Cytosolic events involved in chloroplast protein targeting

Biochimica et Biophysica Acta 1833 (2013) 245–252

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Review

Cytosolic events involved in chloroplast protein targeting☆ Dong Wook Lee a, Chanjin Jung b, Inhwan Hwang a, b,⁎ a b

Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, 790-784, Pohang, Republic of Korea Division of Molecular and Life Sciences, Pohang University of Science and Technology, 790-784, Pohang, Republic of Korea

a r t i c l e

i n f o

Article history: Received 20 January 2012 Received in revised form 24 February 2012 Accepted 8 March 2012 Available online 16 March 2012 Keywords: Chloroplast targeting signal Cytosolic import factors Outer envelope membrane Chloroplast precursors in cytosol

a b s t r a c t Chloroplasts are unique organelles that are responsible for photosynthesis. Although chloroplasts contain their own genome, the majority of chloroplast proteins are encoded by the nuclear genome. These proteins are transported to the chloroplasts after translation in the cytosol. Chloroplasts contain three membrane systems (outer/inner envelope and thylakoid membranes) that subdivide the interior into three soluble compartments known as the intermembrane space, stroma, and thylakoid lumen. Several targeting mechanisms are required to deliver proteins to the correct chloroplast membrane or soluble compartment. These mechanisms have been extensively studied using purified chloroplasts in vitro. Prior to targeting these proteins to the various compartments of the chloroplast, they must be correctly sorted in the cytosol. To date, it is not clear how these proteins are sorted in the cytosol and then targeted to the chloroplasts. Recently, the cytosolic carrier protein AKR2 and its associated cofactor Hsp17.8 for outer envelope membrane proteins of chloroplasts were identified. Additionally, a mechanism for controlling unimported plastid precursors in the cytosol has been discovered. This review will mainly focus on recent findings concerning the possible cytosolic events that occur prior to protein targeting to the chloroplasts. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Despite the presence of a chloroplast genome, the majority of chloroplast proteins are encoded by the nucleus. These proteins are targeted to the chloroplasts after they are synthesized by cytosolic ribosomes [1,2]. The chloroplast contains six compartments: the outer envelope, inner envelope, intermembrane space, stroma, thylakoid membrane, and thylakoid lumen. With the exception of proteins that are targeted to the outer envelope of chloroplasts, most chloroplast proteins are imported into the stroma via an Nterminal targeting signal known as a transit peptide [1,2]. Cytosolic factors such as 14-3-3 protein, Hsp70, Hsp90 and FKBP bind to certain transit peptides and are thought to facilitate protein targeting to chloroplasts (Fig. 1) [3–5]. However, the functions of these cytosolic factors have not been clearly demonstrated in vivo. After navigating through the cytoplasm to chloroplasts, preproteins encounter the TOC (translocon at the outer envelope of chloroplasts)/TIC (translocon at the inner envelope of chloroplasts) complexes present in the outer and inner envelopes, respectively, for import into chloroplasts. During or after translocation, the transit

☆ This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. ⁎ Corresponding author at: Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, 790-784, Pohang, Republic of Korea. Tel.: + 82 54 279 2128; fax: + 82 54 279 8159. E-mail address: [email protected] (I. Hwang). 0167-4889/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2012.03.006

peptide is cleaved off by SPP (stromal processing peptidase) in the stroma, and the mature proteins are then released and folded in the stroma [1,2]. The events that occur at the TOC/TIC complexes and other chloroplast compartments have been extensively discussed in other literature [1,2,6–8]. This review will focus on the cytosolic events that are necessary for chloroplast targeting. 2. Cytosolic events required for protein targeting into interior regions of chloroplasts 2.1. Transit peptides and their sequence motifs for chloroplast targeting Proteins that are imported into chloroplasts contain an Nterminal cleavable signal sequence called a transit peptide [1,9,10]. However, unlike other targeting signals, the lengthy transit peptides of chloroplast proteins vary greatly and do not possess any consensus sequence [11]. To understand the nature of the sequence information encoded in the transit peptide, various approaches have been employed. These include amino acid composition analysis, deletion and substitution mutagenesis, and structural analysis of transit peptides. These studies have revealed that transit peptides exhibit a few characteristic features such as a high content of hydroxylated amino acids and alanines, lack of acidic amino acids, the presence of short sequence motifs, and a propensity to form α-helical structures in hydrophobic environments [11]. In addition, transit peptides exhibit an ability to bind to chloroplast-specific lipid MGDG and contain sequence motifs for interaction with Toc159 or Toc34 [11–13].

