Once upon a Time – Chloroplast Protein Import Research from Infancy to Future Challenges

Molecular Plant Review Article

Once upon a Time – Chloroplast Protein Import Research from Infancy to Future Challenges Bettina Bo¨lter1,2,* and Ju¨rgen Soll1,2 1

Department Biologie I-Botanik, Ludwig-Maximilians-Universita¨t, Großhaderner Straße 2-4, 82152 Planegg-Martinsried, Germany

2

Munich Center for Integrated Protein Science CiPSM, Ludwig-Maximilians-Universita¨t, Feodor-Lynen-Strasse 25, 81377 Munich, Germany

*Correspondence: Bettina Bo¨lter ([email protected]) http://dx.doi.org/10.1016/j.molp.2016.04.014

ABSTRACT Protein import into chloroplasts has been a focus of research for several decades. The first publications dealing with this fascinating topic appeared in the 1970s. From the initial realization that many plastid proteins are being encoded for in the nucleus and require transport into their target organelle to the identification of import components in the cytosol, chloroplast envelopes, and stroma, as well as elucidation of some mechanistic details, more fascinating aspects are still being unraveled. With this overview, we present a survey of the beginnings of chloroplast protein import research, the first steps on this winding road, and end with a glimpse into the future. Key words: chloroplast protein import, historical perspective, Toc, Tic, evolution, envelope membranes Bo¨lter B. and Soll J. (2016). Once upon a Time – Chloroplast Protein Import Research from Infancy to Future Challenges. Mol. Plant. 9, 1–15.

THE VERY BEGINNING – EVOLUTIONARY ASPECTS Chloroplasts are the characteristic organelles of all photosynthetic eukaryotic organisms from unicellular algae to vascular plants. In addition to their hallmark function, the conversion of light into chemical energy through photosynthesis, they perform other vital functions such as synthesis of fatty acids, amino acids, and secondary metabolites. Their evolutionary origin lies in an endosymbiotic event, in which a free-living cyanobacterium was enslaved by an ancestral eukaryotic host (Gould et al., 2008). Within the course of evolution, the majority of genes from the cyanobacterial endosymbiont were transferred to the nuclear genome, rendering the host in control over its newly acquired permanent guest. However, about 100 protein-coding genes were kept in the genome of the chloroplast, called plastome (Martin et al., 1998), so that their expression required tight coordination with nuclear gene expression. Even more drastic were the consequences of all other plastid proteins now being translated on cytosolic ribosomes: an efficient targeting mechanism as well as dedicated translocation machineries in the chloroplast membranes needed to be established. From comparative sequence analyses, we know that some components of the import complexes were already present in the cyanobacterial progenitor and were adapted to the new function of transporting preproteins into chloroplasts, e.g., the import channel in the outer envelope (Bo¨lter et al., 1998a, 1998b; Reumann and Keegstra, 1999), whereas other constituents were commandeered from the eukaryotic host and provided with a completely new function (for review, see Kalanon and McFadden, 2008).

The above process is called primary endosymbiosis and led to the generation of three lineages with primary plastids: chlorophytes (including green algae and vascular plants), rhodophytes (red algae), and glaucophytes. Uptake of a eukaryotic green or red alga by independent eukaryotic hosts (secondary endosymbiosis) led to the emergence of secondary plastids found in euglenophytes, chlorarachniophytes, and chromalveolates. In contrast to primary plastids, these secondary endosymbionts kept not only their original envelope membranes but at least one of the eukaryotic membranes surrounding them after engulfment (Gould et al., 2008). Protein translocation machineries are found in all membranes to ensure that these complex plastids are provided with their specific proteome (Figure 1). Secondary endosymbiosis happened several times independently while primary endosymbiosis was successfully established only once.

NUCLEUS-ENCODED CHLOROPLAST PROTEINS First reports on chloroplast-residing proteins being encoded for in the nucleus appeared in the early 1970s (Kung et al., 1972). At that time, the identity of the proteins was not clear although, from today’s point of view, we can make an educated guess that the authors were looking at light-harvesting complex proteins. A few years later, the same authors identified the small subunit of Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

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Historical Survey of Chloroplast Protein Import Figure 1. Plastid Evolution. An event termed primary endosymbiosis resulted in the formation of the ancestor of all chloroplastcontaining organisms; a eukaryotic host already harboring a nucleus and mitochondria (primary host) engulfed a cyanobacterium, which was eventually integrated into the cell as an organelle. This gave rise to the three algal lineages, Glaucophyta, Rhodophyta, and Chlorophyta, all accommodating the newly acquired photosynthetic organelle, the plastid. The land plants eventually evolved from the Chlorophyta. Later on, further independent endosymbiotic uptakes took place: either a non-photosynthetic host enclosed a red alga, leading to the formation of Cryptophyta; or another picked up a green alga, which resulted in the genesis of Chlorarachniophyta. Both groups split up into different subgroups (not shown here), some of which kept a residual nucleus of the engulfed algae, which is named nucleomorph. In contrast to primary plastids, which only feature the original outer and inner membrane of the cyanobacterial ancestor, the secondary plastids kept one or two membranes from the secondary host, resulting in complex plastids with three or four membranes.

ribulose-1,5-bisphosphate-carboxylase/oxygenase (pSSU), still the favorite model protein for import studies today, as the product of a nuclear gene that is translated on 80S ribosomes in the cytosol and consequently needs to be transported into chloroplasts where it forms an L8S8 holo-complex with a plastome-encoded large subunit (LSU) (Kung, 1976). The same protein from the green alga Chlamydomonas was found to be synthesized as a larger precursor in a wheat germ extract, which is cleaved by an algal extract (Dobberstein et al., 1977). Further characterization of this process in the alga was hampered by the impossibility of isolating intact chloroplasts. Therefore, several groups endeavored to enlighten cytosolic translation and transport into chloroplasts in a system derived from higher plants, choosing pea and spinach as models that readily yielded intact chloroplasts (Morgenthaler et al., 1975). Translation of total mRNA in a wheat germ lysate and further incubation with isolated chloroplasts could then demonstrate that higher plant SSU is also a precursor (pSSU) and proteolytically processed upon entrance into the organelle to yield the mature form SSU (Chua and Schmidt, 1978; Highfield and Ellis, 1978). The number of active import sites per chloroplast was estimated to be around 3500 by counting radioactive mature proteins inside the organelles after import (Cline et al., 1985; Friedman and Keegstra, 1989) and about 30 000 by immuno-gold-labeling of ultrathin sections with antibodies against the main import components (Morin and Soll, 1997). Apart from the processing activity being soluble, nothing more was known about the translocation and processing event. This changed in 1984, when Robinson and Ellis (1984) reported the isolation of a single enzyme responsible for the specific N-terminal cleavage of pSSU to its mature form. Smeekens et al. (1986) showed that the N-terminal extension, now called the transit peptide, is responsible for intra-plastidial targeting of pFD (precursor of ferredoxin) and pPC (precursor of plastocyanin), thus establishing the general principle for chloroplast targeting, which turned out to apply to the majority of chloroplast proteins (von 2

