New Perspectives on Chloroplast Protein Import

New Perspectives on Chloroplast Protein Import Abstract Virtually all chloroplasts in extant photosynthetic eukaryotes derive from a single endosymbiotic event that probably occurred more than a billion years ago between a host eukaryotic cell and a cyanobacterium-like ancestor. Many endosymbiont genes were subsequently transferred to the host nuclear genome, concomitant with the establishment of a system for protein transport through the chloroplast double-membrane envelope. Presently, 2,000–3,000 different nucleus-encoded chloroplast proteins must be imported into the chloroplast following their synthesis in the cytosol. The TOC (translocon at the outer envelope membrane of chloroplasts) and TIC (translocon at the inner envelope membrane of chloroplasts) complexes are protein translocation machineries at the outer and inner envelope membranes, respectively, that facilitate this chloroplast protein import with the aid of a TIC-associated ATP-driven import motor. All the essential components of this protein import system seemed to have been identified through biochemical analyses and subsequent genetic studies that initiated in the late 1990s. However, in 2013, the Nakai group reported a novel inner envelope membrane TIC complex, for which a novel ATP-driven import motor associated with this TIC complex is likely to exist. In this mini review, I will summarize these recent discoveries together with new, or reanalyzed, data presented by other groups in recent years. Whereas the precise concurrent view of chloroplast protein import is still a matter of some debate, it is anticipated that the entire TOC/TIC/ATP motor system, including any novel components, will be conclusively established in the next decade. Such findings may lead to an extensively revised view of the evolution and molecular mechanisms of chloroplast protein import. Introduction While chloroplasts in plants and algae retain their own genome and synthesize approximately 100 proteins within the organelle, >2,000 different nuclear-encoded proteins are synthesized in the cytosol and then transported across the double-membrane chloroplast envelope (Fig. 1A) (Shi and Theg 2013a). Given these numbers, a system of post-translational protein transport into the chloroplast is absolutely essential for chloroplast biogenesis and function (Jarvis and López-Juez 2013, Nakai 2015a, Richardson et al. 2017). Fig. 1 View largeDownload slide Chloroplast protein import mediated by TOC and TIC translocons at the double envelope membranes of the chloroplast together with the associated ATP-driven import motor. (A) Overview of chloroplast protein import. (B) Comparison between the classical long-held model (adapted from Richardson et al. 2017) and the recently revised model for TIC translocon (Nakai 2015a, Nakai 2015b), and its associated putative ATP-driven import motor. For details, see text. Fig. 1 View largeDownload slide Chloroplast protein import mediated by TOC and TIC translocons at the double envelope membranes of the chloroplast together with the associated ATP-driven import motor. (A) Overview of chloroplast protein import. (B) Comparison between the classical long-held model (adapted from Richardson et al. 2017) and the recently revised model for TIC translocon (Nakai 2015a, Nakai 2015b), and its associated putative ATP-driven import motor. For details, see text. Overview of Chloroplast Protein Import In general, a nucleus-encoded chloroplast protein is synthesized as a larger pre-protein with an N-terminal amino acid sequence known as a transit peptide (Fig. 1A). Transit peptides carry intracellular targeting information and are recognized by receptors in the TOC (translocon at the outer envelope membrane of chloroplasts) such as Toc159 and/or Toc33/34, which are GTP-binding proteins exposed to the cytoplasm yet bound to the outer envelope membrane. There are differentially expressed isoproteins of these TOC receptors, namely Toc132/120/90, some of which are likely to contribute to enhanced import efficiency and/or selection of certain subsets of proteins, especially in non-photosynthetic plastids (Demarsy et al. 2014). Pre-proteins recognized by the TOC receptors are translocated across the outer envelope through the Toc75 channel, a β-barrel Omp85 family protein. Toc159, Toc33/34 and Toc75 form a rigid 800–1,000 kDa complex at the outer envelope membrane (Kikuchi et al. 2006, Chen and Li 2007). Pre-proteins that pass through the TOC channel into the intermembrane space are then consecutively translocated across the inner envelope membrane through the TIC (translocon at the inner envelope membrane of chloroplasts) complex (Fig. 1A). In terms of the energy used for chloroplast protein import, GTP binding to TOC receptors triggers the initiation of pre-protein translocation, whereas an ATP-derived pulling force produced from the stromal side of the inner envelope membrane appears to drive protein import (Shi and Theg 2013b). This pulling force probably arises from an import motor ATPase associated with the TIC complex. The Classical Model of Protein Translocation across the Chloroplast Inner Envelope Membrane Whereas a decent consensus regarding TOC complex composition has been reached among different research groups, the accepted molecular model for the TIC complex was called into question in 2013, when my group identified a novel 1 MDa inner envelope membrane protein complex that functions as a TIC complex (Fig. 1B) (Kikuchi et al. 2013). The identification of a directly participating TIC complex raised questions about what acts as the import motor, which should function in close concert with the TIC complex. Despite this discovery, numerous papers and textbooks still present the classical model of the TIC complex and its associated import motor (Vitale et al. 2015). In the latter model, Tic110, an inner envelope membrane protein exposed to the stroma, is thought to form at least part of the protein-conducting channel and to be present in complex with Tic40, another inner envelope membrane protein (Shi and Theg 2013a, Richardson et al. 2017). The model also assumes that Tic110/Tic40 bind various stromal molecular chaperones such as Hsp93/ClpC, Hsp70 and Hsp90C, which function as the above-mentioned ATP-driven import motor (Flores-Pérez and Jarvis 2013). Thus, this classical model is based on the long-held assumption that Tic110, as the first identified TIC protein, functions as a central component of the TIC complex (Fig. 1B) (Sjuts et al. 2017). However, there seems to be no definitive evidence that supports a direct interaction between Tic110 and translocating pre-proteins per se (Chou et al. 2006). Instead, recent studies have revealed some critical weaknesses underlying the classical TIC complex model, including a lack of solid biochemical evidence that Tic110 forms a complex with any other known TIC proteins, including its proposed partner protein Tic40 (Kikuchi et al. 2009, Kikuchi et al. 2013, Nakai 2015a). Recently, some evidence was presented to suggest that Tic110 might be included in the TOC/TIC supercomplex from a Blue Native–PAGE (BN–PAGE) separation of chloroplast lysates (Chen and Li 2017). However, it can be argued that the observed Tic110 signal near the 1 MDa location might correspond to a mere tailing from the abundant Tic110, the majority of which migrated to the 200–400 kDa area. Moreover, under the BN–PAGE conditions used, TOC and TIC separately migrated around the 1 MDa area (Kikuchi et al. 2006, Kikuchi et al. 2009, Kikuchi et al. 2013). Previous observations have similarly shown that Tic110 purified from Arabidopsis chloroplasts does not specifically associate with other proteins, but exhibits a smeared BN–PAGE migration profile that is clearly distinct from the profile of Tic40 (Kikuchi et al. 2013). Thus, additional experiments are required to show conclusively that Tic110 is a component of the TOC/TIC supercomplex. It was also reported that anti-Tic110 antibodies caused a supershift of translocation intermediates in BN–PAGE experiments (Chen and Li 2017). However, this observation may require some reinterpretation as slight retardations in the migration of translocation intermediates were also, to some extent, uniformly observed following the addition of different antibodies. Similarly, control IgGs in previous studies have also appeared to cause a certain degree of supershift (Chen and Li 2007). Given that the number of epitopes in the different antigen proteins available for the different antibodies is expected to vary significantly, it is expected that different antibodies would cause significant degrees of supershift, as we and others have demonstrated previously (Kikuchi et al. 2009, Kikuchi et al. 2013, Chen and Li 2007). A number of review articles, including those published recently, repeatedly claim that Tic110 acts in close contact with Tic20, a central channel component of the TIC complex (see below) (Fig. 1B) (Shi and Theg 2013a, Demarsy et al. 2014, Paila et al. 2015, Richardson et al. 2017). However, there seems to be no direct evidence in the literature for a specific interaction, either physically or functionally, between Tic110 and Tic20, or between Tic110 and the Tic20-containing complex (Kikuchi et al. 2009, Kikuchi et al. 2013). Thus, we argue for a re-evaluation of the classical TIC model regarding the molecular components required for pre-protein translocation across the chloroplast inner envelope membrane, and their interactions. The 1 MDa TIC Complex at the Inner Envelope Membrane Contains Tic20 at the Core with Three Additional Constituents, Tic56, Tic100 and Tic214/YCF1 Our research has focused on identifying the actual components of the TIC complex and its functionally associated ATP-driven import motor (Kikuchi et al. 2009, Hirabayashi et al. 2011, Kikuchi et al. 2013, Nakai 2015a). We first found a 1 MDa translocation intermediate complex at the inner envelope membrane using in vitro import experiments in combination with BN–PAGE (Kikuchi et al. 2009). We attempted to determine the central component of this 1 MDa complex and eventually identified the 20 kDa intrinsic inner envelope membrane protein Tic20. Tic20 is a long-known TIC complex candidate that possesses four transmembrane helices (Chen et al. 2002), and we and others found it to interact directly with translocating pre-proteins (Kouranov et al. 1998, Kikuchi et al. 2009). We subsequently demonstrated that the 1 MDa complex is composed of Tic20 and three additional previously uncharacterized proteins termed Tic56, Tic100 and Tic214 (Fig. 1B) (Kikuchi et al. 2013). These proteins are exclusively localized at the chloroplast inner envelope membrane and together form a rigid 1 MDa complex. Knockouts of Tic20, Tic56 or Tic100 cause an albino seedling-lethal phenotype. Furthermore, it should be noted that Tic214 is encoded by the enigmatic plastid YCF1 gene, an open reading frame encoding an essential protein (Boudreau et al. 1997, Drescher et al. 2000), which has raised interesting questions about the evolution of this complex (see below) (Kikuchi et al. 2013, Nakai 2015b). Strong evidence for these proteins functioning as an actual TIC complex was obtained by purifying the entire translocation intermediate complexes following in vitro import experiments (Kikuchi et al. 2013). Subsequent biochemical identification of specifically associated proteins showed that Tic20, Tic56, Tic100 and Tic214 were stoichiometrically purified with different pre-proteins and, importantly, with well-established TOC components, namely Toc33, Toc75 and Toc159 (Fig. 1A). Controversially, neither Tic110 nor Tic40 was found to be associated with the various analyzed translocating pre-proteins. This lack of association indicates that neither Tic110 nor Tic40 participates in pre-protein translocation involving the above-mentioned TOC/TIC proteins and also discounts the possibility that Tic110 or Tic40 forms an alternative translocon, as has been long proposed, to act in parallel at the inner envelope membrane (Sjuts et al. 2017). Hence, we concluded that the newly identified TIC complex consisting of Tic20, Tic56, Tic100 and Tic214 constitutes a main import route across the inner envelope membrane that, most importantly, functions in close concert with the well-established TOC complex (Fig. 1B) (Kikuchi et al. 2013). After reconstitution into lipid bilayers, the purified 1 MDa complex composed of Tic20, Tic56, Tic100 and Tic214 was demonstrated to form a membrane channel with which pre-proteins specifically interacted. Whereas Tic20 probably forms a primarily α-helical channel in the inner envelope membrane (Kovács-Bogdán et al. 2011, Campbell et al. 2014), precise roles for the other TIC complex constituents remain to be elucidated. An Alternative TIC System Functions in the Absence of, or in Parallel to, the 1 MDa TIC Complex A remarkable implication of the discovery of the Tic20/Tic56/Tic100/Tic214 TIC complex is the revelation that an alternative TIC system probably functions in the absence of, or possibly in parallel to, the main TIC complex (Fig. 2). The presence of an alternative TIC system is indicated by