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Fig. 1. Overview of cytosolic events for the targeting of proteins with a transit peptide into chloroplasts. The N-terminal transit peptides of chloroplast precursor proteins are recognized by several cytosolic factors posttranslationally. Cytosolic Hsp70 and Hsp90 were identified as protein factors that bind to chloroplast precursors in vitro. The transit peptides of some chloroplast proteins such as RbcS and OE23 contain consensus motifs for cytosolic 14-3-3 proteins, which escort preproteins to Toc34. The complex formation of precursors with 14-3-3 and Hsp70 may increase their import competency. On the other hand, Toc159 is known to be a primary receptor for preproteins, although it is not known whether a cytosolic factor (denoted as X) is involved in this pathway. In the cases of non-photosynthetic plastid proteins, such as the E1α subunit of pyruvate dehydrogenase (E1α), proteins are imported into chloroplasts through TOC with Toc132/120. For this pathway, it remains to be elucidated whether any specific cytosolic import factor (denoted as Y) is required. Finally, in the cytosol, Hsp90 binds to the transit peptide and the N-terminal region of the mature portion, and escorts preproteins to Toc64. This is followed by translocation through the TOC/TIC complex for import into chloroplasts.

However, not all transit peptides exhibit the same characteristic features. Recently, to characterize transit peptide sequences, a combined approach was employed using an in vivo targeting experiment in Arabidopsis protoplasts along with bioinformatics [10]. Hierarchical clustering using the transit peptides of 208 authentic chloroplast proteins showed that these 208 transit peptides can be grouped into multiple subgroups. These subgroups are named RbcS (rubisco small subunit), Cab (chlorophyll a/b-binding protein), BCCP (biotin carboxyl carrier protein), DnaJ-J8, PORA (NADPH:protochlorophyllide oxidoreductase A), TOCC (tocopherol cyclase), and Glu2 (ferredoxin-dependent glutamate synthase-2). Interestingly, experimentally identified functional sequence motifs were significantly identical or overlapped with the motifs identified by bioinformatics analysis. Moreover, the accuracy of distinguishing chloroplast proteins from non-chloroplast proteins greatly increased when the transit peptides of chloroplast proteins were considered to contain multiple subgroups with distinctive sequence motifs [10]. These results raise an intriguing question of how these diverse transit peptides can be imported by a few import receptors. One possibility is that import receptors such as Toc159 may have great flexibility for recognizing multiple subgroups of transit peptides. Consistent with this hypothesis, both RbcS and Cab proteins, whose transit peptides are quite different, are imported into chloroplasts in a Toc159-dependent manner [10,14]. Detailed analyses of several transit peptides revealed multiple sequence motifs that are involved in steps such as the binding of preproteins to chloroplasts and receptors, and translocation across the chloroplast envelopes during protein import [9,10,15]. Although much sequence dissimilarity was observed among the different subgroups of transit peptides, certain common features were identified. As an example, the hydrophobic residues located within the first 10-amino acid segment of the transit peptides of RbcS, PORA, TOCC,

and DnaJ-J8 were identified as important for targeting to chloroplasts. However, it is not known whether a cytosolic factor recognizes the Nterminal hydrophobicity of the transit peptide [9,10]. The transit peptides of certain chloroplast proteins such as RbcS, OE23 (oxygenevolving complex 23), OE33 (oxygen-evolving complex 33), and the mature region of PORA possess motifs for interaction with a cytosolic protein 14-3-3 [3,16]. 2.2. Cytosolic factors for chloroplast precursors Organellar proteins can be delivered either by individual direct targeting from the cytoplasm to the target organelles or en mass via vesicle trafficking from a donor compartment to an acceptor compartment. The direct targeting approach is employed for the ER, mitochondria, nucleus, chloroplasts, and peroxisomes, whereas vesicle trafficking is utilized for the Golgi apparatus, plasma membrane, vacuole, protein storage vacuole and endosomes as well as secretion into the apoplasts of plant cells. In general, the direct targeting of organellar proteins from the cytoplasm is mediated by specific cytosolic factors that recognize the targeting signals. For example, the hydrophobic signal sequences of ER proteins are recognized by the signal recognition particle (SRP) cotranslationally or TRC40/Get3 posttranslationally, depending upon the signal sequence position [17–19]. The NLS (nuclear localization signal) of nuclear proteins is recognized by importins in the cytosol [20,21]. The PTS-1 (peroxisomal targeting signal-1) at the Cterminal end of peroxisomal matrix proteins is recognized by cytosolic PEX5, whereas the N-terminal PTS-2 (peroxisomal targeting signal-2) is recognized by cytosolic PEX7 [22,23]. Some cytosolic factors have also been demonstrated to participate in protein targeting to mitochondria. For example, mitochondrial targeting stimulating factor (MSF), which is a heterodimer composed of large (32 kD) and small (30 kD)

