Further in vivo studies on the role of the molecular chaperone, Hsp93, in plastid protein import

In Arabidopsis, Hsp93 is encoded by two genes, atHSP93-V and atHSP93-III. We identified two T-DNA mutants for atHSP93-III: one being a partial 'knockdown' (hsp93-III-1) and the other a complete 'knockout' (hsp93-III-2). Homozygotes for both mutants were indistinguishable from wild type. We crossed each mutant to an atHSP93-V knockout, and identified double mutants with strongly chlorotic phenotypes. This implied redundancy, which was confirmed by the complementation of mildly chlorotic hsp93-V plants by atHSP93-III over-expression. While the hsp93-V hsp93-III-1 mutant was doubly homozygous, the second double mutant was heterozygous for hsp93-III-2 (genotype: hsp93-V/hsp93-V; +/hsp93-III-2). Attempts to identify an hsp93-V hsp93-III-2 double homozygote were unsuccessful, indicating that the Hsp93 pool is essential for viability. Consistently, siliques of the second double mutant contained aborted seeds (because of a block in the zygote-embryo transition) and failed ovules (because of a moderate defect in female gametophytes). Double-mutant plants were chlorophyll-deficient, contained under-developed chloroplasts, and exhibited stunted growth. In import assays using a chimeric pre-protein (plastocyanin transit peptide fused to dihydrofolate reductase; PC-DHFR), a clear defect was observed in hsp93-V hsp93-III-1 chloroplasts. Interestingly, while denaturation or stabilization of the DHFR moiety had a strong effect on import efficiency in the wild type, no such effects were observed with double-mutant (or tic40) chloroplasts. This indicated that pre-protein unfolding is not rate-limiting for import into mutant chloroplasts, and suggested that (unlike the situation in mitochondria) the inner membrane import machinery does not contribute to pre-protein unfolding at the organellar surface.     

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.     

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.     

Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis thaliana

Toc64/OEP64 was identified biochemically in pea as a putative component of the chloroplast protein import apparatus. In Arabidopsis, three paralogous genes (atTOC64-III, atTOC64-V and atTOC64-I) encode Toc64-related proteins, and these have been reported to localize in chloroplasts, mitochondria and the cytosol, respectively. To assess the role of the atToc64-III protein in chloroplast protein import in an in vivo context, we identified and characterized Arabidopsis knockout mutants. The absence of detectable defects in toc64-III single mutants raised the possibility of redundancy, and prompted us to also identify toc64-V and toc64-I mutants, cross them to toc64-III, and generate double- and triple-mutant combinations. The toc64 mutants were analysed carefully with respect to a variety of criteria, including chlorophyll accumulation, photosynthetic performance, organellar ultrastructure and chloroplast protein accumulation. In each case, the mutant plants were indistinguishable from wild type. Furthermore, the efficiency of chloroplast protein import was not affected by the toc64 mutations, even when a putative substrate of the atToc64-III protein (wheatgerm-translated precursor of the 33 kDa subunit of the oxygen-evolving complex, OE33) was examined. Moreover, under various stress conditions (high light, osmotic stress and cold), the toc64 triple-mutant plants were not significantly different from wild type. These results demonstrate that Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis, and draw into question the functional significance of this component.     

