Analysis of the Interactions of Preproteins with the Import Machinery over the Course of Protein Import into Chloroplasts

We have investigated the interactions of two nuclear-encoded preproteins with the chloroplast protein import machinery at three stages in import using a label-transfer crosslinking approach. During energy-independent binding at the outer envelope membrane, preproteins interact with three known components of the outer membrane translocon complex, Toc34, Toc75, and Toc86. Although Toc75 and Toc86 are known to associate with preproteins during import, a role for Toc34 in preprotein binding previously had not been observed. The interaction of Toc34 with preproteins is regulated by the binding, but not hydrolysis of GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the function of GTP as a regulator of preprotein recognition. Toc75 and Toc86 are the major targets of cross-linking upon insertion of preproteins across the outer envelope membrane, supporting the proposal that both proteins function in translocation at the outer membrane as well as preprotein recognition. The inner membrane proteins, Tic(21) and Tic22, and a previously unidentified protein of 14 kD are the major targets of crosslinking during the late stages in import. These data provide additional support for the roles of these components during protein translocation across the inner membrane. Our results suggest a defined sequence of molecular interactions that result in the transport of nuclear-encoded preproteins from the cytoplasm into the stroma of chloroplasts.     

Toc, Tic, and chloroplast protein import.

The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.     

Toc,tic, and chloroplast protein import

The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.     

The transit sequence of ferredoxin contains different domains for translocation across the outer and inner membrane of the chloroplast envelope

Deletion mutants in the transit sequence of preferredoxin were used in label transfer cross-linking assays to map the interactions of the transit sequence with the import machinery. The deletion mutants gave distinct cross-linking patterns to the Toc and Tic components of the import machinery, consistent with the binding and import properties obtained in in vitro import assays. The cross-linking results revealed two separate properties of the transit peptide: first the presentation of specific binding domains for the initial interaction with outer membrane components, and second the presence of different domains for interaction with the outer and inner membrane components of the transport machinery for full envelope translocation. The N-terminal Delta6-14 deletion blocked import of the precursor at the Toc components, whereas the more internal deletion Delta15-25 blocked import at the Tic components. The information for association with the outer and inner membrane components therefore resides in two separate but partly overlapping domains in the first 25 amino acids of the transit sequence.     

Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts

Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.     

The chloroplastic protein import machinery contains a Rieske-type iron-sulfur cluster and a mononuclear iron-binding protein.

Transport of precursor proteins across the chloroplastic envelope membranes requires the interaction of protein translocons localized in both the outer and inner envelope membranes. Analysis by blue native gel electrophoresis revealed that the translocon of the inner envelope membranes consisted of at least six proteins with molecular weights of 36, 45, 52, 60, 100 and 110 kDa, respectively. Tic110 and ClpC, identified as components of the protein import apparatus of the inner envelope membrane, were prominent constituents of this complex. The amino acid sequence of the 52 kDa protein, deduced from the cDNA, contains a predicted Rieske-type iron-sulfur cluster and a mononuclear iron-binding site. Diethylpyrocarbonate, a Rieske-type protein-modifying reagent, inhibits the translocation of precursor protein across the inner envelope membrane, whereas binding of the precursor to the outer envelope membrane is still possible. In another independent experimental approach, the 52 kDa protein could be co-purified with a trapped precursor protein in association with the chloroplast protein translocon subunits Toc86, Toc75, Toc34 and Tic110. Together, these results strongly suggest that the 52 kDa protein, named Tic55 due to its calculated molecular weight, is a member of the chloroplastic inner envelope protein translocon.     

Stable association of chloroplastic precursors with protein translocation complexes that contain proteins from both envelope membranes and a stromal Hsp100 molecular chaperone.

