Insights into the Clp/HSP100 chaperone system from chloroplasts of Arabidopsis thaliana

HSP100 proteins are molecular chaperones involved in protein quality control. They assist in protein (un)folding, prevent aggregation, and are thought to participate in precursor translocation across membranes. Caseinolytic proteins ClpC and ClpD from plant chloroplasts belong to the HSP100 family. Their role has hitherto been investigated by means of physiological studies and reverse genetics. In the present work, we employed an in vitro approach to delve into the structural and functional characteristics of ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). They were expressed in Escherichia coli and purified to near-homogeneity. The proteins were detected mainly as dimers in solution, and, upon addition of ATP, the formation of hexamers was observed. Both proteins exhibited basal ATPase activity (K(m), 1.42 mm, V(max), 0.62 nmol/(min × μg) for AtClpC2 and K(m) ～19.80 mm, V(max) ～0.19 nmol/(min × μg) for AtClpD). They were able to reactivate the activity of heat-denatured luciferase (～40% for AtClpC2 and ～20% for AtClpD). The Clp proteins tightly bound a fusion protein containing a model transit peptide. This interaction was detected by binding assays, where the chaperones were selectively trapped by the transit peptide-containing fusion, immobilized on glutathione-agarose beads. Association of HSP100 proteins to import complexes with a bound transit peptide-containing fusion was also observed in intact chloroplasts. The presented data are useful to understand protein quality control and protein import into chloroplasts in plants.     

Precursors with altered affinity for Hsp70 in their transit peptides are efficiently imported into chloroplasts

Protein import into chloroplasts is postulated to occur with the involvement of molecular chaperones. We have determined that the transit peptide of ferredoxin-NADP(H) reductase precursor binds preferentially to an Hsp70 from chloroplast stroma. To investigate the role of Hsp70 molecular chaperones in chloroplast protein import, we analyzed the import into pea chloroplasts of preproteins with decreased Hsp70 binding affinity in their transit peptides. Our results indicate that the precursor with the lowest affinity for Hsp70 molecular chaperones in its transit peptide was imported to chloroplasts with similar apparent Km as the wild type precursor and a 2-fold increase in Vmax. Thus, a strong interaction between chloroplast stromal Hsp70 and the transit peptide seems not to be essential for protein import. These results indicate that in chloroplasts the main unfolding force during protein import may be applied by molecular chaperones other than Hsp70s. Although stromal Hsp70s undoubtedly participate in chloroplast biogenesis, the role of these molecular chaperones in chloroplast protein translocation differs from the one proposed in the mechanisms postulated up to date.     

Toward a unified model of the action of CLP/HSP100 chaperones in chloroplasts

In chloroplasts, Hsp70 and Hsp100 chaperones have been long suspected to be the motors that provide the necessary energy for the import of precursor proteins destined to the organelle. The chaperones associate with the import translocon and meet the transit peptides as they emerge through the channel. After decades of active research, recent findings demonstrated that Hsp100 chaperones recognize transit peptides both in vitro and in vivo. Moreover, Hsp70 also plays a part in precursor import. The updated model of protein translocation into chloroplasts now presents new questions about the role of the chaperones in the process.     

Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process

Three stromal chaperone ATPases, cpHsc70, Hsp90C, and Hsp93, are present in the chloroplast translocon, but none has been shown to directly bind preproteins in vivo during import, so it remains unclear whether any function as a preprotein-translocating motor and whether they have different functions during the import process. Here, using protein crosslinking followed by ionic detergent solubilization, we show that Hsp93 directly binds to the transit peptides of various preproteins undergoing active import into chloroplasts. Hsp93 also binds to the mature region of a preprotein. A time course study of import, followed by coimmunoprecipitation experiments, confirmed that Hsp93 is present in the same complexes as preproteins at an early stage when preproteins are being processed to the mature size. In contrast, cpHsc70 is present in the same complexes as preproteins at both the early stage and a later stage after the transit peptide has been removed, suggesting that cpHsc70, but not Hsp93, is important in translocating processed mature proteins across the envelope.Most chloroplast proteins are encoded by the nuclear genome as higher M_r preproteins that are fully synthesized in the cytosol before being imported into the chloroplast. The import process is initiated by binding of the N-terminal transit peptide of the preprotein to the translocon at the outer envelope membrane of chloroplasts (TOC) complex, in which Toc159 and Toc34 function as receptors and Toc75 is the outer membrane channel. This step is followed by binding of the transit peptide to the translocon at the inner envelope membrane of chloroplasts (TIC) machinery, the central components of which include the Tic20/Tic56/Tic100/Tic214 channel complex and Tic110. Tic110 functions as the stromal receptor for transit peptides and also as a scaffold for tethering other translocon components (for reviews, see Li and Chiu, 2010; Shi and Theg, 2013; Paila et al., 2015). The actual translocation of the bound preproteins across the envelope is powered by hydrolysis of ATP in the stroma (Pain and Blobel, 1987; Theg et al., 1989), and it is therefore assumed that some stromal ATPase motor proteins bind the preproteins as they emerge from the inner membrane and use the energy of ATP hydrolysis to translocate the preproteins across the envelope into the stroma.Three stromal ATPases have been identified in the translocon complex: cpHsc70 (chloroplast heat shock cognate protein 70 kD), Hsp90C (chloroplast heat shock protein 90), and Hsp93/ClpC (93-kD heat shock protein). Hsp93, the first to be identified, belongs to the Hsp100 subfamily of AAA+ proteins (ATPases associated with various cellular activities) and was detected in coimmunoprecipitation experiments in complexes containing other translocon components and preproteins undergoing import (Akita et al., 1997; Nielsen et al., 1997; Chou et al., 2003; Rosano et al., 2011). In Arabidopsis (Arabidopsis thaliana), Hsp93 exists as two isoforms encoded by the genes HSP93III and HSP93V. Removal of the more abundant Hsp93V results in protein import defects, while double knockout of the two genes causes lethality (Constan et al., 2004; Kovacheva et al., 2007; Chu and Li, 2012; Lee et al., 2015). Purified recombinant Hsp93III can bind to the transit peptide of pea (Pisum sativum) ferredoxin-NADP+ reductase in vitro (Rosano et al., 2011). In addition, the N-terminal domain of Hsp93 is critical both for its in vivo functions and its association with chloroplast membranes and Tic110, suggesting that one of the major functions of Hsp93 requires it to be localized at the envelope with Tic110 (Chu and Li, 2012). However, because many prokaryotic Hsp100 family proteins function as the regulatory components of the Clp proteases (Kress et al., 2009; Nishimura and van Wijk, 2015), and, in Arabidopsis, some Clp proteolytic core components have also been found at the envelope fraction, it has been proposed that Hsp93 is involved in degradation of misfolded or damaged proteins at the envelope (Sjögren et al., 2014). However, whether the Clp proteolytic core can form a stable complex with Hsp93 in higher plant chloroplasts remains to be shown.In mitochondria and the endoplasmic reticulum, protein import is driven by the Hsp70 family of proteins. In chloroplasts, accumulating evidence also supports that Hsp70 is important for chloroplast protein import. Purified recombinant Hsp70 can bind in vitro to the transit peptide of the small subunit of RuBP carboxylase preprotein (prRBCS; Ivey et al., 2000). Stromal Hsp70 can be coimmunoprecipitated with preproteins undergoing import and with other translocon components, and mutations resulting in reduced or altered stromal Hsp70 activity cause protein import defects (Shi and Theg, 2010; Su and Li, 2010). Recently, it has been shown, in moss, that increasing the K_m for Hsp70 ATP hydrolysis results in an increased K_m for ATP usage in chloroplast protein import, indicating that stromal Hsp70 is indeed one of the proteins supplying ATP-derived energy to power import (Liu et al., 2014). Finally, stromal Hsp90C has been shown to be part of active translocon complexes in coimmunoprecipitation experiments (Inoue et al., 2013). As further evidence that Hsp90 is important for protein import into chloroplasts, the Hsp90 ATPase activity inhibitor radicicol reversibly inhibits the import of preproteins into chloroplasts (Inoue et al., 2013).Presence of the three ATPases in the translocon was demonstrated by coimmunoprecipitation after solubilization of chloroplast membranes under conditions that preserve the large membrane protein complexes, either by solubilization with nonionic detergents or by treating chloroplasts with crosslinkers that link all proteins in a complex together (Akita et al., 1997; Nielsen et al., 1997; Shi and Theg, 2010; Su and Li, 2010; Inoue et al., 2013). These complexes contain translocon components that directly bind to preproteins, and also other proteins that are associated with these translocon components but have no direct contacts with the preproteins. For example, Nielsen et al. (1997) demonstrated the presence of Hsp93 in the translocon by binding of prRBCS to isolated pea chloroplasts and then solubilization of chloroplast membranes with the nonionic detergent decylmaltoside. Under these conditions, an anti-Hsp93 antibody specifically immunoprecipitated Hsp93 together with Toc159, Toc75, Toc34, Tic110, and prRBCS (Nielsen et al., 1997). The result showed that Hsp93 is in the same complexes with these proteins but did not provide information whether Hsp93 directly binds to them. It is possible that Hsp93 only has direct contacts with, for example, Tic110, which then binds to prRBCS. Direct binding, in particular to the transit peptide region, would provide strong evidence that an ATPase functions as a protein translocating motor, rather than in assisting the assembly of other translocon components or in the folding or degradation of imported proteins. Furthermore, if all three ATPases were found to be involved in preprotein translocation, it would be important to understand how they work together; for example, whether they preferentially bind different preproteins, bind to different regions of a preprotein, or act at different stages of the import process.Here, we examined whether Hsp93 can directly bind to preproteins undergoing import into chloroplasts, and compared the timing of the binding of Hsp93 and cpHsc70 to the preproteins. We used isolated pea chloroplasts, rather than isolated Arabidopsis chloroplasts, because pea chloroplasts exhibit more robust import ability (Fitzpatrick and Keegstra, 2001). Various crosslinkers that react with cysteines were then used to achieve more specific crosslinkings, followed by solubilization with the ionic detergent lithium dodecyl sulfate (LDS) to thoroughly solubilize chloroplast membranes and to disrupt noncovalent protein-protein interactions. Our results show that Hsp93 directly binds to preproteins undergoing import. Import time course experiments further revealed that Hsp93 functions primarily during the early stage of import, whereas cpHsc70 associates with substrates being imported at both the early stage and a later stage after transit peptide removal.     

Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope

The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone’s interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import.Chloroplasts are essential organelles in plant cells as they are responsible for performing a variety of functions (Jarvis and López-Juez, 2013). Although chloroplasts have their own genome (encoding approximately 100 proteins), the majority of the proteins found in these organelles are nucleus-encoded (approximately 3,000) (Leister, 2003), synthesized in the cytosol, and imported into the chloroplast as precursor proteins (preproteins), each one with a cleavable N-terminal extension or transit peptide (Shi and Theg, 2013a; Paila et al., 2015). The preprotein import mechanism is initiated by the interaction of the transit peptide with the translocon at the outer envelope membrane of chloroplasts (TOC) complex and subsequently involves transport through the translocon at the inner envelope membrane of chloroplasts (TIC) machinery in an energy-dependent process (Theg et al., 1989; Shi and Theg, 2013b). The Tic110 and Tic40 components have long been described as central TIC components, but these proteins were absent from a recently described 1-MD TIC complex (consisting of Tic20, Tic56, Tic100, and Tic214; Kovács-Bogdan et al., 2010; Nakai, 2015). One possible explanation is that two TIC complexes act sequentially during protein import (e.g. a Tic110-containing complex may act downstream of the 1-MD complex). A TIC complex associated import motor is proposed to exist at the stromal side of the inner envelope, and several stromal chaperones, including Hsp93/ClpC and Hsp70, have been proposed to act as motors to drive protein translocation into the stroma (for review, see Flores-Pérez and Jarvis, 2013).Hsp93 is closely related to bacterial ClpC and is a member of the Class I subfamily of Hsp100 chaperones, which themselves belong to the wider AAA+ (ATPases associated with various cellular activities) superfamily (Hanson and Whiteheart, 2005; Flores-Pérez and Jarvis, 2013). AAA+ enzymes are involved in a variety of cellular processes, such as protein folding, unfolding for proteolysis, and disassembly of protein aggregates or protein complexes. Although AAA+ chaperones are well characterized in bacteria, they are found in all kingdoms (Hanson and Whiteheart, 2005). Such proteins possess one or two nucleotide binding domains, both of which contain conserved Walker A and B motifs. These chaperones may also contain a conserved ClpP-binding motif (PBM), or P-loop, which is essential for interaction with the unrelated, proteolytic ClpP subunit (Weibezahn et al., 2004; Hanson and Whiteheart, 2005).In the chloroplast, Hsp93/ClpC partitions between the inner envelope membrane and the chloroplast stroma. Most Hsp93/ClpC protein is located in the stroma. Nonetheless, a large proportion of the total chloroplast Hsp93/ClpC pool (30%) associates with the envelope (Sjögren et al., 2014). Hsp93 has frequently been copurified with TIC and TOC complex components, which led to the hypothesis that it provides the driving force for preprotein import (Akita et al., 1997; Nielsen et al., 1997). Also, Hsp93 was found to specifically coimmunoprecipitate with preproteins under limiting ATP conditions and to stably bind to transit peptides in vitro (Nielsen et al., 1997; Rosano et al., 2011). Genetic and molecular studies have suggested that it functions in close association with Tic110 and Tic40 (Chou et al., 2003; Kovacheva et al., 2005; Chou et al., 2006). More recently, it was shown that the N-terminal domain of Hsp93 is important for its membrane association (Chu and Li, 2012). Despite all this evidence, the nature of the interaction between Hsp93 and the TIC apparatus has not been fully characterized.Analysis of mutants also supported the involvement of the Hsp93 chaperone in protein import. In Arabidopsis (Arabidopsis thaliana), two homologous genes, atHSP93-V (CLPC1) and atHSP93-III (CLPC2), code for Hsp93/ClpC, and the resulting protein isoforms share 91% amino acid sequence identity (Kovacheva et al., 2007). The Hsp93-V protein is the most abundant isoform, and mutations in the atHSP93-V gene lead to a pale-green plant phenotype with protein import defective chloroplasts. In contrast, atHSP93-III knockout plants are indistinguishable from the wild type, most likely due to the compensatory presence of functionally redundant and abundant atHsp93-V (Kovacheva et al., 2005, 2007). Complete loss of both proteins in Arabidopsis is lethal during embryo development, whereas double mutants lacking Hsp93-V but retaining partial Hsp93-III activity are viable but exhibit severe chlorosis and protein import defects (Kovacheva et al., 2007).More typically, as expected by its close relationship to bacterial orthologs, Hsp93/ClpC is a functional component of the caseinolytic protease (Clp) in the chloroplast stroma, where it recognizes and unfolds substrates for degradation (Shanklin et al., 1995). Significantly, the Clp proteolytic core is also bound to the envelope membranes, in quantities which are sufficient to bind to all of the similarly localized Hsp93/ClpC (Sjögren et al., 2014). This recent finding suggested a role for the Clp protease in protein quality control at the envelope. The structure of the Clp protease complex comprises a cylinder-like protease core and an AAA+ chaperone ring complex, and it is generally conserved throughout evolution (Nishimura and van Wijk, 2015). In Arabidopsis, the plastid Clp proteolytic core contains two distinct heptameric rings (the P-ring consisting of ClpP3-P6 and the R-ring consisting of ClpP1 and ClpR1-R4; Sjögren et al., 2006), and attached to this are accessory ClpT proteins involved in core assembly (Sjögren and Clarke, 2011). Several studies have shown that deficiency of the proteolytic subunits of the core complex leads to sick plant phenotypes (Sjögren et al., 2004; Rudella et al., 2006; Sjögren et al., 2006), highlighting the essential nature of Clp proteolytic activity to chloroplast function and plant viability.As described above, the putative interacting partners of Hsp93 at the envelope are Tic110 and Tic40. Tic110 is a highly abundant protein and is essential for plastid biogenesis (Inaba et al., 2005; Kovacheva et al., 2007). It has two N-terminal transmembrane α-helices, and it projects a large C-terminal hydrophilic domain into the stroma (Jackson et al., 1998; Inaba et al., 2003). A stromal region proximal to the second transmembrane helix selectively associates with transit peptides, serving as a docking site for preproteins as they emerge from the TIC channel (Inaba et al., 2003). The hydrophilic domain of algal Tic110 possesses a rod-shaped helix-repeat structure similar to HEAT-repeat domains (and plant Tic110 proteins are predicted to be similar), and these typically function as scaffolds for protein-protein interactions (Tsai et al., 2013). Tic40 is topologically similar to Tic110 and is proposed to act as a cochaperone in the preprotein import motor (Chou et al., 2003). In the corresponding model, a transit peptide emerging from the TIC channel binds to the stromal domain of Tic110; this binding causes a conformational change of Tic110 to recruit Tic40, which in turn triggers transit peptide release to enable association of the preprotein with Hsp93 (Inaba et al., 2003; Chou et al., 2006). Finally, Tic40 is proposed to stimulate ATP hydrolysis by Hsp93 so that the chaperone pulls the preprotein into the stroma (Chou et al., 2006).Although there is good evidence that Hsp93 is involved in protein import, the ability of Hsp93 to associate with the Clp protease core means that, in principle, any aspect of the hsp93 mutant phenotype could be due to disruption of the ClpP-linked functions of the protein. Bearing this in mind, we aimed to further characterize the role of Hsp93 at the inner envelope membrane. First, we analyzed the putative interactions of Hsp93 with the TIC components, Tic110 and Tic40, in a complementary set of in vitro and in vivo studies. Second, we evaluated the proposed role of Hsp93 in protein import independently of its role in proteolysis by creating a PBM mutant of the major Hsp93 isoform, atHsp93-V, and studying its activity in planta.     

