A novel serine/proline-rich domain in ... - Wiley Online Library

The Plant Journal (2007) 52, 824–838

doi: 10.1111/j.1365-313X.2007.03279.x

A novel serine/proline-rich domain in combination with a transmembrane domain is required for the insertion of AtTic40 into the inner envelope membrane of chloroplasts Joanna Tripp1, Kentaro Inoue2, Kenneth Keegstra1 and John E. Froehlich1,* 1 MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48823, USA, and 2 Department of Plant Science, College of Agriculture & Environmental Science, University of California, Davis, CA 95616, USA Received 15 June 2007; accepted 25 July 2007. *For correspondence (fax þ1 517 353 9168; e-mail [email protected]).

Summary AtTic40 is part of the chloroplastic protein import apparatus that is anchored in the inner envelope membrane by a single N-terminal transmembrane domain, and has a topology in which the bulk of the C-terminal domain is oriented toward the stroma. The targeting of AtTic40 to the inner envelope membrane involves two steps. Using an in vitro import assay, we showed that the sorting of AtTic40 requires a bipartite transit peptide, which was first cleaved by the stromal processing peptidase (SPP), thus generating a soluble AtTic40 stromal intermediate (iAtTic40). iAtTic40 was further processed by a second unknown peptidase, which generates its mature form (mAtTic40). Using deletion mutants, we identified a sequence motif N-terminal of the transmembrane domain that was essential for reinsertion of iAtTic40 into the inner envelope membrane. We have designated this region a serine/proline-rich (S/P-rich) domain and present a model describing its role in the targeting of AtTic40 to the inner envelope membrane. Keywords: chloroplasts, protein import, inner envelope membrane.

Introduction Plastids are metabolically important organelles that perform many essential functions within plant cells. One common feature of all plastids is that most of their proteins are encoded in nuclear genes, synthesized on cytosolic ribosomes – generally as larger precursor proteins – and transported into the organelle via a translocation apparatus located in the envelope membranes (Jarvis and Robinson, 2004; Jarvis and Soll, 2002). Transport across the two envelope membranes is thought to occur via a single transport system, sometimes referred to as the general import pathway (GIP; Jarvis and Soll, 2001; Keegstra and Cline, 1999; Keegstra and Froehlich, 1999). The general translocation complex is composed of components located in the outer envelope membrane, termed Toc (translocon at the outer envelope membrane of chloroplasts) proteins, and components at the inner envelope membrane, termed Tic (translocon at the inner envelope membrane of chloroplasts) proteins. Chloroplasts contain six distinct subcompartments to which a protein may be ultimately targeted. The subcompartments are defined as 824

follows: the outer envelope membrane (OEM), the inner envelope membrane (IEM), the thylakoid membrane, and three aqueous subcompartments – the intermembrane space (IMS), the stroma, and the thylakoid lumen. To ensure the efficient and correct targeting of proteins to their final destination, chloroplasts have evolved numerous internal protein routing systems. Following complete translocation into the stroma, newly imported proteins can either remain in the stroma or utilize one of four sorting pathways that are responsible for the further targeting of proteins from the stroma to the thylakoids (Cline and Henry, 1996; Cline et al., 1993). One targeting pathway receiving renewed interest is the sorting mechanism used by proteins destined for the IEM. These proteins are thought to utilize, initially, the GIP, but are then diverted to the IEM via a poorly understood process. Based on analogy with mitochondria, where targeting to the inner membrane has been extensively studied (Koehler, 2004; Neupert, 1997; Truscott et al., 2003), two hypotheses explaining targeting to the chloroplastic IEM have been proposed. (i) In the stop-transfer hypothesis, ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd

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Figure 2. AtTic40 uses a stromal intermediate upon targeting to the inner envelope membrane, and is processed in a two-step mechanism. Radiolabeled precursors of AtTic40 (a and b), ARC6 (c), tp110-110N (d), and the precursor of the small subunit of RUBISCO (prSS; e) were incubated with intact pea chloroplasts for 15 and 30 min (a and b) or 30 min (c–e). Chloroplasts were treated with (+) or without ()) either trypsin or thermolysin. Digestion was performed at 0C for 30 min and was subsequently terminated by adding protease inhibitor or EDTA, respectively. Intact chloroplasts were recovered, lysed and fractionated into soluble (S) and total membrane (P) fractions, and analyzed by SDS-PAGE and fluorography. *Truncated ARC6 fragment after treatment with trypsin; i, intermediate; m, mature portion of protein; pr, precursor.

iTic40 Figure 1. Hypothetical model of the two targeting pathways for proteins destined for the inner envelope of chloroplasts. (a) Called the stop-transfer pathway, transport across the inner envelope membrane (IEM) is halted, leading to the lateral insertion of the protein into the IEM during the import process. (b) The ‘post-import’ pathway predicts that import into the stroma is completed before insertion into the IEM occurs as a discrete second step. Accumulating evidence indicates that AtTic40 uses the ‘post-import’ pathway for its insertion into the IEM. Abbreviations: GIP, general import pathway; i, intermediate; IEM, inner envelope membrane; IMS, intermembrane space; m, mature forms of AtTic40; OEM, outer envelope membrane; pr, precursor; TMD, transmembrane domain.

transport across the IEM is halted, leading to insertion into the IEM during the import process (Glaser et al., 1990; Glick et al., 1992; Figure 1a). For example, it has been shown that the yeast cytochrome oxidase subunit Va protein uses a ‘stop-transfer’ mechanism for its insertion into the mitochondrial inner membrane (Glick et al., 1992; Miller and Cumsky, 1993). (ii) The ‘post-import’ hypothesis predicts that import into the stroma is completed before insertion into the IEM occurs as a discrete second step (Figure 2b).

Recently, Li and Schnell (2006) have shown that AtTic40, a component of the chloroplastic import apparatus, utilizes the ‘post-import’ pathway for its insertion into the IEM. Also, another component of the chloroplastic import apparatus, Tic110 has been shown to utilize the ‘post-import’ pathway for its insertion into the chloroplastic IEM (Lu¨beck et al., 1997). The proposed chloroplastic ‘post-import’ pathway is analogous to the conservative sorting pathway taken by certain proteins targeted to the mitochondrial inner membrane (Gruhler et al., 1995; Herrmann et al., 1997; Herrmann and Neupert, 2003; see Figure 1b). As an example, Rojo et al. (1995) have shown that the sorting of subunit 9 of Fo-ATPase to the inner membrane of Neurospora mitochondria uses the conservative sorting pathway. Hence, it appears that chloroplastic IEM proteins can utilize at least two different pathways for their targeting to the IEM. The targeting determinants that direct chloroplastic IEM proteins to use either the stop-transfer or the ‘post-import’ pathways still need to be determined.

ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 824–838

826 Joanna Tripp et al. In this report, we attempt to define the targeting determinants that indicate whether proteins destined for the IEM of chloroplasts use either the stop-transfer pathway or a twostep mechanism involving a stromal intermediate, that is the ‘post-import’ pathway (Li and Schnell, 2006; Figure 1a and b, respectively). For this investigation, we examined the multistep import pathway of AtTic40, a component of the chloroplastic import apparatus localized to the IEM, in greater detail (Chou et al., 2003; Li and Schnell, 2006; Stahl et al., 1999). AtTic40 serves as a good model IEM protein for our investigation, because it contains a single transmembrane domain (TMD) located near its N-terminal, and upon insertion into the IEM has a proposed topology in which the large C-terminal domain of the protein is oriented toward the stroma (Li and Schnell, 2006). Consequently, the final topology of AtTic40 (i.e. N-termout–C-termin) dictates that AtTic40 uses a two-step pathway for its insertion into the IEM. However, the details of the targeting determinants that redirect the stromal-localized intermediate of AtTic40 (iAtTic40) back to the IEM are poorly understood. In this report we show that: (i) AtTic40 possesses a bipartite transit peptide that is first cleaved by the stromal processing peptidase (SPP), thus generating a soluble stromal intermediate (iAtTic40) that is subsequently retargeted to the IEM and processed to maturity; (ii) a poly serine/proline-rich (S/Prich) domain between the bipartite transit peptide and the TMD of AtTic40 is critical for reinsertion of AtTic40 into the IEM, and (iii) we propose from our data that AtTic40 may utilize the formation of an ‘insertion loop’ composed of the S/P-rich domain in combination with its TMD for its insertion into the IEM. Results AtTic40 is targeted to the inner envelope membrane via the ‘post-import’ pathway Previously, Chou et al. (2003), using either thermolysin or trypsin, examined the susceptibility of endogenous AtTic40 for protease digestion. These proteases were selected because thermolysin cannot penetrate the OEM and thus cannot gain access to the IMS, whereas trypsin can penetrate the OEM and can enter the IMS (Jackson et al., 1998; Kouranov et al., 1998, 1999). From this study, it was demonstrated that AtTic40 was oriented in the IEM in an Ntermout–C-Termin topology (Figure 1). Subsequently, this topology was confirmed by thermolysin treatment of inverted inner envelope vesicles (Li and Schnell, 2006). Supporting evidence was provided by studies about functional interactions of C-terminal domains of Tic40 with the stromal chaperone Hsp93 (Chou et al., 2006). In this investigation we wanted to know how AtTic40 is targeted to the IEM in a way that achieves this topology. In vitro import assays confirmed that AtTic40 was targeted and inserted into the membrane,

and was inaccessible to both thermolysin and trypsin digestion (Figure 2a,b, lanes 1, 3, and 5), confirming that newly imported AtTic40 has the same topology as endogenous AtTic40. Additionally, we confirmed that radiolabeled AtTic40 was specifically targeted to the envelope membrane by separating total membranes into thylakoid and mixed envelope membrane fractions, and analyzing these fractions by SDS-PAGE (Figure S1a). Finally, treatment of isolated total membrane fractions with Na2CO3, pH 11.5 (Figure S2a) demonstrated that AtTic40 was stably inserted into the envelope membrane when using our import assay system. Although the majority of AtTic40 was found to be processed to its mature form (mAtTic40) within 30 min of import (Figure 2b, see mAtTic40), we observed the appearance of a processing intermediate of about 44 kDa at an earlier time point (Figure 2a, lanes 2, 4, and 6, iAtTic40). This intermediate form could be partially recovered in both the membrane and soluble fractions (Figure 2a, see iAtTic40). Protease protection assays (Figure 2) confirmed that soluble iAtTic40 resided within the stroma of chloroplasts and not in the IMS. We base this conclusion on the following observations. (i) iAtTic40 localized in the soluble fraction was resistant to thermolysin and trypsin digestion (Figure 2a, lanes 2, 4, and 6). (ii) In a control import assay the plastid division protein ARC6, which is anchored in the IEM by a single TMD and has an N-termin–C-termout orientation (Vitha et al., 2003), was shown to be inaccessible to thermolysin; however, upon trypsin treatment the approximately 18-kDa domain of ARC6 extending into the IMS was partially digested, resulting in a truncated membrane-bound version of ARC6 (Figure 2c, lane 5). The partial cleavage of ARC6 in this experiment confirmed that trypsin did indeed gain access to the IMS. (iii) Two additional control proteins were in this experiment: a truncated version of Tic110 (Lu¨beck et al., 1997), which is anchored in the IEM with the bulk of the protein oriented toward the stroma and designated tp110110N (Figure 2d), and the stroma-localized precursor of the small subunit of RUBISCO (prSS; Figure 2e; Olsen and Keegstra, 1992). Both proteins were inaccessible to either thermolysin or trypsin. From these results, we concluded that soluble iAtTic40 must be localized within the stroma. The presence of soluble stromal iAtTic40 is in agreement with the previous results of Li and Schnell (2006). Thus we likewise concluded that AtTic40 is targeted to the IEM via the ‘post-import’ pathway rather than the stop-transfer pathway (Figure 2b). The targeting determinants that direct AtTic40 to the IEM were investigated further. The bipartite transit peptide of AtTic40 is processed by two different proteases The appearance of an intermediate of AtTic40 in the stroma indicates that the first processing step of AtTic40 may be performed by the SPP. This peptidase is responsible for the


Protein targeting to the IEM 827 removal of N-terminal transit peptides of most proteins targeted to the chloroplast via the GIP (Richter and Lamppa, 1998). Cleavage of the AtTic40 precursor as predicted by ChloroP (Emanuelsson et al., 1999) would produce a fragment of about approximately 44 kDa, the size of the intermediate observed at the 15-min time point (Figure 2a, lanes 2, 4, and 6). To confirm the predicted SPP cleavage site in AtTic40, we performed an in vitro SPP processing assay using translated AtTic40 (Tranel et al., 1995). Following incubation with a stromal extract, AtTic40 was processed to its intermediate form (Figure 3a, lane 5). Furthermore, iAtTic40 generated in this assay co-migrated with iAtTic40

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Toc75 imp – 30 – – min Lep – – + – ΔLep – – – + pr iToc75 mToc75 10 11 12 13 Figure 3. prAtTic40 is sequentially processed by two different proteases. (a) prAtTic40 was processed to its intermediate form (iAtTic40) after incubation using a crude stromal extract (lane 5). For comparison, prAtTic40 was imported into pea chloroplasts for 10 and 20 min in order to generate both iAtTic40 and mAtTic40 (lanes 1–4). prSS was used as a control for our stromal processing peptidase (SPP) assay (lane 8). (b) A type-I signal peptidase was responsible for converting prAtTic40 to its mature form. The indicated translation products were incubated with recombinant leader peptidase (+Lep) or heat-denatured Lep (+DLep) at 37C for 10 min. In the presence of Lep prAtTic40 was processed to its mature form (mAtTic40), whereas mature AtTic40 (mAtTic40) was not cleaved. prToc75, a substrate for a type-I signal peptidase, was used in a control reaction. Abbreviations: i, intermediate; m, mature portion of protein; pr, precursor.

produced after a 10-min import assay (Figure 3a, compare lanes 1 and 2 with lane 5). Additionally, we confirmed the specificity of our SPP assay by incubating two control proteins, prSS and luciferase, with crude stromal extract. Although prSS was processed to its mature form, luciferase, which does not contain a stromal processing site, was neither processed nor aberrantly digested (Figure 3a, compare lane 10 with lanes 7 and 9; unpublished results, respectively). Given that AtTic40 contains a bipartite transit peptide, we further examined the second processing step of AtTic40. The mature processing site was previously determined by the sequencing of Tic40 from pea by Stahl et al. (1999). Two alanine residues at the )3 and )1 positions to the second cleavage site suggest the involvement of a type-I signal peptidase (SPase I) in processing AtTic40 to its mature form (Paetzel et al., 2002). Three SPase Is are predicted to be located in plastids (Howe and Floyd, 2001; Inoue et al., 2005b), and genetic and in vitro import data suggested that at least one of them, named Plsp1, is partially in the envelope membranes, with its active site facing the IMS (Inoue et al., 2005a). To determine whether an SPase I-type protease could process AtTic40 to its mature form, radiolabeled precursor was incubated with a recombinant SPase I from Escherichia coli, leader peptidase (Lep) (Figure 3b). It cleaved AtTic40, generating a product that co-migrated with the mature form found after import and processing of AtTic40 (Figure 3b, compare lanes 3 and 4). Mature AtTic40 (mAtTic40) was not cleaved by Lep (Figure 3b, lane 8), confirming that processing of the precursor occurred at the N-terminus. The outer envelope protein, Toc75, served as a control and was processed to its mature form (Figure 3b, lane 12) as expected (Inoue et al., 2005a). A serine/proline-rich domain N-terminal of the transmembrane domain facilitates retargeting of AtTic40 to the inner envelope membrane The two-step processing of AtTic40 confirmed that AtTic40 contains a bipartite transit peptide. We hypothesize that the first portion of the transit peptide is responsible for import of AtTic40 into the stroma. However, the determinant(s) that route AtTic40 back to the IEM have yet to be defined. We considered the possibility that a targeting determinant within the C-terminal portion of the AtTic40 bipartite transit peptide may be responsible for its retargeting and insertion into the IEM (Figure 1b). To test this possibility, we prepared various hybrid proteins and deletion mutants, as shown in Figure 4a. To examine the behavior of each construct during import (Figure 4b), we performed time-course experiments using the HgCl2 stop method of Reed et al. (1990), which rapidly stops both protein import and processing, yet allows for the recovery of intact chloroplasts. The soluble intermediate of AtTic40 reached a maximum at about 2–4 min