Cytoplasmically synthesized precursors interact with translocation components in both the outer and inner envelope membranes during transport into chloroplasts. Using co-immunoprecipitation techniques, with antibodies specific to known translocation components, we identified stable interactions between precursor proteins and their associated membrane translocation components in detergent-solubilized chloroplastic membrane fractions. Antibodies specific to the outer envelope translocation components OEP75 and OEP34, the inner envelope translocation component IEP110 and the stromal Hsp100, ClpC, specifically co-immunoprecipitated precursor proteins under limiting ATP conditions, a stage we have called docking. A portion of these same translocation components was co-immunoprecipitated as a complex, and could also be detected by co-sedimentation through a sucrose density gradient. ClpC was observed only in complexes with those precursors utilizing the general import apparatus, and its interaction with precursor-containing translocation complexes was destabilized by ATP. Finally, ClpC was co-immunoprecipitated with a portion of the translocation components of both outer and inner envelope membranes, even in the absence of added precursors. We discuss possible roles for stromal Hsp100 in protein import and mechanisms of precursor binding in chloroplasts.     

Identification of Protein Transport Complexes in the Chloroplastic Envelope Membranes via Chemical Cross-Linking

Transport of cytoplasmically synthesized proteins into chloroplasts uses an import machinery present in the envelope membranes. To identify the components of this machinery and to begin to examine how these components interact during transport, chemical cross-linking was performed on intact chloroplasts containing precursor proteins trapped at a particular stage of transport by ATP limitation. Large crosslinked complexes were observed using three different reversible homobifunctional cross-linkers. Three outer envelope membrane proteins (OEP86, OEP75, and OEP34) and one inner envelope membrane protein (IEP110), previously reported to be involved in protein import, were identified as components of these complexes. In addition to these membrane proteins, a stromal member of the hsp100 family, ClpC, was also present in the complexes. We propose that ClpC functions as a molecular chaperone, cooperating with other components to accomplish the transport of precursor proteins into chloroplasts. We also propose that each envelope membrane contains distinct translocation complexes and that a portion of these interact to form contact sites even in the absence of precursor proteins.     

New Insights into the Protein Import Machinery of the Chloroplast's Outer Envelope

A large number of plastid localized proteins are post-translationally imported as precursor proteins from the cytosol into the organelle. Recognition and translocation is accomplished by a subset of chloroplast envelope proteins, which were identified by different but complementary methods. The o uter e nvelope p roteins OEP 86, OEP 75, OEP 70 (a heat shock cognate 70 homologue) and OEP 34 are clearly involved in the import event and can be isolated as one functionally active translocation unit. For three of these proteins cDNA clones have been very recently obtained, namely OEP 86, OEP 75 and OEP 34. OEP 86 seems to be a precursor protein receptor which could be regulated by GTP binding and ATP-dependent phosphorylation-dephosphorylation. OEP 75 is part of the translocation pore traversing the membrane in multiple β-sheets. OEP 34 is tightly associated with OEP 75. It represents a new type of GTP-binding protein which possesses endogenous GTPase activity. Multiple GTP binding and hydrolysis cycles as well as protein phosphorylation-dephosphorylation events might, therefore, regulate the interaction of a precursor protein with the translocation machinery of the outer envelope, making it very distinct from the mitochondrial outer membrane system. Further proteins of the inner envelope membrane, namely IEP 97 and IEP 36, have been implied to function in the translocation event. These recent data allow not only identification of the players in the game but also speculation about mechanisms and regulation of translocation.     

A constituent of the chloroplast import complex represents a new type of GTP-binding protein

The 34 kDa polypeptide of the outer envelope membranes from pea chloroplasts (OEP 34) is a major constituent of this membrane. OEP 34 is detected on polyacrylamide gels under non-reducing condition in association with OEP 75, the putative protein translocation pore. An antiserum against OEP 34 is able to co-immunoprecipitate the precursor of Rubisco small subunit from a partially purified import complex of chloroplast outer envelope membranes. A full-length cDNA clone coding for pea OEP 34 has been isolated. Analysis of the deduced amino acid sequence revealed typical and conserved sequence motifs found in GTP-binding proteins, making it a new and unique member of this superfamily. OEP 34 behaves as an integral constituent of the outer chloroplast envelope, which is anchored by its C-terminus into the membrane, while the majority of the protein projects into the cytoplasm. OEP 34 does not possess a cleavable N-terminal transit sequence but it is targeted to the chloroplasts and integrated into the outer membranes by internal sequence information which seems to be present in the C-terminal membrane anchor region. Productive integration of OEP 34 into the outer envelope requires, in contrast to other OEPs, protease-sensitive chloroplast surface components and is stimulated by ATR. The GTP binding specificity of OEP 34 is demonstrated by photo-affinity labelling in the presence of [α-³²P]GTP. Overexpressed and purified OEP 34 possesses endogenous GTPase activity. These results indicate a possible regulatory function of OEP 34 in protein translocation into chloroplasts.     

