Reconstitution of protein targeting to the inner envelope membrane of chloroplasts

The chloroplast envelope plays critical roles in the synthesis and regulated transport of key metabolites, including intermediates in photosynthesis and lipid metabolism. Despite this importance, the biogenesis of the envelope membranes has not been investigated in detail. To identify the determinants of protein targeting to the inner envelope membrane (IM), we investigated the targeting of the nucleus-encoded integral IM protein, atTic40. We found that pre-atTic40 is imported into chloroplasts and processed to an intermediate size (int-atTic40) before insertion into the IM. Int-atTic40 is soluble and inserts into the IM from the internal stromal compartment. We also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. Collectively, our experiments are consistent with a "postimport" mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.     

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.     

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.     

atTic110 functions as a scaffold for coordinating the stromal events of protein import into chloroplasts

The translocon of the inner envelope membrane of chloroplasts (Tic) mediates the late events in the translocation of nucleus-encoded preproteins into chloroplasts. Tic110 is a major integral membrane component of active Tic complexes and has been proposed to function as a docking site for translocation-associated stromal factors and as a component of the protein-conducting channel. To investigate the various proposed functions of Tic110, we have investigated the structure, topology, and activities of a 97.5-kDa fragment of Arabidopsis Tic110 (atTic110) lacking only the amino-terminal transmembrane segments. The protein was expressed both in Escherichia coli and Arabidopsis as a stable, soluble protein with a high alpha-helical content. Binding studies demonstrate that a region of the atTic110-soluble domain selectively associates with chloroplast preproteins at the late stages of membrane translocation. These data support the hypothesis that the bulk of Tic110 extends into the chloroplast stroma and suggest that the domain forms a docking site for preproteins as they emerge from the Tic translocon.     

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.     

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.     

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.     

A Nuclear-coded Chloroplastic Inner Envelope Membrane Protein Uses a Soluble Sorting Intermediate upon Import into the Organelle

The chloroplastic inner envelope protein of 110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins were constructed to assess the import and sorting pathway of IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chloroplasts, as shown by the mutual exchange of presequences with the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSSU). Sorting information to the chloroplastic inner envelope is contained in an NH₂-proximal part of mature IEP110 (110N). The NH₂-terminus serves to anchor the protein into the membrane. Large COOH-terminal portions of this protein (80–90 kD) are exposed to the intermembrane space in situ. Successful sorting and integration of IEP110 and the derived constructs into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease thermolysin but accessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space. A hybrid protein consisting of the transit sequence of SSU, the NH₂-proximal part of mature IEP110, and mature SSU (tpSSU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be recovered as soluble, terminally processed 110NmSSU. The soluble 110N-mSSU then enters a reexport pathway, which results not only in the insertion of 110N-mSSU into the inner envelope membrane, but also in the extrusion of large portions of the protein into the intermembrane space. We conclude that chloroplasts possess a protein reexport machinery for IEPs in which soluble stromal components interact with a membrane-localized translocation machinery.     

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.     

Protein Import into and Sorting inside the Chloroplast Are Independent Processes

Plastocyanin is a nuclear-encoded chloroplast thylakoid lumen protein that is synthesized in the cytoplasm with a large N-terminal extension (66 amino acids). Transport of plastocyanin involves two steps: import across the chloroplast envelope into the stroma, followed by transfer across the thylakoid membrane into the lumen. During transport the N-terminal extension is removed in two parts by two different processing proteases. In this study we examined the functions of the two cleaved parts, C1 and C2, in the transport pathway of plastocyanin. The results show that C1 mediates import into the chloroplast. C1 is sufficient to direct chloroplast import of mutant proteins that lack C2. It is also sufficient to direct import of a nonplastid protein and can be replaced functionally by the transit peptide of an imported stromal protein. C2 is a prerequisite for intraorganellar routing but is not required for chloroplast import. Deletions in C2 result in accumulation of intermediates in the stroma or on the outside of the thylakoids. The fact that C1 is functionally equivalent to a stromal-targeting transit peptide shows that plastocyanin is imported into the chloroplast by way of the same mechanism as stromal proteins, and that import into and routing inside the chloroplasts are independent processes.     

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.     

