The Omp85-related chloroplast outer envelope protein OEP80 is essential for viability in Arabidopsis

beta-Barrel proteins of the Omp85 (Outer membrane protein, 85 kD) superfamily exist in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Prominent Omp85 proteins in bacteria and mitochondria mediate biogenesis of other beta-barrel proteins and are indispensable for viability. In Arabidopsis (Arabidopsis thaliana) chloroplasts, there are two distinct types of Omp85-related protein: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD) and OEP80 (Outer Envelope Protein, 80 kD). Toc75 functions as a preprotein translocation channel during chloroplast import, but the role of OEP80 remains elusive. We characterized three T-DNA mutants of the Arabidopsis OEP80 (AtOEP80) gene. Selectable markers associated with the oep80-1 and oep80-2 insertions segregated abnormally, suggesting embryo lethality of the homozygous genotypes. Indeed, no homozygotes were identified among >100 individuals, and heterozygotes of both mutants produced approximately 25% aborted seeds upon self-pollination. Embryo arrest occurred at a relatively late stage (globular embryo proper) as revealed by analysis using Nomarski optics microscopy. This is substantially later than arrest caused by loss of the principal Toc75 isoform, atToc75-III (two-cell stage), suggesting a more specialized role for AtOEP80. Surprisingly, the oep80-3 T-DNA (located in exon 1 between the first and second ATG codons of the open reading frame) did not cause any detectable developmental defects or affect the size of the AtOEP80 protein in chloroplasts. This indicates that the N-terminal region of AtOEP80 is not essential for the targeting, biogenesis, or functionality of the protein, in contrast with atToc75-III, which requires a bipartite targeting sequence.     

OEP61 is a chaperone receptor at the plastid outer envelope

Chloroplast precursor proteins encoded in the nucleus depend on their targeting sequences for delivery to chloroplasts. There exist different routes to the chloroplast outer envelope, but a common theme is the involvement of molecular chaperones. Hsp90 (heat-shock protein 90) delivers precursors via its receptor Toc64, which transfers precursors to the core translocase in the outer envelope. In the present paper, we identify an uncharacterized protein in Arabidopsis thaliana OEP61 which shares common features with Toc64, and potentially provides an alternative route to the chloroplasts. Sequence analysis indicates that OEP61 possesses a clamp-type TPR (tetratricopeptide repeat) domain capable of binding molecular chaperones, and a C-terminal TMD (transmembrane domain). Phylogenetic comparisons show sequence similarities between the TPR domain of OEP61 and those of the Toc64 family. Expression of mRNA and protein was detected in all plant tissues, and localization at the chloroplast outer envelope was demonstrated by a combination of microscopy and in vitro import assays. Binding assays show that OEP61 interacts specifically with Hsp70 (heat-shock protein 70) via its TPR clamp domain. Furthermore, OEP61 selectively recognizes chloroplast precursors via their targeting sequences, and a soluble form of OEP61 inhibits chloroplast targeting. We therefore propose that OEP61 is a novel chaperone receptor at the chloroplast outer envelope, mediating Hsp70-dependent protein targeting to chloroplasts.     

A high-conductance solute channel in the chloroplastic outer envelope from Pea.

The pea chloroplastic outer envelope protein OEP24 can function as a general solute channel. OEP24 is present in chloroplasts, etioplasts, and non-green root plastids. The heterologously expressed protein forms a voltage-dependent, high-conductance (Lambda = 1.3 nS in 1 M KCl), and slightly cation-selective ion channel in reconstituted proteoliposomes. The highest open probability (P open approximately 0. 8) is at 0 mV, which is consistent with the absence of a transmembrane potential across the chloroplastic outer envelope. The OEP24 channels allow the flux of triosephosphate, dicarboxylic acids, positively or negatively charged amino acids, sugars, ATP, and Pi. Structure prediction algorithms and circular dichroism spectra indicate that OEP24 contains seven amphiphilic beta strands. The primary structure of OEP24 shows no homologies to mitochondrial or bacterial porins on a primary sequence basis, and OEP24 is functionally not inhibited by cadaverine, which is a potent inhibitor of bacterial porins. We conclude that OEP24 represents a new type of solute channel in the plastidic outer envelope.     

Mechanism of protein import across the chloroplast envelope

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.     

Protein- and energy-mediated targeting of chloroplast outer envelope membrane proteins

While the import of nuclear-encoded chloroplast proteins is relatively well studied, the targeting of proteins to the outer membrane of the chloroplast envelope is not. The insertion of most outer membrane proteins (OMP) is generally considered to occur without the utilization of energy or proteinaceous components. Recently, however, proteins have been shown to be involved in the integration of outer envelope protein 14 (OEP14), whose outer membrane insertion was previously thought to be spontaneous. Here we investigate the insertion of two proteins from Physcomitrella patens, PpOEP64-1 and PpOEP64-2 (formerly known as PpToc64-1 and PpToc64-2), into the outer membrane of chloroplasts. The association of PpOEP64-1 with chloroplasts was not affected by chloroplast pre-treatments. Its insertion into the membrane was affected, however, demonstrating the importance of measuring insertion specifically in these types of assays. We found that the insertion of PpOEP64-1, PpOEP64-2 and two other OMPs, OEP14 and digalactosyldiacylglycerol synthase 1 (DGD1), was reduced by either nucleotide depletion or proteolysis of the chloroplasts. Integration was also inhibited in the presence of an excess of an imported precursor protein. In addition, OEP14 competed with the insertion of the OEP64s and DGD1. These data demonstrate that the targeting of several OMPs involves proteins present in chloroplasts and requires nucleotides. Together with previous reports, our data suggest that OMPs in general do not insert spontaneously.     