828 Joanna Tripp et al. Figure 4. Sequence motifs N-terminal of the transmembrane domain (TMD) are required for the retargeting and insertion of AtTic40 into the inner envelope membrane. (a) Deletion mutants and hybrid constructs used in this study. Abbreviations: tpn, amino acids 1–43 of the bipartite transit peptide of AtTic40; tpc, amino acids 44–76. Arrows indicate the position of the first processing site, creating the intermediate (i) form of AtTic40, and the second processing site, producing the mature (m) form of AtTic40. The gray boxes indicate the location of the TMDs, whereas the black boxes define the serine/proline-rich domains. (b) Translation products (TP) from various deletion mutants and hybrid constructs were incubated with isolated chloroplasts. After the indicated time periods, the import was stopped by addition of 3.3 mM HgCl2. Subsequently, chloroplasts were pelleted, washed with import buffer containing EDTA, repelleted and finally resuspended in import buffer. Intact chloroplasts were then treated with thermolysin, lysed and separated into soluble (S) and total membrane (P) fractions, and were then analyzed by SDS-PAGE and fluorography. Abbreviations: i, intermediate; m, mature portion of protein; pr, precursor.

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(Figure 4b, panel 1). As the level of iAtTic40 declined, the level of membrane-bound mature AtTic40 (mAtTic40) increased. The time course shows that the accumulation of iAtTic40 in the soluble fraction precedes its reinsertion into the membrane fraction. Although it is not completely clear at which step during the import process the second processing

event occurs, mAtTic40 seems to appear predominantly after association with the membrane fraction. To confirm that the N-terminal portion of the AtTic40 bipartite transit peptide (tpn) was involved solely in import (see Figure 4a), but not in the retargeting to the IEM, we constructed a hybrid protein in which tpn was replaced by the transit peptide of prSS, thus creating the hybrid protein tpSS-iAtTic40 (Figure 4a). The kinetics of processing and integration into chloroplastic membranes of tpSS-iAtTic40 was comparable with that of AtTic40 (Figure 4b, panel 2). From the size of the soluble intermediate of tpSS-iAtTic40, we concluded that the first processing event for this construct occurs at the SS processing site, thus generating a soluble intermediate, iAtTic40, which was then retargeted from the stroma to the membrane and processed to mAtTic40. Additionally, we confirmed that tpSS-iAtTic40 was correctly targeted to the envelope membrane by fractionation experiments (Figure S1c), and we verified by carbonate extraction that tpSS-iAtTic40 was inserted into the membrane (Figure S2c). These results taken together demonstrate that the N-terminal portion of the bipartite transit peptide of AtTic40 can be replaced with the transit peptide of SS without changing the import properties of AtTic40, providing evidence that tpn merely contains stromal targeting information and is not involved in the retargeting of AtTic40 to the IEM. To study the function of the C-terminal portion of the bipartite transit peptide of AtTic40, we created a deletion mutant lacking this region (AtTic40Dtpc; see Figure 4a). Because the second processing site was lacking in AtTic40Dtpc, we predicted that it should be cleaved only once, presumably by the SPP, thus producing mature AtTic40. The overall efficiency of import of AtTic40Dtpc into chloroplasts was low when compared with AtTic40 (Figure 4b, compare panels 1 and 3). The retargeting of AtTic40Dtpc was also impaired, resulting in a prolonged accumulation of a soluble form of AtTic40Dtpc, which was slowly inserted into the


Protein targeting to the IEM 829 membrane (Figure 4b). Because of the low efficiency of import of AtTic40Dtpc, we replaced the complete bipartite transit peptide of AtTic40 with the transit peptide of SS, thus creating the construct tpSS-mAtTic40 (see Figure 4a). Although tpSS-mAtTic40 was imported into chloroplasts, the presence of soluble mAtTic40 during the later stages of import showed that mAtTic40 retargeting to the IEM was delayed. After 30 min of import mAtTic40 was finally retargeted, predominately to the membrane. We conclude that the removal of the C-terminal portion of the bipartite transit peptide of AtTic40 merely delayed the retargeting of mAtTic40 to the IEM (Figure 4b). Likewise, deletion of the C-terminal half of the AtTic40 bipartite transit did not alter the targeting of AtTic40 to the envelope membrane (Figure S1b,d). Finally, both AtTic40Dtpc and tpSS-mAtTic40 were shown to insert into chloroplastic membranes, and were not susceptible to carbonate extraction (Figure S2b,d). From the time-course experiments using AtTic40Dtpc, tpSSiAtTic40, and tpSS-mAtTic40, we conclude that although the bipartite transit peptide of AtTic40 contains stromal targeting information, it plays only a minor role in the retargeting of AtTic40 to the IEM. While employing the HgCl2 stop method of Reed et al. (1990), on the constructs AtTic40-WT, AtTic40Dtpc, tpSSiAtTic40, and tpSS-mAtTic40, we observed the appearance of an extraneous band in the pellet fraction during the time course of import, which was slightly smaller than the mature form of Tic40 (Figure 4). To ascertain whether this band originated from actual processing of precursor protein during import or from possible residual thermolysin protease activity, we performed a time-course assay with AtTic40-WT, AtTic40Dtpc, tpSS-iAtTic40, and tpSS-mAtTic40, according to the method of Reed et al. (1990), but without post-treatment with thermolysin (Figure S3). The results from these time-course experiments showed no generation of extraneous bands (i.e. from residual protease treatment) but only similar major bands, as seen in Figure 4b. Hence, the bands generated in Figure 4b for the constructs, AtTic40-WT, AtTic40Dtpc, tpSS-iAtTic40, and tpSS-mAtTic40, appear to be a by-product of the HgCl2 stop method, in combination with the thermolysin treatment (Reed et al., 1990). Nevertheless, by comparing the time-course experiments presented in Figure 4b (with thermolysin) with the time-course experiments presented in Figure S3 (without thermolysin), we show that both experiments support the conclusion that although the bipartite transit peptide of AtTic40 contains stromal targeting information, it plays only a minor role in the retargeting of AtTic40 to the IEM. To identify other possible determinants that may retarget iAtTic40 from the stroma to the IEM, we focused on two regions within mAtTic40. The first was an S/P-rich domain located between the mature processing site of AtTic40 and its TMD (see Figures 4a). The second domain was the TMD of AtTic40 itself (see Figure 4a). Deletion mutants lacking the

