intermediate was a very minor component, both Hsp93 and cpHsc70 showed almost no association with mature OE33. These data support the idea that Hsp93 and cpHsc70 specifically associate with substrates undergoing import at the envelope.

DISCUSSION

To function as a motor for chloroplast protein import, an ATPase should have direct contact with the substrate being imported. We used various Cys crosslinkers and showed, for the first time to our knowledge, that Hsp93 directly bound to both the transit peptide and the mature region of a preprotein during the early stage of import. Direct early binding, in particular to the transit peptide region, provides strong support for the idea that Hsp93 functions in protein translocation, rather than in the folding or turnover of imported proteins. Furthermore, we observed that Hsp93 bound to both the precursor and the processed mature form, suggesting that transit peptide processing may take place while preproteins are bound by Hsp93. The nature of the in vivo association of cpHsc70 with preproteins requires further investigation. We did not detect crosslinking between cpHsc70 and preproteins using any of the tested crosslinkers and under the same

Figure 5. Hypothetical model describing the roles of Hsp93 and cpHsc70 during preprotein import. The model depicts four import stages, and the red and blue lines represent the transit peptide and the mature region, respectively. I, A preprotein only interacts with Toc75 at the very early stage of import. II, After the transit peptide passes through the outer and inner membranes, Hsp93 directly binds to it. III, Hsp93 still binds to the mature region when the transit peptide is cleaved. IV, cpHsc70 is responsible for translocating the processed mature protein across the envelope.

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conditions that gave rise to Hsp93 crosslinking. Other crosslinkers of different arm lengths may need to be tested or the association may need to be stabilized, e.g. by increasing the ADP concentration in the reaction mixture to prolong the binding of cpHsc70 to preproteins. Based on the additive import defects of the cphsc70-1 hsp93V double mutant, we have previously suggested that Hsp93 and cpHsc70 may function in parallel as independent motors (Su and Li, 2010). Recently, it was further shown, for moss chloroplasts, that increasing the Km for ATP hydrolysis by Hsp70 translates into an increased Km for ATP usage for chloroplast protein import (Liu et al., 2014), showing that preproteins are required to pass through an Hsp70-dependent step. We now propose a more detailed model (Fig. 5) that combines our current data, the data on moss Hsp70 (Liu et al., 2014), and our previous genetic phenotype analyses (Su and Li, 2010). We have shown here that Hsp93 directly binds to transit peptides during preprotein import. Since it has also been suggested that Hsp70 can bind to the N terminus of transit peptides (Chotewutmontri et al., 2012; Chotewutmontri and Bruce, 2015), cpHsc70 may also contribute to transit peptide binding. These two ATPases may prefer different residues/motifs in the transit peptide or provide different modes of translocation force, and, therefore, the double mutant would exhibit additive import defects. An additive import defect would also be observed if Hsp93 is the primary motor for the transit peptide and cpHsc70 the primary motor for the mature region. Processing of transit peptides most likely takes place while the preproteins are bound to Hsp93, and, therefore, binding of Hsp93 to the processed mature proteins would also be detected. Once the transit peptide is cleaved, cpHsc70 may be entirely responsible for the remaining import process; this would agree with the Km study in moss, which showed that there is a major ATPconsuming step that can only be performed by Hsp70 and not by other chaperones (Liu et al., 2014). Since the mature region usually constitutes the bulk of a preprotein, translocation of the mature region by the stromal Hsp70 would be the primary determinant of ATP demand for the import reaction. Although we showed in this study that Hsp93 and cpHsc70 were associated with preproteins at different stages of the import process, it is still not clear why chloroplasts require several ATPases. It has been shown that chloroplasts can import tightly folded preproteins (Guéra et al., 1993; Clark and Theg, 1997), and it has been suggested that the chloroplast translocon can actively unfold bound preproteins during import (Guéra et al., 1993; Ruprecht et al., 2010). It is possible that the active pulling force exerted by an AAA-type motor (White and Lauring, 2007), such as Hsp93, can provide the initial unfolding force, and then proteins, such as cpHsc70 and Hsp90C, can take over to complete the translocation process. In bacteria, ClpA and ClpX help feed substrates into the ClpP proteolytic core for degradation in the cytosol (Baker and Sauer, 2006). In chloroplasts, ClpPR was 864

