MINIREVIEW Protein transport in organelles: The composition, function and regulation of the Tic complex in chloroplast protein import J Philipp Benz1,2, Jurgen Soll1,2 and Bettina Bolter1,2 ă ă Plant Biochemistry, Ludwig-Maximilians-Universitat Munchen, Munich, Germany ă ă Munich Center for Integrated Protein Science CiPSM, Ludwig-Maximilians-Universitat Munchen, Munich, Germany ă ¨ Keywords chloroplast; import motor; preprotein channel; redox regulation; Tic complex; translocon Correspondence J Soll, Plant Biochemistry, LudwigMaximilians-Universitat Munchen, ă ¨ Großhaderner Strasse 2-4, D-82152 Munich, Germany Fax: +49 89 2180 74752 Tel: +49 89 2180 74750 E-mail: soll@lmu.de Website: http://www.chloroplasts.de (Received 31 July 2008, accepted 11 December 2008) doi:10.1111/j.1742-4658.2009.06874.x It is widely accepted that chloroplasts derived from an endosymbiotic event in which an early eukaryotic cell engulfed an ancient cyanobacterial prokaryote During subsequent evolution, this new organelle lost its autonomy by transferring most of its genetic information to the host cell nucleus and therefore became dependent on protein import from the cytoplasm The so-called ‘general import pathway’ makes use of two multisubunit protein translocases located in the two envelope membranes: the Toc and Tic complexes (translocon at the outer/inner envelope membrane of chloroplasts) The main function of both complexes, which are thought to work in parallel, is to provide a protein-selective channel through the envelope membrane and to exert the necessary driving force for the translocation To achieve high efficiency of protein import, additional regulatory subunits have been developed that sense, and quickly react to, signals giving information about the status and demand of the organelle These include calcium-mediated signals, most likely through a potential plastidic calmodulin, as well as redox sensing (e.g via the stromal NADP+/NADPH pool) In this minireview, we briefly summarize the present knowledge of how the Tic complex adapted to the tasks outlined above, focusing more on the recent advances in the field, which have brought substantial progress concerning the motor function as well as the regulatory potential of this protein translocation system Introduction To fulfil their functions correctly, plastids permanently communicate with the surrounding cell This requires a substantial traffic of substances such as nutrients, metabolites and proteins into and out of the organelle, which have to be funnelled across the two envelope membranes surrounding all plastid types Among these transport processes, the translocation of proteins is of particular significance Due to the loss of more than 90% of their genetic information to the host nucleus during evolution, plastids have become almost completely dependent on the surrounding cell Of the approximately 3000 proteins present in chloroplasts, typically only 50–250 (dependent on the species) are still encoded for on the plastome [1] The majority of Abbreviations CaM, calmodulin; ClpC, caseinolytic protease C; Cpn, chaperonin; FNR, ferredoxin-NADP+-oxidoreductase; Hip, Hsp70-interacting protein; Hop, Hsp70/Hsp90-organizing protein; Hsp, heat shock protein; IEM, inner envelope membrane; OEM, outer envelope membrane; SDR, short-chain dehydrogenase; SPP, stromal processing peptidase; Tic, translocon at the inner envelope membrane of chloroplasts; Toc, translocon at the outer envelope membrane of chloroplasts; TPR, tetratricopeptide repeat; Trx, thioredoxin 1166 FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS J P Benz et al proteins therefore have to be imported post-translationally from the cytoplasm, which is most generally performed via two translocation machineries present in the outer (OEM) and inner envelope membrane (IEM), called Toc (translocon at the outer envelope of chloroplasts) and Tic (translocon at the inner envelope of chloroplasts), respectively [2–5] In this pathway, nuclear encoded preproteins are translated with an N-terminal extension called transit peptide, which allows targeting of the precursor to the organelle, specific recognition by the receptor proteins on the surface, and subsequent translocation through both membranes After successful import, the transit peptide is cleaved off by the stromal processing peptidase (SPP), resulting in the mature form of the protein The entire process is superficially reminiscent of that mediated by the protein translocases at the outer and inner mitochondrial membranes [6], but plastids have developed their own ways to solve the main three