植物细胞中蛋白质向叶绿体转运的研究进展

植物细胞中叶绿体的功能主要依赖于叶绿体蛋白, 大部分叶绿体蛋白由核基因组编码, 在细胞质中合成并经过正确的分选后, 通过叶绿体外膜上的Toc复合体和/或内膜上的Tic复合体转运到叶绿体的不同部位。该文主要综述可能参与叶绿体蛋白分选的胞质因子以及Toc和Tic组分如何参与叶绿体蛋白转运的研究进展。     

Identification of the pore-forming region of the outer chloroplast envelope protein OEP16

The chloroplast outer envelope protein OEP16 forms a cation-selective high conductance channel with permeability to amines and amino acids. The region of OEP16 directly involved in channel formation has been identified by electrophysiological analysis of a selection of reconstituted OEP16 mutants. Because analysis of these mutants depended on the use of recombinant protein, we evaluated the electrophysiological properties of OEP16 isolated directly from pea chloroplasts and of the recombinant protein produced in Escherichia coli. The results show that the basic properties like conductance, selectivity, and open probability of the channel formed by native pea OEP16 are comparable with the channel activity formed by the recombinant source of the protein. Following electrophysiological analysis of OEP16 mutants we found that point mutations and insertion of additional amino acid residues in the region of the putative helix 1 (Glu(73) to Val(91)) did not change the properties of the OEP16 channel. The only exception was a Cys(71)-->Ser mutation, which led to a loss of the CuCl(2) sensitivity of the channel. Analysis of N- and C-terminal deletion mutants of OEP16 and mutants containing defined shuffled domains indicated that the minimal continuous region of OEP16, which is able to form a channel in liposomes, lies in the first half of the protein between amino acid residues 21 and 93.     

OEP80, an essential protein paralogous to the chloroplast protein translocation channel Toc75, exists as a 70-kD protein in the Arabidopsis thaliana chloroplast outer envelope

Toc75 and OEP80 are paralogous proteins found in the Viridiplantae lineages, and appear to have evolved from a protein in the outer membrane of an ancient cyanobacterium. Toc75 is known to act as a protein translocation channel at the outer membrane of the chloroplast envelope, whereas the exact function of OEP80 is not understood. In Arabidopsis thaliana, each protein is encoded by a single gene, and both are essential for plant viability from embryonic stages onward. Sequence annotation and immunoblotting data with an antibody against its internal sequence (αOEP80(325-337)) indicated that the molecular weight of OEP80 is ca. 80?kD. Here we present multiple data to show that the size of A. thaliana OEP80 is smaller than previously estimated. First, we prepared the antibody against a recombinant protein consisting of annotated full-length A. thaliana OEP80 with an N-terminal hexahistidine tag (αOEP80(1-732)). This antibody recognized a 70-kD protein in the A. thaliana chloroplast membrane fraction which migrated faster than the His-tagged antigen and the protein recognized by the αOEP80(325-337) antibody on SDS-PAGE. Immunoprecipitation followed by LC-MS/MS analysis confirmed that the 70-kD protein was encoded by the OEP80 cDNA. Next, we performed a genetic complementation assay using embryo-lethal oep80-null plants and constructs encoding OEP80 and its variants. The results revealed that the nucleotide sequence encoding the 52 N-terminal amino acids was not required for functional expression of OEP80 and accumulation of the 70-kD protein. The data also indicated that an additional C-terminal T7 tag remained intact without disrupting the functionality of OEP80, and was not exposed to the cytoplasmic surface of the chloroplast envelope. Finally, OEP80-T7 and Toc75 showed distinct migration patterns on blue native-PAGE. This study provides molecular tools to investigate the function of OEP80, and also calls for caution in using an anti-peptide antibody.     

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.     

AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis

In plant cells, chloroplasts have essential roles in many biochemical reactions and physiological responses. Chloroplasts require numerous protein components, but only a fraction of these proteins are encoded by the chloroplast genome. Instead, most are encoded by the nuclear genome and imported into chloroplasts from the cytoplasm post-translationally. Membrane proteins located in the chloroplast outer envelope membrane (OEM) have a critical function in the import of proteins into the chloroplast. However, the biogenesis of chloroplast OEM proteins remains poorly understood. Here, we report that an Arabidopsis ankyrin repeat protein, AKR2A, plays an essential role in the biogenesis of the chloroplast OEM proteins. AKR2A binds to chloroplast OEM protein targeting signals, as well as to chloroplasts. It also displays chaperone activity towards chloroplast OEM proteins, and facilitates the targeting of OEP7 to chloroplasts in vitro. AKR2A RNAi in plants with an akr2b knockout background showed greatly reduced levels of chloroplast proteins, including OEM proteins, and chloroplast biogenesis was also defective. Thus, AKR2A functions as a cytosolic mediator for sorting and targeting of nascent chloroplast OEM proteins to the chloroplast.     