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Heijne and Nishikawa, 1991). These authors also showed that proteins destined for the thylakoid lumen comprise a bipartite targeting signal, where the N-proximal part engages the general import machineries and is processed in the same manner as stromal proteins, and the second part mediates lumenal targeting across the thylakoid membranes (Smeekens et al., 1986). Transport across the thylakoid membranes is achieved by three different pathways, two implemented by the Sec, the Tat machineries, and the third depending on an electrochemical gradient over the thylakoid membrane named spontaneous insertion (Michl et al., 1994). For more details, please refer to excellent recent reviews (Albiniak et al., 2012; Cline, 2015). Several groups dedicated their research to further characterizing the processing enzyme as well as the nature of transit peptides. Lamppa and Abad (1987) elucidated the mechanistic details over the years: the stromal processing peptidase (SPP) is a metallo-enzyme of 140 kDa with a zinc ion in the active center, which recognizes specific motifs within preprotein sequences (VanderVere et al., 1995; Richter and Lamppa, 1998, 2003). The general characterization of transit peptides first published by Keegstra and independently by von Heijne in 1989 (Lubben et al., 1989; von Heijne and Nishikawa, 1991) still holds true: chloroplast transit peptides are rich in serine and threonine residues, lack tyrosine and negatively charged amino acids, and thus feature an overall positive charge (Bruce, 2000). In contrast to mitochondrial transit sequences, which form classical a helices (von Heijne, 1986), the plastid counterparts have no defined structure per se, but could adopt an a-helical conformation upon interaction with either receptor proteins or membrane lipids (von Heijne and Nishikawa, 1991). These quite loosely defined features make it hard to pinpoint chloroplast targeting signal by just looking at the sequence, although prediction programs are constantly evolving by being fed with proteomic data (Kleffmann et al., 2006; Ferro et al., 2010) and might eventually be sufficiently reliable to

Historical Survey of Chloroplast Protein Import correctly predict chloroplast localization (Emanuelsson et al., 1999). Regulation of transit peptide recognition is possibly achieved by phosphorylation of serine or threonine residues within the targeting signal in combination with recognition by cytosolic factors (Waegemann et al., 1990; Waegemann and Soll, 1996). The responsible kinases originally described in wheat germ were elusive for a decade but were finally identified in Arabidopsis (Martin et al., 2006). Further characterization revealed that they comprise a small group of three isoforms and belong to the STY kinase family. A double knockout in combination with a knockdown of the third isoform exhibits a phenotype indicating that these kinases play a role in chloroplast differentiation (Lamberti et al., 2011). The functional relevance of transit peptide phosphorylation could lie in the downstream interaction with specific cytosolic components such as Hsp70 and 143-3 proteins, which form a guidance complex to escort the preproteins to the chloroplast surface (May and Soll, 2000). Other preproteins are shepherded by Hsp90/Hsp70 pairs from ribosomes to plastids (Qbadou et al., 2006). It was noticed quite early in the import field that factors present in the cytosol may not be essential for the in vitro setups to work but definitely increase binding/translocation efficiency (Ceccarelli et al., 1996; Chirico et al., 1988; della-Cioppa and Kishore, 1988). The question of whether cytosolic Hsp70 plays a role in chaperoning preproteins after release from the ribosome and before binding to outer envelope receptors was first investigated by Rial et al. (2000). Their efforts revealed that Hsp70 interacts preferentially with transit peptides, confirming the in silico predicted binding sites for this chaperone family. As described above, Hsp70 can act in concert either with 14-3-3 proteins to deliver preproteins to outer membrane receptors, together with Hsp90 or alone. In the first case, precursors are delivered directly to Toc34, whereas preproteins bound to Hsp90 are initially docked at Toc64, then handed over to Toc34 (Qbadou et al., 2006). A chaperone-like protein dedicated to the assistance of tail-anchored outer envelope residents such as OEP7 was discovered 2 years later and named Akr2 (Bae et al., 2008). Further characterization uncovered that this particular chaperone interacts with a member of the small chaperone family (Kim et al., 2011) and uses lipids in the outer envelope as the initial docking site (Kim et al., 2014), thus establishing a further kind of guidance complex in the cytosol.

IMPORT NEEDS TO BE FUELED Apart from specific targeting information within chloroplast preproteins, energy is needed for the import process to be initiated and completed. It became clear early on in chloroplast import research that energy in form of ATP (generated by photosynthetic light reactions) stimulates import (Grossman et al., 1980). The same was found to be true for mitochondrial preprotein import although it took the mitochondrial research community more than half a decade longer to demonstrate this fact (Pfanner and Neupert, 1986). The cloning of preproteins from cDNA made it feasible to analyze the import requirements of one single precursor at a time, which had not been possible when complex mixtures of translated