tasks of protein translocation: (a) the formation of a preprotein-specific pore in the membrane (the channel); (b) exerting the necessary driving force (the motor); and (c) installing components that allow regulation of the translocation efficiency depending on developmental or environmental conditions (the regulon) Based on biochemical and genetic evidence, eight proteins have been implicated with respect to preprotein import at the IEM of chloroplasts: Tic110, Tic62, Tic55, Tic40, Tic32, Tic22, Tic21 and Tic20 (Figs and 2) For each component, either a direct contact with imported precursor has been demonstrated or, otherwise, a close interaction with one of the established Tic core proteins (usually Tic110) Last but not least, the chaperone heat shock protein (Hsp) 93/ caseinolytic protease C (ClpC) has been demonstrated to be a central constituent of the Tic motor complex (see below) The present minireview provides a short description of recent advances in the understanding of the channel-, motor- and regulatory components of the Tic complex For reference, some of the available knowledge, including the proposed function of all Tic components, is summarized in Table The Tic channel Tic110 is undoubtedly the central protein of the translocon It is not only the largest, most abundant and best studied of all Tic proteins, but also probably the only component involved in translocation steps happening on both sides of the IEM This includes the assembly of Toc–Tic ‘supercomplexes’ [7–9], preprotein recognition [10], translocation, and folding steps of Translocation across the outer chloroplast membrane successfully imported precursor proteins in the stroma [11,12] However, the exact topology of Tic110 within the IEM is still not completely solved There is mutual consent about two transmembrane-helices at the extreme N-terminus, which anchor the protein in the membrane The position and function of the long C-terminal tail on the other hand remains a matter of controversy [10,11,13–15] According to one hypothesis, the hydrophilic Tic110-Ct faces the stroma, where it functions as a scaffold for the organization of the stromal processes occurring during import [10,13,14] These include the recruitment of chaperones to the import apparatus (see below), as well as providing a transit peptide-docking site, which is localized next to the exit site of the translocon [10] Another study demonstrated that the function of Tic110 could extend well beyond this role Full-length protein as well as Tic110Ct was shown to insert into liposomes and form a cation-selective ion channel, which was sensitive to chloroplast transit peptides [11] Interestingly, using structural prediction software, at least two amphipathic a-helices with acidic faces could be located around the proposed transit peptide binding site [10] These structures have been implicated with channel function (e.g in ligand-gated and voltage-gated K+ channels) [16], and thus could provide an explanation for the observed channel activity of Tic110, as well as for the binding of transit peptides in this region (Fig 1) Another putative channel protein is Tic20 Structural predictions place Tic20 within the large group of small hydrophobic proteins with four transmembranedomains (e.g including the channel proteins Tim17 and Tim23) (Fig 1) Distant sequence similarity also exists between Tic20 and two prokaryotic branchedchain amino acid transporters [17] No data have been published demonstrating channel activity but, because Tic20 has prokaryotic ancestors, this suggests that it could have been one of the very early constituents of an evolving protein import translocon [18] By contrast, only eukaryotic homologues have been found for Tic110 However, Tic20 and Tic110 also display some similar features For example, tissue analysis in Arabidopsis thaliana indicated that both proteins can be detected throughout the plant and that expression does not appear to be restricted to photosynthetic tissue, even though absolute expression levels appear to be much lower for Tic20 than for Tic110 [13,19] When expression was silenced by antisense or completely abolished using a T-DNA knockout, both mutants exhibit severe phenotypes in A thaliana [13,19,20] Tic110 was shown to be essential for chloroplast FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS 1167 Translocation across the outer chloroplast membrane J P Benz et al Fig Schematic overview showing the predicted functional domains and topology of all Tic components Transmembrane domains are depicted as columns Regions involved in membrane binding are coloured in