Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane.     

In vivo studies on the roles of Tic110, Tic40 and Hsp93 during chloroplast protein import

A multisubunit translocon of the inner envelope membrane, termed Tic, mediates the late stages of protein import into chloroplasts. Membrane proteins, Tic110 and Tic40, and a stromal chaperone, Hsp93, have been proposed to function together within the Tic complex. In Arabidopsis, single genes, atTIC110 and atTIC40, encode the Tic proteins, and two homologous genes, atHSP93-V and atHSP93-III, encode Hsp93. These four genes exhibited relatively uniform patterns of expression, suggesting important roles for plastid biogenesis throughout development and in all tissues. To investigate the roles played by these proteins in vivo, we conducted a comparative study of T-DNA knockout mutants for each Tic gene, and for the most abundantly expressed Hsp93 gene, atHSP93-V. In the homozygous state, the tic110 mutation caused embryo lethality, implying an essential role for atTic110 during plastid biogenesis. Homozygous tic110 embryos exhibited retarded growth, developmental arrest at the globular stage and a 'raspberry-like' embryo-proper phenotype. Heterozygous tic110 plants, and plants homozygous for the tic40 and hsp93-V mutations, exhibited chlorosis, aberrant chloroplast biogenesis, and inefficient chloroplast-import of both photosynthetic and non-photosynthetic preproteins. Non-additive interactions amongst the mutations occurred in double mutants, suggesting that the three components may cooperate during chloroplast protein import.     

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.     

Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts

Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation.     

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.     

Arabidopsis stromal 70-kD heat shock proteins are essential for plant development and important for thermotolerance of germinating seeds

The 70-kD heat shock proteins (Hsp70s) have been shown to be important for protein folding, protein translocation, and stress responses in almost all organisms and in almost all subcellular compartments. However, the function of plastid stromal Hsp70s in higher plants is still uncertain. Genomic surveys have revealed that there are two putative stromal Hsp70s in Arabidopsis thaliana, denoted cpHsc70-1 (At4g24280) and cpHsc70-2 (At5g49910). In this study, we show that cpHsc70-1 and cpHsc70-2 could indeed be imported into the chloroplast stroma. Their corresponding T-DNA insertion knockout mutants were isolated and designated as Deltacphsc70-1 and Deltacphsc70-2. No visible phenotype was observed in the Deltacphsc70-2 mutant under normal growth conditions. In contrast, Deltacphsc70-1 mutant plants exhibited variegated cotyledons, malformed leaves, growth retardation, and impaired root growth, even though the protein level of cpHsc70-2 was up-regulated in the Deltacphsc70-1 mutant. After heat shock treatment of germinating seeds, root growth from Deltacphsc70-1 seeds was further impaired, indicating that cpHsc70-1 is important for thermotolerance of germinating seeds. No Deltacphsc70-1 Deltacphsc70-2 double mutant could be obtained, suggesting that the Deltacphsc70 double knockout was lethal. Genotype analyses of F(1) seedlings from various crosses indicated that double-knockout mutation was lethal to the female gametes and reduced the transmission efficiency of the male gametes. These results indicate that cpHsc70s are essential for plant development and the two cpHsc70s most likely have redundant but also distinct functions.     

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.     

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.     

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.     

The amino-terminal domain of chloroplast Hsp93 is important for its membrane association and functions in vivo

Chloroplast 93-kD heat shock protein (Hsp93/ClpC), an Hsp100 family member, is suggested to have various functions in chloroplasts, including serving as the regulatory chaperone for the ClpP protease in the stroma and acting as a motor component of the protein translocon at the envelope. Indeed, although Hsp93 is a soluble stromal protein, a portion of it is associated with the inner envelope membrane. The mechanism and functional significance of this Hsp93 membrane association have not been determined. Here, we mapped the region important for Hsp93 membrane association by creating various deletion constructs and found that only the construct with the amino-terminal domain deleted, Hsp93-ΔN, had reduced membrane association. When transformed into Arabidopsis (Arabidopsis thaliana), most atHsp93V-ΔN proteins did not associate with membranes and atHsp93V-ΔΝ failed to complement the pale-green and protein import-defective phenotypes of an hsp93V knockout mutant. The residual atHsp93V-ΔN at the membranes had further reduced association with the central protein translocon component Tic110. However, the degradation of chloroplast glutamine synthetase, a potential substrate for the ClpP protease, was not affected in the hsp93V mutant or in the atHSP93V-ΔN transgenic plants. Hsp93-ΔN also had the same ATPase activity as that of full-length Hsp93. These data suggest that the association of Hsp93 with the inner envelope membrane through its amino-terminal domain is important for the functions of Hsp93 in vivo.     