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subunits, is involved in mitochondrial protein targeting through its interaction with the basic amino acid residues in the presequences [24,25], and ribosome-associated nascent polypeptide-associated complex (NAC) facilitates the import of mitochondrial proteins when preproteins are still bound by ribosomes [26]. Finally, arylhydrocarbon receptor-interacting protein (AIP), which is an FK506-binding protein homologue, was first identified as a Tom20-binding cytosolic protein; however, further study identified it as critical for mitochondrial protein import by preventing the aggregation of substrate proteins. AIP specifically binds to mitochondrial preproteins, as well as Hsc70 and Tom20, to form a ternary complex that maintains import competency [27]. However, these proteins may not be equivalent to cytosolic receptors such as importin and PEX5 in the nuclear and peroxisomal targeting pathways, respectively. In most of these pathways, molecular chaperones such as Hsp70/Hsc70 or Hsp90 also participate in the targeting processes [27,28]. It remains unclear whether any specific cytosolic targeting factors or mediators exist for protein targeting to the chloroplast. Several proteins that interact with transit peptides have been identified (Fig. 1). 14-3-3 binds to specific transit peptides of chloroplast precursors [3]. The chloroplast preproteins synthesized in wheat germ extract interact with 14-3-3 dimer and cytosolic Hsp70 to form a complex, designated the guidance complex, in vitro. When the precursors are in this complex, they are more efficiently imported into chloroplasts compared to the precursors alone in an in vitro import system [3]. Interestingly, animal MSF of mitochondrial targeting is a 14-3-3 protein and has been shown to facilitate the targeting of mitochondrial preproteins [24]. Serine or threonine residues in the 14-3-3 binding motif are phosphorylated by cytosolic protein kinases [29]. Recent studies identified STY8 (AT2G17700), STY17 (AT4G35780), and STY46 (AT4G38470) as cytosolic protein kinases that phosphorylate the transit peptides [30,31]. In studies, the transit peptides of chloroplast preproteins were phosphorylated, while plant mitochondrial or peroxisomal preproteins were not. This indicates that 14-3-3mediated protein targeting is specific to the chloroplast preproteins in plant cells [3]. Phosphorylated preproteins bind to receptors on the outer envelope of chloroplasts, although the transit peptides are dephosphorylated prior to translocation into the chloroplasts. However, a contradictory report demonstrates that the formation of a guidance complex is not crucial to chloroplast targeting [32]. For example, the transit peptides of RbcS and two amino acyl tRNAsynthetases that had alanine substitution of the phosphorylation sites can deliver GFP efficiently into chloroplasts both in vitro and in vivo [32]. The majority of these interacting proteins were identified in vitro using in vitro-translated precursors. Thus, the physiological relevance of these proteins during protein import into chloroplasts needs to be tested in vivo using approaches such as genetics. In addition to the cytosolic proteins involved in the guidance complex formation, Hsp90 was identified as a cytosolic molecular chaperone involved in targeting a different set of chloroplast proteins (Fig. 1) [4]. The guidance complex is thought to dock onto Toc34 in a phosphorylationdependent manner. In contrast, the Hsp90-preprotein complex docks onto Toc64, a different import receptor, through interaction between the TPR domain of Toc64 and a C-terminal conserved motif of Hsp90. Subsequently, preproteins are transferred from Toc64 to Toc75, the import channel, and translocated through the channel of Toc75 [4]. Although the guidance complex formation is specific to chloroplast targeting, the Hsp90–Toc64 system somewhat parallels the Hsp70/Hsp90–Tom70 system involved in mitochondrial protein import in yeast and humans [28]. Interestingly, plant mitochondria do not contain a known homologue of Tom70. Instead, mtOM64, a Toc64 homologue containing a C-terminal TPR domain, is involved in importing a set of proteins into mitochondria [33]. However, genetic studies revealed that Toc64 is not essential for protein import into chloroplasts. In studies of Arabidopsis thaliana and the moss Physcomitrella patens, knockouts of all Toc64 isoforms were not noticeably defective in chloroplast biogenesis or protein import into