red, motifs involved in protein–protein interaction are blue and the dehydrogenase domains of Tic32 and Tic62 are shown in green Tic110 contains two transmembrane domains at the proximal N-terminus The topology of the long C-terminus is still not completely solved In this model, we tentatively tried to combine several views by adding some transmembrane columns having amphipathic character (indicated by red–white colour marked with a ‘?’) Tic20 and Tic21/PIC1 both belong to the big group of four-transmembrane domain proteins Topology-predictions indicate an Nin/Cin orientation Tic62 belongs to the extended family of SDRs and can be divided in two distinct modules The N-terminus contains the dehydrogenase domain (green) and might mediate membrane binding via a hydrophobic patch on the surface of the protein, whereas the C-terminus features a series of Pro/ Ser-rich repeats (blue) that allow specific binding of FNR Tic22 is a soluble protein located in the intermembrane space (IMS) with no functional domains known so far Tic55 is a Rieske [2Fe-2S]-centre containing oxidoreductase with an additional mononuclear iron binding site (both in brown) and two transmembrane helices at the C-terminus The conserved cysteine pair (CXXC) possibly involved in regulation by thioredoxins is indicated The SDR Tic32 contains an NADPH binding site and the active site motifs characteristic for SDRs (green) A CaM binding site was located in the extreme C-terminus (blue) Tic40 consists of an N-terminal transmembrane domain and a soluble C-terminus protruding into the stroma Conserved regions of the C-terminus are the TPR domain, consisting of seven predicted a-helices (blue), and the Sti1-like Hip/Hop domain at the extreme C-terminus (yellow), involved in activation of Hsp93 biogenesis and embryo development In addition, it displays a rare semi-dominant phenotype because plants with a heterozygous knockout are already clearly affected [13] Antisense plants of the pea ortholog and main Arabidopsis isoform of Tic20, AtTic20-I, similarly exhibit pronounced chloroplast defects, and attic20-I knockouts were albino even in the youngest 1168 parts of the seedling [19,20] The presence of at least one other Tic20 isoform (AtTic20-IV) may prevent attic20-I plants from lethality Two more isoforms have been detected in Arabidopsis, which, however, not possess a predicted transit peptide (Table 1) [18] Furthermore, chloroplasts from attic20-I antisense plants, as well as from heterozygous attic110, were FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS J P Benz et al Translocation across the outer chloroplast membrane Fig Schematic illustration of the Toc and Tic chloroplast import machineries with focus on the components involved in preprotein translocation at the IEM Individual Tic components are labelled with their respective names and some key functional domains are additionally indicated (Tic40 and Tic62); Toc components are not labelled The predicted transmembrane domains of Tic40 and Tic55 are shown as small columns protruding into the IEM Components of the channel/motor complex are depicted in yellow (Tic110, Tic40 and Hsp93), redox-regulatory subunits in blue (Tic62 with associated FNR, Tic55 and Tic32), the proposed alternative import channel Tic20 and the intermembrane space (IMS) component Tic22 in red and the second involved chaperone Cpn60 in green A cytoplasmically translated preprotein with an N-terminal transit peptide is shown during its translocation through the Toc and Tic complexes Tic22 may be involved in the stabilization of the Toc/Tic/preprotein supercomplex In this model, Tic110 forms the channel protein and also acts in the recruitment of Hsp93 in concert with the co-chaperone Tic40 The TPR domain of Tic40 is considered to mediate the interaction with Tic110, whereas the Sti1-like Hip/Hop domain was shown to enhance the ATPase activity of the chaperone Hsp93 The motor activity of this AAA+ ATPase probably accounts for most of the ATP requirement of the import reaction, exerting the pulling force on the incoming precursor The SPP is thought to act very early after the preprotein emerges from the Tic channel, and Cpn60 (a GroEL-homologue) is probably involved in folding of the processed precursor The association of the redox-sensing regulatory subunits Tic62 (with the FNR bound to the C-terminus) and Tic32 appears to be quite dynamic (double arrows) It is not known whether this is also true for the Rieske protein Tic55, but a similar behaviour is assumed in this model demonstrated to be defective in preprotein import across the IEM [13,20] Based