Molecular Plant whole mRNA were used. Again, pSSU was one of the first model precursor proteins to be cloned, translated, and applied in import assays. Flu¨gge and Hinz (1986) reported the need of ATP outside chloroplasts but wrongly concluded that in the stroma none is necessary. In parallel, Soll and co-workers studied the ATP requirement of import into both chloroplasts and etioplasts and could demonstrate the energy dependence in nonphotosynthetic organelles, making the ATP requirement for plastid protein import a general scheme (Schindler et al., 1987). Whereas it was established early on that, in mitochondria, an electrochemical potential across the inner membrane is mandatory to achieve import (Schleyer et al., 1982), the situation in chloroplasts was shown to be different: a stromal ATPase seemed to consume ATP (Pain and Blobel, 1987) but no membrane potential was required for import (Flu¨gge and Hinz, 1986; Pain and Blobel, 1987; Schindler et al., 1987). The topic of the site of ATP consumption kept a number of researchers busy for many years, resulting in partially conflicting reports on the necessity of ATP at the surface of chloroplasts; Flu¨gge and Hinz (1986) interpreted their results to suggest that ATP was only necessary at the chloroplast surface, and Schindler et al. (1987) confirmed this view. Pain and Blobel (1987) and Theg et al. (1989) concluded from their data that ATP was exclusively required in the stromal compartment. At some point, GTP entered the game when Olsen and Keegstra (1992) demonstrated that this nucleotide promoted precursor binding, if not import. In the following decades, the picture became clearer, and we know today that dedicated chaperones consume ATP in the cytosol, at the outer envelope, possibly in the intermembrane space, and in the stroma. Outside chloroplasts, Hsp70 and Hsp90 have been shown to be involved in preprotein targeting, while at the envelope, Hsp70 alone seems to play a role, which most likely was the source of energy consumption detected by Flu¨gge and Hinz in 1986. In the stroma, Hsp70, Hsp90, and Hsp93, as well as Cpn60, have been implicated in motor functions although the latter seems responsible for folding of specific substrates (Tro¨sch et al., 2015). Some recent reports favor Hsp70 as a main import motor component in Arabidopsis as well as in the moss Physcomitrella patens (Shi and Theg, 2010; Su and Li, 2010). Stromal Hsp70 evaded being identified in connection with incoming preproteins for a long time, which in hindsight was due to the antibody used in most experiments until Su and Li (2010) changed the approach and could clearly see Hsp70 bound to Tic components as well as to importing precursor proteins. In agreement with this, the study by Shi and Theg (2010) on Physcomitrella, in which they showed biochemically as well as by mutational analyses that Hsp70 in the stroma is critically involved in motor function of the Tic translocon. For more details, please refer to Schwenkert et al. (2011). The latest findings from Huang et al. (2016), however, suggest that Hsp93 also binds to transit peptides as well as to the mature part of incoming preproteins in early import stages. According to their new model, at least some Hsp93 molecules directly participate in motor function, whereas Hsp70 alone is further involved in the final pulling through step. The subject of the import motor is still not completely resolved and future research will need to unravel the mechanisms at work at the stromal side of the import complexes. GTP-binding and hydrolysis take place solely at the outer envelope, which is discussed in detail below. Molecular Plant 9, 1–15, June 2016 ª The Author 2016.

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THE PLAYERS IN THE IMPORT GAME Outer Envelope Constituents The first indication that proteins at the outer envelope at least partially exposed to the cytosol are essential for import was presented as early as in 1978: protease treatment of the chloroplast surface abolished import of pSSU (Chua and Schmidt, 1978). Almost a decade later, a then quite sophisticated crosslink approach revealed that preproteins bind to a protease-sensitive receptor protein in the initial stages of import (Cornwell and Keegstra, 1987). The molecular weight of the crosslink product justified the interpretation that this was the first glimpse of what would later be known as OEP86 although methodology was not sufficiently advanced to reveal the molecular identity of this receptor protein. Pain et al. (1988) reported the identification of a second receptor protein of 30 kDa localized to contact zones between the outer and inner envelopes. The authors applied a combination of the anti-idiotypic antibody approach with radioactive label-transfer labeling. The antibody that inhibited preprotein import recognized a major membrane protein that also seemingly interacted with pSSU as shown by immunoprecipitation. However, the results obtained with this type of antibody were misleading, since the 30 kDa protein was unequivocally identified as the phosphate translocator (TPT) of the inner envelope membrane (Flu¨gge et al., 1991). Obviously, the antibody cross-reacted with the TPT due to overlapping epitopes, thereby fooling the researchers into believing they had found an elusive protein import receptor. The first isolated import complex from the outer envelope membrane was prepared by Waegemann and Soll (1991). Density gradient centrifugation of solubilized outer envelope yielded four main proteins associated with a bound precursor of 34, 75, 86, and 70 kDa, which were confirmed 3 years later (Kessler et al., 1994; Schnell et al., 1994). The latter applied a chimeric precursor protein consisting of the pSSU transit peptide and a protein A moiety, which was used to affinity purify interacting proteins from chloroplast membranes. These authors went a step further and solved the molecular identity of the preprotein bound components: the 75 kDa band represented IAP/OEP75, an integral membrane protein putatively building a channel by transmembrane b sheets (Schnell et al., 1994). Their proposition at that point was that this channel protein was accompanied by regulatory components, one or more of which was dependent on GTP hydrolysis necessary for the presentation of the transit peptide and/or the regulation of the channel’s open state. A parallel study characterized the 34 and 86 kDa proteins as IAP/OEP34 and IAP/OEP86, two GTP-binding receptor components that expose protease-sensitive portions to the chloroplast surface (Kessler et al., 1994; Seedorf et al., 1995). OEP34 was shown to be synthesized as a mature protein, which inserted into the outer envelope independent of any receptor proteins or energy, a feature that was later defined for almost all OE residents (for review, please refer to Hofmann and Theg, 2005; Bo¨lter and Soll, 2011). In contrast, OEP86 was thought to carry an N-terminal extension, which was interpreted as representing a transit peptide (Kessler et al., 1994). Similar results were obtained in parallel by the Soll group (Hirsch et al., 1994; Muckel and Soll, 1996). Today we know that OEP86 was only a proteolytic fragment and the putative TP is 4

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Historical Survey of Chloroplast Protein Import in fact part of the full-length sequence of Toc159 (Bo¨lter et al., 1998a, 1998b; Chen et al., 2000). By then, the nomenclature used by the different research groups was so diverse and thus confusing that a common nomenclature for import components was introduced, naming the proteins residing in the outer membrane Toc (for translocon at the outer envelope of chloroplasts) and the ones sitting in the inner membrane Tic (for translocon at the inner envelope of chloroplasts), plus their apparent molecular weight (Schnell et al., 1997), thereby paving the way for clear-cut communication. More detailed analyses of the GTPase cycles passed through by Toc34 and Toc159 resulted in equivocal conclusions about the function/mechanism: Toc34 was reported to form a homodimer in the GDP bound state (Sun et al., 2002), proposed to regulate precursor binding. The purported hypothesis was that each monomer acted as a GTPase-activating-protein (GAP) for the other, supported by a critical arginine residue present at the dimerization cleft. However, only structural relevance of this residue was shown, and a mutation had little effect on binding (Weibel et al., 2003). Preprotein binding was shown to stimulate GTP hydrolysis (Jelic et al., 2002; Lee et al., 2009a) and changed dimerization kinetics (Oreb et al., 2011), but did not further nucleotide exchange, which now seems to be an indirect effect of dimer dissociation (Lumme et al., 2014). Heterodimerization of Toc34 and Toc159 was observed by different groups (Wallas et al., 2003; Yeh et al., 2007; Rahim et al., 2009) and was reported to be mediated by conserved arginine residues present in both receptor proteins (Schleiff et al., 2002). Possibly, this heteromeric association represents another regulatory element. Still missing in this picture are the GAP and GEF proteins (for details, see Chang et al., 2012), and it will be a challenging task in the future to define their identity. Besides nucleotide binding, hydrolysis receptor activity is regulated by phosphorylation. The first account of a phosphorylated import-related protein in the outer envelope was made by Hinz and Flugge (1988) who detected a 51 kDa protein labeled by a phosphate moiety, which most probably represented a characteristic Toc159 fragment. Later, phosphorylation of Toc34 and Toc159 by envelope-bound kinases was shown (Sveshnikova et al., 2000; Jelic et al., 2002, 2003), which seemed to downregulate GTPase activity as well as Toc34 assembly into the complex (Oreb et al., 2008). The in vivo relevance of receptor phosphorylation is still under debate, because different groups have reported equivocal findings (Aronsson et al., 2006; Oreb et al., 2007, 2008). The responsible kinases are still elusive and represents a further future challenge in understanding receptor regulation. The 70 kDa protein co-fractionating with these Toc core components was found to represent an envelope-bound Hsp70 isoform (Marshall et al., 1990). It is still not fully clear if Hsp70 is only associated on the chloroplast surface, probably by means of preprotein binding, or if another isoform is localized to the intermembrane space (Marshall et al., 1990). It has been shown, however, that a minimal unit of a functional outer envelope translocon consists of Toc75, Toc34, and Toc159 in the 4:4:1 stoichiometry (Schleiff et al., 2003).