S/P-rich domain (AtTic40DS/P) and the TMD (AtTic40DTMD) were created and tested in time-course experiments (Figure 4b). Deletion of the S/P-rich domain (AtTic40DS/P) produced a soluble intermediate that was not inserted into the membrane, despite the presence of a TMD (Figure 4b). The small quantity of AtTic40DS/P that associated with the membrane fraction was not fully integrated into the membrane, as it was partially extractable by carbonate treatment (Figure S2e). To evaluate the possibility that the inability of AtTic40DS/P to integrate into the membrane was caused by secondary structural changes, we replaced the S/P-rich domain with a 29-amino acid peptide from a region adjacent to the ARC6 TMD (AtTic40DS/P-ARC6) that contains four serine yet no proline residues. Similar to the results observed for AtTic40DS/P, AtTic40DS/P-ARC6 was found in the soluble fraction (Figure 4b). The small quantity of AtTic40DS/P-ARC6 associated with the membrane could be removed from the membrane upon extraction with sodium carbonate (Figure S2f). From these experiments we concluded that the S/P-rich domain of AtTic40 plays a critical role in both the retargeting and insertion of AtTic40 into the envelope membrane. Finally, deletion of the TMD of AtTic40 completely converts the imported product into a soluble stromal protein (Figure 4b). It was previously shown that removal of the TMD of ARC6 results in its targeting to the stroma (Vitha et al., 2003). Interestingly, most of AtTic40DTM appears only to be processed to the intermediate form, which is consistent with the hypothesis that the second processing step for AtTic40 may occur on the intermembrane space side of the IEM (see Figure 1b; Inoue et al., 2005a). Tic110 has been previously described as a protein that is first routed to the stroma and then re-exported to the IEM (Lu¨beck et al., 1997). In our time-course experiment we used the C-terminally truncated version of Tic110, tp110-110N, as a control (Lu¨beck et al., 1997). The mature form of tp110110N, 110N, appears in the soluble fraction after only 0.5 min of import and is subsequently retargeted to the IEM, thus supporting the conclusion that the truncated protein tp110-110N likewise uses the ‘post-import’ pathway (see Figure 1b) for targeting to the IEM of chloroplasts. Likewise, during these studies we examined the targeting of ARC6 and could not detect a soluble intermediate of ARC6 throughout the time course of its import (see Figure 4b). Consequently, we postulate that ARC6 may be routed to the IEM via the stop-transfer pathway (Figure 1a), in contrast to Tic40, which utilizes the ‘post-import’ pathway (Figure 1b). Deletion of the S/P-rich domain alters the subchloroplastic localization of AtTic40 From time-course assays (Figure 4b) we observed that the imported products derived from deletion mutants AtTic40DS/P, AtTic40DS/P-ARC6, and AtTic40DTMD were


830 Joanna Tripp et al. found in the soluble fraction (Figure 4b, lane 14). To determine whether these proteins were located in either the IMS or the stroma, we performed an import assay followed by post-treatment with either thermolysin or trypsin. All imported products were inaccessible to protease digestion by either thermolysin or trypsin (Figure 5, lanes 4 and 6). However, a control protein, ARC6, was partially digested by trypsin (Figure 5, lane 5), confirming that the trypsin treatment was working properly. We conclude from these results that the translocation of these mutant precursors into the

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* 1 2 3 4 5 6 Figure 5. Deletion of the serine/proline-rich domain or the transmembrane domain (TMD) prevents the retargeting and insertion of AtTic40 into the inner envelope membrane. The indicated 35S-labeled mutants and hybrid proteins were incubated with isolated chloroplasts in a standard import reaction for 30 min. After the import, intact chloroplasts were incubated either in the presence (+) or absence ()) of either trypsin or thermolysin for 30 min on ice. After the protease digestion was quenched, chloroplasts were re-isolated, lysed, and fractionated into total membrane (P) and soluble (S) fractions. The fractions were subsequently analyzed by SDS-PAGE and fluorography. Abbreviations: i, intermediate; m, mature portion of protein; pr, precursor.

stroma was not compromised, but rather the subsequent insertion of the stromal intermediates into the IEM was impaired. We also examined the subcellular localization and topology of imported products derived from tpSS-iAtTic40, AtTic40Dtpc, and tpSS-mAtTic40 (Figure 5). Although the products following import of tpSS-iAtTic40, AtTic40Dtpc, and tpSS-mAtTic40 were found to be localized in the membrane (Figure 5, lanes 1, 3, and 5), none of these imported products were susceptible to protease digestion by either thermolysin or trypsin (Figure 5). Similarly, the AtTic40 control was also protected from protease digestion by either thermolysin or trypsin (Figure 5). We concluded that the imported products derived from the hybrid proteins obtained the same orientation within the membrane as AtTic40, because an aberrant inverted orientation would have resulted in these products becoming susceptible to trypsin digestion. Finally, the soluble intermediates observed for tpSS-iAtTic40, AtTic40Dtpc, and tpSS-mAtTic40 (Figure 5, lanes 2, 4, and 6) were protected from protease digestion in the same fashion as the soluble intermediates of AtTic40. Thus the hybrid proteins retained essentially the same localization and topology as AtTic40 (Figure 5). The C-terminal half of the S/P-rich domain is necessary for membrane targeting and insertion The data presented above support the idea that the S/P-rich domain of AtTic40 plays a critical role in its retargeting to the IEM. To dissect the role of the S/P-rich domain in greater detail, we generated a series of deletion mutants in which consecutive parts of this domain were removed (Figure 6a). The deletions removed amino acids 77–82, 83–88, 89–97, and 98–105, starting after the second processing site of prAtTic40 and ending before the TMD (Figure 6a). Import experiments with the deletion mutants demonstrated that the precursors were processed to lower molecular weight forms that were resistant to thermolysin treatment, indicating their internalization (Figure 6b). Products of AtTic40D89– 97, AtTic40D98–105, and the control, AtTic40DS/P, were located predominantly in the soluble fraction (Figure 6b,c). The observed size of the processed proteins suggests that both AtTic40D89–97 and AtTic40D98–105 are processed to the intermediate form. We conclude that because iAtTic40D89–97 and iAtTic40D98–105 reside predominantly within the stroma, their second cleavage site was no longer accessible for processing by a type-I signal peptidase, whose catalytic domain is predicted to face the IMS. Conversely, imported AtTic40D77–82 was found predominantly in the membrane fraction, but was processed only to its intermediate form (Figure 6, see lanes 1, 3, and 5). In contrast with the other mutants, which were protected from trypsin, a small fragment of AtTic40D77–82 was cleaved by this protease (Figure 6, lane 5). To explain this result, we


Protein targeting to the IEM 831

(a) S/P-rich domain

TMD

106 1 76 M-//-AFASIFSSSRDQQTTSVASPSVPVPPPSS STIGSPLFWIGVGVGLSALFSYVTS

Tic40 Tic40ΔS/P

M-//-AFA

GSPLFWIGVGVGLSALFSYVTS

Tic40Δ77-82

M-//-AFA

RDQQTTSVASPSVPVPPPSS STIGSPLFWIGVGVGLSALFSYVTS

Tic40Δ83-88

M-//-AFASIFSSS

Tic40Δ89-97

M-//-AFASIFSSSRDQQTT

Tic40Δ98-105

M-//-AFASIFSSSRDQQTTSVASPSVPV

SVASPSVPVPPPSS STIGSPLFWIGVGVGLSALFSYVTS PPPSS STIGSPLFWIGVGVGLSALFSYVTS GSPLFWIGVGVGLSALFSYVTS

(b)

(c) – Thermolysin + Trypsin

kDa TP P S P S P S 50pr Tic40

100 (% of total)

+ –

Distribution

– –

80 60

Tic40

40 20

m

37-

0

P

S

50pr i

Tic40ΔS/P

(% of total)

100

Distribution

Figure 6. The C-terminal portion of the serine/ proline (S/P)-rich domain is necessary for membrane targeting and insertion. (a) Deletion mutants of the S/P-rich domain (residues 77–105) were generated, and (b) incubated with isolated chloroplasts for 30 min. After the import, intact chloroplasts were incubated either in the presence (+) or absence ()) of either trypsin or thermolysin for 30 min on ice. After the digestion was quenched, chloroplasts were re-isolated, lysed, and fractionated into total membrane (P) and soluble (S) fractions. The fractions were subsequently analyzed by SDSPAGE and fluorography. (c) The results from an import assay (lanes 1 and 2) were further quantitated. Abbreviations: i, intermediate; m, mature portion of protein; pr, precursor.