on these similarities, the hypothesis was proposed that Tic20 and Tic110 could both dynamically associate to co-operate in channel formation [10] The only real biochemical indication for this suggestion shows that a minor fraction of Tic110 (approximately 5%) could be coeluted with Tic20 (and Tic22) in a Toc–Tic supercomplex [21] However, no coelution was detected in the absence of the Toc complex, making a direct or permanent interaction unlikely In summary, both Tic20 and Tic110 are clearly important for plant viability and preprotein translocation, but only for Tic110 the electrophysiological and biochemical data indicate direct channel activity as well as involvement in the import motor complex (see below) Similar data for Tic20 are still missing, but it can be speculated that either various translocons exist, or that Tic20 exhibits a different kind of protein translocation activity, which is possibly analogous to the inner membrane of mitochondria, where the Tim23/Tim17 and Tim22 channels exist in parallel, each responsible for translocation of a different subset of precursors [6] Recently, another protein with four predicted transmembrane-domains, similar to Tic20, was identified as a third putative translocon component and named CIA5/Tic21 (Fig 1) [19] The phenotype of attic21 plants resembled that of attic20-I, but the affiliation with the Tic complex was questioned by a second FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS 1169 Translocation across the outer chloroplast membrane J P Benz et al Table Components implicated with the Tic complex, their Arabidopsis isoforms, and the proposed function Tic component Isoforms in A thaliana (AGI) Tic110 Tic62 Tic55 Tic40 AtTic110 (At1g06950) AtTic62 (At3g18890) AtTic55 (At2g24820) AtTic40 (At5g16620) Tic32 AtTic32-IVa (At4g23430) AtTic32-IVb (At4g23420) AtTic32-IVc (At4g11410) AtTic22-IV (At4g33350) AtTic22-III (At3g23710) AtTic21/AtPIC1 (At2g15290) AtTic20-I (At1g04940) AtTic20-IV (At4g03320) AtTic20-V (At5g55710) AtTic20-II (At2g47840) AtHsp93-V (At5g50920) AtHsp93-III (At3g48870) Tic22 Tic21/PIC1 Tic20 Hsp93 (ClpC) Proposed function in the Tic complex Selected references Channel protein; chaperone recruitment in motor complex Redox regulation: sensing of NADP+/NADPH ratio Redox regulation; possibly regulated by thioredoxins Co-chaperone in motor complex; Hsp93 activator; timing device Redox regulation: sensing of NADP+/NADPH ratio; site of Ca2+/CaM regulation [10–15,28,33,57] [47,51,53] [48,49,58] [27,28,32,34,35,59,60] Intermembrane space complex (with Toc12, imsHsp70 and Toc64) Channel protein; Fe-permease [21,62–64] Channel protein [19–21,65] ATPase in motor complex [7,8,28,32,33,35] study demonstrating that the same gene locus does not encode a protein conducting channel, but instead an iron permease (PIC1) [22] Tic motor function Early chloroplast import studies demonstrated that cytoplasmically synthesized preproteins are imported into the organelle in an ATP-dependent process [23] By contrast to mitochondria, the energy is not used to generate a membrane potential for driving the import reaction, but exerts its effect on a stromal ATPase with a different function [24] Chaperones subsequently have been the main candidates for this ATPase activity and, indeed, members of the Hsp60 [chaperonin (Cpn)60] and Hsp100 (Hsp93) families have been found to interact with the Tic translocon [7,8,12] To date, no involvement of Hsp70s or Hsp90s with preprotein import has been reported, although both have homologues present in the chloroplast stroma This is somewhat surprising, given that the analogous motor of mitochondria relies solely on the activity of an Hsp70 [6,25,26] Cpn60 (60 kDa), a homologue to bacterial GroEL, was the first chaperone demonstrated to specifically co-immunoprecipitate with Tic110 in an ATP-dependent manner [12] However, analysis of the interaction between Tic110, Cpn60 and imported preprotein revealed that only the interaction with the mature form is ATP-dependent and thus mediated by Cpn60 This suggests that Tic110 serves in the recruitment of the 1170 [46,61] [19,22] chaperonin, which then acts in the folding of the processed protein All subsequent studies indicated that it is actually the ternary complex of Tic110, Tic40 and Hsp93/ClpC that comprises the import motor at the IEM of chloroplasts (Fig 2) All three proteins function at approximately the same (late) stage of the import process [27] Genetic characterization of double mutants in Arabidopsis revealed non-additive interactions (epistasis) amongst the respective knockout mutations, providing additional