Historical Survey of Chloroplast Protein Import

Molecular Plant

Figure 2. Two Topological Models for Tic110. Two different topologies for Tic110 have been proposed: (A) Tic110 is anchored to the inner envelope by two hydrophobic a helices while the remainder of the protein is located in the stroma as a globular, soluble domain. (B) agrees in terms of the two hydrophobic helices but suggests that the C-terminal part comprises four amphipathic helices, which build a protein-conducting channel in the form of a homodimer.

The last Toc component to be identified to date was Toc64 (Sohrt and Soll, 2000), which was found to crosslink to pSSU as well as to other Toc members, thus rendering it a bona fide import complex constituent. Functionally, Toc64 was shown to act as a primary docking protein for Hsp90-bound precursor proteins via its cytosolic exposed TPR domain (Qbadou et al., 2006, 2007). It could at least partially function in concert with Toc34 as indicated by studying synthetic effects in double mutant plants (Sommer et al., 2013). Toc64 seems to play a role analogous to that of Tom70 in the outer membrane of mammalian mitochondria. Tom70 in concert with Tom20 is involved in precursor recognition in mitochondria of mammals and yeast, where Tom70 likewise binds to chaperone precursor complexes associated with Hsp70 and Hsp90 by means of its TPR domain (Young et al., 2003). Plant mitochondria do not feature a Tom70 homolog. However, a homolog of Toc64 is found in the mitochondrial membrane, which might take over functionally for Tom70 in plants and be involved in precursor recognition (Chew et al., 2004). An equivalent mechanism for recognizing chaperone-bound preproteins is found at the ER, where one or more translocon component contains TPR domains (Schweiger et al., 2012). Thus it seems that Toc64 represents a conserved docking protein involved in binding molecular chaperones carrying preproteins. None of the TPRcontaining chaperone docking proteins is essential in vivo, most likely because the endogenous targeting information contained in the preproteins is also recognized independently by the receptors in the translocon, which results in a bypass pathway.

Members of the Inner Envelope Translocon The first constituent of the inner envelope translocon to obtain a name was Tic110 from pea (formerly named IAP100/IEP110) (Kessler and Blobel, 1996; Lubeck et al., 1996). The protein had been observed as a crosslink product in previous studies (Schnell et al., 1994), but its identity remained elusive for a couple more years. It was seen in connection with p/mSSU by immunoprecipitation, which also provided evidence of a specific interaction with Cpn60 (Kessler and Blobel, 1996).

Tic110 was shown to be an integral membrane protein with two predicted hydrophobic membrane spanning helices. To analyze its membrane topology, several groups conducted protease treatments of chloroplasts as well as inner envelope vesicles with thermolysin and trypsin and got contradicting results: Kessler and Blobel (1996) reported Tic110 to be trypsin resistant in whole chloroplasts and isolated inner envelope vesicles, whereas in a joint publication of the Keegstra and Soll groups yielded data indicating that Tic110 is accessible to trypsin already in intact plastids, and consequently would be partially exposed to the intermembrane space (Lu¨beck et al., 1996), although they all agreed that the N-terminal part was the membrane anchor. Just a year after the joint report, Keegstra and co-workers published contradictory results from trypsin treatments, claiming that Tic110 was resistant to protease after all and the degradation observed in the previous paper was due to improperly quenched protease activity (Jackson et al., 1998). This conflict still persists to the present day, although in 2009 Balsera et al. (2009a) demonstrated unequivocally that even upon perfect quenching of trypsin, Tic110 gets degraded to the same fragments observed a decade earlier by Lu¨beck. Thus, this discrepancy has still not been fully resolved and the topology of Tic110 is still to be vividly discussed. With regard to the spatial structure of Tic110, its function in protein import is as fiercely argued about as its topology. Those propagating the model (Figure 2A and 2B) with the large C terminus solely exposed to the stroma as a globular domain suggesting Tic110’s function in the binding of stromal chaperones at a late stage of import (Inaba et al., 2003). The alternative model, which matches the data from the protease treatments, proposes Tic110 as the counterpart to Toc75, constituting the preprotein conducting channel where the actual pore is formed by eight amphipathic helices in the C terminus of the Tic110 dimer (Heins et al., 2002; Balsera et al., 2009a). Additional experimental data favor the latter model: electrophysiological measurements, where the purified C terminus without the N-terminal hydrophobic helices (amino acids 91–966 in the pea protein, psTic110DN) was reconstituted into liposomes and fused with an artificial bilayer, showed that psTic110DN forms a cation-selective channel of sufficient size to fulfill the task of Molecular Plant 9, 1–15, June 2016 ª The Author 2016.

