Outer Envelope Membrane Protein Research Articles

Pepper mild mottle virus coat protein interacts with pepper chloroplast outer envelope membrane protein OMP24 to inhibit antiviral immunity in plants.

Pepper mild mottle virus (PMMoV) is a devastating viral pathogen of pepper (Capsicum annuum) but it is unclear whether and how peppers protect against PMMoV infection. The expression of the chloroplast outer membrane protein 24 (OMP24) of C. annuum was upregulated under PMMoV infection and it interacted with PMMoV coat protein (CP). Silencing of OMP24 in either C. annuum or Nicotiana benthamiana facilitated PMMoV infection, whereas overexpression of N. benthamiana OMP24 in transgenic plants inhibited PMMoV infection. Both C. annuum OMP24 (CaOMP24) and N. benthamiana OMP24 (NbOMP24) localized to the chloroplast and have a moderately hydrophobic transmembrane domain that is necessary for their localization. Overexpression of CaOMP24 induced stromules, perinuclear chloroplast clustering, and accumulation of reactive oxygen species (ROS), the typical defense responses of chloroplasts transferring the retrograde signaling to the nucleus to regulate resistance genes. The expression of PR1 and PR2 was also upregulated significantly in plants overexpressing OMP24. Self-interaction of OMP24 was demonstrated and was required for OMP24-mediated plant defense. Interaction with PMMoV CP interfered with the self-interaction of OMP24 and impaired OMP24-induced stromules, perinuclear chloroplast clustering and ROS accumulation. The results demonstrate the defense function of OMP24 in pepper during viral infection and suggest a possible mechanism by which PMMoV CP modulates the plant defense to facilitate viral infection.

Ubiquitin-dependent chloroplast-associated protein degradation in plants.

Chloroplasts contain thousands of nucleus-encoded proteins that are imported from the cytosol by translocases in the chloroplast envelope membranes. Proteolytic regulation of the translocases is critically important, but little is known about the underlying mechanisms. We applied forward genetics and proteomics in Arabidopsis to identify factors required for chloroplast outer envelope membrane (OEM) protein degradation. We identified SP2, an Omp85-type β-barrel channel of the OEM, and CDC48, a cytosolic AAA+ (ATPase associated with diverse cellular activities) chaperone. Both proteins acted in the same pathway as the ubiquitin E3 ligase SP1, which regulates OEM translocase components. SP2 and CDC48 cooperated to bring about retrotranslocation of ubiquitinated substrates from the OEM (fulfilling conductance and motor functions, respectively), enabling degradation of the substrates by the 26S proteasome in the cytosol. Such chloroplast-associated protein degradation (CHLORAD) is vital for organellar functions and plant development.

Biogenesis of chloroplast outer envelope membrane proteins.

Most organisms on Earth use glucose, a photosynthetic product, as energy source. The chloroplast, the home of photosynthesis, is the most representative and characteristic organelle in plants and is enclosed by the outer envelope and inner envelope membranes. The chloroplast biogenesis and unique functions are very closely associated with proteins in the two envelope membranes of the chloroplast. Especially, the chloroplast outer envelope membrane proteins have important roles in signal transduction, protein import, lipid biosynthesis and remodeling, exchange of ions and numerous metabolites, plastid division, movement, and host defense. Therefore, biogenesis of these membrane proteins of chloroplast outer envelope membrane is very important for biogenesis of the entire chloroplast proteome as well as plant development. Most proteins among the outer envelope membrane proteins are encoded by the nuclear genome and are post-translationally targeted to the chloroplast outer envelope membrane. In this process, cytoplasmic receptor and import machineries are required for efficient and correct targeting of these membrane proteins. In this review, we have summarized recent advances on the sorting, targeting, and insertion mechanisms of the outer envelope membrane proteins of chloroplasts and also provide future direction of the study on these topics.

Structural insights into the coordination of plastid division by the ARC6-PDV2 complex.

Chloroplasts divide by binary fission, which is accomplished by the simultaneous constriction of the FtsZ ring on the stromal side of the inner envelope membrane, and the ARC5 ring on the cytosolic side of the outer envelope membrane. The two rings are connected and coordinated mainly by the interaction between the inner envelope membrane protein ARC6 and the outer envelope membrane protein PDV2 in the intermembrane space. The underlying mechanism of this coordination is unclear to date. Here, we solved the crystal structure of the intermembrane space region of the ARC6-PDV2 complex. The results indicated that PDV2 inserts its carboxy terminus into a pocket formed in ARC6, and this interaction further induces the dimerization of the intermembrane space regions of two ARC6 molecules. A pdv2 mutant attenuating PDV2-induced ARC6 dimerization showed abnormal morphology of ARC6 rings and compromised chloroplast division in plant cells. Together, our data reveal that PDV2-induced dimerization of ARC6 plays a critical role in chloroplast division and provide insights into the coordination mechanism of the internal and external plastid division machineries.