Functional similarity between the chloroplast translocon component, Tic40, and the human co-chaperone, Hsp70-interacting protein (Hip)

Tic40 is a component of the protein import apparatus of the inner envelope of chloroplasts, but its role in the import mechanism has not been clearly defined. The C terminus of Tic40 shares weak similarity with the C-terminal Sti1 domains of the mammalian Hsp70-interacting protein (Hip) and Hsp70/Hsp90-organizing protein (Hop) co-chaperones. Additionally, Tic40 may possess a tetratricopeptide repeat (TPR) protein-protein interaction domain, another characteristic feature of Hip/Hop co-chaperones. To investigate the functional importance of different parts of the Tic40 protein and to determine whether the homology between Tic40 and co-chaperones is functionally significant, different Tic40 deletion and Tic40:Hip fusion constructs were generated and assessed for complementation activity in the Arabidopsis Tic40 knock-out mutant, tic40. Interestingly, all Tic40 deletion constructs failed to complement tic40, indicating that each part removed is essential for Tic40 function; these included a construct lacking the Sti1-like domain (DeltaSti1), a second lacking a central region, including the putative TPR domain (DeltaTPR), and a third lacking the predicted transmembrane anchor region. Moreover, the DeltaSti1 and DeltaTPR constructs caused strong dominant-negative, albino phenotypes in tic40 transformants, indicating that the truncated Tic40 proteins interfere with the residual chloroplast protein import that occurs in tic40 plants. Remarkably, the Tic40:Hip fusion constructs showed that the Sti1 domain of human Hip is functionally equivalent to the Sti1-like region of Tic40, strongly suggesting a co-chaperone role for the Tic40 protein. Supporting this notion, yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated the in vivo interaction of Tic40 with Tic110, a protein believed to recruit stromal chaperones to protein import sites.     

Requirements for a conservative protein translocation pathway in chloroplasts

The chloroplast inner envelope translocon subunit Tic110 is imported via a soluble stromal translocation intermediate. In this study an in-organellar import system is established which allows for an accumulation of this intermediate in order to analyze its requirements for reexport. All results demonstrate that the re-export of Tic110 from the soluble intermediate stage into the inner envelope requires ATP hydrolysis, which cannot be replaced by other NTPs. Furthermore, the molecular chaperone Hsp93 seems prominently involved in the reexport pathway of Tic110, because other stromal intermediates like that of the oxygen evolving complex subunit OE33 (iOE33) en route to the thylakoid lumen interacts preferentially with Hsp70.     

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.     

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.     

Alternative Processing of Arabidopsis Hsp70 Precursors during Protein Import into Chloroplasts

During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously reported to localize in the intermembrane space of chloroplasts, was found to interact with the translocating precursor protein but the gene for Hsp70-IAP has not been identified yet. In an attempt to identify the Arabidopsis homolog of Hsp70-IAP, we employed an in vitro protein import assay to determine the localization of three Arabidopsis Hsp70 homologs (AtHsp70-6 through 8), predicted for chloroplast targeting. AtHsp70-6 and AtHsp70-7 were imported into chloroplasts and processed into similar-sized mature forms. In addition, a smaller-sized processed form of AtHsp70-6 was observed. All the processed forms of both AtHsp70 proteins were localized in the stroma. Organelle-free processing assays revealed that the larger processed forms of both AtHsp70-6 and AtHsp70-7 were cleaved by stromal processing peptidase, whereas the smaller processed form of AtHsp70-6 was produced by an unspecified peptidase.     

Arabidopsis stromal 70-kDa heat shock proteins are essential for chloroplast development