Chloroplast Hsp70s are not involved in the import of ferredoxin-NADP+ reductase precursor

Heat-shock protein 70 (Hsp70) chaperones function as molecular motors pulling precursor proteins across membranes. Although several Hsp70s have been identified in chloroplasts, their participation in protein translocation is still uncertain. A phylogenetic analysis of the peptide-binding domain from plant Hsp70s shows that they can be classified into defined groups related to their subcellular localizations, allowing differences in substrate specificities to be inferred. Using an algorithm developed by Blond-Elguindi et al. we detected three regions in the transit peptide of the pea ferredoxin-NADP⁺ reductase precursor (preFNR) that are related to binding with immunoglobulin heavy-chain binding protein (BiP), one of the members of the Hsp70 family resident in the endoplasmic reticulum. We constructed a mutant transit peptide in which prolines 18, 20 and 28 were substituted by serines. Thus, the theoretical probability of BiP-type binding of the peptide was abolished without modifying the sites for Hsp70 with DnaK-type binding. The stromal Hsp70 homolog CSS1 displayed lower affinity for this mutant transit peptide than for the wild-type presequence. Nevertheless, preFNR containing the mutant transit peptide was imported into isolated chloroplasts from pea with initial rates similar to that observed for the wild-type precursor, and only an 18% decrease in the total number of imported molecules was observed after 20 min of reaction. Our results support an import model for the preFNR in which neither DnaK- nor BiP-like Hsp70 molecular chaperones play a central role as motor of the translocation machinery in chloroplasts.     

Molecular chaperones involved in chloroplast protein import.

Transport of cytoplasmically synthesized precursor proteins into chloroplasts, like the protein transport systems of mitochondria and the endoplasmic reticulum, appears to require the action of molecular chaperones. These molecules are likely to be the sites of the ATP hydrolysis required for precursor proteins to bind to and be translocated across the two membranes of the chloroplast envelope. Over the past decade, several different chaperones have been identified, based mainly on their association with precursor proteins and/or components of the chloroplast import complex, as putative factors mediating chloroplast protein import. These factors include cytoplasmic, chloroplast envelope-associated and stromal members of the Hsp70 family of chaperones, as well as stromal Hsp100 and Hsp60 chaperones and a cytoplasmic 14-3-3 protein. While many of the findings regarding the action of chaperones during chloroplast protein import parallel those seen for mitochondrial and endoplasmic reticulum protein transport, the chloroplast import system also has unique aspects, including its hypothesized use of an Hsp100 chaperone to drive translocation into the organelle interior. Many questions concerning the specific functions of chaperones during protein import into chloroplasts still remain that future studies, both biochemical and genetic, will need to address.     