the phenotypes of Arabidopsis Tic20, Tic56 or Tic100 single knockout mutants (Hirabayashi et al. 2011, Kasmati et al. 2011). These mutants have albino seedling-lethal phenotypes, but can germinate and grow at least until the seedling stage. Thus, the plastids in these mutants, although lacking photosynthetic proteins, retain the ability to accumulate housekeeping plastid proteins. This residual protein import ability in the absence of the main TIC complex was experimentally demonstrated to be the result of elevated expression of a minor isoform of Tic20-IV present on chromosome IV. Further genetic analyses confirmed the presence of an alternative TIC system that involves Tic20-IV and is clearly independent from the main TIC complex (Kikuchi et al. 2013). Although minor, this alternative TIC system may play an important role in protein import, in particular in non-photosynthetic plastids (Fig. 2). It remains to be elucidated whether the minor TIC system includes any additional components. Further, it will be interesting to determine the evolutionary relationship between the two TIC systems. Fig. 2 View largeDownload slide Most plausible scenario for the occurrence of divergent TIC and associated motor systems during the evolution of photosynthetic eukaryotes. For details, see text. Fig. 2 View largeDownload slide Most plausible scenario for the occurrence of divergent TIC and associated motor systems during the evolution of photosynthetic eukaryotes. For details, see text. Evolutionary Aspects of the TIC Complex Given that a single endosymbiotic event gave rise to all photosynthetic eukaryote chloroplasts, and because the chloroplast protein import system is thought to have been established very early during subsequent evolutionary processes, it has been assumed that TIC complex components are highly conserved among all the chloroplast-containing lineages. However, upon identification of the novel 1 MDa TIC complex components, it was revealed that Tic20 is the only component shared amongst virtually all chloroplast- or plastid-containing lineages, whether photosynthetic or non-photosynthetic (Kikuchi et al. 2013). Surprisingly, while some early lineages including rhodophyta and glaucophyta possess Tic20, they do not appear to contain Tic56, Tic100 or Tic214/YCF1 (Fig. 2) (Nakai 2015b). The observed phylogenetic distributions suggest that the main TIC complex evolved drastically following the initial chloroplast endosymbiotic event and during the establishment of green lineages, and that the complete Tic20/56/100/214 complex seems to have been evolved when land plants appeared. Whereas Tic56, Tic100 and Tic214 are widely conserved among most seed plants, including dicots and monocots, as is Tic20, it is surprising to learn that a recently diverged unique class of monocot grasses appears to have lost all three of these essential TIC complex constituents, yet has retained Tic20 (Fig. 2) (Kikuchi et al. 2013, Nakai 2015b). Is it possible to propose an evolutionary relationship between the uncharacterized rhodophyta- or glaucophyta-type early TIC system, the uncharacterized grass-type TIC system and the novel 1 MDa TIC complex in other green lineages, as exemplified by that in Arabidopsis? Clearly, the functional TIC systems of other organisms should be characterized experimentally via biochemical and genetic methods rather than solely relying on predictions based on in silico analyses (de Vries et al. 2015). Nevertheless, we may initially predict a plausible evolutionary scenario for these TIC systems (Fig. 2) (Nakai 2015a, Nakai 2015b), in which Tic20 as the primary central core component for inner envelope membrane protein import is conserved among all chloroplast/plastid-containing lineages (van Dooren et al. 2008, Topel and Jarvis 2011). Since all grasses retain Tic20 orthologs, the grass-type TIC system may resemble the above-mentioned minor TIC system that we observed in Arabidopsis, which involves Tic20-IV but lacks other TIC complex constituents, namely Tic56, Tic100 and Tic214. In other words, in the green lineages, the main photosynthetic-type TIC has become essential to sustain the high demands of photosynthetic protein import into chloroplasts and as such has not been replaced by the minor non-photosynthetic-type TIC. Presumably, the evolution of the grass-type TIC from the minor non-photosynthetic-type TIC complex was favored over the main TIC. It should be noted that, in grasses, the process of chloroplast development from the non-photosynthetic plastid starting at the base of the leaf blade to the photosynthetic plastid occurs successively during leaf elongation, which thus can be observed as a gradient along their leaf blades. Therefore, continuous utilization of the non-photosynthetic type TIC complex during all stages of leaf/chloroplast development might have some advantages in grasses. Intriguingly, some of the grass-type TIC complex components, including Tic20, are well conserved among all chloroplast/plastid-containing lineages. This supports the possibility that these highly conserved TIC complex components must form the primary TIC complex and have still been retained in rhodophyta and glaucophyta (Fig. 2). Presumably, the key mechanisms of this TIC system are well conserved among all TIC systems irrespective of the different additional components in each system. The molecular identities and evolutionary histories of the actual TIC-associated import motors for these distinct types of TIC systems remain intriguing open questions. Supporting Evidence for the Novel TIC Complex In 2013, Schnell and colleagues reported the possible involvement of Hsp90C, a stromal Hsp90 family protein, in chloroplast protein import as a part of the import motor (see below) (Inoue et al. 2013). In that work, purification of translocation intermediates was carried out after in vitro import of a tagged form of an inner envelope membrane-localized model pre-protein. While Tic20, Tic56, Tic100 and Tic214 were not analyzed, considerable enrichment of Toc159 and Toc75 in the purified fraction was demonstrated. In contrast, neither Tic110, Hsp93, Hsp70 nor Hsp90C was detected at such enriched levels. Although the experimental approach of using an inner envelope membrane-localized model pre-protein for the purification of the translocation intermediates may need independent verification, their experimental data appear in part consistent with other data suggesting that Tic110 is not specifically associated with translocating pre-proteins (Kikuchi et al. 2013). Nevertheless, the data from Schnell and colleagues may potentially explain why Tic110, a fairly abundant inner envelope membrane protein that is readily detectable after purification procedures, has been repeatedly characterized as a specific pre-protein-interacting protein since its initial identification during the early days of this research field (Kessler and Blobel 1996). More detailed arguments on this have been described elsewhere (Nakai 2015a). In their series of papers using combined transcriptomic and proteomic analyses, Baginsky and colleagues reported the importance of Tic56 in the novel TIC complex for the accumulation of various chloroplast proteins (Köhler et al. 2015, Köhler et al. 2016, Agne et al. 2017). While there are slight discrepancies between their work and our own regarding the interpretation of the observed data, the fact remains that the complete absence of Tic56 results in severe defects in chloroplast protein import and is associated with various pleiotropic defects. Importantly, Baginsky and colleagues successfully demonstrated a strong physical interaction between Tic56 (or the Tic56-containing complex) and the TOC complex (Köhler et al. 2015). Similar results showing strong physical interactions between the TOC complex and novel TIC complex components including Tic56, Tic100 (At5g22640) and Tic214/YCF1 can be found in the data set presented recently by Kessler and colleagues (Zufferey et al. 2017). These experimental data, together with our report (Kikuchi et al. 2013), clearly indicate a strong physical interaction and functional co-operation between the TOC complex and the novel TIC complex at the outer and inner envelope membranes. Through their extensive genetic analyses of embryo-defective (emb) mutants, Meinke and colleagues reported that a loss of chloroplast translation in Arabidopsis led to embryo lethality (Parker et al. 2014). They found that the developmental stage associated with embryo arrest varied slightly among natural accessions of Arabidopsis and that hypersensitivity to the loss of chloroplast translation could be attributed to impaired accumulation of ACC2, the alternative chloroplast-localized homodimeric acetyl-CoA carboxylase. ACC2 is essential for fatty acid biosynthesis, and thus plant viability, only in the absence of the major chloroplast-localized acetyl-CoA carboxylase that contains an indispensable subunit (AccD) encoded by the chloroplast genome. Interestingly, the authors observed that loss of Tic20-IV, the above-mentioned minor isoform of Tic20, resulted in similar hypersensitivity to chloroplast translation deficiency and thus proposed that ACC2 import into the chloroplasts occurs exclusively via the protein import pathway that involves Tic20-IV (Parker et al. 2016). The loss of chloroplast translation causes not only AccD deficiency but also a depletion of Tic214, the essential component of the major TIC complex encoded by the chloroplast gene YCF1. Thus, the data and interpretations reported by Meinke and colleagues match well with our proposed model, in that in addition to the major TIC complex consisting of Tic20 together with Tic56, Tic100 and the chloroplast-encoded Tic214/YCF1, there seems to be an alternative TIC system that involves the minor Tic20-IV isoform (Fig. 2) (Hirabayashi et al. 2011, Kikuchi et al. 2013). The roles of Tic110 and Tic40, two well-known classical TIC candidates that are abundant at the inner envelope membrane, are still unclear given that neither appear to be involved in the novel TIC complex. While it remains possible that these proteins may indirectly affect the import process, functions other than protein import should also be considered. This is not uncommon for such proteins; as for instance was the case for the classical TIC candidate, Tic55, which was ultimately demonstrated instead to be a hydroxylase of phyllobilins through detailed analyses performed by Hörtensteiner and colleagues (Hauenstein et al. 2016). In this vein, whereas experimental data concerning Tic110 function seem to be fairly limited, Jarvis and colleagues recently reported that Tic40 may have a functional link with a non-cognate thylakoidal protein sorting system and that both affect the membrane integrity of thylakoids and/or envelopes (Bédard et al. 2017). Further functional studies will undoubtedly shed light on the specific, direct roles of these long-believed classical TIC proteins (Lintala et al. 2014). The Long-Standing Mystery of Which TIC-Associated Import Motor Drives ATP-Dependent Pre-Protein Translocation across the Inner Envelope Membrane A further potential impact of the discovery of the novel TIC complex lies in the molecular identification of the associated ATP-driven import motor that should also function at the inner envelope membrane (Fig. 1A, B). Most past studies focused on classical TIC candidates such as Tic110 and Tic40, and resulted in the identification of several stromal molecular chaperones, including Hsp93/ClpC, cpHsp70 and Hsp90C (Flores-Pérez and Jarvis 2013, Shi and Theg 2013). However, the functional interaction of these components with those of the newly discovered TIC complex requires further elucidation, as discussed below. Hsp93, an import motor or a chaperone for proteolytic machinery? Among the various candidate proteins, Hsp93, a Hsp100-type AAA+ ATPase formerly known as ClpC, is the best-known partner of Tic110 and has been proposed to act as an ATP-driven import motor (Fig. 1B). Depletion of Hsp93 from the chloroplast in Arabidopsis mutant plants causes a strong chlorotic phenotype and, at least in some cases, defects in chloroplast protein import (Kovacheva et al. 2007). However, several lines of recent evidence suggest that Hsp93 primarily functions as a molecular chaperone within the Clp protease machinery consisting of several ClpP and ClpR as well as ClpT proteins, in addition to Hsp93 and its homolog ClpD (Fig. 1B). Clarke and colleagues reported previously that the pale hsp93-V mutant did not show any chloroplast protein import defects (Sjögren et al. 2004) and, in 2014, they further demonstrated by quantitative analysis of various Clp subunits that Hsp93 appeared to function primarily within the Clp protease (Sjögren et al. 2014). In agreement with this, in 2016, Jarvis and colleagues elegantly demonstrated that interaction with the Clp proteolytic core was absolutely required for the essential function of Hsp93 (Flores-Pérez et al. 2016). Furthermore, Bock and colleagues very recently demonstrated through detailed temporal proteomic analyses using inducible tobacco RNA interference (RNAi) lines that Hsp93 depletion causes a significant