support for this functional co-operation [28] The involvement of the AAA+ family ATPase Hsp93/ClpC in preprotein translocation is interesting because it also acts in intracellular degradation and substrate turnover, which it performs in association with its proteolytic counterpart ClpP [29,30] Nevertheless, Hsp93/ClpC was also shown to display intrinsic chaperone activity [31] and thus appears to be capable of performing several tasks in the chloroplast, which are probably dependent on the suborganellar compartment (stromal versus membrane-tethered) and the respective interaction partners Subsequent to the initial demonstration of a specific, ATP-dependent association of Hsp93 with Tic110 and incoming precursor [7,8], considerable progress has been made, especially concerning the role of Tic40 and Hsp93 in the motor complex [27,28,32–34] and the possible order of events [35] Tic40 is an integral membrane protein containing a single transmembrane span within its extreme FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS J P Benz et al N-terminus, anchoring it in the IEM, whereas the C-terminus of the protein projects into the stroma (Fig 1) [34] Two motifs can be located in the C-terminal half of the stromal domain: (a) the last approximately 60 amino acids are weakly similar to a conserved motif of the mammalian co-chaperones Hsp70-interacting protein (Hip) and Hsp70/Hsp90organizing protein (Hop) and (b) the region immediately preceding this domain is predicted to form a structure similar to a tetratricopeptide repeat (TRP) motif Hip and Hop play regulatory roles in Hsp70 and Hsp90 cycles [36–39] and, interestingly, the yeast Hop homologue Sti1p was also shown to associate with Hsp104, which is a member of the Hsp100 family [40] TPR domains are degenerate 34-amino acid repeats forming anti-parallel a-helices known to be involved in an array of protein–protein interactions (generally with non-TPR proteins) [41] Both domains are very characteristic for co-chaperones Using various Tic40-deletion constructs in an attempt to complement the pale green and slow growing tic40 knockout phenotype, it could be shown that the C-terminal Hip/Hop (Sti1-like) domain, as well as the N-terminal transmembranehelix and a central region including the putative TPR motifs, is essential for correct protein activity Only the full-length cDNA clone was able to reverse the phenotype to wild-type growth [32] A more detailed characterization of the single domains provided valuable insight into the possible functional role of Tic40: the Sti1-like region of Tic40 was shown to be functionally equivalent to the Sti1 domain of human Hip, corroborating the role of Tic40 as a bona fide co-chaperone [32] Additionally, in in vitro assays using overexpressed Hsp93 and various Tic40 deletion constructs, the same domain was found to stimulate the ATPase activity of the chaperone [35] Because this stimulating effect was only visible with the Hip/Hop-domain alone and not with the entire stromal domain including the TPR motifs, it was hypothesized that the protein exists in a closed conformation, in which the TPR domain shields the Hip/Hop-domain from the chaperone Surprisingly, the TPR motifs themselves appear to mediate the interaction with Tic110 and not with the chaperone partner (Hsp93), which is in contrast to the function of these motifs in Hop and Hip [40,42,43] Interestingly, binding of Tic40 to Tic110 is favoured when the transit peptide-binding site of Tic110 is occupied by incoming preprotein, but interaction with Tic40 appears to decrease the affinity of Tic110 for the transit peptide, which is subsequently released and therefore accessible for processing by Translocation across the outer chloroplast membrane the SPP and interaction with Hsp93 [35] Conformational changes occurring upon binding of Tic40 to Tic110 presumably also open the Hip/Hop-domain of Tic40, allowing it to stimulate the motor activity of Hsp93 Obviously, the import motor is still functional in the absence of Tic40 because tic40 knockout plants are viable, even though the plants are very pale [27] In addition, dominant-negative phenotypes could be observed in some Tic40 complementation lines, indicating that the overexpressed deletion-constructs interfered with some residual motor activity [32] Thus, Tic40 clearly enhances the operational efficiency of the complex and was proposed to function as a timing device, co-ordinating the sequential steps of translocation (Fig 2) [32,35] The function of the ATPase Hsp93 in protein import was further analyzed using the characterization of Arabidopsis