Roles of Arabidopsis PARC6 in Coordination of the Chloroplast Division Complex and Negative Regulation of FtsZ Assembly.

Chloroplast division is driven by the simultaneous constriction of the inner FtsZ ring (Z ring) and the outer DRP5B ring. The assembly and constriction of these rings in Arabidopsis (Arabidopsis thaliana) are coordinated partly through the inner envelope membrane protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6). Previously, we showed that PARC6 (PARALOG OF ARC6), also in the inner envelope membrane, negatively regulates FtsZ assembly and acts downstream of ARC6 to position the outer envelope membrane protein PLASTID DIVISION1 (PDV1), which functions together with its paralog PDV2 to recruit DYNAMIN-RELATED PROTEIN 5B (DRP5B) from a cytosolic pool to the outer envelope membrane. However, whether PARC6, like ARC6, also functions in coordination of the chloroplast division contractile complexes was unknown. Here, we report a detailed topological analysis of Arabidopsis PARC6, which shows that PARC6 has a single transmembrane domain and a topology resembling that of ARC6. The newly identified stromal region of PARC6 interacts not only with ARC3, a direct inhibitor of Z-ring assembly, but also with the Z-ring protein FtsZ2. Overexpression of PARC6 inhibits FtsZ assembly in Arabidopsis but not in a heterologous yeast system (Schizosaccharomyces pombe), suggesting that the negative regulation of FtsZ assembly by PARC6 is a consequence of its interaction with ARC3. A conserved carboxyl-terminal peptide in FtsZ2 mediates FtsZ2 interaction with both PARC6 and ARC6. Consistent with its role in the positioning of PDV1, the intermembrane space regions of PARC6 and PDV1 interact. These findings provide new insights into the functions of PARC6 and suggest that PARC6 coordinates the inner Z ring and outer DRP5B ring through interaction with FtsZ2 and PDV1 during chloroplast division.

Lipids Guide the Way: Targeting Proteins to the Chloroplast Outer Envelope Membrane

Correct delivery of peptides to appropriate subcellular organelles requires distinct trafficking and targeting mechanisms. In this issue of Developmental Cell, Kim et al. (2014) demonstrate that AKRA2, a targeting receptor for chloroplast outer envelope membrane proteins, binds chloroplast-specific lipids to ensure proper delivery of cargo to the chloroplast outer envelope.

The selective biotin tagging and thermolysin proteolysis of chloroplast outer envelope proteins reveals information on protein topology and association into complexes.

The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma, and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM), based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid, and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM) might need the integrity of a trans-envelope (IEM–OEM) protein complex (e.g., division ring-forming components) or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM–OEM complexes in various physiological contexts and using virtually any other purified membrane organelle.

Isolation, folding and structural investigations of the amino acid transporter OEP16

Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni–NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. 15N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.

Arabidopsis Tic40 Expression in Tobacco Chloroplasts Results in Massive Proliferation of the Inner Envelope Membrane and Upregulation of Associated Proteins

The chloroplast inner envelope membrane (IM) plays essential roles in lipid synthesis, metabolite transport, and cellular signaling in plants. We have targeted a model nucleus-encoded IM protein from Arabidopsis thaliana, pre-Tic40-His, by relocating its expression from the nucleus to the chloroplast genome. Pre-Tic40-His was properly targeted, processed, and inserted. It attained correct topology and was folded and assembled into a TIC complex, where it accounted for up to 15% of the total chloroplast protein. These results confirm the existence of a novel pathway for protein targeting to the IM. Tic40-His overexpression resulted in a massive proliferation of the IM (up to 19 layers in electron micrographs) without significant effects on plant growth or reproduction. Consistent with IM proliferation, the expression levels of other endogenous IM proteins (IEP37, PPT, Tic110) were significantly (10-fold) upregulated but those of outer envelope membrane (Toc159), stromal (hsp93, cpn60), or thylakoid (LHCP, OE23) proteins were not increased, suggesting retrograde signal transduction between chloroplast and nuclear genomes to increase lipid and protein components for accommodation of increased accumulation of Tic40. This study opens the door for understanding the regulation of membrane biogenesis within the organelle and the utilization of transgenic chloroplasts as bioreactors for hyperaccumulation of membrane proteins for biotechnological applications.