enrichment, as opposed to a reduction, of a wide variety of imported proteins inside the chloroplasts (Moreno et al. 2018). A similar pattern of enrichment was observed upon the reduction of other Clp proteolytic core subunits. Thus, the most likely explanation for the involvement of Hsp93 in chloroplast protein import seems to be not in import propulsion as a motor but rather in quality control at the point of or after import. In a recent paper, Li and colleagues demonstrated a direct interaction between pre-proteins and Hsp93 by chemical cross-linking followed by immunoprecipitation with anti-Hsp93 antibodies (Huang et al. 2016). The specificities of the demonstrated immunoprecipitations may need independent verification because in some cases, even after SDS denaturation, uncross-linked pre-proteins appeared to be significantly co-purified with anti-Hsp93 or with other antibodies. Specificity aside, these findings could be interpreted to support the notion of a protein quality control role for Hsp93 at the point of import. cpHsp70, an import motor just like a mitochondrial or ER Hsp70? Because Hsp70 chaperones located in the mitochondrial matrix and in the lumen of the endoplasmic reticulum (ER) have been extensively characterized and are thought to drive post-translation protein import as the import motor (Craig 2018), the chloroplast stromal Hsp70 had long been expected to act as an import motor at the inner envelope membrane (Shi and Theg 2010, Su and Li 2010) and, in most cases, has been proposed to act in parallel to Hsp93 (Fig. 1B). Many papers with this viewpoint are summarized elsewhere (Shi and Theg 2011, Shi and Theg 2013a). In general, Hsp70 reaction cycles require a DnaJ-related protein or a J-domain-containing protein whose binding to Hsp70 stimulates hydrolysis of Hsp70-bound ATP to ADP and stabilizes the interaction between substrate proteins and Hsp70 (Craig 2018). To function as an import motor, mitochondrial and ER luminal Hsp70s each possess dedicated J-domain-containing partner proteins incorporated into the translocation machinery at their respective membranes: Tim14(Pam18) of the PAM complex at the mitochondrial inner membrane associates with the TIM translocon, and Sec63 at the ER membrane associates with the Sec61 translocon together with Sec62. Thus far, the proposed partner protein for chloroplast stromal Hsp70 is Tic40, which probably contains the so-called Hip/Hop domain, another known Hsp70 co-chaperoning domain. While a genetic interaction between Tic40 and Hsp70 was reported, their physical direct interaction in vivo has not been well demonstrated (Bédard et al. 2007). Theg and colleagues reported that an altered Km for ATP hydrolysis of a mutant form of stroma-localized Hsp70 in moss affected chloroplast protein import (Liu et al. 2014). Furthermore, Bruce and colleagues reported the importance of Hsp70-binding motifs in the transit peptides of various pre-proteins (Chotewutmontri and Bruce 2015). However, conclusive biochemical evidence supporting the direct association of Hsp70 with translocating pre-proteins across the inner envelope membrane per se is still lacking. Furthermore, there seems to be no recognizable Hsp70-interacting motif such as a J-like domain in the newly identified TIC complex components (Kikuchi et al. 2013). Given that no direct physical interaction between the TIC complex and the stromal Hsp70 has so far been observed (Fig. 1B), the idea that the stromal Hsp70 acts as the import motor remains an open question. Hsp90C, an import motor or specialized chaperone acting with cpHsp70? Hsp90C, the stromal Hsp90 homolog, has been proposed to be an alternative import motor (Fig. 1B) (Inoue et al. 2013). While some technical problems with the initial characterization of Hsp90C as a translocation intermediate-interacting protein need to be revisited, as already mentioned, the known Hsp90 inhibitor radicicol was shown to inhibit the in vitro import of some pre-proteins into the chloroplast. However, it should be noted that the ATP-binding site of Hsp90C where radicicol binds is known as the Bergerat fold, which is shared by a variety of ATPases including, for example, two-component histidine kinases and topoisomerases as well as citrate lyase (Ki et al. 2000). Together with the fact that a relatively high concentration of radicicol was required to inhibit chloroplast protein import (Inoue et al. 2013), it seems fair to conclude that the proposed motor function of Hsp90C is still unproven. Very recently, Zhao and colleagues reported that Hsp90C interacts with PsbO1 in the stroma together with Hsp70 before its translocation into the thylakoid lumen (Jiang et al. 2017). Vipp1, a protein known to be essential for the maintenance of the chloroplast envelope membranes as well as the thylakoid membranes, is likely to be another protein interacting with Hsp90C (Feng et al. 2014). Thus, Hsp90C might be a specialized stromal chaperone for a specific subset of chloroplast proteins. Are there any other candidates for the novel TIC-associated import motor? As shown for the mitochondrial and ER Hsp70 system, an important aspect of fulfilling the import motor function is whether a candidate protein or complex has the capacity to pull pre-proteins emerging from the TIC channel in a mechanically/physically coupled fashion (Craig 2018). In this way, it is expected that the newly discovered TIC complex should interact with an import motor both physically and functionally. Thus our current work has been focusing on identifying the inner envelope membrane protein complex that can act as an ATP-driven import motor and has revealed an involvement of a completely novel membrane-bound ATPase as the best candidate (submitted) (Fig. 1B). The identification of such a TIC-associated import motor will undoubtedly lead to a further revision of the molecular model of pre-protein translocation across the inner envelope membrane. Perspectives While the discovery of a novel TIC complex has resulted in an extensive revision of the classic TIC complex model and a more comprehensive understanding of the chloroplast protein import system, further unanswered, intriguing questions have arisen. The characterization of the alternative TIC systems, their evolutionary histories and the functional co-operation between TOC and TIC systems together with an as yet unidentified motor complex, as well as with unknown intermembrane space components besides Tic22 (Kasmati et al. 2013, Rudolf et al. 2013), are key research questions that many groups have been tackling (Figs. 1, 2). Structural information already exists for several components involved in the chloroplast protein import system (Tsai et al. 2013, Lumme et al. 2014, Chang et al. 2017, O’Neil et al. 2017). However, in this era of powerful cryo-electron microscopy, abundant structural data should be more easily gleaned, which could both provide us with detailed mechanistic insight and conclusively reveal the long-awaited composition of the genuine TIC complex and its associated import motor. As is evident from an increasing number of cryo-electron microscopy studies of other protein translocation systems, especially for the SEC-type translocons, this technology is capable of determining fine structures even to the point of engaged translocating polypeptides (Gogala et al, 2014, Park et al. 2014, Pfeffer et al. 2015). The successful purification of translocation intermediate complexes, containing both TOC and TIC complexes together with a motor complex, with fairly good yields raises hopes that the entire structure of this chloroplast protein translocation system in action may be fully elucidated in the not so distant future (Kikuchi et al. 2013). Other recently emerging research topics concerning chloroplast protein import include its regulatory aspects, as reported by Jarvis and colleagues, in which ubiquitin–proteasome systems control the chloroplast protein import machinery (Ling et al. 2012, Ling and Jarvis 2015). In addition, several excellent ongoing studies are examining structural information and requirements of the transit peptides of chloroplast or plastid proteins (Hirohashi et al. 2001, Endow et al. 2016, Lee et al. 2018), such as in relation to age/developmental stage-dependent or tissue-specific protein import (Teng et al. 2012, Chu and Li 2015). Finally, functional co-operation between the TIC complex and the system for protein sorting to the thylakoids and the envelope is also another exciting area of research, which thus far remains underdeveloped (Ouyang et al. 2011, Li et al. 2015). We anticipate that these combined research efforts will further clarify our understanding of the elusive chloroplast protein import system, and will enrich our knowledge of the cellular and molecular basis of chloroplast/plastid biogenesis and, ultimately, whole-plant growth and development. Acknowledgments I would like to thank all the present and past collaborators as well as members of the Nakai laboratory for valuable discussions. I apologize to the authors of the many important publications that could not be cited owing to space limitations. Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan [grants-in-aid for scientific research 15H01535, 17H05668 and 17H05725] and the Japan Society for the Promotion of Science [grants-in-aid 26291060 and 26650017]. Disclosures The author has no conflicts of interest to declare. References Agne B. , Köhler D. , Baginsky S. ( 2017 ) Protein import-independent functions of Tic56, a component of the 1-MDa translocase at the inner chloroplast envelope membrane . Plant Signal. Behav . 12 : e1284726. Google Scholar CrossRef Search ADS PubMed Bédard J. , Kubis S. , Bimanadham S. , Jarvis P. ( 2007 ) Functional similarity between the chloroplast translocon component, Tic40, and the human co-chaperone, Hsp70-interacting protein (Hip) . J. Biol. Chem. 282 : 21404 – 21414 . Google Scholar CrossRef Search ADS PubMed Bédard J. , Trösch R. , Wu F. , Ling Q. , Flores-Pérez Ú. , Töpel M. , et al. . ( 2017 ) Suppressors of the chloroplast protein import mutant tic40 reveal a genetic link between protein import and thylakoid biogenesis . Plant Cell 29 : 1726 – 1747 . Google Scholar CrossRef Search ADS PubMed Boudreau E. , Turmel M. , Goldschmidt-Clermont M. , Rochaix J.D. , Sivan S. , Michaels A. , et al. . ( 1997 ) A large open reading frame (orf1995) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein . Mol. Gen. Genet . 253 : 649 – 653 . Google Scholar CrossRef Search ADS PubMed Campbell J.H. , Hoang T. , Jelokhani-Niaraki M. , Smith M.D. ( 2014 ) Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts . BMC Biochem. 15 : 29. Google Scholar CrossRef Search ADS PubMed Chang J.S. , Chen L.J. , Yeh Y.H. , Hsiao C.D. , Li H.-M. ( 2017 ) Chloroplast preproteins bind to the dimer interface of the Toc159 receptor during import . Plant Physiol. 173 : 2148 – 2162 . Google Scholar CrossRef Search ADS PubMed Chen K.Y. , Li H.-M. ( 2007 ) Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts . Plant J 49 : 149 – 158 . Google Scholar CrossRef Search ADS PubMed Chen L.J. , Li H.-M. ( 2017 ) Stable megadalton TOC–TIC supercomplexes as major mediators of protein import into chloroplasts . Plant J. 92 : 178 – 188 . Google Scholar CrossRef Search ADS PubMed Chen X. , Smith M.D. , Fitzpatrick L. , Schnell D.J. ( 2002 ) In vivo analysis of the role of atTic20 in protein import into chloroplasts . Plant Cell 14 : 641 – 654 . Google Scholar CrossRef Search ADS PubMed Chotewutmontri P. , Bruce B.D. ( 2015 ) Non-native, N-terminal Hsp70 molecular motor recognition elements in transit peptides support plastid protein translocation . J. Biol. Chem. 290 : 7602 – 7621 . Google Scholar CrossRef Search ADS PubMed Chou M.L. , Chu C.C. , Chen L.J. , Akita M. , Li H.-M. ( 2006 ) Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts . J. Cell Biol. 175 : 893 – 900 . Google Scholar CrossRef Search ADS PubMed Chu C.C. , Li H.-M. ( 2015 ) Protein import into isolated pea root leucoplasts . Front. Plant Sci. 6 : 690. Google Scholar CrossRef Search ADS PubMed Craig E.A. ( 2018 ) Hsp70 at the membrane: driving protein translocation . BMC Biol. 16 : 11. Google Scholar CrossRef Search ADS PubMed de Vries J. , Sousa F.L. , Bölter B. , Soll J. , Gould S.B. ( 2015 ) YCF1: a green TIC? Plant Cell 27 : 1827 – 1833 . Google Scholar CrossRef Search ADS PubMed Demarsy E. , Lakshmanan A.M. , Kessler F. ( 2014 ) Border control: selectivity of chloroplast protein import and regulation at the TOC-complex . Front. Plant Sci. 5 : 483. Google Scholar CrossRef Search ADS PubMed Drescher A. , Ruf S. , Calsa T. Jr , Carrer H. , Bock R. ( 2000 ) The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes . Plant J. 22 : 97 – 104 . Google Scholar CrossRef Search ADS PubMed Endow J.K. , Rocha A.G. , Baldwin A.J. , Roston R.L. , Yamaguchi T. , Kamikubo H. , et al. . ( 2016 ) Polyglycine acts as a rejection signal for protein transport at the chloroplast envelope . PLoS One 11 : e0167802. Google Scholar CrossRef Search ADS PubMed Feng J. , Fan P. , Jiang P. , Lv S. , Chen X. , Li Y. ( 2014 ) Chloroplast-targeted Hsp90 plays essential roles in plastid development and embryogenesis in Arabidopsis possibly linking with VIPP1 . Physiol. Plant. 