37-

80 60

Tic40ΔS/P

40 20 0

P

S

pr

Tic40Δ77-82

m

37-

(% of total)

50-

Distribution

100 80 60

Tic40Δ77-82

40 20 0

P

S

pr

Tic40Δ83-88

m

37-

80 (% of total)

50-

Distribution

100

60

Tic40Δ83-88

40 20 0

P

50-

S

37-

(% of total)

pr i

Tic40Δ89-97

Distribution

100 80 60

Tic40Δ89-97

40 20 0

P

Tic40Δ98-105 371 2 3 4 5 6

proposed that the second cleavage site of AtTic40D77–82 was slightly modified during the creation of this construct, thus reducing the efficiency of cleavage by a type-I signal peptidase. Consequently, the unprocessed N-terminal portion of iAtTic40D77–82 extended into the IMS, where it was partially digested by trypsin (Figure 6b). Finally, imported AtTic40D83–88 fractionated predominantly to the membrane fraction, and was neither cleaved by thermolysin nor trypsin (Figure 6b). From these results, we conclude that only the C-terminal half (amino acids 89–105) of the S/P-rich domain of AtTic40 is necessary for the retargeting and insertion of AtTic40 into the IEM. The S/P-rich domain in combination with the TMD of AtTic40 can direct GFP to the envelope membrane of chloroplasts Sequence alignment of Tic40 from various plant species shows that the S/P-rich domain is common among all Tic40s

S

80 (% of total)

pr i

100

Distribution

50-

60

Tic40Δ98-105

40 20 0

P

S

analyzed (Chou et al., 2003). To investigate the functionality of the S/P-rich domain for directing a foreign protein to the envelope membrane, we generated Tic40–GFP fusion proteins (Figure 7a) containing different portions of the N-terminal region of AtTic40 (i.e. amino acids 1–128). From these experiments we observed that Tic40 N-term-GFP, containing the complete N-terminal of Tic40 fused to GFP (Figure 7a), could direct the targeting of GFP to the membrane fraction (Figure 7b). Carbonate extraction demonstrated that Tic40 N-term-GFP was indeed inserted into the membrane (data not shown). However, Tic40 DS/P-GFP (N-terminal without S/P-rich domain fused to GFP) and Tic40 DTMD-GFP (N-terminal without TMD fused to GFP) were shown to be imported into the stroma of chloroplasts, but failed to be subsequently ‘post-imported’ into the IEM (Figure 7b). Finally, the tp of Tic40 could facilitate the import of GFP to the stroma in a similar fashion as the tp of SS. This result adds additional support to the notion that the bipartite


832 Joanna Tripp et al. tp tpn tpc

(a)

i

m TMD

Tic40 (WT) 1

44 77106 128

447

GFP(28kDa)

Tic40 N-term-GFP Tic40 ΔS/P-GFP

GFP(28kDa)

Tic40 ΔTMD-GFP

GFP(28kDa)

The TMD contains information about the sorting pathway of AtTic40

GFP(28kDa)

tpTic40-GFP

GFP(28kDa)

tpSS-GFP 1

(b)

transit peptide of AtTic40 contains only stromal targeting information. Finally, the data presented here confirm the prerequisite that the retargeting of AtTic40 to the IEM requires the presence of both the S/P-rich domain and its TMD, and show that no targeting information is present within the C-terminus of the protein.

62

Thermolysin – + – – Trypsin kDa TP P S P S 50-

– + P S pr

Tic40 N-term-GFP 37-

i m

37-

pr i

37-

pr

Tic40 ΔTMD-GFP

Tic40 ΔS/P-GFP

i m 37-

pr i

tpTic40-GFP 37-

pr

tpSS-GFP

prSS

m

252015-

pr m

101 2 3 4

5 6

Figure 7. The serine/proline (S/P)-rich domain in combination with the transmembrane domain (TMD) of AtTic40 can direct GFP to the envelope membrane of chloroplasts. (a) AtTic40 hybrid constructs used in this study: tpn, amino acids 1–43 of the bipartite transit peptide of AtTic40; tpc, amino acids 44–76. Arrows indicate the position of the first processing site, creating the intermediate (i) form of AtTic40, and the second processing site, producing the mature (m) form of AtTic40. The gray boxes indicate the location of the TMDs, whereas the black boxes define the S/P-rich domains. Green fluorescence protein is fused to the C-terminal end of all AtTic40–GFP fusion proteins. (b) Translation products (TP) from various AtTic40–GFP fusion proteins were incubated with isolated chloroplasts for 20 min. Chloroplasts were treated with (+) or without ()) either thermolysin or trypsin. Digestion was performed at 0C for 30 min and was subsequently terminated by adding EDTA or protease inhibitor, respectively. Intact chloroplasts were recovered, lysed, and fractionated into soluble (S) and total membrane (P) fractions, and were analyzed by SDS-PAGE and fluorography. Abbreviations: i, intermediate; m, mature portion of protein; pr, precursor.

In the experiments presented above while we confirm previous observations that AtTic40 uses the ‘post-import’ pathway (Li and Schnell, 2006; Figure 1b), and we further demonstrate that a novel S/P-rich domain is needed for retargeting to the IEM. We have also shown preliminary evidence that ARC6 follows the stop-transfer route to reach the IEM (Figures 1a and 5). Thus, multiple pathways target proteins to the chloroplastic IEM. This observation raises an intriguing question: what sorting signal(s) determine whether a protein is halted at the IEM (stop-transfer) or is allowed to pass (‘post-import’ pathway). One possibility is that the TMD is the important determinant. To test this hypothesis, we created constructs in which only the TMDs of AtTic40 (amino acids 106–128) and ARC6 (amino acids 615– 635) were exchanged (Figure 8a). In an in vitro import experiment using ARC6-AtTic40TMD (ARC6 containing the TMD of AtTic40), the imported products were predominantly localized in the soluble fraction (Figure 8b, lanes 1–6). The portion of the ARC6-AtTic40TMD product that associated with the membrane was carbonate-extractable, indicating that it had not integrated into the IEM (unpublished results). Finally, imported ARC6-AtTic40TMD products associated with the membrane fraction did not produce the 18-kDa truncated fragment after trypsin treatment, unlike ARC6, which becomes truncated after trypsin digestion (Figure 8b, lanes 5 and 6). From these initial experiments, we conclude that (i) replacement of the TMD of ARC6 with the TMD of AtTic40 significantly alters the targeting of ARC6-AtTic40TMD to the stroma when compared with wild-type ARC6 (Figure 8), and (ii) the TMD of ARC6 acts as an authentic stop-transfer domain with respect to wild-type ARC6. In contrast, in AtTic40-ARC6TMD, the TMD of ARC6 alone was not sufficient to act as a genuine stop-transfer signal and thus facilitate the complete arrest of AtTic40 in the IEM (Figure 8b, lanes 1–6). Arrest of AtTic40-ARC6TMD within the IEM would lead to an inverted topology compared with AtTic40, with the bulk of AtTic40-ARC6TMD facing the IMS, thus making AtTic40-ARC6TMD accessible for trypsin digestion (Figure 8b, lanes 5 and 6). This was not the case: imported AtTic40-ARC6TMD was inaccessible to both thermolysin and trypsin digestion. AtTic40-ARC6TMD was also found in both the soluble and membrane fractions (Figure 8b, lanes 2, 4, and 6). However, the portion of imported AtTic40-ARC6TMD that was membrane associated