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts

Hsp70 family proteins function as motors driving protein translocation into mitochondria and the endoplasmic reticulum. Whether Hsp70 is involved in protein import into chloroplasts has not been resolved. We show here Arabidopsis thaliana knockout mutants of either of the two stromal cpHsc70s, cpHsc70-1 and cpHsc70-2, are defective in protein import into chloroplasts during early developmental stages. Protein import was found to be affected at the step of precursor translocation across the envelope membranes. From solubilized envelope membranes, stromal cpHsc70 was specifically coimmunoprecipitated with importing precursors and stoichiometric amounts of Tic110 and Hsp93. Moreover, in contrast with receptors at the outer envelope membrane, cpHsp70 is important for the import of both photosynthetic and nonphotosynthetic proteins. These data indicate that cpHsc70 is part of the chloroplast translocon for general import and is important for driving translocation into the stroma. We further analyzed the relationship of cpHsc70 with the other suggested motor system, Hsp93/Tic40. Chloroplasts from the cphsc70-1 hsp93-V double mutant had a more severe import defect than did the single mutants, suggesting that the two proteins function in parallel. The cphsc70-1 tic40 double knockout was lethal, further indicating that cpHsc70-1 and Tic40 have an overlapping essential function. In conclusion, our data indicate that chloroplasts have two chaperone systems facilitating protein translocation into the stroma: the cpHsc70 system and the Hsp93/Tic40 system.     

In vivo analyses of the roles of essential Omp85-related proteins in the chloroplast outer envelope membrane

Two different, essential Omp85 (Outer membrane protein, 85 kD)-related proteins exist in the outer envelope membrane of Arabidopsis (Arabidopsis thaliana) chloroplasts: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD), encoded by atTOC75-III; and OEP80 (Outer Envelope Protein, 80 kD), encoded by AtOEP80/atTOC75-V. The atToc75-III protein is closely related to the originally identified pea (Pisum sativum) Toc75 protein, and it forms a preprotein translocation channel during chloroplast import; the AtOEP80 protein is considerably more divergent from pea Toc75, and its role is unknown. As knockout mutations for atTOC75-III and AtOEP80 are embryo lethal, we employed a dexamethasone-inducible RNA interference strategy (using the pOpOff2 vector) to conduct in vivo studies on the roles of these two proteins in older, postembryonic plants. We conducted comparative studies on plants silenced for atToc75-III (atToc75-III↓) or AtOEP80 (AtOEP80↓), as well as additional studies on a stable, atToc75-III missense allele (toc75-III-3/modifier of altered response to gravity1), and our results indicated that both proteins are important for chloroplast biogenesis at postembryonic stages of development. Moreover, both are important for photosynthetic and nonphotosynthetic development, albeit to different degrees: atToc75-III↓ phenotypes were considerably more severe than those of AtOEP80↓. Qualitative similarity between the atToc75-III↓ and AtOEP80↓ phenotypes may be linked to deficiencies in atToc75-III and other TOC proteins in AtOEP80↓ plants. Detailed analysis of atToc75-III↓ plants, by electron microscopy, immunoblotting, quantitative proteomics, and protein import assays, indicated that these plants are defective in relation to the biogenesis of both photosynthetic and nonphotosynthetic plastids and preproteins, confirming the earlier hypothesis that atToc75-III functions promiscuously in different substrate-specific import pathways.     

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.     

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.     

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.     

In vivo studies on the roles of two closely related Arabidopsis Tic20 proteins, AtTic20-I and AtTic20-IV

Protein translocation across the inner envelope of plastids is mediated by the TIC (translocon at the inner envelope membrane of chloroplasts) protein translocation machinery. Tic20 has been shown to function as a central component of TIC machinery. The Arabidopsis genome encodes four Tic20 homologous proteins, AtTic20-I, AtTic20-II, AtTIC20-IV and AtTic20-V, among which only AtTic20-I has been extensively characterized and demonstrated to be essential for protein import into chloroplasts. AtTic20-I is more closely related to AtTic20-IV than to AtTic20-II or AtTic20-V, whereas AtTic20-II and AtTic20-V show higher similarities to each other than to AtTic20-I or AtTic20-IV. Here, we show that AtTic20-IV is expressed mainly in roots whereas AtTic20-I is more abundant in shoots than in roots. Although AtTic20-IV is dispensable for viability in the wild-type background, interestingly, expression of AtTic20-IV is markedly elevated in both shoots and roots in the tic20-I knockout mutant that exhibits severe albino and seedling-lethal phenotypes. The albino tic20-I seedlings do not accumulate any of the photosynthetic proteins analyzed, but the plastids can still import non-photosynthetic housekeeping proteins. This residual import ability of the tic20-I mutant can be attributed to partial compensation by the elevated expression of AtTic20-IV, since a double knockout mutant of AtTic20-I and AtTic20-IV exhibits more severe embryonic lethality. Further overexpression of AtTic20-IV in the tic20-I mutant can only marginally rescue the accumulation of photosynthetic proteins in the albino seedlings. These data demonstrate an absolute requirement of at least one of the two closely related Tic20 proteins in protein translocation across the inner envelope of plastids and also suggest their distinct substrate preferences.     