ATP Requirement for Chloroplast Protein Import Is Set by the Km for ATP Hydrolysis of Stromal Hsp70 in Physcomitrella patens

The 70-kD family of heat shock proteins (Hsp70s) is involved in a number of seemingly disparate cellular functions, including folding of nascent proteins, breakup of misfolded protein aggregates, and translocation of proteins across membranes. They act through the binding and release of substrate proteins, accompanied by hydrolysis of ATP. Chloroplast stromal Hsp70 plays a crucial role in the import of proteins into plastids. Mutations of an ATP binding domain Thr were previously reported to result in an increase in the K_m for ATP and a decrease in the enzyme’s k_cat. To ask which chloroplast stromal chaperone, Hsp70 or Hsp93, both of which are ATPases, dominates the energetics of the motor responsible for protein import, we made transgenic moss (Physcomitrella patens) harboring the K_m-altering mutation in the essential stromal Hsp70-2 and measured the effect on the amount of ATP required for protein import into chloroplasts. Here, we report that increasing the K_m for ATP hydrolysis of Hsp70 translated into an increased K_m for ATP usage by chloroplasts for protein import. This thus directly demonstrates that the ATP-derived energy long known to be required for chloroplast protein import is delivered via the Hsp70 chaperones and that the chaperone’s ATPase activity dominates the energetics of the reaction.     

Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase

We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the γ-subunit of chloroplast ATPase-coupling factor 1 (CF₁-γ, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF₁-γ accumulation in vivo. The CF₁-γ transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF₁-γ.     

The Clp chaperones and proteases of the human malaria parasite Plasmodium falciparum

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.     

In vivo and in vitro interaction of DnaK and a chloroplast transit peptide

Chloroplast transit peptides have been proposed to function as substrates for Hsp70 molecular chaperones. Many models of chloroplast protein import depict Hsp70s as the translocation motors that drive protein import into the organelle, but to our knowledge, no direct evidence has demonstrated that transit peptides function either in vivo or in vitro as substrates for the chaperone. In this report, we demonstrate that DnaK binds SStp (the full-length transit peptide for the precursor to the small subunit of Rubisco) in vivo when fused to either glutathione-S-transferase (GST) or to an His6-S-peptide tag (His-S) via an ATP-dependent mechanism. Three independent biophysical and biochemical assays confirm the ability of DnaK and SStp to interact in vitro. The cochaperones, DnaJ and GrpE, were also associated with the DnaK/SStp complex. Therefore, both GST-SStp and His-S-SStp can be used as affinity-tagged substrates to study prokaryotic chaperone/transit peptide interactions as well as to provide a novel functional probe to study the dynamics of DnaK/DnaJ/GrpE interactions in vivo. The combination of these results provides the first experimental support for a transit peptide-dependent interaction between a chloroplast precursor and Hsp70. These results are discussed in light of a general mechanism for protein translocation into chloroplasts and mitochondria.     

The motors of protein import into chloroplasts

Chloroplast function is largely dependent on its resident proteins, most of which are encoded by the nuclear genome and are synthesized in cytosol. Almost all of these are imported through the translocons located in the outer and inner chloroplast envelope membranes. The motor protein that provides the driving force for protein import has been proposed to be Hsp93, a member of the Hsp100 family of chaperones residing in the stroma. Combining in vivo and in vitro approaches, recent publications have provided multiple lines of evidence demonstrating that a stromal Hsp70 system is also involved in protein import into this organelle. Thus it appears that protein import into chloroplasts is driven by two motor proteins, Hsp93 and Hsp70. A perspective on collaboration between these two chaperones is discussed.Key words: stromal Hsp70, chloroplast protein import, stromal motor complex, ATPase, Physcomitrella patens, Hsp93, Toc, Tic, transit peptide, translocationChloroplasts are plant and algal specific organelles where photosynthesis and many other cellular processes take place. Chloroplasts contain ∼3,000 proteins,^1,2 with about 100 encoded by the chloroplast genome. In other words, more than 90% of chloroplast proteins are encoded by nuclear genes, synthesized in the cytosol and post-translationally imported into plastids. Most imported proteins are synthesized as precursors with a cleavable N-terminal signal, called a transit peptide. Such precursors are recognized by receptors in the outer envelope membrane, translocated through translocons in the outer and inner envelope membranes of chloroplasts (Toc and Tic), and processed to either their mature- or intermediate-sized forms in the chloroplast stroma.^3–8 Thylakoid proteins are further transported to their final destinations via one of four pathways, the cpSec, cpSRP, cpTAT and spontaneous pathways.^9–11 It is believed that the precursors are translocated across the envelope membranes in at least partially unfolded conformations and that the import machinery possesses some degree of unfolding activity.¹²Three proteins make up the core Toc complex, Toc159, Toc34 and Toc75. The Toc159 and Toc34 proteins are receptors possessing GTPase activities and recognizing transit peptides. Toc75 is a ß-barrel protein that forms the protein-translocating channel across the outer envelope membrane.¹³ The Tic complex is also formed from multiple subunits. Tic110, Tic21 and Tic20 have each been suggested to function as the channel of the Tic complex.^14–16 A ternary complex containing the stroma-facing domain of Tic110, Tic40 and a stromal factor, Hsp93 (a member of the Hsp100 family, possessing two ATPase domains), interacts with incoming precursor proteins.^17–26 Hsp93 has been proposed to serve as the import motor.²⁷ Other Tic components include regulatory subunits Tic62, Tic55 and Tic32 that are purported to facilitate redox- and calcium/calmodulin-dependent precursor translocation across the inner envelope membrane (reviewed in ref. ³). Tic22 is a peripheral membrane protein associated with the inner envelope and exposed to the intermembrane space.²⁸ It is suggested that Tic22 connects the Toc and Tic translocons during protein import.     