Protein targeting to the IEM 833 TMD

(a) Tic40

TMD

ARC6 Tic40-ARC6TMD ARC6-Tic40TMD – + – Thermolysin – – + Trypsin TP P S P S P S pr

(b)

Tic40

m

pr Tic40-ARC6TMD

i m pr m

ARC6-Tic40TMD

ARC6

*

pr m

1 2 3 4 5 6 Figure 8. Replacement of the transmembrane domain (TMD) of ARC6 with the TMD of AtTic40 leads to mistargeting of ARC6 into the stroma. (a) Schematic representation of mutants used in this TMD exchange experiment. (b) The indicated proteins were imported into isolated pea chloroplasts for 30 min and treated with protease as previously described, fractionated, and analyzed by SDS-PAGE and fluorography. Abbreviations: i, intermediate; m, mature portion of protein; pr, precursor.

could be extracted from the membrane upon carbonate treatment (unpublished results). Thus, AtTic40-ARC6TMD was unable to be retargeted to the IEM, despite the presence of both an ARC6 TMD and the S/P-rich domain of AtTic40. We conclude from this set of results that the TMD of AtTic40 contains specific information, which, in combination with the S/P-rich domain, is required for retargeting of AtTic40 to the IEM. Discussion In this work we have studied the targeting of AtTic40 to the IEM of chloroplasts. Our studies indicate that the targeting of AtTic40 involves two steps. Using an in vitro import assay, we have shown that in the first step AtTic40 is transported across the IEM into the stroma. The resulting soluble stromal intermediate is subsequently retargeted to the IEM by an unknown mechanism (Figures 2 and 5). The sorting of AtTic40 involves a bipartite transit peptide, which is first

cleaved by the SPP, thus generating the stromal intermediate (iAtTic40). iAtTic40 is then further processed by a second peptidase, which generates its mature form (mAtTic40; Figure 3a,b). From the presence of the soluble, stromal intermediate, we conclude that AtTic40 follows the ‘post-import’ pathway (Li and Schnell, 2006; Figure 1b). Our results are consistent with the results of Li and Schnell (2006), who have independently shown that targeting of AtTic40 involves a soluble intermediate that inserts into the inner envelope from the stromal side of the membrane. We further investigated the second processing step of AtTic40 in greater detail, and demonstrated that AtTic40 was processed to its mature form by an SPase I from E. coli, Lep (Figure 3b). Recently, genetic and in vitro import data suggested that an SPase I-type protease, Plsp1, is partially localized to the chloroplastic IEM, with its active site facing the IMS (Inoue et al., 2005a). Mechanistically, if indeed Plsp1 has this unique topology, this protease may well be responsible for the second processing step of AtTic40. Our in vitro processing assays using recombinant Lep cleaved AtTic40 to its mature form (Figure 3b), but Inoue et al. (2005a) have shown that the disruption of the PLSP1 gene affected the processing of only Toc75, whereas processing of AtTic40 remained unaffected in vivo. However, two other SPase I proteins have been predicted to be located in plastids. These plastidic SPase Is, described by Howe and Floyd (2001) and Inoue et al. (2005b), appear to be mainly located in thylakoids (Inoue et al., unpublished data). Nevertheless, there are at least four additional SPase Is of which the subcellular location remains unknown (Inoue et al., 2005b). It is intriguing to speculate that one of them could be located in the chloroplast envelope and be responsible for the processing of AtTic40 to its mature form. We likewise investigated what role if any that the bipartite transit peptide of AtTic40 plays in its targeting and insertion into the IEM. The bipartite organization of the transit peptide of AtTic40 suggested a mechanism analogous to the targeting of proteins destined for the thylakoid membrane or lumen (Mori and Cline, 2001; Mu¨ller and Klo¨sgen, 2005; Robinson and Bolhuis, 2004). In these targeting pathways that utilize a bipartite targeting signal, the first part of the targeting signal is responsible for the transport of the protein into the stroma, whereas the second portion, which often has features similar to bacterial signal peptides, mediates the targeting of the protein to its final destination. A typical feature of signal peptides is the presence of a hydrophobic core sequence. This sequence partially mediates the insertion of proteins into the thylakoid membrane. For instance, for ‘spontaneously’ inserting thylakoid membrane proteins PsbW, PsbX, and CFoII, it has been proposed that the hydrophobic domain core sequence transiently interacts with a transmembrane domain, forming a loop confirmation that facilitates protein insertion into the thylakoid membrane (Thompson et al., 1998).


834 Joanna Tripp et al. Hou et al. (2006) have shown that even when proteinaceous thylakoid transport machinery is involved, unassisted membrane insertion using a loop conformation might be important in the initial stages of the transport process. With the chimeric 16/23 protein, which uses the thylakoid DpH/Tat pathway, Hou et al. (2006) demonstrated that the formation of a loop configuration, created by the interaction between the hydrophobic core domain of the signal peptide with the amphipathic a-helical domain contained within the mature 16/23 protein, was critical for its initial insertion into the thylakoid membrane. However, in the case of AtTic40, the C-terminal portion of the bipartite transit peptide of AtTic40 did not resemble a typical signal peptide (Figure 4). Furthermore, the deletion of the C-terminal portion of the AtTic40 bipartite transit peptide impaired only the initial targeting of AtTic40 to the chloroplast (Figure 4b), whereas retargeting of AtTic40 to the IEM was only moderately impaired. This observation is also in agreement with the results of Li and Schnell (2006). From the deletion mutant experiments we concluded that although the C-terminal portion of the bipartite transit peptide of AtTic40 was not essential for its insertion into the chloroplastic IEM (Figure 4b), it does have a role in the translocation of AtTic40 into the stroma of chloroplasts. Intriguingly, both Toc75 (Tranel et al., 1995) and AtTic40 are the only known components of the chloroplastic import apparatus to have a bipartite transit peptide. However, when the bipartite transit peptide of Toc75 is replaced with tpSS, mToc75 is no longer targeted to the OEM but instead is mistargeted to the stroma (Tranel and Keegstra, 1996). Hence, the C-terminal portion of the bipartite transit peptide of Toc75 contains intraorganellar

targeting information. Conversely, our investigation has demonstrated that when the entire bipartite transit peptide of Tic40 is replaced with tpSS, Tic40 is still correctly targeted to the IEM (Figure 4b). To our surprise, deletion of the sequence positioned between the mature processing site of AtTic40 and its TMD essentially abolished the retargeting of AtTic40 to the IEM, and resulted in the accumulation of iAtTic40 in the stroma (Figure 4b). Based on experiments using deletion mutants (Figure 6b), we defined the sequence critical for reinsertion of AtTic40 to amino acid residues 89–105 (Figure 6a). The first part of this sequence (amino acids 89–97) is rich in serine and hydrophobic residues, whereas the second part (amino acids 98–105) contains a stretch of serine and proline residues. Hence, we have designated this novel motif, the S/P-rich domain. From our analysis, we propose that the S/P-rich domain of AtTic40 forms a unique secondary structure motif essential for the re-insertion of AtTic40 into the IEM. The S/P-rich domain may functions as follows: (i) the cluster of prolines positioned before the TMD of AtTic40 induces a turn within the polypeptide; (ii) the induced turn allows the S/P-rich domain to interact briefly with the TMD of AtTic40, thus forming a ‘transient insertion loop’; and (iii) once AtTic40 is completely inserted into the IEM, the S/P-rich domain undergoes a conformational change allowing the C-terminal portion of the bipartite transit peptide to gain access to the IMS, where it can be processed by a Plsp-type peptidase (Figure 9, steps 1–3). Several proposed features of our S/P-rich domain loop have support from the literature. For instance, it has already been shown that a single proline residue is sufficient to induce a

prTic40 C TMD

TP NCytosol

OEM IMS

SPase?