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Molecular Plant transporting preproteins (Heins et al., 2002; Balsera et al., 2009a). Moreover, cysteine-labeling disclosed that at least one of nine cysteine residues is located in the intermembrane space, implying that apart from the two hydrophobic helices in the N terminus, a minimum of two more transmembrane regions must be present. Taking into account all other published data obtained for Tic110 concerning transit peptide and chaperone binding (Inaba et al., 2003; Inaba et al., 2005; Kovacheva et al., 2005), the topological model proposing that the C terminus transverses the inner envelope membrane four times by means of amphipathic helices makes perfect sense (Balsera et al., 2009b). In addition, the recently published structure of a C-terminal fragment of Cyanidioschyzon merolae Tic110 (Tsai et al., 2013) mostly fits this model, even if a separated sliver of such a large protein is unlikely to adopt the native structure found in the full-length protein; the artificial N terminus of the crystallized portion starts in the middle of the predicted third amphipathic helix, so that folding comparable with the native protein is prevented by the choice of fragment. Consequently, the finding that this crystal structure does not reveal any possibly membrane spanning parts does not exclude the possibility that the full-length C terminus of Tic110 forms a channel via amphipathic helices. Recently, however, the involvement of Tic110 in protein import itself has been challenged (see below). Another Tic component has long been a changeling between the outer and inner envelope. First described by Ko et al. (1995) as Cim/Com44, this protein was found by immunoscreening of a Brassica cDNA library with an antibody against total envelope. An open reading frame encoding a 36 kDa protein was identified, which was not full length compared with the apparent molecular weight of the immunoreactive band found in both envelope fractions on the SDS gel. Import experiments with this truncated protein were clearly not successful, although the data were interpreted differently at that time by the authors. From today’s perspective, it is obvious that a lot of fortuitous results were taken at face value. The same authors renamed the protein as Toc36, again misinterpreting their data (Pang et al., 1997). This controversy was resolved in 1999 by characterizing a cDNA clone from pea, which revealed a 48 kDa protein representing the precursor of the 44 kDa band (Stahl et al., 1999). Immunoprecipitation with a specifically raised antiserum resulted in two bands of 42 and 44 kDa, respectively. N-terminal sequencing by Edman degradation disclosed that the lower band corresponds to a proteolytic fragment of the full-length 44 kDa protein. Thus, ‘‘Toc36’’ from Brassica was indeed a truncated version. This protein was then named Tic40 according to the calculated molecular weight of the mature form. It comprises a single transmembrane helix and a classical transit peptide, consequently following the general import pathway. Subfractionation of the chloroplasts after import as well as immunoblotting and electron microscopic studies clearly confirmed the inner envelope localization. Tic40 can be directly connected to Tic110 by chemical crosslinking using a 0 A˚ crosslinker, demonstrating their close proximity. The C terminus shows clear homologies to Sti1-domains of Hsp70/Hsp90-interacting proteins (Hip), suggesting that Tic40 might act as a co-chaperone, recruiting molecular chaperones such as Hsp93 to the import machinery in conjunction with Tic110 (Stahl et al., 1999). Eukaryotic Sti16

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Historical Survey of Chloroplast Protein Import domains bind to the N- and C-terminal parts of cytosolic Hsp90 and prevent the N-terminal dimerization reaction that is required for efficient ATP hydrolysis by the chaperone (Richter et al., 2003). Throughout the years, Tic40 has repeatedly been reported to interact specifically with Hsp93 (Kovacheva et al., 2007; Shi and Theg, 2011), but novel data imply that Hsp93 does not tightly interact with Tic40 but rather with Tic110 and that the role of Hsp93 in conjunction with the Tic complex is in proteolysis in the context of protein quality control rather than in import (Sjo¨gren et al., 2014; Flores-Pe´rez et al., 2015). Two further Tic members (Tic20 and Tic22) were identified in a label-transfer crosslink approach as interacting with an incoming precursor protein (Kouranov et al., 1998). Tic20 was characterized as an integral membrane protein, and, due to the presence of four predicted hydrophobic helices, was discussed early on as a possible channel protein, whereas Tic22 constitutes a soluble protein in the intermembrane space. The import of Tic22 revealed novel features, indicating that translocation into the intermembrane space (IMs) can be different from the general import pathway (Kouranov et al., 1999; Vojta et al., 2007). A decade later, Tic20 was described as the core component of a 1 MDa complex that did not comprise Tic110 but a protein of 21 kDa, which the authors named Tic21 (Kikuchi et al., 2009). This component, however, had been identified as an iron transporter Pic1 2 years earlier (Duy et al., 2007). The channel activity of Tic20 alone was confirmed by Kovacs-Bogdan et al. (2011) who also found Tic110 and Tic20 to be in two distinct complexes. Affiliated with the core Tic translocon are three proteins that have been termed the redox regulon (Stengel et al., 2007). Originally, two of them were found to be components of a precursorbound Tic complex isolated from blue native PAGE: Tic55 (Caliebe et al., 1997) and Tic62 (Kuchler et al., 2002). The third member, Tic32, was found in direct interaction with the N terminus of Tic110 (Hoermann et al., 2004). All these proteins exhibit redox active features, rendering the idea feasible that they play a role in redox-dependent import regulation; Tic62 and Tic32 constitute active dehydrogenases (Kuchler et al., 2002; Chigri et al., 2006), whereas Tic55 is Rieske protein (Caliebe et al., 1997). The redox regulon has the potential to adapt protein import to the metabolic status of the organelle. We refer the reader to a recent comprehensive review about this topic for more details (Bolter et al., 2015).

ARABIDOPSIS: THE NEW TOOL IN THE GENETIC ERA The introduction of Arabidopsis mutants in a first reverse genetic screen was a milestone in chloroplast research (McKinney et al., 1995). Although this publication did not deal with plastids or protein import, it got the ball rolling for the Arabidopsis genetic era. The first Arabidopsis mutant of an import component was described in 1998: ppi1 = attoc33, the homolog of psToc34 (Jarvis et al., 1998). It showed a pale phenotype during the early developmental stages, whereas after about 2 weeks the plants had a more or less wild-type (WT) appearance. This corresponded to the atToc33 RNA levels, which were high in young plants and rapidly declined with age. This pattern was

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Historical Survey of Chloroplast Protein Import

Figure 3. Schematic Presentation of the General Import Translocon(s). The outer envelope (OE) translocon consists of the two GTP-binding proteins, Toc159 and Toc34, which act as preprotein receptors, the b-barrel channel protein Toc75, and the dynamically associated TPR protein Toc64, which functions as a receptor for Hsp90-bound precursor proteins. The translocation machinery in the inner envelope (IE) comprises Tic110 as the channel counterpart to Toc75, the co-chaperone Tic40 with its Sti1 domains, and the small intermembrane space (IMS) protein Tic22. Associated with these is the redox regulon composed of the two short chain dehydrogenases, Tic32 and Tic62, which features a proline helix in the C terminus, plus the Rieske protein Tic55. The alternatively suggested Tic complex comprising Tic20, Ycf1, Tic100, and Tic56 as described by Kikuchi et al., 2013 and Tic20 as a homo-multimer forms a channel together with Tic214 (Ycf1). Both are associated with Tic56, embedded in the holo-complex, and Tic100 on the intermembrane space side. Both Tic complexes have been reported to interact with the Toc complex, therefore the outer envelope translocation machinery is set in the middle.