Three Sets of Translocation Intermediates Are Formed during the Early Stage of Protein Import into Chloroplasts

During the early stage of protein import into chloroplasts, precursor proteins synthesized in the cytosol irreversibly bind to chloroplasts to form the early translocation intermediate under stringent energy conditions. Many efforts have been made to identify the components involved in protein import by analyzing the early intermediate. However, the state of the precursor within the intermediate has not been well investigated so far. In this study, an attempt was made to evaluate the extent of translocation of the precursor by determining the state of the precursor in the early intermediate under various conditions and analyzing the fragments generated by limited proteolysis of the precursors docked to chloroplasts. Our results indicate that three different sets of early intermediate are formed based on temperature and the hydrolysis of GTP/ATP. These have been identified based on the size of proteolytic fragments of the precursor as "energy-dependent association," "insertion," and "penetration" states. These findings suggest two individual ATP-hydrolyzing steps during the early stage of protein import, one of which is temperature-sensitive. Our results also demonstrate that translocation through the outer envelope membrane is mainly dependent on internal ATP.

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.

PDV1 and PDV2 Mediate Recruitment of the Dynamin-Related Protein ARC5 to the Plastid Division Site

During plastid division, the dynamin-related protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5) is recruited from the cytosol to the surface of the outer chloroplast envelope membrane. In Arabidopsis thaliana arc5 mutants, chloroplasts arrest during division site constriction. Analysis of mutants similar to arc5 along with map-based cloning identified PLASTID DIVISION1 (PDV1), an integral outer envelope membrane protein, and its homolog PDV2 as components of the plastid division machinery. Similar to ARC5, PDV1 localized to a discontinuous ring at the division site in wild-type plants. The midplastid PDV1 ring formed in arc5 mutants and the ARC5 ring formed in pdv1 and pdv2 mutants, but not in pdv1 pdv2. Stromal FtsZ ring assembly occurred in pdv1, pdv2, and pdv1 pdv2, as it does in arc5. Topological analysis showed that the large N-terminal region of PDV1 upstream of the transmembrane helix bearing a putative coiled-coil domain is exposed to the cytosol. Mutation of the conserved PDV1 C-terminal Gly residue did not block PDV1 insertion into the outer envelope membrane but did abolish its localization to the division site. Our results indicate that plastid division involves the stepwise localization of FtsZ, PDV1, and ARC5 at the division site and that PDV1 and PDV2 together mediate the recruitment of ARC5 to the midplastid constriction at a late stage of division.

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.

퉁퉁마디로부터 색소체 외막 단백질 유전자의 분리 및 발현분석

Differential display 방법으로 NaCl에 의하여 발현이 증가되는 cDNA들을 분리하였으며 그중 하나가 식물유래의 outer envelope membrane protein과 높은 유사성을 보였으므로 이를 ShOEP로 명명하였다. ShOEP는 1293 bp 길이에 359개의 아미노산으로 구성된 open reading frame을 포함하고 있으며, 이로부터 추정되는 분자량은 8.9 kDa이었다. ShOEP 단백질은 애기장대의 OEP와는 40.6%, 시금치와는 38%의 유사성을 나타내었다. Northern 분석결과, ShOEP 유전자는 NaCl의 농도가 증가함에 따라 발현량이 급격히 증가하는 것으로 나타났다. 염생식물인 퉁퉁마디의 OEP는 PEG에 의하여 발현이 증가하는 반면 비염생식물인 애기장대의 OEP는 큰 차이를 보이지 않았다. 효모 complementation 실험결과 ShOEP는 NaCl에 특이적이었으며 식물의 염분스트레스 내성 기작에 직접적으로 관여하고 있음을 알 수 있었다. Complementary DNA encoding chloroplast outer envelope membrane protein (OEP) from the halophyte Salicornia herbacea has been cloned and sequenced. The full length cDNA is 596 bp and encodes a polypeptide of 91 amino acid residues with a molecular mass of 8.9 kDa. The expression level of ShOEP increased by salt, drought and ABA treatments. ShOEP expression was largely induced in roots and shoots by high salts. The biological function of ShOEP was examined by yeast complementation. ShOEP can suppress Na<TEX>$^{+}$</TEX> sensitivity of yeast mutant (cnb<TEX>$\Delta$</TEX>) in the presence of salt. These results suggest that ShOEP is a salt inducible gene and may have functions in the regulation of plant salt stress.ant salt stress.

