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1.
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.  相似文献   

2.
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.  相似文献   

3.
Protein import into plant chloroplasts is a fascinating topic that is being investigated by many research groups. Since the majority of chloroplast proteins are synthesised as precursor proteins in the cytosol, they have to be posttranslationally imported into the organelle. For this purpose, most preproteins are synthesised with an N-terminal presequence, which is both necessary and sufficient for organelle recognition and translocation initiation. The import of preproteins is facilitated by two translocation machineries in the outer and inner envelope of chloroplasts, the Toc and Tic complexes, respectively. Translocation of precursor proteins across the envelope membrane has to be highly regulated to react to the metabolic requirements of the organelle. The aim of this review is to summarise the events that take place at the translocation machineries that are known so far. In addition, we focus in particular on alternative import pathways and the aspect of regulation of protein transport at the outer and inner envelope membrane.  相似文献   

4.
The import of nucleus-encoded preproteins into plastids requires the coordinated activities of membrane protein complexes that facilitate the translocation of polypeptides across the envelope double membrane. Tic20 was identified previously as a component of the import machinery of the inner envelope membrane by covalent cross-linking studies with trapped preprotein import intermediates. To investigate the role of Tic20 in preprotein import, we altered the expression of the Arabidopsis Tic20 ortholog (atTic20) by antisense expression. Several antisense lines exhibited pronounced chloroplast defects exemplified by pale leaves, reduced accumulation of plastid proteins, and significant growth defects. The severity of the phenotypes correlated directly with the reduction in levels of atTic20 expression. In vitro import studies with plastids isolated from control and antisense plants indicated that the antisense plastids are defective specifically in protein translocation across the inner envelope membrane. These data suggest that Tic20 functions as a component of the protein-conducting channel at the inner envelope membrane.  相似文献   

5.
Protein import into cyanelles and complex chloroplasts   总被引:5,自引:0,他引:5  
Higher-plant, green and red algal chloroplasts are surrounded by a double membrane envelope. The glaucocystophyte plastid (cyanelle) has retained a prokaryotic cell wall between the two envelope membranes. The complex chloroplasts of Euglena and dinoflagellates are surrounded by three membranes while the complex chloroplasts of chlorarachniophytes, cryptomonads, brown algae, diatoms and other chromophytes, are surrounded by 4 membranes. The peptidoglycan layer of the cyanelle envelope and the additional membranes of complex chloroplasts provide barriers to chloroplast protein import not present in the simpler double membrane chloroplast envelope. Analysis of presequence structure and in vitro import experiments indicate that proteins are imported directly from the cytoplasm across the two envelope membranes and peptidoglycan layer into cyanelles. Protein import into complex chloroplasts is however fundamentally different. Analysis of presequence structure and in vitro import into microsomal membranes has shown that translocation into the ER is the first step for protein import into complex chloroplasts enclosed by three or four membranes. In vivo pulse chase experiments and immunoelectronmicroscopy have shown that in Euglena, proteins are transported from the ER to the Golgi apparatus prior to import across the three chloroplast membranes. Ultrastructural studies and the presence of ribosomes on the outermost of the four envelope membranes suggests protein import into 4 membrane-bounded complex chloroplasts is directly from the ER like outermost membrane into the chloroplast. The fundamental difference in import mechanisms, post-translational direct chloroplast import or co-translational translocation into the ER prior to chloroplast import, appears to reflect the evolutionary origin of the different chloroplast types. Chloroplasts with a two-membrane envelope are thought to have evolved through the primary endosymbiotic association between a eukaryotic host and a photosynthetic prokaryote while complex chloroplasts are believed to have evolved through a secondary endosymbiotic association between a heterotrophic or possibly phototrophic eukaryotic host and a photosynthetic eukaryote.  相似文献   

6.
Lee YJ  Kim DH  Kim YW  Hwang I 《The Plant cell》2001,13(10):2175-2190
Certain small outer envelope membrane proteins of chloroplasts are encoded by the nuclear genome without a cleavable N-terminal transit peptide. We investigated in vivo the targeting mechanism of AtOEP7, an Arabidopsis homolog of the small outer envelope membrane protein. AtOEP7 was expressed as a fusion protein with the green fluorescent protein (GFP) either transiently in protoplasts or stably in transgenic plants. In either case, fluorescence microscopy of transformed cells and protein gel blot analysis of fractionated proteins confirmed that the AtOEP7:GFP fusion protein was targeted to the chloroplast outer envelope membrane. In vivo targeting experiments revealed that two regions, the transmembrane domain (TMD) and its C-terminal neighboring seven-amino acid region, were necessary and sufficient for targeting to the chloroplast outer membrane. Substitution of aspartic acid or lysine residues with glycine residues or scrambling of the amino acid sequence of the seven-amino acid region caused mistargeting to the plasma membrane. Although the amino acid sequence of the TMD is not important for targeting, amino acid residues with large side chains inhibited targeting to the chloroplasts and resulted in the formation of large aggregates in the protoplasts. In addition, introduction of a proline residue within the TMD resulted in inhibition of targeting. Finally, a fusion protein, AtOEP7:NLS:GFP, was targeted efficiently to the chloroplast envelope membranes despite the presence of a nuclear localization signal. On the basis of these results, we conclude that the seven-amino acid region and the TMD are determinants for targeting to the chloroplast outer envelope membrane. The seven-amino acid region plays a critical role in AtOEP7 evading the endomembrane system and entering the chloroplast pathway, and the TMD plays critical roles in migration to the chloroplasts and/or subsequent insertion into the membrane.  相似文献   