similar to the expression of the second isoform, AtToc34, although this was expressed at generally lower levels but was upregulated in attoc33 mutants, which probably accounted for the viability of ppi1 plants. The important role of AtToc33, especially in the very early stages, in cotyledons also became obvious through the finding that in etioplasts the prolamellar body was reduced to 50%. Import efficiency was accordingly low in chloroplasts from very young leaves, whereas those isolated from older leaves showed no defect in import.

import activity mediated by atToc132/atToc120 focuses on non-photosynthetic proteins and that atToc159 is a dedicated receptor for substrates involved in photosynthesis. However, this notion was later rejected due to proteomic data from ppi2 plants, which clearly revealed the presence of nuclear-encoded photosynthetic proteins in the plastids (Bischof et al., 2011). This makes it quite obvious that a grouping into photosynthetic and non-photosynthetic proteins is too simplistic and thus inaccurate. A more reliable grouping is, however, currently not available.

The second Arabidopsis mutant characterized was ppi2, the atToc159 knockout plant (Bauer et al., 2000). In Arabidopsis there are four isoforms belonging to the atToc159 family: atToc159, atToc132, atToc120, and atToc90. All members show a similar domain structure with an N-terminal A domain, a G domain in the middle, and a large M domain at the C terminus (Figure 3) (Cline, 2000; Chang et al., 2012). The main form is atToc159, which is predominantly expressed in green tissue. Consequently, the mutant plants exhibit an albino phenotype and die after the cotyledon stage on soil, making them seedling lethal. Plastids isolated from these plants are devoid of thylakoids and starch, containing structures reminiscent of prolamellar bodies. Obviously, import into such plastids is so drastically impaired that an unknown feedback mechanism results in the downregulation of nuclear genes encoding for chloroplast proteins. The fact that atToc75 and atTic110 amounts in these rudimentary plastids were comparable with WT led to the conclusion that any residual

Also the subject of receptor phosphorylation was addressed by using Arabidopsis mutant plants. In two studies, the phosphomimicking form of atToc33 (AtToc33S181E) exhibited lower GTPase activity in vitro (Aronsson et al., 2006; Oreb et al., 2007), whereas complementation of the ppi1 mutant with three different AtToc33 mutant forms (atToc33S181E/S118A/S181D) representing either phosphomimicking or the non-phosphorylatable version indicated that the phosphorylation status of serine 181 is irrelevant in vivo (Aronsson et al., 2006). A subsequent publication, however, reported a slightly reduced photosynthetic capacity of the atToc33S181E ppi1 mutant (Oreb et al., 2007). This effect was restricted to very early developmental stages under heterotrophic growth conditions, which fits with the requirement of atToc33 in early chloroplast biogenesis (see above). Concerning atToc159, phospho-proteomic profiling revealed that the A domain as well as the full-length atToc159 is highly phosphorylated (Reiland et al., 2009). The hyperphosphorylated A domain was implicated as a possible soluble Molecular Plant 9, 1–15, June 2016 ª The Author 2016.

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Molecular Plant signaling molecule after proteolytic cleavage from atToc159, although experimental evidence for this hypothesis is still lacking (Agne et al., 2010). For Toc64, the first knockout mutant was described in the moss Physcomitrella patens. It did not exhibit any import-related defects, making Toc64 a non-essential protein for preprotein import (Rosenbaum Hofmann and Theg, 2005). A similar finding was described for Arabidopsis attoc64 mutants, in which import was identical to WT plastids (Aronsson et al., 2007). It seems clear that the role of Toc64 is dispensable for plants under all tested growth conditions, indicating that the in vivo function of this outer envelope TPR protein can be bypassed (see above). In contrast to the main Toc receptor proteins, atToc159 and atToc33, which constitute small families in Arabidopsis, the channel protein atToc75 seems to be encoded for by a single gene on chromosome three (atToc75-III) (Arabidopsis Genome Initiative, 2000). Deletion results in embryo lethality as early as the twocell stage of embryo development (Hust and Gutensohn, 2006). A related protein that belongs to the same group of outer membrane b-barrel proteins, which was named atToc75-V due to this structural similarity (Eckart et al., 2002), is obviously not able to complement for the lack of Toc75-III. Further analysis revealed that atToc75-V is part of a family of highly conserved b-barrel proteins that were shown to be involved in membrane protein insertion in bacteria and mitochondria (Schleiff and Soll, 2005). Although it seems to have a comparable structure, it cannot substitute for atToc75-III function. The counterpart to atToc75 in the inner envelope, atTic110, also proved to be essential for plant viability (Inaba et al., 2005). Even the overexpression of N- or C-terminally tagged atTic110 proteins led to a visible phenotype, indicating that this indispensable Tic protein does not like to be interfered with. It seemed that adding a tag meddled with Tic complex assembly, thereby rendering the chloroplasts less able to import preproteins. This effect was partly due to overexpression under the control of a CaMV-35S promoter, but complementation of attic110 heterozygous knockout plants with tagged versions under control of the native promoter likewise failed to restore WT growth (Inaba et al., 2005). Although the exact function of Tic110 is still under debate (see above), the implication of these data is that it plays a crucial role in protein translocation. The presumed partner of Tic110 in recruiting stromal chaperones, Tic40, is a non-essential gene in Arabidopsis, although knockout plants display a severe growth phenotype (Chou et al., 2003). The mutant plants are pale green and grow slowly, which is reflected by reduced chloroplast pigmentation and protein content. Import into these plastids was blocked at the level of the inner envelope at a late stage, supporting the notion of Tic40 being involved in the final steps of translocation (Chou et al., 2006). Complementation of these plants with a chimeric protein consisting of the plant atTic40 N terminus fused to the human Sti1 domain resulted in restored growth, establishing that the conserved domain in the plant protein has the same function as the human homolog. Structural analyses of the atTic40-Sti1 domain confirmed the similarity to the human form (Kao et al., 2012). Stromal Hsp90 was discovered in a complex with Tic and Toc components as well as with incoming preproteins after 8