150 : 292 – 307 . Google Scholar CrossRef Search ADS PubMed Flores-Pérez Ú. , Bédard J. , Tanabe N. , Lymperopoulos P. , Clarke A.K. , Jarvis P. ( 2016 ) Functional analysis of the Hsp93/ClpC chaperone at the chloroplast envelope . Plant Physiol. 170 : 147 – 162 . Google Scholar CrossRef Search ADS PubMed Flores-Pérez Ú. , Jarvis P. ( 2013 ) Molecular chaperone involvement in chloroplast protein import . Biochim. Biophys. Acta 1833 : 332 – 340 . Google Scholar CrossRef Search ADS PubMed Gogala M. , Becker T. , Beatrix B. , Armache J.P. , Barrio-Garcia C. , Berninghausen O. , et al. . ( 2014 ) Structures of the Sec61 complex engaged in nascent peptide translocation or membrane insertion . Nature 506 : 107 – 110 . Google Scholar CrossRef Search ADS PubMed Hauenstein M. , Christ B. , Das A. , Aubry S. , Hörtensteiner S. ( 2016 ) A role for Tic55 as a hydroxylase of phyllobilins, the products of chlorophyll breakdown during plant senescence . Plant Cell 28 : 2510 – 2527 . Google Scholar CrossRef Search ADS PubMed Hirabayashi Y. , Kikuchi S. , Oishi M. , Nakai M. ( 2011 ) In vivo studies on the roles of two closely related Arabidopsis Tic20 proteins, AtTic20-I and AtTic20-IV . Plant Cell Physiol . 52 : 469 – 478 . Google Scholar CrossRef Search ADS PubMed Hirohashi T. , Hase T. , Nakai M. ( 2001 ) Maize non-photosynthetic ferredoxin precursor is missorted to the intermembrane space of chloroplasts in the presence of light . Plant Physiol . 125 : 2154 – 2163 . Google Scholar CrossRef Search ADS PubMed Huang P.K. , Chan P.T. , Su P.H. , Chen L.J. , Li H.-M. ( 2016 ) Chloroplast Hsp93 directly binds to transit peptides at an early stage of the preprotein import process . Plant Physiol. 170 : 857 – 866 . Google Scholar CrossRef Search ADS PubMed Inoue H. , Li M. , Schnell D.J. ( 2013 ) An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts . Proc. Natl. Acad. Sci. USA 110 : 3173 – 3178 . Google Scholar CrossRef Search ADS Jarvis P. , López-Juez E. ( 2013 ) Biogenesis and homeostasis of chloroplasts and other plastids . Nat. Rev. Mol. Cell Biol. 14 : 787 – 802 . Google Scholar CrossRef Search ADS PubMed Jiang T. , Oh E.S. , Bonea D. , Zhao R. ( 2017 ) HSP90C interacts with PsbO1 and facilitates its thylakoid distribution from chloroplast stroma in Arabidopsis . PLoS One 12 : e0190168 . Google Scholar CrossRef Search ADS PubMed Kasmati A.R. , Topel M. , Patel R. , Murtaza G. , Jarvis P. ( 2011 ) Molecular and genetic analyses of Tic20 homologues in Arabidopsis thaliana chloroplasts . Plant J . 66 : 877 – 889 . Google Scholar CrossRef Search ADS PubMed Kasmati A.R. , Töpel M. , Khan N.Z. , Patel R. , Ling Q. , Karim S. , et al. . ( 2013 ) Evolutionary, molecular and genetic analyses of Tic22 homologues in Arabidopsis thaliana chloroplasts . PLoS One 8 : e63863 . Google Scholar CrossRef Search ADS PubMed Kessler F. , Blobel G. ( 1996 ) Interaction of the protein import and folding machineries of the chloroplast . Proc. Natl. Acad. Sci. USA 93 : 7684 – 7689 . Google Scholar CrossRef Search ADS Ki S.W. , Ishigami K. , Kitahara T. , Kasahara K. , Yoshida M. , Horinouchi S. ( 2000 ) Radicicol binds and inhibits mammalian ATP citrate lyase . J. Biol. Chem. 275 : 39231 – 39236 . Google Scholar CrossRef Search ADS PubMed Kikuchi S. , Bédard J. , Hirano M. , Hirabayashi Y. , Oishi M. , Imai M. , et al. . ( 2013 ) Uncovering the protein translocon at the chloroplast inner envelope membrane . Science 339 : 571 – 574 . Google Scholar CrossRef Search ADS PubMed Kikuchi S. , Hirohashi T. , Nakai M. ( 2006 ) Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE . Plant Cell Physiol . 47 : 363 – 371 . Google Scholar CrossRef Search ADS PubMed Kikuchi S. , Oishi M. , Hirabayashi Y. , Lee D.W. , Hwang I. , Nakai M. ( 2009 ) A 1-megadalton translocation complex containing Tic20 and Tic21 mediates chloroplast protein import at the inner envelope membrane . Plant Cell 21 : 1781 – 1797 . Google Scholar CrossRef Search ADS PubMed Köhler D. , Helm S. , Agne B. , Baginsky S. ( 2016 ) Importance of translocon subunit Tic56 for rRNA processing and chloroplast ribosome assembly . Plant Physiol. 172 : 2429 – 2444 . Google Scholar CrossRef Search ADS PubMed Köhler D. , Montandon C. , Hause G. , Majovsky P. , Kessler F. , Baginsky S. , et al. . ( 2015 ) Characterization of chloroplast protein import without Tic56, a component of the 1-megadalton translocon at the inner envelope membrane of chloroplasts . Plant Physiol. 167 : 972 – 990 . Google Scholar CrossRef Search ADS PubMed Kouranov A. , Chen X. , Fuks B. , Schnell D.J. ( 1998 ) Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane . J. Cell Biol. 143 : 991 – 1002 . Google Scholar CrossRef Search ADS PubMed Kovacheva S. , Bédard J. , Wardle A. , Patel R. , Jarvis P. ( 2007 ) Further in vivo studies on the role of the molecular chaperone, Hsp93, in plastid protein import . Plant J . 50 : 364 – 379 . Google Scholar CrossRef Search ADS PubMed Kovács-Bogdán E. , Benz J.P. , Soll J. , Bölter B. ( 2011 ) Tic20 forms a channel independent of Tic110 in chloroplasts . BMC Plant Biol. 11 : 133. Google Scholar CrossRef Search ADS PubMed Lee D.-W. , Yoo Y.-J. , Razzak M.A. , Hwang I. ( 2018 ) Prolines in transit peptides are crucial for efficient preprotein translocation into chloroplasts . Plant Physiol. 176 : 663 – 677 . Google Scholar CrossRef Search ADS PubMed Li Y. , Singhal R. , Taylor I.W. , McMinn P.H. , Chua X.Y. , Cline K. , et al. . ( 2015 ) The Sec2 translocase of the chloroplast inner envelope contains a unique and dedicated SECE2 component . Plant J. 84 : 647 – 658 . Google Scholar CrossRef Search ADS PubMed Ling Q. , Huang W. , Baldwin A. , Jarvis P. ( 2012 ) Chloroplast biogenesis is regulated by direct action of the ubiquitin–proteasome system . Science 338 : 655 – 659 . Google Scholar CrossRef Search ADS PubMed Ling Q. , Jarvis P. ( 2015 ) Regulation of chloroplast protein import by the ubiquitin E3 ligase SP1 is important for stress tolerance in plants . Curr. Biol . 25 : 2527 – 2534 . Google Scholar CrossRef Search ADS PubMed Lintala M. , Schuck N. , Thormählen I. , Jungfer A. , Weber K.L. , Weber A.P. , et al. . ( 2014 ) Arabidopsis tic62 trol mutant lacking thylakoid-bound ferredoxin-NADP+ oxidoreductase shows distinct metabolic phenotype . Mol. Plant 7 : 45 – 57 . Google Scholar CrossRef Search ADS PubMed Liu L. , McNeilage R.T. , Shi L.X. , Theg S.M. ( 2014 ) ATP requirement for chloroplast protein import is set by the Km for ATP hydrolysis of stromal Hsp70 in Physcomitrella patens . Plant Cell. 26 : 1246 – 1255 . Google Scholar CrossRef Search ADS PubMed Lumme C. , Altan-Martin H. , Dastvan R. , Sommer M.S. , Oreb M. , Schuetz D. , et al. . ( 2014 ) Nucleotides and substrates trigger the dynamics of the Toc34 GTPase homodimer involved in chloroplast preprotein translocation . Structure 22 : 526 – 538 . Google Scholar CrossRef Search ADS PubMed Moreno J.C. , Martínez-Jaime S. , Schwartzmann J. , Karcher D. , Tillich M. , Graf A. , et al. . ( 2018 ) Temporal proteomics of inducible RNAi lines of Clp protease subunits identifies putative protease substrates . Plant Physiol. 176 : 1485 – 1508 . Google Scholar CrossRef Search ADS PubMed Nakai M. ( 2015a ) The TIC complex uncovered: the alternative view in the molecular mechanism of protein translocation across the inner envelope membrane of chloroplasts . Biochim. Biophys. Acta 1847 : 957 – 967 . Google Scholar CrossRef Search ADS Nakai M. ( 2015b ) YCF1: a green TIC . Plant Cell 27 : 1834 – 1838 . Google Scholar CrossRef Search ADS O’Neil P.K. , Richardson L. , Paila Y.D. , Piszczek G. , Chakravarthy S. , Noinaj N. , et al. . ( 2017 ) The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts . Proc. Natl. Acad. Sci. USA 114 : E4868 – E4876 . Google Scholar CrossRef Search ADS Ouyang M. , Li X. , Ma J. , Chi W. , Xiao J. , Zou M. , et al. . ( 2011 ) LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway . Nat. Commun. 2 : 277. Google Scholar CrossRef Search ADS PubMed Paila Y.D. , Richardson L.G.L. , Schnell D.J. ( 2015 ) New insights into the mechanism of chloroplast protein import and its integration with protein quality control, organelle biogenesis and development . J. Mol. Biol . 427 : 1038 – 1060 . Google Scholar CrossRef Search ADS PubMed Park E. , Ménétret J.F. , Gumbart J.C. , Ludtke S.J. , Li W. , Whynot A. , et al. . ( 2014 ) Structure of the SecY channel during initiation of protein translocation . Nature 506 : 102 – 106 . Google Scholar CrossRef Search ADS PubMed Parker N. , Wang Y. , Meinke D. ( 2014 ) Natural variation in sensitivity to a loss of chloroplast translation in arabidopsis . Plant Physiol . 166 : 2013 – 2027 . Google Scholar CrossRef Search ADS PubMed Parker N. , Wang Y. , Meinke D. ( 2016 ) Analysis of arabidopsis accessions hypersensitive to a loss of chloroplast translation . Plant Physiol. 172 : 1862 – 1875 . Google Scholar CrossRef Search ADS PubMed Pfeffer S. , Burbaum L. , Unverdorben P. , Pech M. , Chen Y. , Zimmermann R. , et al. . ( 2015 ) Structure of the native Sec61 protein-conducting channel . Nat. Commun. 6 : 8403. Google Scholar CrossRef Search ADS PubMed Richardson L.G.L. , Singhal R. , Schnell D.J. ( 2017 ) The integration of chloroplast protein targeting with plant developmental and stress responses . BMC Biol. 15 : 118. Google Scholar CrossRef Search ADS PubMed Rudolf M. , Machettira A.B. , Groß L.E. , Weber K.L. , Bolte K. , Bionda T. , et al. . ( 2013 ) In vivo function of Tic22, a protein import component of the intermembrane space of chloroplasts . Mol. Plant 6 : 817 – 829 . Google Scholar CrossRef Search ADS PubMed Shi L.X. , Theg S.M. ( 2010 ) A stromal heat shock protein 70 system functions in protein import into chloroplasts in the moss Physcomitrella patens . Plant Cell 22 : 205 – 220 . Google Scholar CrossRef Search ADS PubMed Shi L.X. , Theg S.M. ( 2011 ) The motors of protein import into chloroplasts . Plant Signal. Behav. 6 : 1397 – 1401 . Google Scholar CrossRef Search ADS PubMed Shi L.X. , Theg S.M. ( 2013a ) The chloroplast protein import system: from algae to trees . Biochim. Biophys. Acta 1833 : 314 – 331 . Google Scholar CrossRef Search ADS Shi L.X. , Theg S.M. ( 2013b ) Energetic cost of protein import across the envelope membranes of chloroplasts . Proc. Natl. Acad. Sci. USA 110 : 930 – 935 . Google Scholar CrossRef Search ADS Sjögren L.L. , MacDonald T.M. , Sutinen S. , Clarke A.K. ( 2004 ) Inactivation of the clpC1 gene encoding a chloroplast Hsp100 molecular chaperone causes growth retardation, leaf chlorosis, lower photosynthetic activity, and a specific reduction in photosystem content . Plant Physiol . 136 : 4114 – 4126 . Google Scholar CrossRef Search ADS PubMed Sjögren L.L. , Tanabe N. , Lymperopoulos P. , Khan N.Z. , Rodermel S.R. , Aronsson H. , et al. . ( 2014 ) Quantitative analysis of the chloroplast molecular chaperone ClpC/Hsp93 in Arabidopsis reveals new insights into its localization, interaction with the Clp proteolytic core, and functional importance . J. Biol. Chem. 289 : 11318 – 11330 . Google Scholar CrossRef Search ADS PubMed Sjuts I. , Soll J. , Bölter B. ( 2017 ) Import of soluble proteins into chloroplasts and potential regulatory mechanisms . Front. Plant Sci. 8 : 168. Google Scholar CrossRef Search ADS PubMed Su P.H. , Li H.M. ( 2010 ) Stromal Hsp70 is important for protein translocation into pea and Arabidopsis chloroplasts . Plant Cell 22 : 1516 – 1531 . Google Scholar CrossRef Search ADS PubMed Teng Y.S. , Chan P.T. , Li H.-M. ( 2012 ) Differential age-dependent import regulation by signal peptides . PLoS Biol. 10 : e1001416. Google Scholar CrossRef Search ADS PubMed Topel M. , Jarvis P. ( 2011 ) The Tic20 gene family: phylogenetic analysis and evolutionary considerations . Plant Signal. Behav . 6 : 1046 – 1048 . Google Scholar CrossRef Search ADS PubMed Tsai J.Y. , Chu C.C. , Yeh Y.H. , Chen L.J. , Li H.M. , Hsiao C.D. ( 2013 ) Structural characterizations of the chloroplast translocon protein Tic110 . Plant J. 75 : 847 – 857 . Google Scholar CrossRef Search ADS PubMed van Dooren G.G. , Tomova C. , Agrawal S. , Humbel B.M. , Striepen B. ( 2008 ) Toxoplasma gondii Tic20 is essential for apicoplast protein import . Proc. Natl. Acad. Sci. USA 105 : 13574 – 13579 . Google Scholar CrossRef Search ADS Vitale A. , Schnell D.J. , Raikhel N.V. , Chrispeels M.J. ( 2015 ) Protein sorting and vesicle traffic. In Biochemistry & Molecular Biology of Plants , 2nd edn . Edited by Buchanan B.B. , Gruissem W. , Jones R.L. pp. 151 – 190 . John Wiley & Sons, Ltd ., Chichester . Zufferey M. , Montandon C. , Douet V. , Demarsy E. , Agne B. , Baginsky S. , et al. . ( 2017 ) The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery . J. Biol. Chem. 292 : 6952 – 6964 . Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations BN–PAGE Blue Native–PAGE ER endoplasmic reticulum TIC translocon at the inner envelope membrane of chloroplasts TOC translocon at the outer envelope membrane of chloroplasts © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

New Perspectives on Chloroplast Protein Import

Plant and Cell Physiology , Volume Advance Article (6) – Apr 19, 2018

Loading next page...