knockout mutants [28,33] In Arabidopsis, two homologues of Hsp93 exist (Hsp93-III and Hsp93V), sharing high (approximately 91%) sequence identity Hsp93-V is thought to be the main isoform with a several-fold higher expression rate than Hsp93-III Nevertheless, some degree of redundancy appears to exist among both proteins because the mildly chlorotic hsp93-V knockout phenotype can be complemented by overexpression of the other isoform However, analysis of double knockouts of both Hsp93 homologues did not result in the identification of double homozygotes, establishing that Hsp93 function is essential for viability, just as is the case for Tic110 [28,33] This observation indicates that Hsp93 and Tic110 are of similar importance for the organelle Another finding concerns the significance of the Hsp93-related motor activity on the overall import of preproteins through both envelope membranes It is known that Tic110 and Hsp93 are constituents of Toc–Tic supercomplexes that are associated with precursor protein [7,8] Therefore, it could be possible that the ATPase activity of Hsp93 exerts a pulling effect also at the level of the OEM, similar to the situation in mitochondria When performing import experiments with tightly folded as well as unfolded preprotein in viable hsp93-III/-V double-mutant (knockdown) chloroplasts, the use of an unfolded preprotein did not alleviate the decreased import efficiency in hsp93-III/-V (and tic40) plants This implies that the rate-limiting step for protein import in the mutant chloroplasts is not precursor unfolding [33,44] and could be interpreted as an indication for separate unfolding forces (and thus motor activities) in the outer and inner membranes of the chloroplast envelope during preprotein import FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS 1171 Translocation across the outer chloroplast membrane J P Benz et al Possible ways of regulation As outlined above, a great amount of protein traffic has to take place at the envelope membranes of chloroplasts, which has to be tightly regulated to ensure that the supply correlates with the demand of the organelle at any given time Logically, translocation across the envelope is surely a bottleneck in the path of transported proteins from the cytosol to their final destination in the chloroplast The Tic and Toc translocons are therefore perfectly situated to impose a regulatory control over incoming preproteins Additionally, because the demand of the chloroplast is ‘sensed’ inside the organelle, the IEM is closest to the origin of the signal, and thus regulation at the Tic complex could be one of the fastest ways to react efficiently To our current knowledge, at least two types of signals convene at the Tic complex: (a) the stromal NADP+/NADPH ratio sensed via Tic62 and Tic32, giving information about the metabolic state of the chloroplast and (b) a calcium signal, which is mediated by a still elusive chloroplast calmodulin (CaM), associated with Tic32 (Fig 3) Redox regulation Redox regulation is long known to play a prominent role in the chloroplast metabolism, and also at least two preproteins (the nonphotosynthetic ferredoxin FdIII and the ferredoxin-NADP+-oxidoreductase isoform II of maize) were demonstrated to be differentially imported in the light compared to the dark [45] Diurnal changes in the thylakoids or, more generally, the stromal redox system (e.g the NADP+/NADPH pool) thus appear to have an impact on the import characteristics of the organelle It is therefore not surprising to find proteins with redox-active domains as Tic constituents Up to now, the ‘regulon’ of the Tic complex comprises three proteins: Tic62, Tic32 and Tic55 The former two proteins belong to the (extended) family of short-chain dehydrogenases/reductases (SDRs) and have already been demonstrated to possess dehydrogenase activity in vitro [46,47] Less is known about the redox properties of Tic55 Sequence analysis revealed the presence of a Rieske-type [2Fe2S] cluster and a mononuclear iron-binding site [48] Database research classifies Tic55 as a member of the chlorophyll a oxygenase/pheophorbide a oxygenaselike oxygenases, which act for example in chlorophyll biogenesis or oxygen-dependent degradation pathways Rieske proteins generally play important roles in electron transfer (e.g in the cytochromes present in the respiratory chain of mitochondria or in the thylakoids of chloroplasts) Whether Tic55 acts as an oxygenase in vitro or in vivo has not been studied to date, but the close proximity of the Rieske protein Tic55 and the two bona fide dehydrogenases Tic32 and Tic62 