originally found to be mostly in the stroma (Peltier et al., 2004), while Hsp93 resides both in the envelope and the stroma (Moore and Keegstra, 1993; Akita et al., 1997; Nielsen et al., 1997; Chu and Li, 2012), so the envelope-localized Hsp93 most likely has a function independent of ClpPR. However, recently in Arabidopsis chloroplasts, some ClpPR core complexes have been detected in the envelope (Sjögren et al., 2014). Additionally, a Hsp93V-I772E (Ile at residue 772 changed to Glu) construct was recently used to complement the Arabidopsis hsp93v mutants (Flores-Perez et al., 2015). Similar residue changes in bacteria resulted in impaired interaction of ClpC or ClpX with ClpP. The Arabidopsis Hsp93V-I772E mutation did not affect binding of Hsp93 with Tic110 and the plants still showed reduced degradation of a presumed substrate for the ClpPR protease. It was therefore suggested that the data agree more with their proposal that Hsp93, together with envelope-localized ClpPR, may have some novel functions; for example, during the folding of newly imported proteins, if misfolding is detected, Hsp93 and ClpPR would be responsible for degradation of the damaged proteins (Sjögren et al., 2014). However, the Hsp93V-I772E complemented plants still showed severe protein import defect like the original hsp93v mutants (Flores-Perez et al., 2015). Therefore, whether the I772E mutation affects Hsp93 ATPase activity or oligomerization, which would then affect all functions of Hsp93, needs to be investigated. Also, whether the envelope ClpPR core complex is associated with Hsp93 or with the TIC machinery remains to be elucidated. Our data showed direct crosslinking of Hsp93 to transit peptides, including the N terminus of the transit peptide, during the early stage of import. Our import time course experiments further showed there is at least one more step after the action of Hsp93 that is carried out by cpHsc70. We suggest that at least some Hsp93 molecules in the envelope function in pulling the transit peptide and the mature region across the envelope membranes during the early stage of import. MATERIALS AND METHODS Plant Growth, Chloroplast Isolation, and in Vitro Translation of Preproteins Pea (Pisum sativum ‘Green Arrow’) seeds were imbibed overnight and grown on vermiculite for 7 to 10 d under a 12-h photoperiod at 20°C with a light intensity of approximately 150 mmol m22 s21. Pea seedlings were harvested and chloroplasts isolated as described previously (Perry et al., 1991). [35S]Metlabeled preproteins were in vitro transcribed/translated using the TNT Coupled Wheat Germ Extract System and SP6 RNA polymerase (Promega). The preproteins used were prRBCS (Lubben and Keegstra, 1986) and Arabidopsis (Arabidopsis thaliana) prOE33 (At5g66570).

Crosslinking Assays Using SMCC, LC-SPDP, or BMH, and Immunoprecipitation [35S]Met-labeled preproteins were bound to chloroplasts in the presence of 50 mM ATP on ice for 5 min, then the reaction mixture was loaded onto a 40% Percoll cushion and centrifuged at 2,900g for 6 min at 4°C to recover intact Plant Physiol. Vol. 170, 2016

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chloroplasts. After washing the pellet with import buffer (IB; 50 mM HEPESKOH, pH 8.0, and 0.33 M sorbitol), the chloroplasts were chased by 3 mM ATP in IB for 2 or 4 min at room temperature, then the reaction was stopped by diluting the reaction mixture with cold IB and removing the supernatant after centrifugation at 1,500g for 3 min at 4°C. The pellet was resuspended in IB, and a crosslinker (BMH, SMCC, or LC-SPDP; Thermo Scientific) was added to a final concentration of 0.5 mM and the mixture incubated at 4°C for 30 min. The optimal chase time and crosslinker concentration were determined by pilot experiments. Crosslinking reactions were terminated by adding Cys (for LC-SPDP) or DTT (for SMCC and BMH) to a final concentration of 50 mM, and the samples incubated at 4°C for 15 min. The chloroplasts were lysed by three freeze-thaw cycles. All subsequent steps were performed at 4°C. To collect the total membrane fraction, the lysate was centrifuged at 3,000g for 10 min to collect the thylakoid membranes, and the supernatant was further centrifuged at 100,000g for 45 min to obtain the crude envelope membranes. The two membrane fractions were combined and solubilized using LDS co-IP buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, and 1% LDS) for 30 min at 4°C, then the solution was clarified by centrifugation at 100,000g for 5 min and the supernatant collected and used in immunoprecipitation studies. For immunoprecipitation, the solubilized membranes were diluted with 9 volumes of co-IP buffer without LDS, then antiserum or antibody was added and the mixture incubated overnight, and then antibodies were precipitated using protein A-agarose beads (Pierce). The beads were washed four times with co-IP buffer containing 0.1% LDS and bound proteins eluted using 23 SDSPAGE sample buffer containing 126 mM DTT in all cases except those indicated for the LC-SPDP crosslinking products. Samples were analyzed by SDS-PAGE (NuPAGE 4–12% gradient gel system; Invitrogen). The antisera against pea Toc75, Arabidopsis Toc159 M domain (both described in Tu et al., 2004), and pea Hsp93 (Chou et al., 2006) were produced in-house in rabbits, while the anti-FLAG tag antibody (mouse monoclonal M2, F3165) was purchased from Sigma and the rabbit anti-cpHsc70 antiserum (AS08 348) from Agrisera.