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.     

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.     

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 protein import inhibition by a soluble factor from wheat germ lysate

Protein import into chloroplasts occurs post-translationally in vitro. The precursor proteins are generally synthesised in a reticulocyte lysate- or wheat germ lysate-derived system and imported out of this system into chloroplast. These complex soluble protein mixtures are likely to contain factors, which influence somehow the import competence and import efficiency. Here we describe a heat-stable soluble proteinaceaous factor, which inhibits protein import into chloroplasts in vitro. The inhibitor interacts directly with the precursor protein and renders it import incompetent. This mode of action is supported by two observations: firstly, binding of the precursor to the chloroplast surface is diminished in the presence of the inhibitor. Secondly, when chloroplasts were loaded with precursor proteins under conditions, which allow only binding but not import the inhibitor was unable to abolish the subsequent translocation step.     

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.     

Analysis of chloroplast transit peptide function using mutations in the carboxyl-terminal region

Protein import into chloroplasts requires a transit peptide, which interacts with the chloroplast transport apparatus and leads to translocation of the protein across the chloroplast envelope. While the amino acid sequences of many transit peptides are known, functional domains have been difficult to identify. Previous studies suggest that the carboxyl terminus of the transit peptide for ribulose bisphosphate carboxylase small subunit is important for both translocation across the chloroplast envelope and proper processing of the precursor protein. We dissected this region using in vitro mutagenesis, creating a set of mutants with small changes in primary structure predicted to cause alterations in secondary structure. The import behavior of the mutant proteins was assessed using isolated chloroplasts. Our results show that removal of a conserved arginine residue in this region results in impaired processing, but does not necessarily affect import rates. In contrast, substituting amino acids with low reverse turn or amphiphilic potential for other original residues affected import rate but not processing.     

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.     

Fatty acid export from the chloroplast. Molecular characterization of a major plastidial acyl-coenzyme A synthetase from Arabidopsis

Acyl-coenzyme A (CoA) synthetases (ACSs, EC 6.2.1.3) catalyze the formation of fatty acyl-CoAs from free fatty acid, ATP, and CoA. Essentially all de novo fatty acid synthesis occurs in the plastid. Fatty acids destined for membrane glycerolipid and triacylglycerol synthesis in the endoplasmic reticulum must be first activated to acyl-CoAs via an ACS. Within a family of nine ACS genes from Arabidopsis, we identified a chloroplast isoform, LACS9. LACS9 is highly expressed in developing seeds and young rosette leaves. Both in vitro chloroplast import assays and transient expression of a green fluorescent protein fusion indicated that the LACS9 protein is localized in the plastid envelope. A T-DNA knockout mutant (lacs9-1) was identified by reverse genetics and these mutant plants were indistinguishable from wild type in growth and appearance. Analysis of leaf lipids provided no evidence for compromised export of acyl groups from chloroplasts. However, direct assays demonstrated that lacs9-1 plants contained only 10% of the chloroplast long-chain ACS activity found for wild type. The residual long-chain ACS activity in mutant chloroplasts was comparable with calculated rates of fatty acid synthesis. Although another isozyme contributes to the activation of fatty acids during their export from the chloroplast, LACS9 is a major chloroplast ACS.