Import pathways of chloroplast interior proteins and the outer-membrane protein OEP14 converge at Toc75

Most chloroplast outer-membrane proteins are synthesized at their mature size without cleavable targeting signals. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. It has therefore been assumed that insertion of outer-membrane proteins proceeds through a different pathway from import into the interior of chloroplasts, which requires a thermolysin-sensitive translocon complex and ATP. Here, we show that a model outer-membrane protein, OEP14, competed with the import of a chloroplast interior protein, indicating that the two import pathways partially overlapped. Cross-linking studies showed that, during insertion, OEP14 was associated with Toc75, a thermolysin-resistant component of the outer-membrane protein-conducting channel that mediates the import of interior-targeted precursor proteins. Whereas almost no OEP14 inserted into protein-free liposomes, OEP14 inserted into proteoliposomes containing reconstituted Toc75 with a high efficiency. Taken together, our data indicate that Toc75 mediates OEP14 insertion, and therefore plays a dual role in the targeting of proteins to the outer envelope membrane and interior of chloroplasts.     

Protein import into chloroplasts: new aspects of a well-known topic

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.     

Lipid composition of outer leaflet of chloroplast outer envelope determines topology of OEP7.

OEP7, a 6.7-kDa outer envelope protein of spinach chloroplasts inserts into the outer envelope of the organelle independent of a classical cleavable targeting signal. The insertion of OEP7 was studied to describe the determinants for association with, integration into, and orientation of the protein in the outer envelope of chloroplasts. The insertion of OEP7 into the membrane is independent of outer membrane channel proteins and can be reconstituted with the use of protein-free liposomes. In situ, the binding of OEP7 to the membrane surface is not driven by electrostatic interaction because reduction of phosphatidylglycerol or phosphatidylinositol did not reduce the association with the liposomes. The positively charged amino acids flanking the transmembrane domain at the C terminus are essential to retain the native N(in)-C(out) orientation during insertion into chloroplasts. OEP7 inserts with reversed orientation into liposomes containing the average lipid composition of the outer envelopes. The native like N(in)-C(out) orientation is achieved by reduction of the phoshpatidylglycerol concentration mimicking the composition of the outer leaflet of the outer envelope of chloroplasts. We conclude that the unique lipid composition of the outer leaflet due to lipid asymmetry of the outer envelope is essential for the correct topology of OEP7.     

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.     

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     

Calcium regulation of chloroplast protein import

The majority of chloroplast proteins is nuclear-encoded and therefore synthesized on cytosolic ribosomes. In order to enter the chloroplast, these proteins have to cross the double-membrane surrounding the organelle. This is achieved by means of two hetero-oligomeric protein complexes in the outer and inner envelope, the Toc and Tic translocon. The process of chloroplast import is highly regulated on both sides of the envelope membranes. Our studies indicate the existence of an undescribed mode of control for this process so far, at the same time providing further evidence that the chloroplast is integrated into the calcium-signalling network of the cell. In pea chloroplasts, the calmodulin inhibitor Ophiobolin A as well as the calcium ionophores A23187 and Ionomycin affect the translocation of those chloroplast proteins that are imported with an N-terminal cleavable presequence. Import of these proteins is inhibited in a concentration-dependent manner. Addition of external calmodulin or calcium can counter the effect of these inhibitors. Translocation of chloroplast proteins that do not possess a cleavable transit peptide, that is outer envelope proteins or the inner envelope protein Tic32, is not affected. These results suggest that the import of a certain subset of chloroplast proteins is regulated by calcium. Our studies furthermore indicate that this regulation occurs downstream of the Toc translocon either within the intermembrane space or at the inner envelope translocon. A potential promoter of the calcium regulation is calmodulin, a protein well known as part of the plant's calcium signalling system.     

A rectifying ATP-regulated solute channel in the chloroplastic outer envelope from pea.

Phosphorylated carbohydrates are the main photoassimilated export products from chloroplasts that support the energy household and metabolism of the plant cell. Channels formed by the chloroplastic outer envelope protein OEP21 selectively facilitate the translocation of triosephosphate, 3-phosphoglycerate and phosphate, central intermediates in the source-sink relationship between the chloroplast and the cytosol. The anion selectivity and asymmetric transport properties of OEP21 are modulated by the ratio between ATP and triosephosphates, 3-phosphoglycerate and phosphate in the intermembrane space. Conditions that lead to export of triosephosphate from chloroplasts, i.e. photosynthesis, result in outward-rectifying OEP21 channels, while a high ATP to triosephosphate ratio, e.g. dark metabolism, leads to inward-rectifying OEP21 channels with a less pronounced anion selectivity. We conclude that solute exchange between plastids and cytosol can already be regulated at the level of the organellar outer membrane.     

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.