The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space.

Protein import into chloroplasts is initiated by a binding interaction between a precursor protein and the surface of the outer envelope. The binding step was previously shown to be energy-dependent (Olsen, L. J., Theg, S. M., Selman, B. R., and Keegstra, K. (1989) J. Biol. Chem. 264, 6724-6729). We took advantage of the broad nucleotide specificity of the energy requirement for binding to investigate the site of the nucleoside triphosphate (NTP) requirement. GTP supported precursor binding to chloroplasts. It was not converted to ATP, as determined by direct ATP measurements, and was not transported across the inner envelope. Thus, GTP supported binding from either the intermembrane space or outside the outer membrane. To distinguish between an intermembrane space and an external NTP requirement, we experimentally manipulated the NTP levels inside and outside chloroplasts. Internally generated ATP was able to support binding in the presence of an external membrane-impermeant ATP trap. Therefore, since GTP supported binding from either the intermembrane space or outside the chloroplast, and ATP supported binding from either the intermembrane space or the stroma, we concluded that the site of NTP utilization for precursor binding to chloroplasts was the intermembrane space between the two envelope membranes.     

Transport of proteins into chloroplasts

The import of cytoplasmically synthesized proteins into chloroplasts involves an interaction between at least two components; the precursor protein, and the import apparatus in the chloroplast envelope membrane. This review summarizes the information available about each of these components. Precursor proteins consist of an amino terminal transit peptide attached to a passenger protein. Transit peptides from various precurosrs are diverse with respect to length and amino acid sequence; analysis of their sequences has not revealed insight into their mode of action. A variety of foreign passenger proteins can be imported into chloroplasts when a transit peptide is present at the amino terminus. However, foreign passenger proteins are not imported as efficiently as natural passenger proteins, and some chimeric precursor proteins are not imported into chloroplasts at all. Therefore, the passenger protein, as well as the transit peptide, influences the import process. Import begins by binding of the precursor to the chloroplast surface. It has been suggested that this binding is mediated by a receptor, but evidence to support this hypothesis remains incomplete and a receptor protein has not yet been characterized. Protein translocation requires energy derived from ATP hydrolysis, although there are conflicting reports as to where hydrolysis occurs and it is unclear how this energy is utilized. The mechanism(s) whereby proteins are translocated across either the two envelope membranes or the thylakoid membrane is not known.Abbreviations EPSP 5-enolpyruvyulshikimate-3-phosphate - LHCP Chlorophyll a/b binding protein of the light-harvesting complex - NPT-II Neomycin phosphotransferase II - PC Plastocyanin - Pr Precursor - Rubisco Ribulose-1,5,-bisphosphate carboxylase/oxygenase - SS Small subunit of Rubisco     

Envelope membrane proteins that interact with chloroplastic precursor proteins.

The post-translational transport of cytoplasmically synthesized precursor proteins into chloroplasts requires proteins in the envelope membranes. To identify some of these proteins, label transfer cross-linking was performed using precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase (prSSU) that was blocked at an early stage of the transport process. Two envelope proteins were identified: an 86-kD protein and a 75-kD protein, both present in the outer membrane. Labeling of both proteins required prSSU and could not be accomplished with SSU lacking a transit peptide. Labeling of the 75-kD protein occurred only when low levels of ATP were present, whereas labeling of the 86-kD protein occurred in the absence of exogenous ATP. Although both labeled proteins were identified as proteins of the outer envelope membrane, the labeled form of the 75-kD protein could only be detected in fractions containing mixed envelope membranes. Based on these observations, we propose that prSSU first binds in an ATP-independent fashion to the 86-kD protein. The energy-requiring step is association with the 75-kD protein and assembly of a translocation contact site between the inner and outer membrane of the chloroplastic envelope.     