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chloroplasts [34,35]. This raises an intriguing possibility that the lack of Toc64 may be compensated by an alternative pathway. To date, two different pathways for protein import into chloroplasts have been identified in Arabidopsis [1,2,8,15]. One is the Toc159/Toc33-dependent pathway for photosynthetic chloroplast proteins (e.g. RbcS), and the other is Toc132 or Toc120/Toc34-dependent pathway for housekeeping plastid proteins (e.g., the E1α subunit of pyruvate dehydrogenase) (Fig. 1). Although transit peptides are directly recognized by Toc159 or Toc132, little is known about the existence of specific factors (denoted as X and Y in Fig. 1) that facilitate these interactions. A previous report identified multiple sequence motifs in the RbcS transit peptide as motifs that specify the Toc159dependent pathway [15]. One possibility is that a certain cytosolic factor binds to these sequence motifs and mediates the interaction between the transit peptide and Toc159. The cytosolic proteins that are identified as transit peptide binding proteins (Hsp70, Hsp90 and 14-3-3) are members of well-known protein families whose functions are not directly related to protein targeting to chloroplasts [36,37]. Hsp70 and Hsp90 are involved in various protein biogenesis-related processes including translation, protein quality control and protein targeting to organelles [36,38–40]. In addition, 14-3-3 proteins are known to function as regulators of a wide range of biological processes through direct interactions with numerous target proteins [24,37,41]. In addition to protein import into chloroplasts, these target proteins are involved in protein shuttling between the nucleus and cytoplasm and protein import into mitochondria. Thus, unlike protein targeting to the ER, nucleus or peroxisomes, specific cytosolic factors responsible for targeting transit peptide-containing proteins to chloroplasts have not been identified. As an intriguing possibility, the targeting of transit peptide-containing proteins may not require specific targeting factors. Another possibility is that the specific targeting factor exists but remains to be identified. As evidence for the latter possibility, fusion constructs containing both an N-terminal transit peptide (60 or 70 amino acids long) and C-terminal PTS-1 were almost exclusively targeted to chloroplasts. This indicates that the N-terminal transit peptide was able to override peroxisomal targeting, which is mediated by a cytosolic PEX5 import receptor [42,43]. Additional evidence for the presence of a cytosolic sorting factor comes from an in vitro study that demonstrated that purified plant mitochondria are able to import chloroplast precursors in vitro with the same efficiency as chloroplasts [44]. In addition to soluble stromal proteins, many membrane proteins are imported into chloroplasts and inserted into the inner envelope or thylakoid membranes [45,46]. These proteins present a different challenge in protein targeting to chloroplasts. The membrane proteins imported into the chloroplasts contain the N-terminal transit peptide along with variable numbers of hydrophobic TMDs for insertion into membranes within the chloroplast. For example, a maltose transporter localized to the inner envelope membrane contains 9 TMDs [47]. Thus, intriguing questions are how these TMD-containing proteins escape SRP-mediated cotranslational targeting to the ER and how these hydrophobic TMDs are maintained in the cytosol without forming non-specific aggregates. These results strongly suggest that certain cytosolic factor(s) may function in guiding these targets to the chloroplasts but not the ER. Currently, the cytosolic processes for the chloroplast membrane proteins are largely unknown at the molecular level. 2.3. Unimported chloroplast precursor responses In the cytosol, unfolded proteins are generated from various sources. Translation of new proteins may generate unfolded proteins due to the inability of certain proteins to fold spontaneously into a mature form after translation. Various abiotic stresses such as UV and heat cause the denaturation of folded proteins [38–40]. Unfolded proteins in the cytosol are prone to form non-specific aggregates that

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can potentially threaten cell viability [38–40]. Therefore, the level of unfolded proteins should be maintained at minimal levels. Indeed, a cytosolic protein quality control mechanism exists in the cell and handles unfolded proteins in two different ways: assisting correct folding and removing unfolded proteins through 26S proteasomemediated degradation after polyubiquitination [39,40]. Another compartment well known for protein quality control is the ER. The protein quality control mechanism in the ER has been extensively studied [48,49]. The nascent proteins targeted to the ER cotranslationally must be folded correctly prior to transport to other endomembrane compartments via lipid vesicle-mediated protein trafficking. Proteins imported into chloroplasts are likely unfolded prior to their import across the two envelope membranes of the chloroplasts [50]. This is similar to protein import into mitochondria, although in contrast to protein targeting into the ER, nucleus and peroxisomes [27]. During nuclear targeting, proteins are transported through NPCs (nuclear pore complexes) that are tightly embedded in the nuclear envelopes [51]. Since the pore size is flexible and can accommodate very large particles, protein targeting to the nucleus does not require protein unfolding. Similarly, targeting peroxisomal matrix proteins across the peroxisomal membrane does not require unfolding [52], yet the translocation mechanism appears to be different from that of nuclear proteins. For the translocation of large, folded, or even oligomeric peroxisomal matrix proteins, a dynamic transient pore is assembled at the peroxisomal membrane by a proteinaceous peroxisomal importomer composed of a PTS1 receptor, PEX5 and PEX14. This pore creates a gated ion-conducting channel of 9 nm to accommodate the large peroxisomal cargoes [53]. During protein targeting to the ER, a different mechanism is employed: proteins are translocated into the ER cotranslationally [17]. SRP binding to the signal sequences of ER proteins causes translational pausing. The ribosome/nascent polypeptide/SRP/mRNA complex is recruited to the ER membrane through interactions between SRP and SRP receptors. Ribosomes on the ER membrane connect with the import channels composed of Sec61. Subsequently, translation resumes and nascent proteins are directly translocated through the import channels [54]. In this manner, unfolded ER proteins are not exposed to the cytosol. In contrast, the translation of chloroplast precursors is completed in the cytosol before import into chloroplasts. Thus, these precursors exist as unfolded proteins in the cytosol. The unfolded chloroplast precursors in the cytosol may potentially harm cell viability. Thus, to prevent the aggregate formation of unfolded precursors, the precursors are thought to associate with chaperones in the cytosol. Indeed, when chloroplast precursors were translated in vitro, they associate with Hsp70 [3]. Similarly, mitochondrial precursors also exist in association with various chaperones such as Hsp70 family member Hsc70 and DnaJ homologues dj2 and/or dj3 in mammals, and Hsp70 family member (Ssz1p) and a DnaJ homologue (zuotin) in yeast [55–57]. Despite these measures, it is likely that the potential danger of unfolded proteins in the cytosol remains. In particular, when the expression levels of chloroplast proteins do not match the capacity for protein import into the chloroplasts, unfolded chloroplast precursors may accumulate to high levels, leading to the formation of non-specific cytotoxic aggregates in the cytosol. However, the questions of whether and how unfolded chloroplast precursor levels are controlled in the cytosol have received little attention. One of the main reasons could be the use of an in vitro import system to study the mechanism of protein import into chloroplasts where in vitro translated precursors were mixed with purified chloroplasts in vitro [58]. Recently, Lee et al. [14] reported that unimported chloroplast proteins are removed by cytosolic Hsc70-4 and CHIP-mediated 26S proteasomal degradation (Fig. 2). Interestingly, two regions of the transit peptide of RbcS and Cab contain sequence motifs for Hsc70-4 binding for degradation. The sequence motif VASPA is located at the N-terminus of RbcS and was identified as an Hsc70-4 binding motif.