Ligand Recognition by the TPR Domain of the Import Factor Toc64 from Arabidopsis thaliana

The specific targeting of protein to organelles is achieved by targeting signals being recognised by their cognate receptors. Cytosolic chaperones, bound to precursor proteins, are recognized by specific receptors of the import machinery enabling transport into the specific organelle. The aim of this study was to gain greater insight into the mode of recognition of the C-termini of Hsp70 and Hsp90 chaperones by the Tetratricopeptide Repeat (TPR) domain of the chloroplast import receptor Toc64 from Arabidopsis thaliana (At). The monomeric TPR domain binds with 1∶1 stoichiometry in similar micromolar affinity to both Hsp70 and Hsp90 as determined by isothermal titration calorimetry (ITC). Mutations of the terminal EEVD motif caused a profound decrease in affinity. Additionally, this study considered the contributions of residues upstream as alanine scanning experiments of these residues showed reduced binding affinity. Molecular dynamics simulations of the TPR domain helices upon peptide binding predicted that two helices within the TPR domain move backwards, exposing the cradle surface for interaction with the peptide. Our findings from ITC and molecular dynamics studies suggest that AtToc64_TPR does not discriminate between C-termini peptides of Hsp70 and Hsp90.     

The amyloplast-targeting transit peptide of the waxy protein of maize also mediates protein transport in vitro into chloroplasts

Summary The transit peptide of the waxy protein of maize which in the maize plant targets this protein only into amyloplasts was used for in vitro protein transport experiments with isolated amyloplasts from maize and chloroplasts from maize, pea and potato. In the presence of both intact and disrupted amyloplasts an artificial preprotein (TP30), consisting of the waxy transit peptide plus the first 34 amino acids of the mature waxy protein fused in-frame to the -glucuronidase of Escherichia coli, is processed to the size expected when the transit peptide is cleaved off. The chloroplasts studied show in vitro import and correct processing of both TP30 and the authentic waxy protein, but not of the -glucuronidase without the waxy transit peptide. The in vitro import of TP30 into chloroplasts is almost as efficient as that of the precursor of the small subunit of pea ribulose-1,5-bisphosphate carboxylase, a nuclear-encoded chloroplast protein, whereas the waxy protein accumulates to a lesser extent in the chloroplasts. Since the amino-terminal transit peptides of TP30 and the waxy precursor are the same, this difference must be due to the mature part of the waxy protein. One possible explanation is the observed instability of the waxy protein in the presence of chloroplasts.     

Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide.

Phosphorylation of the transit peptide of several chloroplast-targeted proteins enables the binding of 14-3-3 proteins. The complex that forms, together with Hsp70, has been demonstrated to be an intermediate in the chloroplast protein import pathway in vitro[May, T. & Soll, J. (2000) Plant Cell 12, 53-63]. In this paper we report that mutagenesis (in order to remove the phosphorylation site) of the transit peptide of the small subunit of ribulose bisphosphate carboxylase/oxygenase did not affect its ability to target green fluorescent protein to chloroplasts in vivo. We also found no mistargeting to other organelles such as mitochondria. Similar alterations to the transit peptides of histidyl- or cysteinyl-tRNA synthetase, which are dual-targeted to chloroplasts and mitochondria, had no effect on their ability to target green fluorescent protein in vivo. Thus, phosphorylation of the transit peptide is not responsible for the specificity of chloroplast import.     

Pea Chloroplast DnaJ-J8 and Toc12 Are Encoded by the Same Gene and Localized in the Stroma