Immunogold labelling of cryosectioned pea chloroplasts and initial localization of the proteins associated with the protein import machinery

The electron-microscopic technique for immunogold labelling of thawed cryosectioned material (K.T. Tokuyasu, 1989, Histochem J 21: 163–171) has been adapted for use with isolated chloroplasts. Percoll-purified pea (Pisum Sativum L. cv Feltham First) chloroplasts were fixed in a buffered glutaraldehyde solution and then infiltrated with a buffered solution of 10% polyvinylpyrrolidone in 2.07 M sucrose prior to freezing in liquid nitrogen and sectioning in an ultracryomicrotome. Sections were thawed, immunolabelled, and stained with ammonium molybdate in methyl cellulose on Formvar/carbon-coated Cu or Cu/Pd electron-microscope grids. Cryosectioning gave excellent structural preservation and retained antigenicity. The effectiveness of this technique in localizing proteins to their specific chloroplast compartment was assayed using antibodies raised against: (i) the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a stromal protein, (ii) the chloroplast ATP synthase (CF₁), a peripheral thylakoid protein, and (iii) different envelope membrane proteins. Antibodies raised against three members of the chloroplasticouterenvelopeprotein (OEP) import machinery, a 34-kDa protein (OEP34 or IAP34), the channel-forming 75-kDa protein (OEP75 or IAP75), and the 86-kDa precursor protein receptor (OEP86 or IAP86) were tested for their localization. The previous localization of OEP86, OEP75 and OEP34 to the outer envelope by biochemical methods was confirmed by our immuno electronmicroscopic analysis. Additionally, a constituent of the chloroplastic inner envelope protein (IEP) import machinery IEP 110 (IAP 100) was clearly localized to this membrane. Therefore, cryosectioning and immunogold labelling of intact chloroplasts provides a method for studying the localization of chloroplast proteins, especially those residing in the inner and outer envelope membranes.Abbreviations FCS fetal calf serum - IAP import intermediate associated protein - IEP inner envelope protein - OEP outer envelope protein (numbers signifying the relative molecular mass in kilodaltons) - PBS phosphate buffered saline - PVP polyvinyl pyrrolidone - Rubisco ribulose-1,5-biophosphate carboxylase/oxygenase     

A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway.

A chloroplastic outer envelope membrane protein of 75 kDa (OEP75) was identified previously as a component of the protein import machinery. Here we provide additional evidence that OEP75 is a component of protein import, present the isolation of a cDNA clone encoding this protein, briefly describe its developmental expression and tissue specificity, and characterize its insertion into the outer envelope membrane. OEP75 was synthesized as a higher molecular weight precursor (prOEP75) which bound to isolated chloroplasts in an in vitro import assay and subsequently was processed to the mature form (mOEP75). During this import assay, two proteins intermediate in size between prOEP75 and mOEP75 were detected. One of these intermediates was also detected in chloroplast envelopes isolated from young pea leaves. Binding and processing of prOEP75 required ATP and one or more surface-exposed proteinaceous components, and was competed by prSSU, a stromal-targeted protein. We propose that the N-terminus of the prOEP75 transit peptide acts as a stromal-targeting domain and a central, hydrophobic region of this transit peptide acts as a stop-transfer domain. A complex route of insertion and processing of prOEP75 may exist to ensure high fidelity targeting of this import component.     

A functionally active protein import complex from

Isolated outer chloroplast envelope membranes were solubilized by digitonin and separated on linear sucrose density gradients. A membrane complex was recovered from the gradients and exhibited characteristics of a protein import apparatus, i.e. the interaction of the complex with the precursor polypeptides depends on the presence of a transit sequence, ATP and protease-sensitive components. Furthermore, trans-location intermediates detected in the organellar system are also found after interaction of the precursor polypeptide with the isolated import complex.     

A mutant deficient in the plastid lipid DGD is defective in protein import into chloroplasts

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol with N-terminal extensions called transit peptides. Transit peptides function as the import signal to chloroplasts. The import process requires several protein components in the envelope and stroma and also requires the hydrolysis of ATP. Lipids have been implicated in the import process based on theories or experiments with in vitro model systems. We show here that chloroplasts isolated from an Arabidopsis mutant deficient in the plastid lipid digalactosyl diacylglycerol (DGD) were normal in importing a chloroplast outer membrane protein, but were defective in importing precursor proteins targeted to the interior of chloroplasts. The impairment includes the binding, or docking, step of the import process that is supported by 100 μM ATP.     