Based on previous sequence analyses, transit peptides were proposed to contain multiple Hsp70 binding sites [59]. However, it is unclear if these potential Hsp70 binding sites are the same as the sites for the Hsc70-4-mediated unimported chloroplast precursor response. Since these motifs are present in the transit peptide, an intriguing possibility is that during the evolution of the protein import mechanism for chloroplasts, the protein import systems also incorporated a mechanism by which excess chloroplast precursors could be eliminated through 26S proteasome-mediated protein degradation. Indeed, in the ppi2 mutant, a knockout of AtTOC159, which encodes a major import receptor for preproteins at the outer envelope of chloroplasts, the expression levels of both Hsc70-4 and AtCHIP E3 ligase are highly upregulated. This indicates that plant cells contain a mechanism to sense the level of unimported precursors and this signal is used to activate genes involved in the removal of unimported precursors from the cytosol [14]. AtCHIP was also identified as binding to Cab and RbcS using yeast two hybrid screening [60]. In addition, AtCHIP is involved in the degradation of ClpP4 and FtsH1, subunits of chloroplast Clp protease and FtsH protease, respectively. In this manner, AtCHIP affects chloroplast function [61,62]. The importance of this unimported chloroplast precursor response was demonstrated by Hsc70-4 RNAi mutant plants. These plants exhibit a seedling lethal phenotype, presumably due to cytotoxic cell death caused by unimported cytosolic chloroplast proteins [14]. During embryogenesis, every cell plays a crucial role in pattern formation. Therefore, the death of even a single cell can result in severe developmental defects. Comparative transcriptomic and proteomic analyses using wild-type and ppi2 plants support the presence of an unimported chloroplast precursor response in plant cells [63]. Interestingly, these cytosolic unimported chloroplast proteins are modified by N-terminal acetylation at the amino acid position 2. This modification is utilized for 60–90% of all proteins in eukaryotic cells. In yeast, this modification is a degradation signal for the ubiquitin–proteasome system [64]. Thus, it is conceivable that the N-terminal acetylation functions as a signal for preprotein degradation through the Hsc70-4 and CHIP-mediated 26S proteasomal pathway. Notably, a cytosolic N-acetyltransferase is known to play a role in photosynthesis efficiency. A mutation at AtMAK3, the gene that encodes the N-acetyltransferase, causes a minor defect in the formation of thylakoid multiprotein complexes. This potentially implicates N-terminal acetylation of chloroplast precursors in efficient chloroplast biogenesis [65]. However, the manner in which cytosolic proteins affect the thylakoid multiprotein complexes remains unclear. 3. Cytosolic events required for targeting to the outer envelope of chloroplasts 3.1. Targeting signals of chloroplast outer membrane proteins Outer envelope membrane proteins are classified into two groups based on structure: α-helical TMD proteins and β-barrel proteins [66]. These proteins are thought to be targeted to chloroplasts by different mechanisms. Unlike proteins imported into chloroplasts, these outer envelope membrane proteins do not contain a cleavable transit peptide, with the sole exception of Toc75 [1,66]. Many different targeting pathways have been proposed for individual membrane proteins containing α-helical TMD. For example, E6.7 and OEP21 are targeted spontaneously to purified pea chloroplasts in vitro. In contrast, the targeting of OEP14 and OEP64/Toc64 is mediated in an NTP-dependent manner [66]. OEP14 and OEP7 are inserted into liposomes at a higher efficiency when in the presence of Toc75 in vitro. Thus, Toc75 is proposed to serve as a receptor for the insertion of certain membrane proteins into the chloroplast outer envelope membrane [67]. However, the targeting mechanisms and signals responsible for sending β-barrel proteins into the outer envelope membranes are less understood. One exception is the import channel protein Toc75, which utilizes the general import pathway using a