70 kDa heat shock proteins (Hsp70s) act as molecular chaperones involved in essential cellular processes such as protein folding and protein transport across membranes. They also play a role in the cell’s response to a wide range of stress conditions. The Arabidopsis family of Hsp70s homologues includes two highly conserved proteins, cpHsc70-1 and cpHsc70-2 which are both imported into chloroplasts (Su and Li in Plant Physiol 146:1231–1241, 2008). Here, we demonstrate that YFP-fusion proteins of both cpHsc70-1 and cpHsc70-2 are predominantly stromal, though low levels were detected in the thylakoid membrane. Both genes are ubiquitously expressed at high levels in both seedlings and adult plants. We further show that both cpHsc70-1 and cpHsc70-2 harbour ATPase activity which is essential for Hsp70 chaperone activity. A previously described T-DNA insertion line for cpHsc70-1 (ΔcpHsc70-1) has variegated cotyledons, malformed leaves, growth retardation, impaired root growth and sensitivity to heat shock treatment. In addition, under stress conditions, this mutant also exhibits unusual sepals, and malformed flowers and sucrose concentrations as low as 1% significantly impair growth. cpHsc70-1/cpHsc70-2 double-mutants are lethal. However, we demonstrate through co-suppression and artificial microRNA (amiRNA) approaches that transgenic plants with severely reduced levels of both genes have a white and stunted phenotype. Interestingly, chloroplasts in these plants have an unusual morphology and contain few or no thylakoid membranes. Our data show that cpHsc70-1 and cpHsc70-2 are essential ATPases, have overlapping roles and are required for normal plastid structure.     

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.     

The localization of Tic20 proteins in Arabidopsis thaliana is not restricted to the inner envelope membrane of chloroplasts

Tic20 is a central, membrane-embedded component of the precursor protein translocon of the inner envelope of chloroplasts (TIC). In Arabidopsis thaliana, four different isoforms of Tic20 exist. They are annotated as atTic20-I, -II, -IV and -V and form two distinct phylogenetic subfamilies in embryophyta. Consistent with atTic20-I being the only essential isoform for chloroplast development, we show that the protein is exclusively targeted to the chloroplasts inner envelope. The same result is observed for atTic20-II. In contrast, atTic20-V is localized in thylakoids and atTic20-IV dually localizes to chloroplasts and mitochondria. These results together with the previously established expression profiles explain the recently described phenotypes of Tic20 knockout plants and point towards a functional diversification of these proteins within the family. For all Tic20 proteins a 4-helix topology is proposed irrespective of the targeted membrane, which in part could be confirmed in vivo by application of a self-assembling GFP-based topology approach. By the same approach we show that the inner envelope localized Tic20 proteins expose their C-termini to the chloroplast stroma. This localization would be consistent with the positive inside rule considering a stromal translocation intermediate as discussed.     

Alternative processing of Arabidopsis Hsp70 precursors during protein import into chloroplasts

During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously reported to localize in the intermembrane space of chloroplasts, was found to interact with the translocating precursor protein but the gene for Hsp70-IAP has not been identified yet. In an attempt to identify the Arabidopsis homolog of Hsp70-IAP, we employed an in vitro protein import assay to determine the localization of three Arabidopsis Hsp70 homologs (AtHsp70-6 through 8), predicted for chloroplast targeting. AtHsp70-6 and AtHsp70-7 were imported into chloroplasts and processed into similar-sized mature forms. In addition, a smaller-sized processed form of AtHsp70-6 was observed. All the processed forms of both AtHsp70 proteins were localized in the stroma. Organelle-free processing assays revealed that the larger processed forms of both AtHsp70-6 and AtHsp70-7 were cleaved by stromal processing peptidase, whereas the smaller processed form of AtHsp70-6 was produced by an unspecified peptidase.     

A stromal Hsp100 protein is required for normal chloroplast development and function in Arabidopsis

Molecular chaperones are required for the translocation of many proteins across organellar membranes, presumably by providing energy in the form of ATP hydrolysis for protein movement. In the chloroplast protein import system, a heat shock protein 100 (Hsp100), known as Hsp93, is hypothesized to be the chaperone providing energy for precursor translocation, although there is little direct evidence for this hypothesis. To learn more about the possible function of Hsp93 during protein import into chloroplasts, we isolated knockout mutant lines that contain T-DNA disruptions in either atHSP93-V or atHSP93-III, which encode the two Arabidopsis (Arabidopsis thaliana) homologs of Hsp93. atHsp93-V mutant plants are much smaller and paler than wild-type plants. In addition, mutant chloroplasts contain less thylakoid membrane when compared to the wild type. Plastid protein composition, however, seems to be largely unaffected in atHsp93-V knockout plants. Chloroplasts isolated from the atHsp93-V knockout mutant line are still able to import a variety of precursor proteins, but the rate of import of some of these precursors is significantly reduced. These results indicate that atHsp93-V has an important, but not essential, role in the biogenesis of Arabidopsis chloroplasts. In contrast, knockout mutant plants for atHsp93-III, the second Arabidopsis Hsp93 homolog, had a visible phenotype identical to the wild type, suggesting that atHsp93-III may not play as important a role as atHsp93-V in chloroplast development and/or function.