at the Tic complex holds the intriguing possibility of a Fig Schematic model of the proposed regulatory signals sensed by the Tic complex and their effect on the involved subunits Three signals are thought to convene at the Tic complex: (1) information about the chloroplast metabolic redox state, represented by the stromal NADP+/NADPH ratio and sensed by the two dehydrogenases Tic62 and Tic32; (2) a calcium signal, mediated by a still unknown plastidic CaM or CaM-like protein binding to Tic32; and (3) a second redox-related signal, in which a stromal thioredoxin interacts with a conserved cysteine pair (CXXC) of the Rieske protein Tic55 The redox state of the NADP+/NADPH pool was demonstrated to have a drastic effect on the association of Tic62 and Tic32 with the Tic complex Both components dissociate from the complex at high NADPH concentrations Tic62 was shown to reversibly shuttle between the stroma the IEM dependent on the NADP+/NADPH ratio For Tic32, a similar relocalization as for Tic62 is assumed in this model 1172 FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS J P Benz et al small electron transfer chain being present at the Tic translocon [47] In addition, a very recent study identified Tic55 as a target of stromal thioredoxins (Trx) in barley chloroplasts [49] Trxs are small ubiquitous proteins with redox-active disulfide bridges that regulate enzyme activities (e.g in the Calvin cycle or the oxidative pentose phosphate cycle) by dithiol oxidoreduction of their target proteins [50] Necessary for this reaction is a conserved pair of cysteines, which can be detected in Tic55 (CXXC motif; Fig 1) However, no further conclusion about how the oxidoreduction affects Tic55 function in the Tic complex could be drawn from this analysis Investigation of the Tic complex under changing redox conditions revealed a high degree of dynamics For example, addition of NADPH leads to dissociation of the two dehydrogenases Tic32 and Tic62 from the complex, indicating that the metabolic state of the organelle appears to have a profound influence on Tic composition [46] Further studies with Tic62 corroborated this finding and revealed that this protein shuttles between the chloroplast membrane compartment and the stroma dependent on the stromal NADP+/ NADPH ratio [47] (Fig 3) Oxidizing conditions lead to fast membrane binding and integration into the Tic complex Reducing conditions on the other hand lead to solubilization into the stroma and increased interaction with its other known interaction partner ferredoxin-NADP+-oxidoreductase (FNR) Interestingly, this membrane binding was found to be reversible, and is assumed to be mediated by a hydrophobic patch on the protein surface, located in the N-terminal half of the protein, including the dehydrogenase domain Specific binding of the FNR is mediated by a unique series of proline/serine-rich repeat motifs located in the C-terminus For the integration into the Tic complex finally, a central region of the protein was shown to be sufficient, which contains parts of both, the N-terminus and C-terminus (Fig 1) These results demonstrate that Tic62 is able to react very sensitively to redox changes in the chloroplast stroma and that it adjusts its localization accordingly These features would allow it to fulfil its proposed role as a redox-sensor protein in the chloroplast [47,51] How exactly changes in the redox state of the chloroplast affect the translocation is not yet known, but it has been suggested that the dynamic Tic composition could influence the import characteristics of a certain subset of preproteins, which might also act in redox-dependent pathways [47] The reason for the strong association of Tic62 with the FNR still remains one of many open questions Because flavin-containing proteins have already been described to be present in redox chains in chloroplast Translocation across the outer chloroplast membrane envelope membranes [52], one possibility is the recruitment of FNR from the stroma or even thylakoids to the Tic complex in order to become part of the hypothetical electron transfer chain mentioned above However, the involvement of the FNR appears to be an evolutionary young mode of regulation This notion derives from an extensive database analysis of the Tic62 protein looking for homologues in other sequenced organisms [53] It was found that the N-terminal half of the protein, comprising the dehydrogenase domain, is highly conserved in all oxyphototrophs, and homologues can be found even in green sulfur bacteria The C-terminus, containing the FNR binding repeats, on the other