Insertion of atToc34 into the chloroplastic outer membrane is assisted by at least two proteinaceous components in the import system.

Toc34 is a member of the outer membrane translocon complex that mediates the initial stage of protein import into chloroplasts. Toc34, like most outer membrane proteins, is synthesized in the cytosol at its mature size without a cleavable transit peptide. The majority of outer membrane proteins do not require thermolysin-sensitive components on the chloroplastic surface or ATP for their insertion into the outer membrane. However, different results have been obtained concerning the factors required for Toc34 insertion into the outer membrane. Using an Arabidopsis homologue of pea Toc34, atToc34, we show that the insertion of atToc34 was greatly reduced by thermolysin pretreatment of chloroplasts as assayed either by protease digestion or by alkaline extraction. The insertion was also dependent on the presence of ATP or GTP. A mutant of atToc34 with the GTP-binding domain deleted still required ATP for optimal insertion, indicating that ATP was used by other protein components in the import system. The ATP-supported insertion was observed even in thermolysin-pretreated chloroplasts, suggesting that the protein component responsible for ATP-stimulated insertion is a different protein from the thermolysin-sensitive component that assists atToc34 insertion.     

Cytometric analysis of an epitope-tagged transit peptide bound to the chloroplast translocation apparatus

Chloroplast transit peptides are necessary and sufficient for the targeting and translocation of precursor proteins across the chloroplast envelope. However, the mechanism by which transit peptides engage the translocation apparatus has not been investigated. To analyse this interaction, we have developed a novel epitope-tagged transit peptide derived from the precursor of the small subunit of pea Rubisco. The recombinant transit peptide, His-S-SStp, contains a removable dual-epitope tag, His-S, at its N-terminus that permits both rapid purification via immobilized metal affinity chromatography and detection by blotting, flow cytometry and laser-scanning confocal microscopy. Unlike other chimeric precursors, which place the passenger protein C-terminal to the transit peptide, His-S-SStp bound to the translocation apparatus yet did not translocate across the chloroplast envelope. This early translocation intermediate allowed non-radioactive detection using fluorescent and chemiluminescent reporters. The physiological relevance of this interaction was confirmed by protein import competitions, sensitivity to pre- and post-import thermolysin treatment, photochemical cross-linking and organelle fractionation. The interaction was specific for the transit peptide since His-S alone did not engage the chloroplast translocation apparatus. Quantitation of the bound transit peptide was determined by flow cytometry, showing saturation of binding yet only slight ATP-dependence. The addition of GTP showed inhibition of the binding of His-S-SStp to the chloroplasts indicating an involvement of GTP in the formation of this early translocation intermediate. In addition, direct visualization of His-S-SStp and Toc75 by confocal microscopy revealed a patch-like labeling, suggesting a co-ordinate localization to discrete regions on the chloroplast envelope. These findings represent the first direct visualization of a transit peptide interacting with the chloroplast translocation apparatus. Furthermore, identification of a chloroplast-binding intermediate may provide a novel tool to dissect interactions between a transit peptide and the chloroplast translocation apparatus.     

Interaction of plant mitochondrial and chloroplast signal peptides with the Hsp70 molecular chaperone.

Most mitochondrial and chloroplast proteins are synthesized on cytosolic polyribosomes as precursor proteins, with an N-terminal signal sequence that targets the precursor to the correct organelle. In mitochondria, the chaperone Hsp70 functions as a molecular motor, pulling the precursor across the mitochondrial membranes; 97.0% of plant mitochondrial presequences contain an Hsp70 binding site. In chloroplasts, the outer envelope, intermembrane space and a stromal Hsp70 are thought to participate in protein import; 82.5% of chloroplast transit peptides have an Hsp70 binding site. The interaction of signal peptides with Hsp70 during the import process is supported by biochemical and bioinformatic studies.