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Historical Survey of Chloroplast Protein Import immunoprecipitation and proved to be vital for plant survival. Further support for Hsp90 playing a role in translocation comes from the observation that a specific inhibitor (radicicol) interferes with import, arresting precursor proteins in an early state of transport. Whether complex formation is mediated by direct binding of Hsp90 to Tic40 Sti1 domains remains to be investigated. Since in eukaryotic cells cytosolic Sti1 domains are responsible for interaction with Hsp90 (Richter et al., 2003; Lee et al., 2012), this most recent addition to the stromal chaperone club discussed as an import motor fits into the picture (Inoue et al., 2013). The only Tic component in the intermembrane space, Tic22, resisted detailed characterization for quite some time. Fourteen years after its discovery in pea, Glaser et al. (2012) performed a structural and functional study on Tic22 from Plasmodium falciparum (PfTic22). This apicoplastic homolog was shown to be essential for parasite survival and possibly function as a holdase chaperone, keeping incoming preproteins fit for further translocation. A year later, two separate research groups published the characterization of an Arabidopsis double mutant, where both atTic22 paralogs carried T-DNA insertions (attic22IIxIV). These plants featured a slight growth phenotype pronounced under high light conditions, and import efficiency into attic22IIxIV plastids seemed to be less efficient, which led to diverse metabolic changes hinting at disturbed carbon metabolism (Kasmati et al., 2013; Rudolf et al., 2013). The authors concluded that this IMS constituent is predominantly important under conditions where high import rates are required, such as prolonged high light treatment. The exact molecular function of atTic22 is still not resolved and needs to be investigated further. First hints at the importance of atTic20 for plant viability came from the generation of antisense plants (Chen et al., 2002). All plants with reduced levels of atTic20 expression exhibited strong chloroplast defects reflected by albinotic phenotypes, decreased plastid protein amounts, and severely stunted growth depending on the degree of RNA downregulation. Import into plastids isolated from these antisense plants was strongly inhibited at the level of the inner envelope membrane, leading the authors to conclude that atTic20 is critically involved in channel formation, which was corroborated by Kovacs-Bogdan et al. (2011) (see above). In line with this, a study on an apicoplastic isoform, this time from Toxoplasma gondii (TgTic20), came to the conclusion that TgTic20 is essential for parasite growth (van Dooren et al., 2008). By using a conditional null mutant, the authors elegantly established that loss of TgTic20 leads to serious impairment of apicoplast protein import across the innermost membrane, which in turn led to loss of apicoplast and death of the parasites. In Arabidopsis, four genes are annotated as coding for Tic20-like proteins, but only the main isoform expressed in leaves and shoots, atTic20-I, is essential for plant survival. AtTic20-IV, which is predominantly expressed in roots, can partly complement for the loss of atTic20-I but only for non-photosynthetic proteins (Hirabayashi et al., 2011). These results were confirmed by the Jarvis group in the same year (Kasmati et al., 2011). Since atTic20-I obviously has a pivotal function in chloroplast biogenesis and had been detected in a large complex (see

Historical Survey of Chloroplast Protein Import above), Kikuchi et al. (2009) tried to identify further members of this complex. To this end, attic20-I loss of function plants were complemented with atTic20-I-TEV-ProtA, a construct that featured IgG-binding domains of Protein A at the C terminus of the protein and could be cleaved off by the TEV protease (Kikuchi et al., 2013). Affinity purification of solubilized chloroplasts on Protein G Sepharose yielded three proteins that formed a complex with atTic20-I: atTic56, atTic100, and atTic214 (Ycf1). While the two former proteins are nuclear encoded, Ycf1/ Tic214 is encoded for by the plastid genome and has long been known as essential for chloroplast biogenesis although its function remains elusive. Applying the same tag on pSSU got the identical three proteins as specific interaction partners, leading the authors to the conclusion that the only true Tic translocon is represented by atTic20, atTic56, atTic100, and Ycf1. Curiously, the same construct was used by Schnell et al. (1994) to identify Tic110 as a major import component, which cannot be easily explained. Kikuchi et al. (2013) presented analyses of knockout mutants for atTic56 and atTic100 in comparison with attic20-I and ppi2 (attoc159), interpreting their embryo lethality as proof of their crucial function in protein import. This notion was, however, refuted by a proteomic analysis, which showed that many chloroplast proteins are present in the attic56 deficient plastids (Ko¨hler et al., 2015). Thus, protein import into these chloroplasts is functional to a certain degree, implying the presence of other translocation machineries in the inner envelope membrane. The biggest question left open by this publication is how all the species devoid of ycf1 in the plastome manage to import their proteins. The Poaceae (grasses), like all crop plants, do not feature YCF1, either in the plastome or in the nucleus. Kikuchi et al.’s hypothesis that grasses developed a dedicated Tic machinery instead of the Ycf1 complex does not solve this problem satisfactorily; looking at other plant sequences, many lineages also from dicots have lost the Ycf1 gene completely. We have discussed this topic in detail in a recent publication (de Vries et al., 2015), thus will not elaborate on it here. In a response, Nakai (2015) claimed that his reevaluation was contrary to ours, which is not entirely true. The manual re-analysis of individual chloroplast genomes came to pretty much the same result but a slightly different interpretation. The additional YCF1 genes that were identified by Nakai (2015) were also identified by us, but not included because they did not pass the common Hidden Markov Models (HMM) quality scores and cut-offs. Since none of this was mentioned in the response and no e-values or sequence identity scores were provided, it is difficult for the reader to properly assess the presented data (Nakai, 2015) and to form an educated opinion. On that note, it is also hard to understand the argument of why some chloroplast genomes are listed as ‘‘sequencing is incomplete’’ (Nakai, 2015) - as was done for example for Urticularia gibba — while database entries prove the contrary. It remains a fact that a ‘‘canonical Ycf100 does not exist and that several lineages have lost it independently. Nakai’s analysis reported differences in Ycf1 length that range from 393 to 4186 amino acids. That difference is largely due to a highly variable C terminus that reaches into the stroma. What is its function when it is part of a translocon? Whatever that function might be, it must be very important. There is no question that chloroplast genomes are under heavy selection and experience evolutionary reduction (Allen et al., 2011) and that protein translation comes at a cost. If we assume that it takes four ATP molecules per peptide bond

Molecular Plant (Stouthamer, 1973), then the difference in the YCF1s so far sequenced would cost some chloroplasts up to 15 172 ATP molecules per YCF1 translation extra. So there must be a good reason for that organelle to keep that stromal tail, which can make up to more than 90% of a Ycf1 protein. Furthermore, one has to keep in mind that, with regard to available chloroplast genomes, there is a great bias toward angiosperms and in particular crops. We speculate that, with broader sampling, the sequence diversity of YCF1 and the amounts of independent losses will increase (Barnard-Kubow et al., 2014). Thus, a conserved function as a general import component could only be fulfilled by the N-terminal part of Ycf1, which is predicted to comprise six transmembrane helices and could contribute to channel formation. However, the question persists how all the independent plant lineages completely lacking Ycf1 manage to perform import across the inner envelope, since Ycf1 is dispensable in so many plant species (de Vries et al., 2015). It seems therefore more likely that the Tic complex around Ycf1 (also containing atTic20, atTic100, and atTic56, Figure 3) is not the new general import translocon as suggested by Nakai’s group, but could rather represent an alternative machinery in addition to the Tic110 complex, which is conserved in all vascular plant genomes sequenced so far. The fact that the function of Ycf1 is still less than clear is backed up by a very recent publication, which convincingly implicates Ycf1 as a target for a nuclear-encoded translational activator (Yang et al., 2016). This interaction was shown to be critical for the biogenesis of NDH, PSI, and Cytb6f complexes at the thylakoids. Although the authors did not exclude a function of Ycf1 at the inner envelope in import, they presented an alternative role for this protein. Clearly, more experimental work will need to be done to resolve the discrepancies surrounding Ycf1.