hand, is present only in higher plants This C-terminal extension therefore appears to have been added only recently in evolution, which could make Tic62 one of the youngest Tic constituents Ca2+/CaM regulation Calcium is a common secondary messenger that regulates many biochemical processes (e.g relaying environmental signals to various cellular response pathways) This is generally achieved through binding to calcium sensing proteins such as CaM, which subsequently change their affinities to downstream target proteins, leading to further responses [54,55] Even though regulation by calcium/CaM is considered to be a eukaryotic trait, import analyses into chloroplasts could demonstrate that organellar processes have been integrated into the calcium signalling network of the cell [56] Calcium ionophores as well as the CaMinhibitor ophiobolin A affected the translocation of preproteins containing a cleavable N-terminal transit peptide This indicates that: (a) the general Toc/Tic pathway is involved in calcium regulation and (b) a CaM or CaM-like protein is the most likely mediator of this regulation In an attempt to isolate CaM-binding proteins, Tic32 was identified as the only IEM protein specifically interacting with CaM in a calciumdependent manner, corroborating the idea that the Tic complex is the site of calcium regulation (Fig 3) Further binding assays employing several Tic32-deletion constructs allowed the localization of the CaM-binding site to the 26 most C-proximal amino acids (Fig 1) This region was predicted to form a basic amphipathic helical structure characteristic for CaM-binding domains, and contains at least one conserved potential CaM-binding motif [46] Additionally, the binding of CaM at the C-terminus and the binding of NADPH at the extreme N-terminus appear to be mutually exclusive, suggesting that two different signalling pathways FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS 1173 Translocation across the outer chloroplast membrane J P Benz et al convene at Tic32 and are integrated at the Tic complex Conclusions Increasing evidence is accumulating to suggest that we experience not only the one Tic complex, but also that the composition and activity of the Tic machinery can be adapted (regulated) Distinct regulatory circuits might sense distinct organellar requirements via: (a) a Ca2+/CaM; (b) a metabolic NADP+/NADPH; or (c) an environmental Trx mediated signal These signals, either alone or in combination, could influence the import of preproteins A prominent and difficult task for future studies will therefore be to determine how organelle metabolism and physiology influences protein import by the Tic complex and by the Toc–Tic translocon as a whole Acknowledgements We would like to thank our colleagues from the laboratory for helpful discussions, and especially Anna Stengel for critical reading of the manuscript Financial support was provided by the Deutsche Forschungsgemeinschaft Grant SFB594 and the Elite Network of Bavaria (to J P Benz) References Gould SB, Waller RF & McFadden GI (2008) Plastid evolution Annu Rev Plant Biol 59, 491–517 Benz P, Soll J & Bolter B (2007) The role of the tic machinery in chloroplast protein import In The Enzymes – Molecular Machines Involved in 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translocon of chloroplasts Mol Biol Cell 15, 5130–5144 63 Fulda S, Norling B, Schoor A & Hagemann M (2002) The Slr0924 protein of Synechocystis sp strain PCC 6803 resembles a subunit of the chloroplast protein import complex and is mainly localized in the thylakoid lumen Plant Mol Biol 49, 107–118 64 Qbadou S, Becker T, Bionda T, Reger K, Ruprecht M, Soll J & Schleiff E (2007) Toc64 – a preprotein-receptor at the outer membrane with bipartide function J Mol Biol 367, 1330–1346 65 Ma Y, Kouranov A, LaSala SE & Schnell DJ (1996) Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope J Cell Biol 134, 315–327 FEBS Journal 276 (2009) 1166–1176 ª 2009 The Authors Journal compilation ª 2009 FEBS ... in protein? ? ?protein interaction are blue and the dehydrogenase domains of Tic3 2 and Tic6 2 are shown in green Tic1 10 contains two transmembrane domains at the proximal N-terminus The topology of. .. surprising to find proteins with redox-active domains as Tic constituents Up to now, the ‘regulon’ of the Tic complex comprises three proteins: Tic6 2, Tic3 2 and Tic5 5 The former two proteins belong... and the two bona fide dehydrogenases Tic3 2 and Tic6 2 at the Tic complex holds the intriguing possibility of a Fig Schematic model of the proposed regulatory signals sensed by the Tic complex and