 
/lp/ou_press/new-perspectives-on-chloroplast-protein-import-2qF840txzq
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
ISSN
0032-0781
eISSN
1471-9053
D.O.I.
10.1093/pcp/pcy083
Publisher site
See Article on Publisher Site

Abstract

Abstract Virtually all chloroplasts in extant photosynthetic eukaryotes derive from a single endosymbiotic event that probably occurred more than a billion years ago between a host eukaryotic cell and a cyanobacterium-like ancestor. Many endosymbiont genes were subsequently transferred to the host nuclear genome, concomitant with the establishment of a system for protein transport through the chloroplast double-membrane envelope. Presently, 2,000–3,000 different nucleus-encoded chloroplast proteins must be imported into the chloroplast following their synthesis in the cytosol. The TOC (translocon at the outer envelope membrane of chloroplasts) and TIC (translocon at the inner envelope membrane of chloroplasts) complexes are protein translocation machineries at the outer and inner envelope membranes, respectively, that facilitate this chloroplast protein import with the aid of a TIC-associated ATP-driven import motor. All the essential components of this protein import system seemed to have been identified through biochemical analyses and subsequent genetic studies that initiated in the late 1990s. However, in 2013, the Nakai group reported a novel inner envelope membrane TIC complex, for which a novel ATP-driven import motor associated with this TIC complex is likely to exist. In this mini review, I will summarize these recent discoveries together with new, or reanalyzed, data presented by other groups in recent years. Whereas the precise concurrent view of chloroplast protein import is still a matter of some debate, it is anticipated that the entire TOC/TIC/ATP motor system, including any novel components, will be conclusively established in the next decade. Such findings may lead to an extensively revised view of the evolution and molecular mechanisms of chloroplast protein import. Introduction While chloroplasts in plants and algae retain their own genome and synthesize approximately 100 proteins within the organelle, >2,000 different nuclear-encoded proteins are synthesized in the cytosol and then transported across the double-membrane chloroplast envelope (Fig. 1A) (Shi and Theg 2013a). Given these numbers, a system of post-translational protein transport into the chloroplast is absolutely essential for chloroplast biogenesis and function (Jarvis and López-Juez 2013, Nakai 2015a, Richardson et al. 2017). Fig. 1 View largeDownload slide Chloroplast protein import mediated by TOC and TIC translocons at the double envelope membranes of the chloroplast together with the associated ATP-driven import motor. (A) Overview of chloroplast protein import. (B) Comparison between the classical long-held model (adapted from Richardson et al. 2017) and the recently revised model for TIC translocon (Nakai 2015a, Nakai 2015b), and its associated putative ATP-driven import motor. For details, see text. Fig. 1 View largeDownload slide Chloroplast protein import mediated by TOC and TIC translocons at the double envelope membranes of the chloroplast together with the associated ATP-driven import motor. (A) Overview of chloroplast protein import. (B) Comparison between the classical long-held model (adapted from Richardson et al. 2017) and the recently revised model for TIC translocon (Nakai 2015a, Nakai 2015b), and its associated putative ATP-driven import motor. For details, see text. Overview of Chloroplast Protein Import In general, a nucleus-encoded chloroplast protein is synthesized as a larger pre-protein with an N-terminal amino acid sequence known as a transit peptide (Fig. 1A). Transit peptides carry intracellular targeting information and are recognized by receptors in the TOC (translocon at the outer envelope membrane of chloroplasts) such as Toc159 and/or Toc33/34, which are GTP-binding proteins exposed to the cytoplasm yet bound to the outer envelope membrane. There are differentially expressed isoproteins of these TOC receptors, namely Toc132/120/90, some of which are likely to contribute to enhanced import efficiency and/or selection of certain subsets of proteins, especially in non-photosynthetic plastids (Demarsy et al. 2014). Pre-proteins recognized by the TOC receptors are translocated across the outer envelope through the Toc75 channel, a β-barrel Omp85 family protein. Toc159, Toc33/34 and Toc75 form a rigid 800–1,000 kDa complex at the outer envelope membrane (Kikuchi et al. 2006, Chen and Li 2007). Pre-proteins that pass through the TOC channel into the intermembrane space are then consecutively translocated across the inner envelope membrane through the TIC (translocon at the inner envelope membrane of chloroplasts) complex (Fig. 1A). In terms of the energy used for chloroplast protein import, GTP binding to TOC receptors triggers the initiation of pre-protein translocation, whereas an ATP-derived pulling force produced from the stromal side of the inner envelope membrane appears to drive protein import (Shi and Theg 2013b). This pulling force probably arises from an import motor ATPase associated with the TIC complex. The Classical Model of Protein Translocation across the Chloroplast Inner Envelope Membrane Whereas a decent consensus regarding TOC complex composition has been reached among different research groups, the accepted molecular model for the TIC complex was called into question in 2013, when my group identified a novel 1 MDa inner envelope membrane protein complex that functions as a TIC complex (Fig. 1B) (Kikuchi et al. 2013). The identification of a directly participating TIC complex raised questions about what acts as the import motor, which should function in close concert with the TIC complex. Despite this discovery, numerous papers and textbooks still present the classical model of the TIC complex and its associated import motor (Vitale et al. 2015). In the latter model, Tic110, an inner envelope membrane protein exposed to the stroma, is thought to form at least part of the protein-conducting channel and to be present in complex with Tic40, another inner envelope membrane protein (Shi and Theg 2013a, Richardson et al. 2017). The model also assumes that Tic110/Tic40 bind various stromal molecular chaperones such as Hsp93/ClpC, Hsp70 and Hsp90C, which function as the above-mentioned ATP-driven import motor (Flores-Pérez and Jarvis 2013). Thus, this classical model is based on the long-held assumption that Tic110, as the first identified TIC protein, functions as a central component of the TIC complex (Fig. 1B) (Sjuts et al. 2017). However, there seems to be no definitive evidence that supports a direct interaction between Tic110 and translocating pre-proteins per se (Chou et al. 2006). Instead, recent studies have revealed some critical weaknesses underlying the classical TIC complex model, including a lack of solid biochemical evidence that Tic110 forms a complex with any other known TIC proteins, including its proposed partner protein Tic40 (Kikuchi et al. 2009, Kikuchi et al. 2013, Nakai 2015a). Recently, some evidence was presented to suggest that Tic110 might be included in the TOC/TIC supercomplex from a Blue Native–PAGE (BN–PAGE) separation of chloroplast lysates (Chen and Li 2017). However, it can be argued that the observed Tic110 signal near the 1 MDa location might correspond to a mere tailing from the abundant Tic110, the majority of which migrated to the 200–400 kDa area. Moreover, under the BN–PAGE conditions used, TOC and TIC separately migrated around the 1 MDa area (Kikuchi et al. 2006, Kikuchi et al. 2009, Kikuchi et al. 2013). Previous observations have similarly shown that Tic110 purified from Arabidopsis chloroplasts does not specifically associate with other proteins, but exhibits a smeared BN–PAGE migration profile that is clearly distinct from the profile of Tic40 (Kikuchi et al. 2013). Thus, additional experiments are required to show conclusively that Tic110 is a component of the TOC/TIC supercomplex. It was also reported that anti-Tic110 antibodies caused a supershift of translocation intermediates in BN–PAGE experiments (Chen and Li 2017). However, this observation may require some reinterpretation as slight retardations in the migration of translocation intermediates were also, to some extent, uniformly observed following the addition of different antibodies. Similarly, control IgGs in previous studies have also appeared to cause a certain degree of supershift (Chen and Li 2007). Given that the number of epitopes in the different antigen proteins available for the different antibodies is expected to vary significantly, it is expected that different antibodies would cause significant degrees of supershift, as we and others have demonstrated previously (Kikuchi et al. 2009, Kikuchi et al. 2013, Chen and Li 2007). A number of review articles, including those published recently, repeatedly claim that Tic110 acts in close contact with Tic20, a central channel component of the TIC complex (see below) (Fig. 1B) (Shi and Theg 2013a, Demarsy et al. 2014, Paila et al. 2015, Richardson et al. 2017). However, there seems to be no direct evidence in the literature for a specific interaction, either physically or functionally, between Tic110 and Tic20, or between Tic110 and the Tic20-containing complex (Kikuchi et al. 2009, Kikuchi et al. 2013). Thus, we argue for a re-evaluation of the classical TIC model regarding the molecular components required for pre-protein translocation across the chloroplast inner envelope membrane, and their interactions. The 1 MDa TIC Complex at the Inner Envelope Membrane Contains Tic20 at the Core with Three Additional Constituents, Tic56, Tic100 and Tic214/YCF1 Our research has focused on identifying the actual components of the TIC complex and its functionally associated ATP-driven import motor (Kikuchi et al. 2009, Hirabayashi et al. 2011, Kikuchi et al. 2013, Nakai 2015a). We first found a 1 MDa translocation intermediate complex at the inner envelope membrane using in vitro import experiments in combination with BN–PAGE (Kikuchi et al. 2009). We attempted to determine the central component of this 1 MDa complex and eventually identified the 20 kDa intrinsic inner envelope membrane protein Tic20. Tic20 is a long-known TIC complex candidate that possesses four transmembrane helices (Chen et al. 2002), and we and others found it to interact directly with translocating pre-proteins (Kouranov et al. 1998, Kikuchi et al. 2009). We subsequently demonstrated that the 1 MDa complex is composed of Tic20 and three additional previously uncharacterized proteins termed Tic56, Tic100 and Tic214 (Fig. 1B) (Kikuchi et al. 2013). These proteins are exclusively localized at the chloroplast inner envelope membrane and together form a rigid 1 MDa complex. Knockouts of Tic20, Tic56 or Tic100 cause an albino seedling-lethal phenotype. Furthermore, it should be noted that Tic214 is encoded by the enigmatic plastid YCF1 gene, an open reading frame encoding an essential protein (Boudreau et al. 1997, Drescher et al. 2000), which has raised interesting questions about the evolution of this complex (see below) (Kikuchi et al. 2013, Nakai 2015b). Strong evidence for these proteins functioning as an actual TIC complex was obtained by purifying the entire translocation intermediate complexes following in vitro import experiments (Kikuchi et al. 2013). Subsequent biochemical identification of specifically associated proteins showed that Tic20, Tic56, Tic100 and Tic214 were stoichiometrically purified with different pre-proteins and, importantly, with well-established TOC components, namely Toc33, Toc75 and Toc159 (Fig. 1A). Controversially, neither Tic110 nor Tic40 was found to be associated with the various analyzed translocating pre-proteins. This lack of association indicates that neither Tic110 nor Tic40 participates in pre-protein translocation involving the above-mentioned TOC/TIC proteins and also discounts the possibility that Tic110 or Tic40 forms an alternative translocon, as has been long proposed, to act in parallel at the inner envelope membrane (Sjuts et al. 2017). Hence, we concluded that the newly identified TIC complex consisting of Tic20, Tic56, Tic100 and Tic214 constitutes a main import route across the inner envelope membrane that, most importantly, functions in close concert with the well-established TOC complex (Fig. 1B) (Kikuchi et al. 2013). After reconstitution into lipid bilayers, the purified 1 MDa complex composed of Tic20, Tic56, Tic100 and Tic214 was demonstrated to form a membrane channel with which pre-proteins specifically interacted. Whereas Tic20 probably forms a primarily α-helical channel in the inner envelope membrane (Kovács-Bogdán et al. 2011, Campbell et al. 2014), precise roles for the other TIC complex constituents remain to be elucidated. An Alternative TIC System Functions in the Absence of, or in Parallel to, the 1 MDa TIC Complex A remarkable implication of the discovery of the Tic20/Tic56/Tic100/Tic214 TIC complex is the revelation that an alternative TIC system probably functions in the absence of, or possibly in parallel to, the main TIC complex (Fig. 2). The presence of an alternative TIC system is indicated by the phenotypes of Arabidopsis Tic20, Tic56 or Tic100 single knockout mutants (Hirabayashi et al. 2011, Kasmati et al. 2011). These mutants have albino seedling-lethal phenotypes, but can germinate and grow at least until the seedling stage. Thus, the plastids in these mutants, although lacking photosynthetic proteins, retain the ability to accumulate housekeeping plastid proteins. This residual protein import ability in the absence of the main TIC complex was experimentally demonstrated to be the result of elevated expression of a minor isoform of Tic20-IV present on chromosome IV. Further genetic analyses confirmed the presence of an alternative TIC system that involves Tic20-IV and is clearly independent from the main TIC complex (Kikuchi et al. 2013). Although minor, this alternative TIC system may play an important role in protein import, in particular in non-photosynthetic plastids (Fig. 2). It remains to be elucidated whether the minor TIC system includes any additional components. Further, it will be interesting to determine the evolutionary relationship between the two TIC systems. Fig. 2 View largeDownload slide Most plausible scenario for the occurrence of divergent TIC and associated motor systems during the evolution of photosynthetic eukaryotes. For details, see text. Fig. 2 View largeDownload slide Most plausible scenario for the occurrence of divergent TIC and associated motor systems during the evolution of photosynthetic eukaryotes. For details, see text. Evolutionary Aspects of the TIC Complex Given that a single endosymbiotic event gave rise to all photosynthetic eukaryote chloroplasts, and because the chloroplast protein import system is thought to have been established very early during subsequent evolutionary processes, it has been assumed that TIC complex components are highly conserved among all the chloroplast-containing lineages. However, upon identification of the novel 1 MDa TIC complex components, it was revealed that Tic20 is the only component shared amongst virtually all chloroplast- or plastid-containing lineages, whether photosynthetic or non-photosynthetic (Kikuchi et al. 2013). Surprisingly, while some early lineages including rhodophyta and glaucophyta possess Tic20, they do not appear to contain Tic56, Tic100 or Tic214/YCF1 (Fig. 2) (Nakai 2015b). The observed phylogenetic distributions suggest that the main TIC complex evolved drastically following the initial chloroplast endosymbiotic event and during the establishment of green lineages, and that the complete Tic20/56/100/214 complex seems to have been evolved when land plants appeared. Whereas Tic56, Tic100 and Tic214 are widely conserved among most seed plants, including dicots and monocots, as is Tic20, it is surprising to learn that a recently diverged unique class of monocot grasses appears to have lost all three of these essential TIC complex constituents, yet has retained Tic20 (Fig. 2) (Kikuchi et al. 2013, Nakai 2015b). Is it possible to propose an evolutionary relationship between the uncharacterized rhodophyta- or glaucophyta-type early TIC system, the uncharacterized grass-type TIC system and the novel 1 MDa TIC complex in other green lineages, as exemplified by that in Arabidopsis? Clearly, the functional TIC systems of other organisms should be characterized experimentally via biochemical and genetic methods rather than solely relying on predictions based on in silico analyses (de Vries et al. 2015). Nevertheless, we may initially predict a plausible evolutionary scenario for these TIC systems (Fig. 2) (Nakai 2015a, Nakai 2015b), in which Tic20 as the primary central core component for inner envelope membrane protein import is conserved among all chloroplast/plastid-containing lineages (van Dooren et al. 2008, Topel and Jarvis 2011). Since all grasses retain Tic20 orthologs, the grass-type TIC system may resemble the above-mentioned minor TIC system that we observed in Arabidopsis, which involves Tic20-IV but lacks other TIC complex constituents, namely Tic56, Tic100 and Tic214. In other words, in the green lineages, the main photosynthetic-type TIC has become essential to sustain the high demands of photosynthetic protein import into chloroplasts and as such has not been replaced by the minor non-photosynthetic-type TIC. Presumably, the evolution of the grass-type TIC from the minor non-photosynthetic-type TIC complex was favored over the main TIC. It should be noted that, in grasses, the process of chloroplast development from the non-photosynthetic plastid starting at the base of the leaf blade to the photosynthetic plastid occurs successively during leaf elongation, which thus can be observed as a gradient along their leaf blades. Therefore, continuous utilization of the non-photosynthetic type TIC complex during all stages of leaf/chloroplast development might have some advantages in grasses. Intriguingly, some of the grass-type TIC complex components, including Tic20, are well conserved among all chloroplast/plastid-containing lineages. This supports the possibility that these highly conserved TIC complex components must form the primary TIC complex and have still been retained in rhodophyta and glaucophyta (Fig. 2). Presumably, the key mechanisms of this TIC system are well conserved among all TIC systems irrespective of the different additional components in each system. The molecular identities and evolutionary histories of the actual TIC-associated import motors for these distinct types of TIC systems remain intriguing open questions. Supporting Evidence for the Novel TIC Complex In 2013, Schnell and colleagues reported the possible involvement of Hsp90C, a stromal Hsp90 family protein, in chloroplast protein import as a part of the import motor (see below) (Inoue et al. 2013). In that work, purification of translocation intermediates was carried out after in vitro import of a tagged form of an inner envelope membrane-localized model pre-protein. While Tic20, Tic56, Tic100 and Tic214 were not analyzed, considerable enrichment of Toc159 and Toc75 in the purified fraction was demonstrated. In contrast, neither Tic110, Hsp93, Hsp70 nor Hsp90C was detected at such enriched levels. Although the experimental approach of using an inner envelope membrane-localized model pre-protein for the purification of the translocation intermediates may need independent verification, their experimental data appear in part consistent with other data suggesting that Tic110 is not specifically associated with translocating pre-proteins (Kikuchi et al. 2013). Nevertheless, the data from Schnell and colleagues may potentially explain why Tic110, a fairly abundant inner envelope membrane protein that is readily detectable after purification procedures, has been repeatedly characterized as a specific pre-protein-interacting protein since its initial identification during the early days of this research field (Kessler and Blobel 1996). More detailed arguments on this have been described elsewhere (Nakai 2015a). In their series of papers using combined transcriptomic and proteomic analyses, Baginsky and colleagues reported the importance of Tic56 in the novel TIC complex for the accumulation of various chloroplast proteins (Köhler et al. 2015, Köhler et al. 2016, Agne et al. 2017). While there are slight discrepancies between their work and our own regarding the interpretation of the observed data, the fact remains that the complete absence of Tic56 results in severe defects in chloroplast protein import and is associated with various pleiotropic defects. Importantly, Baginsky and colleagues successfully demonstrated a strong physical interaction between Tic56 (or the Tic56-containing complex) and the TOC complex (Köhler et al. 2015). Similar results showing strong physical interactions between the TOC complex and novel TIC complex components including Tic56, Tic100 (At5g22640) and Tic214/YCF1 can be found in the data set presented recently by Kessler and colleagues (Zufferey et al. 2017). These experimental data, together with our report (Kikuchi et al. 2013), clearly indicate a strong physical interaction and functional co-operation between the TOC complex and the novel TIC complex at the outer and inner envelope membranes. Through their extensive genetic analyses of embryo-defective (emb) mutants, Meinke and colleagues reported that a loss of chloroplast translation in Arabidopsis led to embryo lethality (Parker et al. 2014). They found that the developmental stage associated with embryo arrest varied slightly among natural accessions of Arabidopsis and that hypersensitivity to the loss of chloroplast translation could be attributed to impaired accumulation of ACC2, the alternative chloroplast-localized homodimeric acetyl-CoA carboxylase. ACC2 is essential for fatty acid biosynthesis, and thus plant viability, only in the absence of the major chloroplast-localized acetyl-CoA carboxylase that contains an indispensable subunit (AccD) encoded by the chloroplast genome. Interestingly, the authors observed that loss of Tic20-IV, the above-mentioned minor isoform of Tic20, resulted in similar hypersensitivity to chloroplast translation deficiency and thus proposed that ACC2 import into the chloroplasts occurs exclusively via the protein import pathway that involves Tic20-IV (Parker et al. 2016). The loss of chloroplast translation causes not only AccD deficiency but also a depletion of Tic214, the essential component of the major TIC complex encoded by the chloroplast gene YCF1. Thus, the data and interpretations reported by Meinke and colleagues match well with our proposed model, in that in addition to the major TIC complex consisting of Tic20 together with Tic56, Tic100 and the chloroplast-encoded Tic214/YCF1, there seems to be an alternative TIC system that involves the minor Tic20-IV isoform (Fig. 2) (Hirabayashi et al. 2011, Kikuchi et al. 2013). The roles of Tic110 and Tic40, two well-known classical TIC candidates that are abundant at the inner envelope membrane, are still unclear given that neither appear to be involved in the novel TIC complex. While it remains possible that these proteins may indirectly affect the import process, functions other than protein import should also be considered. This is not uncommon for such proteins; as for instance was the case for the classical TIC candidate, Tic55, which was ultimately demonstrated instead to be a hydroxylase of phyllobilins through detailed analyses performed by Hörtensteiner and colleagues (Hauenstein et al. 2016). In this vein, whereas experimental data concerning Tic110 function seem to be fairly limited, Jarvis and colleagues recently reported that Tic40 may have a functional link with a non-cognate thylakoidal protein sorting system and that both affect the membrane integrity of thylakoids and/or envelopes (Bédard et al. 2017). Further functional studies will undoubtedly shed light on the specific, direct roles of these long-believed classical TIC proteins (Lintala et al. 2014). The Long-Standing Mystery of Which TIC-Associated Import Motor Drives ATP-Dependent Pre-Protein Translocation across the Inner Envelope Membrane A further potential impact of the discovery of the novel TIC complex lies in the molecular identification of the associated ATP-driven import motor that should also function at the inner envelope membrane (Fig. 1A, B). Most past studies focused on classical TIC candidates such as Tic110 and Tic40, and resulted in the identification of several stromal molecular chaperones, including Hsp93/ClpC, cpHsp70 and Hsp90C (Flores-Pérez and Jarvis 2013, Shi and Theg 2013). However, the functional interaction of these components with those of the newly discovered TIC complex requires further elucidation, as discussed below. Hsp93, an import motor or a chaperone for proteolytic machinery? Among the various candidate proteins, Hsp93, a Hsp100-type AAA+ ATPase formerly known as ClpC, is the best-known partner of Tic110 and has been proposed to act as an ATP-driven import motor (Fig. 1B). Depletion of Hsp93 from the chloroplast in Arabidopsis mutant plants causes a strong chlorotic phenotype and, at least in some cases, defects in chloroplast protein import (Kovacheva et al. 2007). However, several lines of recent evidence suggest that Hsp93 primarily functions as a molecular chaperone within the Clp protease machinery consisting of several ClpP and ClpR as well as ClpT proteins, in addition to Hsp93 and its homolog ClpD (Fig. 1B). Clarke and colleagues reported previously that the pale hsp93-V mutant did not show any chloroplast protein import defects (Sjögren et al. 2004) and, in 2014, they further demonstrated by quantitative analysis of various Clp subunits that Hsp93 appeared to function primarily within the Clp protease (Sjögren et al. 2014). In agreement with this, in 2016, Jarvis and colleagues elegantly demonstrated that interaction with the Clp proteolytic core was absolutely required for the essential function of Hsp93 (Flores-Pérez et al. 2016). Furthermore, Bock and colleagues very recently demonstrated through detailed temporal proteomic analyses using inducible