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Fig. 2. Quality control of unimported chloroplast precursors. For efficient protein import into chloroplasts, several requirements exist. These include a functional targeting signal (transit peptide), available cytosolic factors, and translocation machineries. When chloroplast precursor proteins are not efficiently imported, they are recognized by Hsc70-4, one of the cytosolic Hsc70s, and CHIP, an E3 ligase, for degradation through 26S proteasome in the cytosol. Furthermore, the high levels of unimported chloroplast precursors activate a signaling pathway that leads to the induction of Hsc70-4 and CHIP, which are involved in proteasomal degradation of aggregated proteins.

bipartite transit peptide at the N-terminus [68]. After import into the stroma, the bipartite transit peptide is cleaved off sequentially by SPP (stromal processing peptidase) and Plsp-I (plastid type I signal peptidase). The mature Toc75 is then translocated in a retrograde fashion to the outer envelope membrane where it is inserted [69]. However, the details of the targeting and insertion of Toc75 are not fully understood. In general, any protein that contains an N-terminal or internal TMD is a substrate for SRP and thereby targeted to the ER cotranslationally. Thus, the targeting of TMD-containing proteins to the chloroplasts poses an intrinsic problem: these proteins must first evade the SRP-mediated ER targeting. This is also true for the mitochondrial outer envelope membrane proteins that contain an N-terminal or internal TMD [70]. The targeting signal for chloroplast outer envelope membrane proteins with an N-terminal TMD is clearly defined. Both the TMD and positively charged amino acid residues (three to five lysine and arginine residues) located at the C-terminal flanking region of the TMD, named the CPR (C-terminal positively-charged flanking region), are required for protein targeting to the chloroplast outer envelope membrane and, thus, constitute the targeting signal [71,72]. Similarly, the TMD and CPR are also critical for the targeting of membrane proteins to mitochondria in both animal and plant cells [70,72]. When the CPR is substituted with neutral amino acids, the chloroplast membrane proteins are targeted to the ER [73]. This indicates that although the TMD of chloroplast membrane proteins can be recognized by SRP, the CPR prevents SRP from recognizing the TMD. This results in the inhibition of ER targeting. However, the presence of CPR is not sufficient to target a membrane protein to the chloroplast. Many ER proteins also contain a CPR-like motif. In ER membrane proteins, the positive charges that flank the TMD play an important role in determining membrane protein topology, as summarized by the positiveinside rule. Another important characteristic for the chloroplast targeting of membrane proteins is the hydrophobicity value of the TMDs. In general, chloroplast proteins contain a TMD with a lower hydrophobicity value than ER proteins. In previous studies on mitochondrial outer envelope membrane proteins, the TMDs had moderate hydrophobicity values [73,74]. When various hydrophobicity scales were employed to investigate the hydrophobicity differences in the TMDs of ER and chloroplast proteins, the Wimley and White hydrophobicity (WWH) scale [75] produced the most clear separation between them. The majority (over 85%) of chloroplast proteins have a TMD with a hydrophobicity value below 0.4 using the WWH

scale. In contrast, the majority (over 85%) of ER proteins have a TMD with a hydrophobicity value greater than 0.4 using the WWH scale [73]. In particular, to target proteins with a CPR-like motif to the ER, a TMD with a hydrophobicity value greater than 0.4 on the WWH scale is needed. One possible explanation would be that higher TMD hydrophobicity may increase SRP binding affinity to the TMD. This could perhaps overcome the inhibitory effect of the CPR on SRP binding to the TMD. This characteristic hydrophobicity value of the TMDs of chloroplast membrane proteins is also exhibited by the TMDs of mitochondrial proteins. Thus, the targeting signals, the CPR and the low hydrophobicity value of the TMD are common to both chloroplast membrane proteins and mitochondria membrane proteins. This raises another critical and unique question in plant cells concerning the determination of membrane protein targeting specificity for chloroplasts and mitochondria. The means for distinguishing these proteins is currently unknown. In addition, the tail-anchored chloroplast outer envelope membrane proteins also contain short flanking regions after the TMD, and their targeting specificities depend upon the characteristics of their flanking sequences in plant cells [76]. 3.2. Cytosolic receptor/chaperone for targeting to chloroplast outer membrane Ankyrin repeat protein 2A (AKR2A) was identified as a cytosolic factor for targeting N-terminal TMD-containing (signal-anchored) proteins to the chloroplast outer envelope membrane (Fig. 3) [77]. AKR2A was identified in a yeast two hybrid screen using chloroplast outer envelope membrane protein OEP7 as bait [77]. AKR2A is a member of a large family of proteins containing an ankyrin repeat domain, which mediates protein–protein interactions [78]. Interaction of AKR2A with OEP7 depends on the presence of both the TMD and CPR. Accordingly, AKR2A does not possess a high affinity for membrane proteins that do not contain the CPR and thus are targeted to other organelles such as the ER. AKR2A and its close homologue AKR2B facilitate the targeting of signal-anchored proteins to the chloroplast outer envelope membrane in protoplasts (Fig. 3). In addition, AKR2A exhibits chaperone activity towards chloroplast outer envelope membrane proteins, which may be important for the posttranslational targeting of membrane proteins. Indeed, similar activity was observed with TRC40 and Get3, proteins involved in the posttranslational targeting of tail-anchored ER proteins in animal or yeast cells,