GIP Export

Export Stroma

N IEM

N S/PC Rich Domain

C

C

Figure 9. Model for the stepwise import and insertion of AtTic40 into the inner envelope membrane (IEM) of chloroplasts. The bipartite transit peptide of prAtTic40 interacts with the general import pathway (GIP). prAtTic40 is translocated into chloroplasts and is cleaved by the stromal processing peptidase (SPP) to generate iAtTic40, which is located in the stroma (step 1). The serine/proline (S/P)-rich domain interacts with the transmembrane domain (TMD) of iAtTic40 to form a transient insertion loop (step 2). With the aid of the insertion loop and some undefined export machinery, iAtTic40 inserts into the IEM. Finally, the S/P-rich domain undergoes a conformational change upon complete insertion into the IEM, forcing the second portion of the AtTic40 bipartite transit peptide to extend into the intermembrane space (IMS). There, it undergoes a final cleavage, generating mAtTic40 (step 3).

N

N N-

C SPP

1

2

3

iTic40

iTic40

mTic40


Protein targeting to the IEM 835 turn in a hydrophobic segment, leading to the formation of a ‘helical hairpin’ (Nilsson and von Heijne, 1998). Furthermore, Dawson et al. (2002) have shown that motifs of serines and threonines can drive the homo-oligomerization of transmembrane helices. Additional experiments will certainly be necessary to confirm whether the formation of a transient loop is essential for the insertion of AtTic40 into the IEM (Figure 9). Likewise, it remains to be determined how prevalent the S/P-rich domain motif is amongst other IEM proteins. Sequence alignment of Tic40s from different plant species (Chou et al., 2003) shows that the S/P-rich domain is present in variant forms. By creating Tic40–GFP fusion proteins, we show that the S/P-rich domain can serve as part of a functional membranetargeting motif (Figure 7). Simply attaching just the N-terminal half of Tic40, containing both the S/P-rich domain and its TMD, to GFP resulted in the targeting and insertion of GFP to membranes. Hence, the S/P-rich domain of Tic40 in combination with its TMD appears to function as an authentic membrane-targeting motif. A variant of this membrane-targeting motif is also seen in Tic110 (Lu¨beck et al., 1997). Sequence analysis of Tic110 shows the presence of an S/P-rich-like domain present between the transit peptide of Tic110 and its TMD (Lu¨beck et al., 1997). Whether the S/P-rich-like domain of Tic110 can function in a similar fashion as the S/P-rich domain of Tic40 needs additional investigation. Finally, future experiments designed to define the S/P-rich domain in greater detail should allow us to search various protein databases, in order to find additional candidate IEM proteins that have this motif and thus may use the same multistep IEM targeting pathway as AtTic40. The model in Figure 9 outlining a working hypothesis regarding the AtTic40 targeting pathway can be used to integrate the current results and to design further investigations. One significant unresolved problem regarding the AtTic40 targeting pathway is identifying the targeting determinants that specifically direct AtTic40 to the IEM, rather than to the thylakoid membrane. Ensuring that iAtTic40 is specifically targeted to the IEM may involve the interaction of unique AtTic40 retargeting signals with novel export machinery. Recently, Li and Schnell (2006) demonstrated that the reinsertion of AtTic40 into the IEM requires proteinaceous components, but does not involve the hydrolysis of nucleoside triphosphates and is independent of soluble, stromal factors. The nature of the export machinery, however, has yet to be identified. We speculate that an export mechanism of bacterial origin exists in the IEM of chloroplasts, and that this mechanism may be involved in retargeting AtTic40 (Kilian and Kroth, 2003; Reumann et al., 2005). The membrane insertion of most plasma (inner) membrane proteins in E. coli is facilitated by the SecYEG machinery, assisted by the ATPase SecA. YidC supports the function of the Sec translocase, but can also mediate the

membrane insertion of proteins in a Sec-independent manner (Samuelson et al., 2000) by directly catalyzing the insertion of proteins into the lipid bilayer (Serek et al., 2004). In mitochondria, no homologs of the Sec translocase can be found, but a homolog of YidC, Oxa1, is involved in the insertion of inner membrane proteins. In chloroplasts, homologs of SecY, SecE, and SecA are involved in thylakoid targeting (Di Cola et al., 2005), and two thylakoid-localized homologs of YidC, Alb3, and Alb4 have been identified (Gerdes et al., 2006; Sundberg et al., 1997). No homolog of YidC or the Sec translocase has yet been identified in a sequencing of the plastidic envelope membrane proteome (Ferro et al., 2003; Froehlich et al., 2003; Sun et al., 2004). Future investigation is needed to identify the export machinery involved in the retargeting and insertion of AtTic40 into the IEM. However, although it would be interesting to find a novel chloroplastic export apparatus, we must consider the possibility that already defined chloroplastic protein import machinery components at the IEM (i.e. Tic components) might be utilized for the insertion of AtTic40 itself, and for the general export of proteins from the stroma to the IMS and/or cytosol. Finally, our results demonstrate that, analogous to the situation in mitochondria, two pathways are employed for targeting proteins to the IEM of chloroplasts. As discussed previously, AtTic40 uses the ‘post-import’ pathway, whereas our results indicate that ARC6 appears to utilize the stop-transfer pathway (Figures 1 and 4). In mitochondria, the hydrophobicity of the TMD and the presence of proline residues within the TMD serve as sorting signals that decide which pathway a protein takes to the inner membrane (Meier et al., 2005). For instance, Meier et al. (2005) found that proline residues within the TMD of a protein seem to disfavor the use of the stop-transfer pathway. Referencing the Aramemnon database (Schwacke et al., 2003), we found no significant difference in the calculated hydrophobicity of the TMDs of the two proteins Tic40 (‘post-import’ sorted) and ARC6 (sorted by the stoptransfer pathway) (Figures 2 and 4b). However, in contrast to the TMD of ARC6, the TMD of AtTic40 does contain a proline residue. An exchange of the TMD of ARC6 for the TMD of AtTic40 caused sorting of the hybrid protein ARC6AtTic40TMD to the stroma. This indicates that some features of the TMD of AtTic40, possibly the presence of the single proline residue, allowed the hybrid protein to be translocated across the membrane (Figure 8b). To assess the potential impact of this single proline residue on the translocation of AtTic40 into the stroma, we changed the proline residue in the TMD of AtTic40 to a leucine. Despite this modification, AtTic40 was still targeted to the stroma and subsequently redirected to the IEM (unpublished results). We concluded from these experiments (Figure 8b) that additional features of the TMD are essential in preventing translocation arrest in the IEM. In contrast, the


836 Joanna Tripp et al. replacement of the TMD of AtTic40 with the TMD of ARC6 in AtTic40-ARC6TMD did not lead to the complete arrest of this hybrid protein in the envelope membrane (Figure 7b). From the results obtained with the hybrid proteins ARC6AtTic40TMD and AtTic40-ARC6TMD, we concluded that the TMD of ARC6 contains part of the information necessary for utilizing the stop-transfer mechanism. However, additional information is required for the complete arrest of the protein at the envelope membrane. For instance, charged residues at the C-terminal of the TMD appear to be an important topogenic signal required for translocation arrest of D-lactate dehydrogenase, which spans the inner envelope of mitochondria once in an N-termin–C-termout orientation (Rojo et al., 1998). The TMD of ARC6 is flanked by several charged residues on both sites, whereas Tic40 contains charged residues only close to the C-terminal end of the TMD. In our TMD swapping experiments, these charged residues were essentially retained, except for a single lysine residue at the C-terminal end of the ARC6 TMD that was carried over. At this time, we propose that the charged residues flanking the ARC6 TMD may sufficiently slow down the translocation of ARC6 in order to allow its TMD to be laterally released into the IEM. However, at this preliminary stage, additional experiments will be required to determine which features of a TMD direct a chloroplastic IEM protein to use either the ‘postimport’ or the stop-transfer pathway (Figure 1).