tobacco RNA interference (RNAi) lines that Hsp93 depletion causes a significant enrichment, as opposed to a reduction, of a wide variety of imported proteins inside the chloroplasts (Moreno et al. 2018). A similar pattern of enrichment was observed upon the reduction of other Clp proteolytic core subunits. Thus, the most likely explanation for the involvement of Hsp93 in chloroplast protein import seems to be not in import propulsion as a motor but rather in quality control at the point of or after import. In a recent paper, Li and colleagues demonstrated a direct interaction between pre-proteins and Hsp93 by chemical cross-linking followed by immunoprecipitation with anti-Hsp93 antibodies (Huang et al. 2016). The specificities of the demonstrated immunoprecipitations may need independent verification because in some cases, even after SDS denaturation, uncross-linked pre-proteins appeared to be significantly co-purified with anti-Hsp93 or with other antibodies. Specificity aside, these findings could be interpreted to support the notion of a protein quality control role for Hsp93 at the point of import. cpHsp70, an import motor just like a mitochondrial or ER Hsp70? Because Hsp70 chaperones located in the mitochondrial matrix and in the lumen of the endoplasmic reticulum (ER) have been extensively characterized and are thought to drive post-translation protein import as the import motor (Craig 2018), the chloroplast stromal Hsp70 had long been expected to act as an import motor at the inner envelope membrane (Shi and Theg 2010, Su and Li 2010) and, in most cases, has been proposed to act in parallel to Hsp93 (Fig. 1B). Many papers with this viewpoint are summarized elsewhere (Shi and Theg 2011, Shi and Theg 2013a). In general, Hsp70 reaction cycles require a DnaJ-related protein or a J-domain-containing protein whose binding to Hsp70 stimulates hydrolysis of Hsp70-bound ATP to ADP and stabilizes the interaction between substrate proteins and Hsp70 (Craig 2018). To function as an import motor, mitochondrial and ER luminal Hsp70s each possess dedicated J-domain-containing partner proteins incorporated into the translocation machinery at their respective membranes: Tim14(Pam18) of the PAM complex at the mitochondrial inner membrane associates with the TIM translocon, and Sec63 at the ER membrane associates with the Sec61 translocon together with Sec62. Thus far, the proposed partner protein for chloroplast stromal Hsp70 is Tic40, which probably contains the so-called Hip/Hop domain, another known Hsp70 co-chaperoning domain. While a genetic interaction between Tic40 and Hsp70 was reported, their physical direct interaction in vivo has not been well demonstrated (Bédard et al. 2007). Theg and colleagues reported that an altered Km for ATP hydrolysis of a mutant form of stroma-localized Hsp70 in moss affected chloroplast protein import (Liu et al. 2014). Furthermore, Bruce and colleagues reported the importance of Hsp70-binding motifs in the transit peptides of various pre-proteins (Chotewutmontri and Bruce 2015). However, conclusive biochemical evidence supporting the direct association of Hsp70 with translocating pre-proteins across the inner envelope membrane per se is still lacking. Furthermore, there seems to be no recognizable Hsp70-interacting motif such as a J-like domain in the newly identified TIC complex components (Kikuchi et al. 2013). Given that no direct physical interaction between the TIC complex and the stromal Hsp70 has so far been observed (Fig. 1B), the idea that the stromal Hsp70 acts as the import motor remains an open question. Hsp90C, an import motor or specialized chaperone acting with cpHsp70? Hsp90C, the stromal Hsp90 homolog, has been proposed to be an alternative import motor (Fig. 1B) (Inoue et al. 2013). While some technical problems with the initial characterization of Hsp90C as a translocation intermediate-interacting protein need to be revisited, as already mentioned, the known Hsp90 inhibitor radicicol was shown to inhibit the in vitro import of some pre-proteins into the chloroplast. However, it should be noted that the ATP-binding site of Hsp90C where radicicol binds is known as the Bergerat fold, which is shared by a variety of ATPases including, for example, two-component histidine kinases and topoisomerases as well as citrate lyase (Ki et al. 2000). Together with the fact that a relatively high concentration of radicicol was required to inhibit chloroplast protein import (Inoue et al. 2013), it seems fair to conclude that the proposed motor function of Hsp90C is still unproven. Very recently, Zhao and colleagues reported that Hsp90C interacts with PsbO1 in the stroma together with Hsp70 before its translocation into the thylakoid lumen (Jiang et al. 2017). Vipp1, a protein known to be essential for the maintenance of the chloroplast envelope membranes as well as the thylakoid membranes, is likely to be another protein interacting with Hsp90C (Feng et al. 2014). Thus, Hsp90C might be a specialized stromal chaperone for a specific subset of chloroplast proteins. Are there any other candidates for the novel TIC-associated import motor? As shown for the mitochondrial and ER Hsp70 system, an important aspect of fulfilling the import motor function is whether a candidate protein or complex has the capacity to pull pre-proteins emerging from the TIC channel in a mechanically/physically coupled fashion (Craig 2018). In this way, it is expected that the newly discovered TIC complex should interact with an import motor both physically and functionally. Thus our current work has been focusing on identifying the inner envelope membrane protein complex that can act as an ATP-driven import motor and has revealed an involvement of a completely novel membrane-bound ATPase as the best candidate (submitted) (Fig. 1B). The identification of such a TIC-associated import motor will undoubtedly lead to a further revision of the molecular model of pre-protein translocation across the inner envelope membrane. Perspectives While the discovery of a novel TIC complex has resulted in an extensive revision of the classic TIC complex model and a more comprehensive understanding of the chloroplast protein import system, further unanswered, intriguing questions have arisen. The characterization of the alternative TIC systems, their evolutionary histories and the functional co-operation between TOC and TIC systems together with an as yet unidentified motor complex, as well as with unknown intermembrane space components besides Tic22 (Kasmati et al. 2013, Rudolf et al. 2013), are key research questions that many groups have been tackling (Figs. 1, 2). Structural information already exists for several components involved in the chloroplast protein import system (Tsai et al. 2013, Lumme et al. 2014, Chang et al. 2017, O’Neil et al. 2017). However, in this era of powerful cryo-electron microscopy, abundant structural data should be more easily gleaned, which could both provide us with detailed mechanistic insight and conclusively reveal the long-awaited composition of the genuine TIC complex and its associated import motor. As is evident from an increasing number of cryo-electron microscopy studies of other protein translocation systems, especially for the SEC-type translocons, this technology is capable of determining fine structures even to the point of engaged translocating polypeptides (Gogala et al, 2014, Park et al. 2014, Pfeffer et al. 2015). The successful purification of translocation intermediate complexes, containing both TOC and TIC complexes together with a motor complex, with fairly good yields raises hopes that the entire structure of this chloroplast protein translocation system in action may be fully elucidated in the not so distant future (Kikuchi et al. 2013). Other recently emerging research topics concerning chloroplast protein import include its regulatory aspects, as reported by Jarvis and colleagues, in which ubiquitin–proteasome systems control the chloroplast protein import machinery (Ling et al. 2012, Ling and Jarvis 2015). In addition, several excellent ongoing studies are examining structural information and requirements of the transit peptides of chloroplast or plastid proteins (Hirohashi et al. 2001, Endow et al. 2016, Lee et al. 2018), such as in relation to age/developmental stage-dependent or tissue-specific protein import (Teng et al. 2012, Chu and Li 2015). Finally, functional co-operation between the TIC complex and the system for protein sorting to the thylakoids and the envelope is also another exciting area of research, which thus far remains underdeveloped (Ouyang et al. 2011, Li et al. 2015). We anticipate that these combined research efforts will further clarify our understanding of the elusive chloroplast protein import system, and will enrich our knowledge of the cellular and molecular basis of chloroplast/plastid biogenesis and, ultimately, whole-plant growth and development. Acknowledgments I would like to thank all the present and past collaborators as well as members of the Nakai laboratory for valuable discussions. I apologize to the authors of the many important publications that could not be cited owing to space limitations. Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan [grants-in-aid for scientific research 15H01535, 17H05668 and 17H05725] and the Japan Society for the Promotion of Science [grants-in-aid 26291060 and 26650017]. Disclosures The author has no conflicts of interest to declare. References Agne B. , Köhler D. , Baginsky S. ( 2017 ) Protein import-independent functions of Tic56, a component of the 1-MDa translocase at the inner chloroplast envelope membrane . Plant Signal. Behav . 12 : e1284726. Google Scholar CrossRef Search ADS PubMed Bédard J. , Kubis S. , Bimanadham S. , Jarvis P. ( 2007 ) Functional similarity between the chloroplast translocon component, Tic40, and the human co-chaperone, Hsp70-interacting protein (Hip) . J. Biol. Chem. 282 : 21404 – 21414 . Google Scholar CrossRef Search ADS PubMed Bédard J. , Trösch R. , Wu F. , Ling Q. , Flores-Pérez Ú. , Töpel M. , et al. . ( 2017 ) Suppressors of the chloroplast protein import mutant tic40 reveal a genetic link between protein import and thylakoid biogenesis . Plant Cell 29 : 1726 – 1747 . Google Scholar CrossRef Search ADS PubMed Boudreau E. , Turmel M. , Goldschmidt-Clermont M. , Rochaix J.D. , Sivan S. , Michaels A. , et al. . ( 1997 ) A large open reading frame (orf1995) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein . Mol. Gen. Genet . 253 : 649 – 653 . Google Scholar CrossRef Search ADS PubMed Campbell J.H. , Hoang T. , Jelokhani-Niaraki M. , Smith M.D. ( 2014 ) Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts . BMC Biochem. 15 : 29. Google Scholar CrossRef Search ADS PubMed Chang J.S. , Chen L.J. , Yeh Y.H. , Hsiao C.D. , Li H.-M. ( 2017 ) Chloroplast preproteins bind to the dimer interface of the Toc159 receptor during import . Plant Physiol. 173 : 2148 – 2162 . Google Scholar CrossRef Search ADS PubMed Chen K.Y. , Li H.-M. ( 2007 ) Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts . Plant J 49 : 149 – 158 . Google Scholar CrossRef Search ADS PubMed Chen L.J. , Li H.-M. ( 2017 ) Stable megadalton TOC–TIC supercomplexes as major mediators of protein import into chloroplasts . Plant J. 92 : 178 – 188 . Google Scholar CrossRef Search ADS PubMed Chen X. , Smith M.D. , Fitzpatrick L. , Schnell D.J. ( 2002 ) In vivo analysis of the role of atTic20 in protein import into chloroplasts . Plant Cell 14 : 641 – 654 . Google Scholar CrossRef Search ADS PubMed Chotewutmontri P. , Bruce B.D. ( 2015 ) Non-native, N-terminal Hsp70 molecular motor recognition elements in transit peptides support plastid protein translocation . J. Biol. Chem. 290 : 7602 – 7621 . Google Scholar CrossRef Search ADS PubMed Chou M.L. , Chu C.C. , Chen L.J. , Akita M. , Li H.-M. ( 2006 ) Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts . J. Cell Biol. 175 : 893 – 900 . Google Scholar CrossRef Search ADS PubMed Chu C.C. , Li H.-M. ( 2015 ) Protein import into isolated pea root leucoplasts . Front. Plant Sci. 6 : 690. Google Scholar CrossRef Search ADS PubMed Craig E.A. ( 2018 ) Hsp70 at the membrane: driving protein translocation . BMC Biol. 16 : 11. Google Scholar CrossRef Search ADS PubMed de Vries J. , Sousa F.L. , Bölter B. , Soll J. , Gould S.B. ( 2015 ) YCF1: a green TIC? Plant Cell 27 : 1827 – 1833 . Google Scholar CrossRef Search ADS PubMed Demarsy E. , Lakshmanan A.M. , Kessler F. ( 2014 ) Border control: selectivity of chloroplast protein import and regulation at the TOC-complex . Front. Plant Sci. 5 : 483. Google Scholar CrossRef Search ADS PubMed Drescher A. , Ruf S. , Calsa T. Jr , Carrer H. , Bock R. ( 2000 ) The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes . Plant J. 22 : 97 – 104 . Google Scholar CrossRef Search ADS PubMed Endow J.K. , Rocha A.G. , Baldwin A.J. , Roston R.L. , Yamaguchi T. , Kamikubo H. , et al. . ( 2016 ) Polyglycine acts as a rejection signal for protein transport at the chloroplast envelope . PLoS One 11 : e0167802. Google Scholar CrossRef Search ADS PubMed Feng J. , Fan P. , Jiang P. , Lv S. , Chen X. , Li Y. ( 2014 ) Chloroplast-targeted Hsp90 plays essential roles in plastid development and embryogenesis in Arabidopsis possibly linking with VIPP1 . Physiol. Plant. 