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respectively. The binding of TRC40 and Get3 to the TMD prevents non-specific aggregate formation that might result from the hydrophobic TMD [17,79]. The importance of AKR2s-mediated protein targeting to chloroplasts was demonstrated using AKR2A RNAi plants that exhibit a severe albino phenotype with decreased amounts of various chloroplast proteins [77]. In addition to the N-terminal signal-anchored chloroplast proteins, tail-anchored chloroplast proteins such as atToc33/34 and a putative tail-anchored protein, OEP9, also interact with AKR2A [77,80]. However, it remains unknown whether AKR2s also function in the targeting of these proteins. Moreover, the mechanisms underlying chloroplast targeting of these tailanchored proteins are still unclear. In the case of OEP9, which has a single TMD in the internal region and a C-terminal hydrophilic sequence comprising 32 amino acids, chloroplast targeting depends on the existence of both the TMD and C-terminal flanking regions, in which the net charge and/or charge distribution may be important for determining the targeting specificity [80]. However, the mechanism by which AKR2A binds to the internal TMD of OEP9 and delivers it to chloroplasts remains unknown. Chloroplast targeting of Toc33/ 34, which has its TMD at the C-terminal region, depends on the existence of an almost complete protein sequence rather than just the TMD and its flanking region; this is different from the targeting of signal-anchored proteins and OEP9, raising the possibility that chloroplast targeting of tail-anchored proteins may not solely depend on AKR2A [80]. In ER, nuclear, and peroxisomal targeting, where the targeting factors have been identified, multiple cofactors are involved in the targeting processes [17,23,51,52]. Searches for an AKR2 cofactor involved in protein targeting to chloroplasts led to the identification of Hsp17.8 as a binding partner of AKR2A (Fig. 3) [81]. Hsp17.8 is a member of the small heat shock proteins (sHsps) that play critical roles in various physiological processes, particularly in folding denatured proteins that are generated under various stress conditions [82]. In this process, sHsps are assembled into high molecular weight complexes with as many as 32 subunits. By contrast, Hsp17.8 binds to chloroplasts as a dimer. Thus, when Hsp17.8 serves as a cofactor of AKR2s during protein targeting to the chloroplasts, it may behave differently from sHsps that function in protein quality control under stress conditions.

Interestingly, Hsp17.8 also binds to chloroplasts in vitro. Through these interactions, Hsp17.8 facilitates AKR2A-mediated targeting of OEP7:GFP to the chloroplasts. Together, these results strongly suggest that Hsp17.8 functions as a cofactor of AKR2A during protein targeting to the chloroplast outer envelope membrane [81]. However, it is not known whether Hsp17.8 is the only member of the sHsp family involved in protein targeting to the outer envelope membranes. Other sHsps may function in protein targeting to the outer envelope membrane as another sHsp (Hsp17.4) was also shown to interact strongly with AKR2A. In understanding the detailed mechanism of AKR2s-mediated protein targeting to chloroplasts, many questions remain. One of which is how AKR2s capture their cargo in the cytosol. The N-terminal domain of AKR2A is involved with binding cargo proteins [77], yet how and when the AKR2s capture their cargo proteins in the cytosol is still unknown. The AKR2s-mediated chloroplast targeting of signal-anchored proteins is similar to SRP-mediated targeting to the ER with respect to the targeting signal position. This raises the possibility that AKR2s may capture cargo proteins at the ribosomes during translation. For cotranslational ER targeting, SRP captures cargo from ribosomes during translation via an interaction between the M domain of SRP54 and ribosomal protein Rlp23a located near the ribosomal exit tunnel [83,84]. For tail-anchored ER proteins, the targeting signal is located at the C-terminal end of the protein and, thus, it does not appear until the completion of translation. Interestingly, the BAG6 complex binds to ribosomes. This complex is composed of BAG6, TRC35 and UBL4A, and these proteins are thought to function as pre-targeting cofactors for substrate loading onto TRC40 [79,85,86]. In addition, Get4 and Get5, the yeast homologues of TRC35 and UBL4A, respectively, interact weakly with ribosomes [87]. This indicates that the tail-anchored ER proteins may also be captured at ribosomes. Thus, one possibility is that AKR2 may interact with ribosomes directly or through a binding factor to capture the cargo proteins. Another important question in AKR2s-mediated protein targeting to chloroplasts is how the AKR2s are recruited to chloroplasts. The C-terminal ankyrin repeat domain of AKR2A is able to bind purified chloroplasts [77,81]. In addition, as described above, Hsp17.8 also helps in the AKR2A binding to the chloroplast outer envelope membranes [81].