7.
Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved in transit peptide recognition. The cytosolic region of Toc34 reveals 34% alpha-helical and 26% beta-strand structure and is stabilized by intramolecular electrostatic interaction. Toc34 binds both chloroplast preproteins and isolated transit peptides in a guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that the soluble, cytosolic domain of Toc34 (Toc34deltaTM) functions as receptor in vitro and is capable to compete with the import of the preprotein of the small subunit (preSSU) of ribulose-1,5-bisphosphate carboxylase-oxygenase into chloroplasts in a GTP-dependent manner. We have developed a biosensor assay to study the interaction of Toc34deltaTM with purified preproteins and transit peptides. The results are compared with the interactions of both a full-size preprotein and the transit peptide of preSSU with the translocon of the outer envelope of chloroplasts (Toc complex) in situ. Several mutants of the transit peptide of preSSU were evaluated to identify amino acid segments that are specifically recognized by Toc34. We present a model of how Toc34 may recognize the transit peptide and discuss how this interaction may facilitate interaction and translocation of preproteins via the Toc complex in vivo.  相似文献   

8.
The chloroplast division machinery is composed of numerous proteins that assemble as a large complex to divide double‐membraned chloroplasts through binary fission. A key mediator of division‐complex formation is ARC6, a chloroplast inner envelope protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. ARC6 connects stromal and cytosolic contractile rings across the two membranes through interaction with an outer envelope protein within the intermembrane space (IMS). The ARC6 IMS region bears a structurally uncharacterized domain of unknown function, DUF4101, that is highly conserved among ARC6 and Ftn2 proteins. Here we report the crystal structure of this domain from Arabidopsis thaliana ARC6. The domain forms an α/β barrel open towards the outer envelope membrane but closed towards the inner envelope membrane. These findings provide new clues into how ARC6 and its homologs contribute to chloroplast and cyanobacterial cell division.  相似文献   

9.
The chloroplast membranes are highly regulated and biological active regions of the living plant cell, which carry numerous essential proteinaceous components. For example, in the thylakoid membrane the photosynthesis apparatus, one of the most life-relevant biological machineries, is located. How these membrane proteins are targeted to and inserted into their target membranes was one of the questions we aimed to understand in the last few years. Fifteen years ago little to nothing was known about the targeting and translocation of outer envelope proteins (G.W. Schmidt and L.M. Mishkind, Annu. Rev. Biochem. 55 (1986)). Although several protein assisted pathways for translocation of proteins across the membranes have been characterised, only recent results gave insight into how membrane proteins are inserted into the chloroplast membranes. Here we will focus on the mode of insertion of a class of proteins into the outer envelope and the thylakoid membranes, which share a unique feature: they insert apparently directly into the lipid bilayer, i.e. without the help of a proteinaceous translocation pore.  相似文献   

10.
The biogenesis of chloroplasts requires the coordinated interplay with the nucleus and the cytoplasm. The majority of chloroplast proteins are encoded by the nuclear genome and must be faithfully and efficiently delivered to the organelle upon completion of translation in the cytosol. This high-fidelity targeting is accomplished by specific chloroplast targeting signal peptides. Several cytoplasmic factors recognise, modify, and bind this targeting sequence, and deliver the preproteins to the chloroplast translocation machinery. The multisubunit translocation complex at the outer envelope contains receptor proteins, a translocation channel, and accessory subunits. Complete import into the stroma utilizes both outer and inner envelope translocons and molecular chaperones in the intermembrane space and in the stroma. The entire import process appears to be regulated by phosphorylation, nucleotide binding, and hydrolysis. Recent evidence indicates that several subunits of the chloroplast import machinery may have evolved from cyanobacterial ancestors.  相似文献   