Tic40, a new "old" subunit of the chloroplast protein import translocon

The protein import translocon at the inner envelope of chloroplasts (Tic complex) is a heteroligomeric multisubunit complex. Here, we describe Tic40 from pea as a new component of this complex. Tic40 from pea is a homologue of a protein described earlier from Brassica napus as Cim/Com44 or the Toc36 subunit of the translocon at the outer envelope of chloroplasts, respectively (Wu, C., Seibert, F. S., and Ko, K. (1994) J. Biol. Chem. 269, 32264-32271; Ko, K., Budd, D., Wu, C., Seibert, F., Kourtz, L., and Ko, Z. W. (1995) J. Biol. Chem. 270, 28601-28608; Pang, P., Meathrel, K., and Ko, K. (1997) J. Biol. Chem. 272, 25623-25627). Tic40 can be covalently connected to Tic110 by the formation of a disulfide bridge under oxidizing conditions, indicating its close physical proximity to an established translocon component. The Tic40 protein is synthesized in the cytosol as a precursor with an N-terminal cleavable chloroplast targeting signal and imported into the organelle via the general import pathway. Immunoblotting and immunogold-labeling studies exclusively confine Tic40 to the chloroplastic inner envelope, in which it is anchored by a single putative transmembrane span.     

Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677–1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.     

Tic21 is an essential translocon component for protein translocation across the chloroplast inner envelope membrane

An Arabidopsis thaliana mutant defective in chloroplast protein import was isolated and the mutant locus, cia5, identified by map-based cloning. CIA5 is a 21-kD integral membrane protein in the chloroplast inner envelope membrane with four predicted transmembrane domains, similar to another potential chloroplast inner membrane protein-conducting channel, At Tic20, and the mitochondrial inner membrane counterparts Tim17, Tim22, and Tim23. cia5 null mutants were albino and accumulated unprocessed precursor proteins. cia5 mutant chloroplasts were normal in targeting and binding of precursors to the chloroplast surface but were defective in protein translocation across the inner envelope membrane. Expression levels of CIA5 were comparable to those of major translocon components, such as At Tic110 and At Toc75, except during germination, at which stage At Tic20 was expressed at its highest level. A double mutant of cia5 At tic20-I had the same phenotype as the At tic20-I single mutant, suggesting that CIA5 and At Tic20 function similarly in chloroplast biogenesis, with At Tic20 functioning earlier in development. We renamed CIA5 as Arabidopsis Tic21 (At Tic21) and propose that it functions as part of the inner membrane protein-conducting channel and may be more important for later stages of leaf development.     

Arabidopsis tic110 is essential for the assembly and function of the protein import machinery of plastids

The translocon at the inner envelope membrane of chloroplasts (Tic) plays a central role in plastid biogenesis by coordinating the sorting of nucleus-encoded preproteins across the inner membrane and coordinating the interactions of preproteins with the processing and folding machineries of the stroma. Despite these activities, the precise roles of known Tic proteins in translocation, sorting, and preprotein maturation have not been defined. In this report, we examine the in vivo function of a major Tic component, Tic110. We demonstrate that Arabidopsis thaliana Tic110 (atTic110) is essential for plastid biogenesis and plant viability. The downregulation of atTic110 expression results in the reduced accumulation of a wide variety of plastid proteins. The expression of dominant negative mutants of atTic110 disrupts assembly of Tic complexes and the translocation of preproteins across the inner envelope membrane. Together, these data suggest that Tic110 plays a general role in the import of nuclear-encoded preproteins as a common component of Tic complexes.     

Stable megadalton TOC–TIC supercomplexes as major mediators of protein import into chloroplasts

Preproteins are believed to be imported into chloroplasts through membrane contact sites where the translocon complexes of the outer (TOC) and inner (TIC) envelope membranes are assembled together. However, a single TOC–TIC supercomplex containing preproteins undergoing active import has not yet been directly observed. We optimized the blue native polyacrylamide gel electrophoresis (PAGE) (BN‐PAGE) system to detect and resolve megadalton (MD)‐sized complexes. Using this optimized system, the outer‐membrane channel Toc75 from pea chloroplasts was found in at least two complexes: the 880‐kD TOC complex and a previously undetected 1‐MD complex. Two‐dimensional BN‐PAGE immunoblots further showed that Toc75, Toc159, Toc34, Tic20, Tic56 and Tic110 were all located in the 880‐kD to 1.3‐MD region. During active preprotein import, preproteins were transported mostly through the 1‐MD complex and a smaller amount of preproteins was also detected in a complex of 1.25 MD. Antibody‐shift assays showed that the 1‐MD complex is a TOC–TIC supercomplex containing at least Toc75, Toc159, Toc34 and Tic110. Results from crosslinking and import with Arabidopsis chloroplasts suggest that the 1.25‐MD complex is also a supercomplex. Our data provide direct evidence supporting that chloroplast preproteins are imported through TOC–TIC supercomplexes, and also provide the first size estimation of these supercomplexes. Furthermore, unlike in mitochondria where translocon supercomplexes are only transiently assembled during preprotein import, in chloroplasts at least some of the supercomplexes are preassembled stable structures.     