Toc12 is a novel J domain-containing protein identified in pea (Pisum sativum) chloroplasts. It was shown to be an integral outer membrane protein localizing in the intermembrane space of the chloroplast envelope. Furthermore, Toc12 was shown to associate with an intermembrane space Hsp70, suggesting that Toc12 is important for protein translocation across the chloroplast envelope. Toc12 shares a high degree of sequence similarity with Arabidopsis (Arabidopsis thaliana) DnaJ-J8, which has been suggested to be a soluble protein of the chloroplast stroma. Here, we isolated genes encoding DnaJ-J8 from pea and found that Toc12 is a truncated clone of one of the pea DnaJ-J8s. Protein import analyses indicate that Toc12 and DnaJ-J8s possess a cleavable transit peptide and are localized in the stroma. Arabidopsis mutants with T-DNA insertions in the DnaJ-J8 gene show no defect in chloroplast protein import. Implications of these results in the energetics and mechanisms of chloroplast protein import are discussed.Most chloroplast proteins are encoded by the nuclear genome and synthesized in the cytosol as higher molecular mass precursors with an N-terminal extension known as the transit peptide. Precursor proteins are imported into chloroplasts through a translocon complex located at the chloroplast envelope. Translocon components associated with the outer membrane are called Toc (for translocon of the outer envelope membrane of chloroplast) proteins, and those associated with the inner membrane are called Tic (for translocon of the inner envelope membrane of chloroplast) proteins. Cleavage of the transit peptide from the precursor by a specific stromal processing peptidase during translocation results in the production of the lower molecular mass mature protein. Various translocon components have been assigned functions in the basic steps of the import process (for review, see Inaba and Schnell, 2008; Jarvis, 2008; Li and Chiu, 2010). For example, Toc159 (the no. indicates the calculated molecular mass of the protein) and Toc34 are receptors for the transit peptides, and Toc75 is the protein-translocating channel across the outer membrane. Toc64, on the other hand, has a dual function: it serves as a docking site for the cytosolic Hsp90 through its cytosolic domain and as a scaffold for translocon components located in the intermembrane space through its intermembrane space domain (Qbadou et al., 2007).Protein import into chloroplasts involves at least two distinct ATP-consuming steps. The first step is called “early import intermediate” or “docking,” in which less than 100 μm ATP is required and precursors are translocated across the outer membrane and come into contact with translocon components in the inner membrane (Olsen et al., 1989; Kouranov and Schnell, 1997; Inaba et al., 2003; Inoue and Akita, 2008). It has been shown that the ATP is used in the intermembrane space (Olsen and Keegstra, 1992), most likely by a yet unidentified intermembrane space Hsp70 called imsHsp70 or Hsp70-IAP (ims for “intermembrane space” and IAP for “import intermediate-associated protein”; Marshall et al., 1990; Schnell et al., 1994; Qbadou et al., 2007). The second ATP-consuming step is the complete translocation of precursors across the two envelope membranes into the stroma. This step requires about 1 mm ATP. The ATP is most likely used by the stromal Hsp93 and chloroplast Hsc70 associated with the translocon to drive protein translocation into the stroma (Nielsen et al., 1997; Shi and Theg, 2010; Su and Li, 2010).Hsp70 family proteins are involved in many cellular processes, including protein folding, protein translocation across membranes, and regulation of protein degradation. Hsp70 proteins are often recruited to perform a certain function by specifically localized J domain-containing proteins. The J domain-containing proteins interact with Hsp70 when Hsp70 is bound to ATP and stimulate ATP hydrolysis by Hsp70. The specific J domain-containing cochaperone that recruits the stromal chloroplast Hsc70 to the inner envelope membrane to assist in protein translocation has not been identified. The specific J domain-containing cochaperone for imsHsp70 for its function in protein import into chloroplasts is proposed to be a protein named Toc12 (Becker et al., 2004).Toc12 was identified as a novel J domain-containing protein from pea (Pisum sativum) chloroplasts. It belongs to the type III J domain proteins containing only the J domain without the Gly- and Phe-rich domain (G/F domain) and the zinc-finger domain originally found in Escherichia coli DnaJ. It has been shown that the protein is synthesized at its mature size of 103 amino acids without a cleavable transit peptide. After import, the protein has been shown to anchor in the outer membrane by its N-terminal part, which has been suggested to form a β-barrel-type domain. Its C-terminal part, composed of the J domain, has been shown to localize in the intermembrane space. Toc12 has been shown to associate with imsHsp70. Toc12 and imsHsp70 interact with the intermembrane space domain of Toc64, which in turn associates with another intermembrane space translocon component, Tic22. It is proposed that the Toc12-imsHsp70-Toc64-Tic22 complex mediates protein translocation across the intermembrane space through specific precursor binding and ATP hydrolysis (Becker et al., 2004; Qbadou et al., 2007). However, the existence of imsHsp70 has only been shown on immunoblots by its reactivity to the monoclonal antibody SPA820 raised against human Hsp70. Its encoding gene has never been identified. The Arabidopsis (Arabidopsis thaliana) Hsp70 gene family has 14 members. Only two of them are localized in chloroplasts, and both have been shown to locate in the stroma (Ratnayake et al., 2008; Su and Li, 2008). A recent study has further shown that the major protein recognized by the SPA820 antibody in pea chloroplasts is located in the stroma, indicating that imsHsp70 is most likely a stromal protein (Ratnayake et al., 2008).Most translocon components were originally identified from pea chloroplasts. While all translocon components identified from pea have easily recognizable Arabidopsis homologs, Toc12 seems to be an exception. The Arabidopsis gene suggested to be the pea TOC12 homolog, At1g80920 (Inoue, 2007; Jarvis, 2008), encodes a protein that is much larger than pea Toc12 and is annotated as J8 (referred to as AtJ8 herein). The entire pea Toc12 has a high sequence similarity to the N-terminal two-thirds of AtJ8. AtJ8 contains an extra C-terminal domain of 60 amino acids that is highly conserved among J8 proteins from other higher plants. However, in contrast to pea Toc12, AtJ8 is predicted to locate in the stroma (Miernyk, 2001; www.arabidopsis.org). Indeed, a fusion protein consisting of the first 80 amino acids of AtJ8 fused at the N terminus of GFP was imported into the chloroplast stroma, and approximately 46 amino acids from the N terminus were processed after import (Lee et al., 2008), indicating that the first 46 amino acids of AtJ8 function as a cleavable stroma-targeting transit peptide. A T-DNA insertion in the AtJ8 gene that causes the truncation of the last three amino acids results in no visible phenotype. However, detailed analyses indicate that the mutant has lower CO₂ assimilation and Rubisco activity than the wild type (Chen et al., 2010).We are interested in identifying J domain-containing proteins interacting with stromal Hsp70. As part of the initial effort, we investigated the suborganellar location of J8 and examined the relationship between Toc12 and J8. We found that, in pea, there are at least two genes encoding J8, which we named PsJ8a and PsJ8b. TOC12 represents part of PsJ8b. Toc12, AtJ8, and the two PsJ8 proteins could be imported into chloroplasts and processed to stromally localized soluble mature proteins. Four alleles of AtJ8 mutants were analyzed, but none of them showed any defect in the import of various chloroplast precursor proteins.     