Identification of a signal that distinguishes between the chloroplast outer envelope membrane and the endomembrane system in vivo.

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.

The N-terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts.

Toc75 is an outer envelope membrane protein of chloroplasts. It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide. The N-terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts. We prepared a 45 amino-acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein-mediated system, and purified it by reverse-phase HPLC. Its identity was confirmed by N-terminal amino-acid sequencing and matrix assisted laser desorption ionization mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine. However, it did not insert into other chloroplastic lipids, such as mono- and digalactosyl diacylglycerol, and phosphatidylcholine. Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase. The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase.

A protochlorophyllide-dependent protein import complex of chloroplasts

Chloroplasts synthesize an abundance of different tetrapyrrole compounds. Among them are chlorophyll and its precursor protochlorophyllide, which accumulate in light- and dark-grown plants, respectively. Protochlorophyllide is bound to two NADPH:Pchlide oxidoreductases (POR), termed PORA and PORB, which in angiosperms form higher molecular weight complexes in the prolamellar body of etioplasts. Both POR proteins are nuclear gene products which must be imported from the cytosol.. Import of the precursor of the PORA requires Pchlide and is due to the operation of a second, substrate-dependent protein import site in the plastid envelope. Here we report the isolation of components of the Pchlide-dependent import (PIM) complex and show that it is composed of at least 12 different polypeptides. Among them is a Pchlide a oxygenase which provides Pchlide b as the import substrate of the pPORA, and a tyrosine amino transferase presumably involved in conferring photoprotection onto the PIM complex. Two other constituents were found to be related to previously characterized outer envelope membrane proteins: a 33K protein to TOC33 which is a GTP binding protein, and a 16K protein to a voltage-dependent, amino acid-sective channel protein in the outer plastid envelope.

Insertion of OEP14 into the outer envelope membrane is mediated by proteinaceous components of chloroplasts.

Most chloroplastic outer envelope membrane proteins are synthesized in the cytosol at their mature size without a cleavable targeting signal. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. The insertion has been assumed to be mediated by a spontaneous mechanism or by interaction solely with the lipid components of the outer membrane. However, we show here that insertion of an outer membrane protein requires some trypsin-sensitive and some N-ethylmaleimide-sensitive components of chloroplasts. Association and insertion of the outer membrane protein are saturable and compete with the import of another outer membrane protein. These data suggest that import of chloroplastic outer membrane proteins occurs at specific proteinaceous sites on chloroplasts.

Targeting of proteins into and within the chloroplast

Chloroplasts are semiautonomous organelles that cannot be made de novo. They possess their own DNA, which encodes only a small part of chloroplastic proteins. The nucleotide sequences for ten different chloroplast genomes: liverwort, maize, tobacco, rice, black pine, Euglena, Epifagus, ChloreIla vulgaris, Cyanidum caldarium, Odontella sinensis, Toxoplasma gondii, have been determined (see NCBI, Entrez, Genomes, Database). There are about 130 structural genes in the chloroplast genome, coding for about 100 different proteins (Sugita and Sugiura 1996). Most proteins in chloroplast are encoded by the nuclear genome and synthesised in the cytosol. With the exception of most outer envelope membrane proteins, nuclearencoded chloroplastic proteins are synthesised with N-terminal extension that contains the chloroplast targeting information of these proteins. This Nterminal extension is referred to as a transit peptide (also termed transit sequence) to distinguish it from other sorting signals such as the signal sequence involved in the secretion of protein in prokaryotes. Transit sequence is necessary and sufficient to target a protein to the chloroplast and is removed after translocation by a stromal processing protease. Import is initiated by binding of the precursor protein to proteinaceous receptor localised on the surface of the chloroplast fol lowed by translocation through the outer and inner envelope membranes. Protein transpor t into chloroplast usually requires ATP and GTP for binding and translocation. Sometimes cytosolic factors are required for keeping the precursor soluble and to provide an import competent conformation. Beside the transit sequence there is information present in the protein for proper routing inside the chloroplast. Proteins that have to cross the thylakoid membrane contain a second Nterminal extension behind the transit peptide that is cleaved by a thylakoid processing peptidase. Protein transport and routing inside chloroplast are outlined on Fig. 1. Chloroplasts are structurally complex organelles with three different membranes: the

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 cross-linked 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.