IMPORT REGULATION FROM THE CYTOSOL Besides the regulation of translocation efficiency by posttranslational modifications of some Toc components such as phosphorylation on the cytosolic side, it was found that targeted protein removal by the ubiquitin-proteasome system plays an important role in import adjustment (Ling et al., 2012). Ling and Jarvis (2016) characterized an outer membrane E3 ligase named SP1, which directs ubiquitination of Toc components especially in developmental phases and stress conditions. Their data disclose a novel role for ubiquitination in adapting the protein import machinery to imminent production of reactive oxygen species. A second mechanism relying on the addition of ubiquitin was described earlier by Lee et al. (2009b). They showed that accumulating precursor proteins, e.g., in the ppi2 mutant with greatly impaired import efficiency, are recognized by a specific Hsp70 isoform (Hsp70-4), which in turn interacts with the E3 ligase CHIP. Ubiquitination then leads to proteasomal degradation and thereby removal of potentially toxic aggregating preproteins in the cytosol. Recognition of non-import competent preproteins by Hsp70-4 is mediated via specific sequence motifs within the transit peptide, although it is still unclear how the chaperone distinguishes between import Molecular Plant 9, 1–15, June 2016 ª The Author 2016.

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Molecular Plant competent and non-competent preproteins. Alternatively, in case of decreased chloroplast import capacity, superfluous preproteins can be removed from the cytosol by Hsp70-4/CHIP. This hypothesis is supported by the fact that Hsp70-4 and CHIP are upregulated in ppi2 plants as well as by artificially induced high levels of precursor proteins (Lee et al., 2009b).

EVOLUTIONARY ORIGIN OF TRANSLOCON COMPONENTS The Toc complex features a single conserved protein, Toc75, which belongs to the ubiquitous Omp85 (BamA) family (Bo¨lter et al., 1998a, 1998b; Reumann et al., 1999; Gentle et al., 2005; To¨pel et al., 2012). A second representative of this family is located in the outer envelope, OEP80, the function of which is less clear (Patel et al., 2008). Both components are found in all organisms studied so far, although their specific molecular function might have been adapted over time (To¨pel et al., 2012; Sommer et al., 2011). The inner envelope membrane translocon features several conserved proteins that have been kept from the cyanobacterial endosymbiont: Tic20, Tic22, and Tic55. It is not completely clear yet if Tic55 as such has been conserved or if a Rieske-type protein with a formerly different function has been adjusted to play a role in preprotein import (Boij et al., 2009). It seems that all import complexes evolved from common ancestors, which are all nuclear encoded, including Toc and Tic in Glaucophytes and Rhodophytes, which makes the proposed function of the plastome-encoded Ycf1 (not present in the above-mentioned organisms) as a general import component even more unlikely (de Vries et al., 2015).

FUTURE PROSPECTS AND CHALLENGES Research has made tremendous headway in the field of chloroplast protein import over the last decades. The sequencing and assembly of whole genomes, including plastomes, has led to much greater understanding of evolutionary aspects and sophisticated genetic tools have enabled scientists to generate and analyze mutant plants, thereby gaining valuable knowledge about gene function. New techniques are constantly being developed and will continue to improve our toolkit. All the -omics procedures provide us with detailed information about, e.g., the proteome of the plant compartments (proteomics), let us follow metabolic changes under different conditions or in mutants compared with WT plants (metabolomics), or study the effect of mutations on the expression of all other genes (transcriptomes/genomics). It would go beyond the scope of this article to describe all of these methods in detail. One outstanding example was the engineering of TALENs (Cermak et al., 2011), which allowed directed mutagenesis in the genome of higher plants for the first time, until then only susceptible to non-directed T-DNA insertion or RNA interference approaches. The latest hallmark in the genetic toolbox is the CRISPR/Cas technique, which is even more effective and supposed to more easily handled (Feng et al., 2013). This will enable scientists to, e.g., study functions of essential genes that result in lethal plants upon deletion and analyze the effect of point mutations in vivo. On the basis of our growing knowledge and constantly improving techniques, we will eventually be able to solve the persisting questions concerning regulation of the import process and paint a clearer picture of the components involved. Remaining issues to be 10

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Historical Survey of Chloroplast Protein Import investigated certainly include the detailed working mode of recognition at the chloroplast surface and the role of posttranslational modifications of the Toc components, especially phosphorylation and the GTPase cycles. The so far enigmatic function of import factors in the intermembrane space will need to be addressed in the future, particularly in view of the quite complex redox-dependent folding machinery in the intermembrane space of mitochondria (Herrmann et al., 2009). It seems inconceivable that the inter-envelope space of chloroplasts should be a void except for the presence of Tic22, an Hsp70, and Mgd1 (Vojta et al., 2007). Are there more (import-related) proteins in this compartment? Do they require redox-mediated folding? How is signaling across this gap between the envelope membranes achieved? All these questions will need to be answered if we want to fully understand protein import into chloroplasts. A further pressing puzzle is the identity of the Tic translocation channel(s). As elaborated above, there are several candidates for this role. The data presented in the literature most certainly indicate that there is more than one channel, but how these interact with the Toc complex and which substrates are transported is still a mystery. Finally, we have still only scratched the surface of regulatory features of the import process. We need to figure out how signals from the chloroplast are transferred across the envelopes, which factors are involved here, and how these signals are perceived and translated into action in the cytosol.

FUNDING J.S. received funding from the Munich Center for Integrated Protein Science (CiPSM) and the SFB1035.

ACKNOWLEDGMENTS No conflict of interest declared. Received: December 15, 2015 Revised: April 25, 2016 Accepted: April 27, 2016 Published: April 30, 2016

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