Protein binding and disruption by Clp/Hsp100 chaperones

Clp/Hsp100 chaperones work with other cellular chaperones and proteases to control the quality and amounts of many intracellular proteins. They employ an ATP-dependent protein unfoldase activity to solubilize protein aggregates or to target specific classes of proteins for degradation. The structural complexity of Clp/Hsp100 proteins combined with the complexity of the interactions with their macromolecular substrates presents a considerable challenge to understanding the mechanisms by which they recognize and unfold substrates and deliver them to downstream enzymes. Fortunately, high-resolution structural data is now available for several of the chaperones and their functional partners, which together with mutational data on the chaperones and their substrates has provided a glimmer of light at the end of the Clp/Hsp100 tunnel.     

A Stromal Heat Shock Protein 70 System Functions in Protein Import into Chloroplasts in the Moss Physcomitrella patens

Heat shock protein 70s (Hsp70s) are encoded by a multigene family and are located in different cellular compartments. They have broad-ranging functions, including involvement in protein trafficking, prevention of protein aggregation, and assistance in protein folding. Hsp70s work together with their cochaperones, J domain proteins and nucleotide exchange factors (e.g., GrpEs), in a functional cycle of substrate binding and release accompanied by ATP hydrolysis. We have taken advantage of the gene targeting capability of the moss Physcomitrella patens to investigate the functions of chloroplast Hsp70s. We identified four Hsp70 genes and two GrpE cochaperone homolog genes (CGE) in moss that encode chloroplast proteins. Disruption of one of the Hsp70 genes, that for Hsp70-2, caused lethality, and protein import into heat-shocked chloroplasts isolated from temperature-sensitive hsp70-2 mutants was appreciably impaired. Whereas the double cge null mutant was not viable, we recovered a cge1 null/cge2 knock down mutant in which Hsp70-2 was upregulated. Chloroplasts isolated from this mutant demonstrated a defect in protein import. In addition, two different precursors staged as early import intermediates could be immunoprecipitated with an Hsp70-2–specific antibody. This immunoprecipitate also contained Hsp93 and Tic40, indicating that it represents a precursor still in the Toc/Tic translocon. Together, these data indicate that a stromal Hsp70 system plays a crucial role in protein import into chloroplasts.     

The plastid-encoded protein Orf2971 is required for protein translocation and chloroplast quality control

Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control.

Repression of Orf2971 induces accumulation of chloroplast precursor proteins and impaired chloroplast quality indicating that Orf2971 is required for protein import and chloroplast quality control.

IN A NUTSHELL Background: The chloroplast is an important bioreactor as well as a photosynthetic site. Approximately 3,000 plastid proteins encoded in the nucleus are translocated into the chloroplast envelope via the TOC (translocon at the outer chloroplast envelope) and TIC machineries. Most nucleus-encoded preproteins that enter the plastid are unfolded as they traverse the TOC–TIC import complexes. To prevent these unfolded or misfolded proteins from causing chloroplast damage, a quality control mechanism comprising molecular chaperones and proteases ensures that all polypeptides entering chloroplasts are either correctly folded or degraded. However, there is still no consensus on the TIC complex’s components, motor proteins, or mechanism for refolding proteins entering the chloroplast. Question: What is the precise function of each of the proteins in the TIC complex? What is the composition of the chloroplast protein import machinery motor? How are the newly imported chloroplast proteins refolded and assembled into functional complexes? Findings: We found that Orf2971, encoded by the largest gene in the Chlamydomonas reinhardtii chloroplast genome and proposed to be an ortholog of Ycf2, is directly associated with the protein import machinery and plays a crucial role in ensuring the quality of proteins targeted to the chloroplast. Orf2971 deficiency induces protein precursor accumulation, partial proteolysis and protein aggregation, increased expression of chaperones and proteases, and autophagy. We hypothesize that Orf2971 is intimately linked to the protein import machinery and plays a critical role in chloroplast protein quality control. Next steps: The next challenge is to identify the sorting components associated with this complex on the stromal side. Furthermore, additional experimental evidence is required to investigate the relationship between different import machineries, including the analysis of the accumulation of precursor proteins in the various import mutants.