Purification and MBP Conjugation of Pre-atTic40-3xFLAG (415C/S), Crosslinking Assays Using the MBP-Conjugated Preprotein, Immunoprecipitation, and Immunoblotting Using Anti-Flag Antibody The pre-atTic40-3xFLAG construct has been described previously (Inoue et al., 2013). The only Cys in the mature region was mutated to Ser by site-directed mutagenesis, creating the construct pre-atTic40-3xFLAG(415C/S) in the plasmid pET21d, and the resultant plasmid was transformed into the Escherichia coli BL21 (DE3) pLysS strain and protein expression induced using 1 mM IPTG. After incubation at 37°C for 3 h, the bacteria were harvested and lysed using an M-110P Microfluidizer (Microfluidics). The target recombinant protein was mainly in inclusion bodies, so, after centrifugation at 12,000g for 10 min, the pellet was collected and washed sequentially twice with washing buffer (20 mM Tris-HCl, pH 7.9, and 500 mM NaCl), once with washing buffer containing 0.5% Triton X-100, once with solubilization buffer (S buffer; 25 mM HEPES-KOH, pH 7.5, 50 mM KCl, 2 mM MgCl2), and twice with S buffer containing 4 M urea. Finally, the pellet was solubilized in S buffer containing 8 M urea (S-8M) and the solution centrifuged at 20,000g for 5 min at 4°C, and the supernatant collected and concentrated using Amicon Ultra-15 centrifugal filter devices. Purified pre-atTic40-3xFLAG(415C/S) was concentrated to 0.1 mM in S-8M buffer. MBP (Sigma) conjugation was performed as described previously (Akita and Inoue, 2009), then the MBP-conjugated pre-atTic40-3xFLAG(415C/S) was precipitated using trichloroacetic acid and the pellet dissolved in S-8M buffer. For immunoprecipitation studies, isolated pea chloroplasts were incubated with 100 nM MBP-conjugated preprotein in the presence of 2.4 mM ATP and 160 mM urea at room temperature in the dark for 1 min, then the reaction was stopped by adding cold IB. After resuspending the reisolated chloroplasts in IB, the sample was divided into two equal aliquots, one of which was exposed to 312 nm UV for 5 min on ice at a distance of 2 cm to initiate the crosslinking reaction, while the other was kept in the dark on ice as a negative control. Crosslinking was followed by chloroplast lysis, membrane solubilization with the LDS co-IP buffer, and immunoprecipitation as described above. Immunoprecipitation was performed using anti-FLAG agarose beads (Sigma A2220) or various antisera followed by protein A agarose beads. The immunoprecipitated proteins were eluted using 23 SDS-PAGE sample buffer containing 126 mM DTT and analyzed by SDS-PAGE (NuPAGE 4–12% gradient gel system; Invitrogen), followed by immunoblotting with antibodies against the FLAG tag (Sigma F3165) and alkaline phosphatase-coupled goat antimouse IgG antibodies (Jackson ImmunoResearch) and detection using the NBT and 5-bromo-4chloro-3-indolyl phosphate colorimetric system. Plant Physiol. Vol. 170, 2016

Import Time Course Assays Using DSP and Coimmunoprecipitation Isolated pea chloroplasts were incubated with [35S]Met-labeled preprotein and 0.1 mM ATP on ice for 5 min to accumulate bound preproteins, then the chloroplasts were reisolated by loading onto a 40% Percoll cushion and centrifugation at 2,900g for 6 min at 4°C. The chloroplasts were washed and resuspended in IB and chased with 3 mM ATP in IB for 0 to 24 min at room temperature, then the chase was stopped by dilution with ice-cold IB. Crosslinking reactions were initiated by adding DSP (Pierce) to a final concentration of 1 mM and incubation for 15 min in the dark at 4°C, then the reaction was terminated by addition of Gly to a final concentration of 100 mM and incubation on ice for 15 min to quench the free DSP. After washing the chloroplasts with IB, they were resuspended and lysed by three freeze-thaw cycles. The lysate was centrifuged at 3,000g for 10 min at 4°C to collect the thylakoid membrane fraction, then the supernatant was centrifuged at 100,000g for 45 min at 4°C to obtain the envelope membrane fraction. The two membrane pellets were solubilized separately in DM-co-IP buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 4 mM MgCl2, 1% decylmaltoside, and 10% glycerol), then the samples were pooled and clarified by centrifuging at 100,000g for 5 min. The supernatant was designated as total “input.” For immunoprecipitation, antisera against Toc75, Tic110, Hsp93, or cpHsc70 were incubated with the total input for at least 2.5 h in the cold room, then protein A-agarose beads (Pierce) were added and the samples incubated for at least 1.5 h in the cold room. The beads were then washed four times with DM-co-IP buffer and the precipitated proteins eluted using 23 SDS-PAGE sample buffer containing 126 mM DTT and analyzed by SDS-PAGE. Experiments with both prRBCS and prOE33 were performed multiple times, and the same difference in the association patterns of Toc75, Hsp93, and cpHsc70 with import substrates were consistently observed. Sequence data from this article can be found in the GenBank/EMBL/TAIR data libraries under accession numbers At5g16620 (Arabidopsis pre-atTic40) and At5g66570 (Arabidopsis prOE33).

ACKNOWLEDGMENTS We thank Dr. Danny Schnell for supplying the pre-atTic40-3xFLAG construct. We are grateful for the assistance of Dr. Tom Barkas in English editing, and we acknowledge assistance of the Institute of Molecular Biology Scientific English Editing Core in preparing this article. Received November 23, 2015; accepted December 16, 2015; published December 16, 2015.

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