11.
Plastids are a diverse group of plant organelles that perform essential functions including important steps in many biosynthetic pathways. Chloroplasts are the best characterized type of plastid, and constitute the site of oxygenic photosynthesis in plants, a process essential to all higher life forms. It is well established that the majority (>90%) of chloroplast proteins are nucleus-encoded and must be post-translationally imported into these envelope-bound compartments. Most nucleus-encoded chloroplast proteins are translated in precursor form on cytosolic ribosomes, targeted to the chloroplast surface, and then imported across the double-membrane envelope by translocons in the outer and inner envelope membranes of the chloroplast, termed TOC and TIC, respectively. Recently, significant progress has been made in our understanding of how proteins are targeted to the chloroplast surface and translocated across the chloroplast envelope into the stroma. Evidence suggesting the existence of multiple import pathways at the outer envelope membrane for different classes of precursor proteins has been presented. These pathways appear to utilize similar TOC complexes equipped with different combinations of homologous GTPase receptors, providing preprotein recognition specificity.  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
Chloroplasts have evolved an elaborate system of membrane and soluble subcompartments to organize and regulate photosynthesis and essential aspects of amino acid and lipid metabolism. The biogenesis and maintenance of organellar architecture rely on protein subunits encoded by both nuclear and plastid genomes. Import of nuclear-encoded proteins is mediated by interactions between the intrinsic N-terminal transit sequence of the nuclear-encoded preprotein and a common import machinery at the chloroplast envelope. Recent investigations have shown that there are two unique membrane-bound translocation systems, in the outer and inner envelope membranes, which physically associate during import to transport preproteins from the cytoplasm to the internal stromal compartment. This review discusses current understanding of these translocation systems and models for the way in which they might function.  相似文献   

16.
A Caliebe  R Grimm  G Kaiser  J Lübeck  J Soll    L Heins 《The EMBO journal》1997,16(24):7342-7350
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.  相似文献   

17.
Protein import into chloroplasts   总被引:2,自引:0,他引:2  
Most chloroplastic proteins are encoded in the nucleus, synthesized on cytosolic ribosomes and subsequently imported into the organelle. In general, proteins destined for the chloroplast are synthesized as precursor proteins with a cleavable N-terminal presequence that mediates routing to the inside of the chloroplast. These precursor proteins have to be targeted to the correct organellar membrane surface after their release from the ribosome and furthermore they have to be maintained in a conformation suitable for translocation across the two envelope membranes. Recognition and import of most chloroplastic precursor proteins are accomplished by a jointly used translocation apparatus. Different but complementary studies of several groups converged recently in the identification of the outer envelope proteins OEP86, OEP75, OEP70 (a Hsp 70-related protein), OEP34, and of the inner envelope protein IEP110 as components of this translocation machinery. None of these proteins, except for OEP70, shows any homology to components of other protein translocases. The plastid import machinery thus seems to be an original development in evolution. Following translocation into the organelle, chloroplastic proteins are sorted to their suborganellar destination, i.e., the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen. This structural and evolutionary complexity of chloroplasts is reflected by a variety of routing mechanisms by which proteins reach their final location once inside the organelle. This review will focus on recent advances in the identification of components of the chloroplastic protein import machinery, and new insights into the pathways of inter-and intraorganellar sorting.  相似文献   

18.
Proteins in the chloroplast outer envelope membrane are nuclear encoded and post-translationally targeted to the chloroplast. The targeting and membrane insertion of these proteins is not well understood. Although early work suggested otherwise, the best-studied outer membrane proteins (OMPs) use both proteins within the chloroplast and NTPs for insertion. There have been conflicting reports in the field regarding protein targeting and insertion, which have probably arisen because of differences in experimental methodology and different interpretations of reduction (versus abolition) of integration. This review summarizes what is known to date about the mechanism of chloroplast OMP targeting.  相似文献   

19.
Energy dependence of protein translocation into chloroplasts   总被引:25,自引:0,他引:25  
The translocation of in vitro synthesized precursor proteins into intact spinach chloroplasts was investigated with respect to its energy requirement. It was demonstrated that MgATP itself, and not a transmembrane electrochemical gradient across the envelope membrane, promotes protein import. By manipulating the external and the stromal level of MgATP, we provided evidence that MgATP energized the protein import not within the chloroplast but at the outside of the envelope membrane. It is postulated that an MgATP-dependent phosphorylation/dephosphorylation cycle at the outer membrane face was involved in the course of protein translocation into the chloroplast.  相似文献   

20.
Chen K  Chen X  Schnell DJ 《Plant physiology》2000,122(3):813-822
Two integral outer envelope GTPases, Toc34 and Toc86, are proposed to regulate the recognition and translocation of nuclear-encoded preproteins during the early stages of protein import into chloroplasts. Defining the precise roles of Toc86 and Toc34 has been complicated by the inability to distinguish their GTPase activities. Furthermore, the assignment of Toc86 function is rendered equivocal by recent reports suggesting that the standard protocol for the isolation of chloroplasts results in significant proteolysis of Toc86 (B. Bolter, T. May, J. Soll [1998] FEBS Lett 441: 59-62; G. Schatz [1998] Nature 395: 439-440). We demonstrate that Toc86 corresponds to a native protein of 159 kD in pea (Pisum sativum), designated Toc159. We take advantage of the proteolytic sensitivity of Toc159 to selectively remove its 100-kD cytoplasmic GTPase domain and thereby distinguish its activities from other import components. Proteolysis eliminates detectable binding of preproteins at the chloroplast surface, which is consistent with the proposed role of Toc159 as a receptor component. Remarkably, preprotein translocation across the outer membrane can occur in the absence of the Toc159 cytoplasmic domain, suggesting that binding can be bypassed. Translocation remains sensitive to GTP analogs in the absence of the Toc159 GTP-binding domain, providing evidence that Toc34 plays a key role in the regulation of translocation by GTP.  相似文献   

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