Fig. 3. Sorting and targeting of chloroplast outer envelope proteins in the cytosol. SRP, the cytosolic carrier for N-terminal TMD-containing ER proteins, recognizes ER proteins via binding to the hydrophobic TMD during translation. AKR2 functions as a cytosolic carrier for N-terminal signal-anchored proteins that are targeted to chloroplast outer envelope membranes. Binding of AKR2 to the membrane proteins requires both the TMD and its C-terminal positively charged flanking region (CPR) for chloroplast outer envelope membrane proteins. When AKR2 captures the cargo proteins and how AKR2 is recruited to the chloroplast outer envelope membranes remain unknown. Small heat shock protein Hsp17.8 functions as a cofactor for AKR2-mediated chloroplast targeting. Both AKR2 and Hsp17.8 bind to the chloroplast outer envelope membrane. In addition, it is not known which factor in the chloroplast outer envelope functions as a docking site. Furthermore, it is unclear whether any cytosolic carrier protein is required for the targeting of signalanchored proteins to the outer envelope membranes of mitochondria.

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However, even in the absence of Hsp17.8, AKR2A binds to purified chloroplasts. This raises the possibility that AKR2A may directly interact with chloroplasts. In ER targeting, the SRP receptor is necessary for recruiting SRP to the ER membrane [88]. In addition, for the ER targeting of tail-anchored proteins, ER membrane proteins Get1 and Get2 interact with Get3 and may play a role in recruiting Get3 to the ER membranes [19,79]. Thus, ER membrane proteins play a critical role in recruiting cytosolic targeting factors to the ER membrane as part of the ER targeting mechanism. This strongly suggests that certain factors may exist at the chloroplast envelope membrane for AKR2 recruitment. The import channel Toc75 plays a role in the insertion of OEP14 into the chloroplast outer envelope membrane [67]. Toc75 may function as the receptor for AKR2s at the envelope membrane. However, it is equally possible that Toc75 may assist in the insertion of membrane proteins into the outer envelope membrane rather than recruiting the AKR2s to the chloroplasts. 4. Concluding remarks and future directions Recent findings have significantly advanced current knowledge of the cytosolic events required for protein targeting to chloroplasts. However, these findings uncover new questions regarding the cytosolic process involved in chloroplast targeting. The entire process of protein targeting to the ER can be divided into three steps: the recognition of ER proteins during and/or after translation, targeting of cytosolic carriers bound with cargo proteins to the ER membrane, and release of cargo proteins from the cytosolic carrier and their insertion into the membranes. The big question would be whether the steps for protein targeting to the ER are also applicable to chloroplasts. The involvement of AKR2 with targeting signal-anchored proteins to chloroplasts strongly suggests this possibility. If protein targeting to the chloroplast occurs similarly to that for ER proteins, the second question would be: what is the carrier for chloroplast proteins and how does sorting of chloroplast proteins occur in the cytosol? AKR2 is identified as a carrier protein for signal-anchored chloroplast proteins. However, any carrier proteins for proteins containing a transit peptide remain elusive. During the sorting process, one fascinating aspect is that ER proteins appear to be captured at the ribosomes regardless of whether they are targeted to the ER cotranslationally or posttranslationally. Thus, it is of interest to determine whether the sorting of chloroplast membrane proteins in AKR2-mediated targeting occurs at the ribosomes. The third question asks: what is the receptor at the chloroplast envelope membrane? The Toc receptors, such as Toc159 and Toc33, at the envelope membrane act as the receptors for chloroplast proteins possessing a transit peptide. In contrast, the factor involved in the recruitment of AKR2s to the chloroplast membrane is not fully understood. The final question, unrelated to the import process, would be how the unfolded chloroplast proteins or TMD-containing chloroplast membrane proteins are managed by the cytosolic quality control mechanisms. A hint for this process comes from the Hsc70/CHIP-mediated unimported chloroplast precursor response. However, a thorough understanding of this question is far from complete. These questions could serve as the basis for further research into protein targeting to the chloroplasts and will provide important clues for understanding protein targeting and biogenesis of chloroplasts in plant cells. Acknowledgements This work was supported in part by grants from the World Class University Project (R31-2008-000-10105); from the National Research Foundation (20110000025), Ministry of Education, Science and Technology; and from IPET, the Ministry of Agriculture, Food and Fishery (Korea) (609004-05-3-HD240). Dong Wook Lee was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2011-355-C00148).

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