150 : 292 – 307 . Google Scholar CrossRef Search ADS PubMed Flores-Pérez Ú. , Bédard J. , Tanabe N. , Lymperopoulos P. , Clarke A.K. , Jarvis P. ( 2016 ) Functional analysis of the Hsp93/ClpC chaperone at the chloroplast envelope . Plant Physiol. 170 : 147 – 162 . Google Scholar CrossRef Search ADS PubMed Flores-Pérez Ú. , Jarvis P. ( 2013 ) Molecular chaperone involvement in chloroplast protein import . Biochim. Biophys. Acta 1833 : 332 – 340 . Google Scholar CrossRef Search ADS PubMed Gogala M. , Becker T. , Beatrix B. , Armache J.P. , Barrio-Garcia C. , Berninghausen O. , et al. . ( 2014 ) Structures of the Sec61 complex engaged in nascent peptide translocation or membrane insertion . Nature 506 : 107 – 110 . Google Scholar CrossRef Search ADS PubMed Hauenstein M. , Christ B. , Das A. , Aubry S. , Hörtensteiner S. ( 2016 ) A role for Tic55 as a hydroxylase of phyllobilins, the products of chlorophyll breakdown during plant senescence . Plant Cell 28 : 2510 – 2527 . Google Scholar CrossRef Search ADS PubMed Hirabayashi Y. , Kikuchi S. , Oishi M. , Nakai M. ( 2011 ) In vivo studies on the roles of two closely related Arabidopsis Tic20 proteins, AtTic20-I and AtTic20-IV . Plant Cell Physiol . 52 : 469 – 478 . Google Scholar CrossRef Search ADS PubMed Hirohashi T. , Hase T. , Nakai M. ( 2001 ) Maize non-photosynthetic ferredoxin precursor is missorted to the intermembrane space of chloroplasts in the presence of light . Plant Physiol . 125 : 2154 – 2163 . Google Scholar CrossRef Search ADS PubMed Huang P.K. , Chan P.T. , Su P.H. , Chen L.J. , Li H.-M. ( 2016 ) Chloroplast Hsp93 directly binds to transit peptides at an early stage of the preprotein import process . Plant Physiol. 170 : 857 – 866 . Google Scholar CrossRef Search ADS PubMed Inoue H. , Li M. , Schnell D.J. ( 2013 ) An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts . Proc. Natl. Acad. Sci. USA 110 : 3173 – 3178 . Google Scholar CrossRef Search ADS Jarvis P. , López-Juez E. ( 2013 ) Biogenesis and homeostasis of chloroplasts and other plastids . Nat. Rev. Mol. Cell Biol. 14 : 787 – 802 . Google Scholar CrossRef Search ADS PubMed Jiang T. , Oh E.S. , Bonea D. , Zhao R. ( 2017 ) HSP90C interacts with PsbO1 and facilitates its thylakoid distribution from chloroplast stroma in Arabidopsis . PLoS One 12 : e0190168 . Google Scholar CrossRef Search ADS PubMed Kasmati A.R. , Topel M. , Patel R. , Murtaza G. , Jarvis P. ( 2011 ) Molecular and genetic analyses of Tic20 homologues in Arabidopsis thaliana chloroplasts . Plant J . 66 : 877 – 889 . Google Scholar CrossRef Search ADS PubMed Kasmati A.R. , Töpel M. , Khan N.Z. , Patel R. , Ling Q. , Karim S. , et al. . ( 2013 ) Evolutionary, molecular and genetic analyses of Tic22 homologues in Arabidopsis thaliana chloroplasts . PLoS One 8 : e63863 . Google Scholar CrossRef Search ADS PubMed Kessler F. , Blobel G. ( 1996 ) Interaction of the protein import and folding machineries of the chloroplast . Proc. Natl. Acad. Sci. USA 93 : 7684 – 7689 . Google Scholar CrossRef Search ADS Ki S.W. , Ishigami K. , Kitahara T. , Kasahara K. , Yoshida M. , Horinouchi S. ( 2000 ) Radicicol binds and inhibits mammalian ATP citrate lyase . J. Biol. Chem. 275 : 39231 – 39236 . Google Scholar CrossRef Search ADS PubMed Kikuchi S. , Bédard J. , Hirano M. , Hirabayashi Y. , Oishi M. , Imai M. , et al. . ( 2013 ) Uncovering the protein translocon at the chloroplast inner envelope membrane . Science 339 : 571 – 574 . Google Scholar CrossRef Search ADS PubMed Kikuchi S. , Hirohashi T. , Nakai M. ( 2006 ) Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE . Plant Cell Physiol . 47 : 363 – 371 . Google Scholar CrossRef Search ADS PubMed Kikuchi S. , Oishi M. , Hirabayashi Y. , Lee D.W. , Hwang I. , Nakai M. ( 2009 ) A 1-megadalton translocation complex containing Tic20 and Tic21 mediates chloroplast protein import at the inner envelope membrane . Plant Cell 21 : 1781 – 1797 . Google Scholar CrossRef Search ADS PubMed Köhler D. , Helm S. , Agne B. , Baginsky S. ( 2016 ) Importance of translocon subunit Tic56 for rRNA processing and chloroplast ribosome assembly . Plant Physiol. 172 : 2429 – 2444 . Google Scholar CrossRef Search ADS PubMed Köhler D. , Montandon C. , Hause G. , Majovsky P. , Kessler F. , Baginsky S. , et al. . ( 2015 ) Characterization of chloroplast protein import without Tic56, a component of the 1-megadalton translocon at the inner envelope membrane of chloroplasts . Plant Physiol. 167 : 972 – 990 . Google Scholar CrossRef Search ADS PubMed Kouranov A. , Chen X. , Fuks B. , Schnell D.J. ( 1998 ) Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane . J. Cell Biol. 143 : 991 – 1002 . Google Scholar CrossRef Search ADS PubMed Kovacheva S. , Bédard J. , Wardle A. , Patel R. , Jarvis P. ( 2007 ) Further in vivo studies on the role of the molecular chaperone, Hsp93, in plastid protein import . Plant J . 50 : 364 – 379 . Google Scholar CrossRef Search ADS PubMed Kovács-Bogdán E. , Benz J.P. , Soll J. , Bölter B. ( 2011 ) Tic20 forms a channel independent of Tic110 in chloroplasts . BMC Plant Biol. 11 : 133. Google Scholar CrossRef Search ADS PubMed Lee D.-W. , Yoo Y.-J. , Razzak M.A. , Hwang I. ( 2018 ) Prolines in transit peptides are crucial for efficient preprotein translocation into chloroplasts . Plant Physiol. 176 : 663 – 677 . Google Scholar CrossRef Search ADS PubMed Li Y. , Singhal R. , Taylor I.W. , McMinn P.H. , Chua X.Y. , Cline K. , et al. . ( 2015 ) The Sec2 translocase of the chloroplast inner envelope contains a unique and dedicated SECE2 component . Plant J. 84 : 647 – 658 . Google Scholar CrossRef Search ADS PubMed Ling Q. , Huang W. , Baldwin A. , Jarvis P. ( 2012 ) Chloroplast biogenesis is regulated by direct action of the ubiquitin–proteasome system . Science 338 : 655 – 659 . Google Scholar CrossRef Search ADS PubMed Ling Q. , Jarvis P. ( 2015 ) Regulation of chloroplast protein import by the ubiquitin E3 ligase SP1 is important for stress tolerance in plants . Curr. Biol . 25 : 2527 – 2534 . Google Scholar CrossRef Search ADS PubMed Lintala M. , Schuck N. , Thormählen I. , Jungfer A. , Weber K.L. , Weber A.P. , et al. . ( 2014 ) Arabidopsis tic62 trol mutant lacking thylakoid-bound ferredoxin-NADP+ oxidoreductase shows distinct metabolic phenotype . Mol. Plant 7 : 45 – 57 . Google Scholar CrossRef Search ADS PubMed Liu L. , McNeilage R.T. , Shi L.X. , Theg S.M. ( 2014 ) ATP requirement for chloroplast protein import is set by the Km for ATP hydrolysis of stromal Hsp70 in Physcomitrella patens . Plant Cell. 26 : 1246 – 1255 . Google Scholar CrossRef Search ADS PubMed Lumme C. , Altan-Martin H. , Dastvan R. , Sommer M.S. , Oreb M. , Schuetz D. , et al. . ( 2014 ) Nucleotides and substrates trigger the dynamics of the Toc34 GTPase homodimer involved in chloroplast preprotein translocation . Structure 22 : 526 – 538 . Google Scholar CrossRef Search ADS PubMed Moreno J.C. , Martínez-Jaime S. , Schwartzmann J. , Karcher D. , Tillich M. , Graf A. , et al. . ( 2018 ) Temporal proteomics of inducible RNAi lines of Clp protease subunits identifies putative protease substrates . Plant Physiol. 176 : 1485 – 1508 . Google Scholar CrossRef Search ADS PubMed Nakai M. ( 2015a ) The TIC complex uncovered: the alternative view in the molecular mechanism of protein translocation across the inner envelope membrane of chloroplasts . Biochim. Biophys. Acta 1847 : 957 – 967 . Google Scholar CrossRef Search ADS Nakai M. ( 2015b ) YCF1: a green TIC . Plant Cell 27 : 1834 – 1838 . Google Scholar CrossRef Search ADS O’Neil P.K. , Richardson L. , Paila Y.D. , Piszczek G. , Chakravarthy S. , Noinaj N. , et al. . ( 2017 ) The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts . Proc. Natl. Acad. Sci. USA 114 : E4868 – E4876 . Google Scholar CrossRef Search ADS Ouyang M. , Li X. , Ma J. , Chi W. , Xiao J. , Zou M. , et al. . ( 2011 ) LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway . Nat. Commun. 2 : 277. Google Scholar CrossRef Search ADS PubMed Paila Y.D. , Richardson L.G.L. , Schnell D.J. ( 2015 ) New insights into the mechanism of chloroplast protein import and its integration with protein quality control, organelle biogenesis and development . J. Mol. Biol . 427 : 1038 – 1060 . Google Scholar CrossRef Search ADS PubMed Park E. , Ménétret J.F. , Gumbart J.C. , Ludtke S.J. , Li W. , Whynot A. , et al. . ( 2014 ) Structure of the SecY channel during initiation of protein translocation . Nature 506 : 102 – 106 . Google Scholar CrossRef Search ADS PubMed Parker N. , Wang Y. , Meinke D. ( 2014 ) Natural variation in sensitivity to a loss of chloroplast translation in arabidopsis . Plant Physiol . 166 : 2013 – 2027 . Google Scholar CrossRef Search ADS PubMed Parker N. , Wang Y. , Meinke D. ( 2016 ) Analysis of arabidopsis accessions hypersensitive to a loss of chloroplast translation . Plant Physiol. 172 : 1862 – 1875 . Google Scholar CrossRef Search ADS PubMed Pfeffer S. , Burbaum L. , Unverdorben P. , Pech M. , Chen Y. , Zimmermann R. , et al. . ( 2015 ) Structure of the native Sec61 protein-conducting channel . Nat. Commun. 6 : 8403. Google Scholar CrossRef Search ADS PubMed Richardson L.G.L. , Singhal R. , Schnell D.J. ( 2017 ) The integration of chloroplast protein targeting with plant developmental and stress responses . BMC Biol. 15 : 118. Google Scholar CrossRef Search ADS PubMed Rudolf M. , Machettira A.B. , Groß L.E. , Weber K.L. , Bolte K. , Bionda T. , et al. . ( 2013 ) In vivo function of Tic22, a protein import component of the intermembrane space of chloroplasts . Mol. Plant 6 : 817 – 829 . Google Scholar CrossRef Search ADS PubMed Shi L.X. , Theg S.M. ( 2010 ) A stromal heat shock protein 70 system functions in protein import into chloroplasts in the moss Physcomitrella patens . Plant Cell 22 : 205 – 220 . Google Scholar CrossRef Search ADS PubMed Shi L.X. , Theg S.M. ( 2011 ) The motors of protein import into chloroplasts . Plant Signal. Behav. 6 : 1397 – 1401 . Google Scholar CrossRef Search ADS PubMed Shi L.X. , Theg S.M. ( 2013a ) The chloroplast protein import system: from algae to trees . Biochim. Biophys. Acta 1833 : 314 – 331 . Google Scholar CrossRef Search ADS Shi L.X. , Theg S.M. ( 2013b ) Energetic cost of protein import across the envelope membranes of chloroplasts . Proc. Natl. Acad. Sci. USA 110 : 930 – 935 . Google Scholar CrossRef Search ADS Sjögren L.L. , MacDonald T.M. , Sutinen S. , Clarke A.K. ( 2004 ) Inactivation of the clpC1 gene encoding a chloroplast Hsp100 molecular chaperone causes growth retardation, leaf chlorosis, lower photosynthetic activity, and a specific reduction in photosystem content . Plant Physiol . 136 : 4114 – 4126 . Google Scholar CrossRef Search ADS PubMed Sjögren L.L. , Tanabe N. , Lymperopoulos P. , Khan N.Z. , Rodermel S.R. , Aronsson H. , et al. . ( 2014 ) Quantitative analysis of the chloroplast molecular chaperone ClpC/Hsp93 in Arabidopsis reveals new insights into its localization, interaction with the Clp proteolytic core, and functional importance . J. Biol. Chem. 289 : 11318 – 11330 . Google Scholar CrossRef Search ADS PubMed Sjuts I. , Soll J. , Bölter B. ( 2017 ) Import of soluble proteins into chloroplasts and potential regulatory mechanisms . Front. Plant Sci. 8 : 168. Google Scholar CrossRef Search ADS PubMed Su P.H. , Li H.M. ( 2010 ) Stromal Hsp70 is important for protein translocation into pea and Arabidopsis chloroplasts . Plant Cell 22 : 1516 – 1531 . Google Scholar CrossRef Search ADS PubMed Teng Y.S. , Chan P.T. , Li H.-M. ( 2012 ) Differential age-dependent import regulation by signal peptides . PLoS Biol. 10 : e1001416. Google Scholar CrossRef Search ADS PubMed Topel M. , Jarvis P. ( 2011 ) The Tic20 gene family: phylogenetic analysis and evolutionary considerations . Plant Signal. Behav . 6 : 1046 – 1048 . Google Scholar CrossRef Search ADS PubMed Tsai J.Y. , Chu C.C. , Yeh Y.H. , Chen L.J. , Li H.M. , Hsiao C.D. ( 2013 ) Structural characterizations of the chloroplast translocon protein Tic110 . Plant J. 75 : 847 – 857 . Google Scholar CrossRef Search ADS PubMed van Dooren G.G. , Tomova C. , Agrawal S. , Humbel B.M. , Striepen B. ( 2008 ) Toxoplasma gondii Tic20 is essential for apicoplast protein import . Proc. Natl. Acad. Sci. USA 105 : 13574 – 13579 . Google Scholar CrossRef Search ADS Vitale A. , Schnell D.J. , Raikhel N.V. , Chrispeels M.J. ( 2015 ) Protein sorting and vesicle traffic. In Biochemistry & Molecular Biology of Plants , 2nd edn . Edited by Buchanan B.B. , Gruissem W. , Jones R.L. pp. 151 – 190 . John Wiley & Sons, Ltd ., Chichester . Zufferey M. , Montandon C. , Douet V. , Demarsy E. , Agne B. , Baginsky S. , et al. . ( 2017 ) The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery . J. Biol. Chem. 292 : 6952 – 6964 . Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations BN–PAGE Blue Native–PAGE ER endoplasmic reticulum TIC translocon at the inner envelope membrane of chloroplasts TOC translocon at the outer envelope membrane of chloroplasts © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Plant and Cell PhysiologyOxford University Press

Published: Apr 19, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off