Experimental procedures Plasmid construction The cDNA encoding wild-type AtTic40 (accession no. At5g16620; RIKEN Institute, http://www.riken.go.jp/engn/index.html; Seki et al., 1998, 2002) was amplified using the primers AtTic40(WT)-F1 (5¢-CACCATGGAGAACCTTACCCTAGTT-3¢) and AtTic40(WT)-R1 (5¢-GTGAGCTTTTTCAACCCGTCATTC-3¢) and cloned into pENTR/ SD/D-TOPO (Invitrogen, http://www.invitrogen.com). The following deletion mutants and hybrid constructs used for in vitro transcription/translation were cloned in pCRII-TOPO (Invitrogen) using the KpnI and XhoI sites of the vector. For construction of AtTic40DTMD, the sequences encoding residues 1–105 and 128–447 were amplified with the primer pairs AtTic40F1(KpnI) (5¢-AGCCCGGTACCATGGAGAACC-3¢)/AtTic40R1(SpeI) (5¢-GATCCACTAGTTGATGAAGATGG-3¢) and AtTic40F2(SpeI) (5¢-GTAACTACTAGTTTAAAGAAATAT-3¢)/AtTic40R2(XhoI) (5¢-TGTGACTCGAGT CAACCCGTCATT-3¢). The resulting fragments were cut with KpnI, SpeI, and XhoI. To generate the AtTic40Dtpc construct, we used the primer pairs AtTic40F1(KpnI)/tAtTic40-R(SpeI) (5¢-TTGGAACTAGTGAGGACAATATTAGG-3¢) and mAtTic40-F(SpeI) (5¢-CTTTTACTAGTATATTTTCTTCGA-3¢)/AtTic40R2(XhoI) to amplify sequences encoding residues 1–43 and 77–447, respectively. The PCR products were digested with KpnI, SalI, and XhoI. Sequences encoding tpSS (residues 1–57) and residues 77–447 of AtTic40 were amplified with the primer pairs tSS-F(KpnI) (5¢-AGTCAGGTACCATGGCTTCTATGAT-3¢)/tSS-R(SpeI) (5¢-ACCTGACTAGTCTTTACTCTTCCACC-3¢) and mAtTic40-F(SpeI)/AtTic40R2(XhoI) to create the tpSS-mAtTic40

construct. PCR products were cut with KpnI, SpeI, and XhoI. Likewise, for generation of tpSS-iAtTic40, the primer pair tSS-F(KpnI)/tSS-R(SpeI) was used, in combination with primers iAtTic40(SpeI)-F (5¢-TCCTCACTAGTTCCAAAATATCTGC-3¢) and AtTic40R2(XhoI). For cloning of AtTic40DS/P, the PCR fragments encoding residues 1–77 and 106–447 were generated using the primer pairs AtTic40-F1(KpnI)/mAtTic40(SpeI)-R (5¢-AAATATACTAGTAAAAGCTTTGCTTC-3¢) and TMAtTic40(SpeI)-F (5¢-TCAACCACTAGTTCACCACTTTTCTG-3¢)/AtTic40R2(XhoI). For generation of AtTic40DS/P-ARC6, a PCR fragment coding for residues 586–614 of ARC6 was amplified with the primers ARC6(SpeI)-F (5¢-TTGAAACTAGTGATTATGCAATTCGAGCTG-3¢) and ARC6(SpeI)-R (5¢-ACACTACTAGTCTTTAACATATCAGCAACGG-3¢), digested with SpeI, and ligated into the AtTic40DSP construct cut with SpeI. The amino acid exchanges caused by the introduction of restriction sites were restored using the GeneTailor Site-Directed Mutagenesis System (Invitrogen). The GeneTailor Site-Directed Mutagenesis System was used to generate the deletion mutants AtTic40D77–82, AtTic40D83–88, AtTic40D89–97, and AtTic40D98–105, with wild-type AtTic40 in pDEST14 as a template. Primers were designed according to the manufacturer’s recommendations.

Preparation of AtTic40–GFP fusion proteins The cDNA encoding wild-type AtTic40 was used as the template to generate most of the AtTic40–GFP fusion proteins. All PCR fragments were subsequently cloned into the vector pcDNA3.1/CT-GFP (Invitrogen). AtTic40 S/P TMD-GFP, encoding amino acids 1–128, was amplified using the primers AtTic40 S/P-GFP-F (5¢-ATGGAGAACCTTACCCTAGTT-3¢) and AtTic40-S/PTMD-GFP-R (5¢GTAA ATTTGAAGTTACATATGA-3¢). AtTic40 S/P-GFP was prepared using the primers, AtTic40 S/P-GFP-F (5¢-ATGGAGAACCTTACCCTAGTT-3¢) and AtTic40 S/P-GFP-R (5¢-TGGTTGATGAAGATGGTGGTGG-3¢). tpAtTic40–GFP was prepared using the primers AtTic40 S/P-GFP-F (5¢-ATGGAGAACCTTACCCTAGTT-3¢) and AtTic40-TPGFP-R (5¢-ATATACTTGCAAAAGCTTT-3¢). Using the template AtTic40 DS/P (construct previously described), AtTic40 TMD-GFP was prepared using the primers AtTic40 S/P-GFP-F (5¢-ATGGAGAACCTTACCCTAGTT-3¢) and AtTic40-S/PTMD-GFP-R (5¢-GTAAATTTGAAGTTACATATGA-3¢). The construct tpSS-GFP was prepared by using the pea cDNA encoding prSSU as the template, and with the following primers: tpSS-GFP-F (5¢-ATGGCTTCTATGATATCCTCT-3¢) and tpSS-GFP-R (5¢-GCACCTGCATGCACTTTACTCTTCC-3¢).

Isolation of pea chloroplasts Intact chloroplasts were isolated from 8- to 12-day-old pea seedlings or 10-day-old tomato seedlings and purified over a Percoll gradient as previously described (Bruce et al., 1994). Intact pea chloroplasts were reisolated and resuspended in import buffer (330 mM sorbitol, 50 mM HEPES/KOH, pH 8.0) at a concentration of 1 mg chlorophyll ml)1. Imports were performed as described by Bruce et al. (1994).

In vitro translation of precursor protein The precursor proteins used in this study were radiolabeled using [35S]-methionine and translated with TNT Coupled Reticulocyte Lysate System (Promega, http://www.promega.com) according to the manufacturer’s protocol.


Protein targeting to the IEM 837 Import assays, extraction, and fractionation experiments Import assays, extraction, and fractionation experiments were performed as previously described in Tranel et al. (1995).

Acknowledgements The authors would like to thank Karen Bird for her efforts in preparing this manuscript. This study was supported by NSF grant MCB-0316262 to JEF and KK.

Supplementary Material The following supplementary material is available for this article online: Figure S1. N-terminal deletion mutants are not mistargeted to the thylakoid membrane Figure S2. Deletions of sequence motifs N-terminal of the transmembrane domain impair insertion of Tic40 into the inner envelope membrane Figure S3. A modified time course of import protocol does not alter the conclusion that the bipartite transit peptide of AtTic40 contains stromal targeting information, but plays only a minor role in the retargeting of AtTic40 to the inner envelope membrane (IEM) This material is available as part of the online article from http:// www.blackwell-synergy.com

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