An imported thylakoid protein accumulates in the stroma when insertion into thylakoids is inhibited

The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP) that is imported into chloroplasts and assembled into thylakoid membranes. Under appropriate conditions, either pLHCP or LHCP will integrate into isolated thylakoids. We have identified two situations that inhibit integration in this assay. Ionophores and uncouplers inhibited integration up to 70%. Carboxyl-terminal truncations of pLHCP also interfered with integration. A 22-residue truncation reduced integration to about 25% of control, whereas a 93 residue truncation completely abolished it. When pLHCP was imported into chloroplasts in the presence of uncouplers or when truncated forms of pLHCP were used, significant amounts of the imported proteins failed to insert into thylakoids and instead accumulated in the aqueous stroma. Accumulation of stromal LHCP occurred at uncoupler concentrations required to dissipate the trans-thylakoid proton electrochemical gradient and was enhanced at reduced levels of ATP. The latter effect may be a secondary consequence of a reduction in ATP-dependent degradation within the stroma. These results indicate that the stroma is an intermediate location in the LHCP assembly pathway and provide the first evidence for a soluble intermediate during biogenesis of a chloroplast membrane protein.     

Arabidopsis thaliana Tic110, involved in chloroplast protein translocation, contains at least fourteen highly divergent heat-like repeated motifs

Endosymbiotic theory suggests that plastids originated from a photosynthetic bacterium that was engulfed by a primitive eukaryotic cell. In consequence, the chloroplast genome remains affected by this ancestral event, although it is reduced in size and the number of constituent genes. Most parts of the plastid genome have been transferred to the host cell nuclear genome and are nuclear-encoded. Thus, chloroplast proteins are synthesized in the cytosol as precursors with N-terminal extensions called transit peptides. The evolution of import machinery was required to transfer transit peptides to the stroma. Until the present, two protein complexes have been found to mediate the import process: the Toc (outer) and Tic (inner) envelope membrane translocons. The evolutionary origin of many Tic and Toc proteins has been established, but not for the Tic110 subunit. Tic110 binds signal peptides and serves as a scaffold for the recruitment of stromal components. In this study, we analyzed hydrophobic clusters, protein folds, and protein structure homology and we conclude that Tic110 is composed of fourteen repeated motifs related to HEAT-repeats. The explanation for the presence of such repeats in Tic110 is that membrane arrangement is found in separate domains and their probable function in the chloroplast import process is discussed.     

Tic22 is targeted to the intermembrane space of chloroplasts by a novel pathway.

Tic22 previously was identified as a component of the general import machinery that functions in the import of nuclear-encoded proteins into the chloroplast. Tic22 is peripherally associated with the outer face of the inner chloroplast envelope membrane, making it the first known resident of the intermembrane space of the envelope. We have investigated the import of Tic22 into isolated chloroplasts to define the requirements for targeting of proteins to the intermembrane space. Tic22 is nuclear-endoded and synthesized as a preprotein with a 50-amino acid N-terminal presequence. The analysis of deletion mutants and chimerical proteins indicates that the precursor of Tic22 (preTic22) presequence is necessary and sufficient for targeting to the intermembrane space. Import of preTic22 was stimulated by ATP and required the presence of protease-sensitive components on the chloroplast surface. PreTic22 import was not competed by an excess of an authentic stromal preprotein, indicating that targeting to the intermembrane space does not involve the general import pathway utilized by stromal preproteins. On the basis of these observations, we conclude that preTic22 is targeted to the intermembrane space of chloroplasts by a novel import pathway that is distinct from known pathways that target proteins to other chloroplast subcompartments.     

Characterization of Tic110, a channel-forming protein at the inner envelope membrane of chloroplasts, unveils a response to Ca(2+) and a stromal regulatory disulfide bridge

Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92-959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca(2+) has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin.