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.     

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.     

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 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.     

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.     

Neofunctionalization within the Omp85 protein superfamily during chloroplast evolution

The Toc75 and OEP80 proteins reside in the chloroplast outer envelope membrane. Both are members of the Omp85 superfamily of β-barrel proteins, and both are essential in Arabidopsis plants with important roles throughout development. Toc75 forms the translocation channel of the TOC complex, which is responsible for importing nucleus-encoded proteins into chloroplasts, while the function of OEP80 remains uncertain. Deficiency of Toc75 in plants that have artificially reduced OEP80 levels suggests that the latter may be involved in the biogenesis of β-barrel proteins, in similar fashion to Omp85-related proteins in other systems. To elucidate the evolutionary relationship between the two proteins, we conducted a phylogenetic analysis using 48 sequences from diverse species. This indicated that Toc75 and OEP80 belong to sister groups in the Omp85 superfamily, and originate from a gene duplication in an ancient eukaryotic organism > 1.2 billion years ago. Our analysis also supports the notion that the Toc75 family has undergone a phase of neofunctionalization to accommodate the organelle’s newly acquired need to import proteins.     

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.     

The targeting of the atToc159 preprotein receptor to the chloroplast outer membrane is mediated by its GTPase domain and is regulated by GTP

The multimeric translocon at the outer envelope membrane of chloroplasts (Toc) initiates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two Toc GTPases, Toc159 and Toc33/34, mediate preprotein recognition and regulate preprotein translocation. Although these two proteins account for the requirement of GTP hydrolysis for import, the functional significance of GTP binding and hydrolysis by either GTPase has not been defined. A recent study indicates that Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, raising the possibility that it might cycle between the cytoplasm and chloroplast as a soluble preprotein receptor. In the present study, we examined the mechanism of targeting and insertion of the Arabidopsis thaliana orthologue of Toc159, atToc159, to chloroplasts. Targeting of atToc159 to the outer envelope membrane is strictly dependent only on guanine nucleotides. Although GTP is not required for initial binding, the productive insertion and assembly of atToc159 into the Toc complex requires its intrinsic GTPase activity. Targeting is mediated by direct binding between the GTPase domain of atToc159 and the homologous GTPase domain of atToc33, the Arabidopsis Toc33/34 orthologue. Our findings demonstrate a role for the coordinate action of the Toc GTPases in assembly of the functional Toc complex at the chloroplast outer envelope membrane.     

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.     

Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

Background

Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope.

Methodology/Principal Findings

Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein.

Conclusions/Significance

Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA proteins to their proper intracellular locations in general.     

Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE

The preprotein translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two receptor components, Toc159 and Toc34, and the channel Toc75 form the Toc complex. In this study, we have analyzed the molecular architecture and organization of the Toc complex by blue native PAGE (BN-PAGE), which is a high-resolution method for separating membrane protein complexes under non-denaturing conditions. Pea chloroplasts isolated in the presence of a protease inhibitor cocktail were directly solubilized in detergent solution and analyzed by BN-PAGE and size exclusion chromatography. Subsequent immunoblot analyses indicated that the complex composed of Toc75, Toc159 and Toc34 has a molecular mass of 800-1,000 kDa. Limited proteolysis revealed a core of the Toc complex, which was resistant to proteases and detergent treatments. The stoichiometry of the three Toc proteins was calculated as approximately 1 : 3 : 3 between Toc159 : Toc75 : Toc34. We have also analyzed the Toc complex of etioplasts and root plastids. These plastids were found to have essentially the same sized Toc complex as that of the chloroplast.     

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 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.     

In vivo analyses of the roles of essential Omp85-related proteins in the chloroplast outer envelope membrane

Two different, essential Omp85 (Outer membrane protein, 85 kD)-related proteins exist in the outer envelope membrane of Arabidopsis (Arabidopsis thaliana) chloroplasts: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD), encoded by atTOC75-III; and OEP80 (Outer Envelope Protein, 80 kD), encoded by AtOEP80/atTOC75-V. The atToc75-III protein is closely related to the originally identified pea (Pisum sativum) Toc75 protein, and it forms a preprotein translocation channel during chloroplast import; the AtOEP80 protein is considerably more divergent from pea Toc75, and its role is unknown. As knockout mutations for atTOC75-III and AtOEP80 are embryo lethal, we employed a dexamethasone-inducible RNA interference strategy (using the pOpOff2 vector) to conduct in vivo studies on the roles of these two proteins in older, postembryonic plants. We conducted comparative studies on plants silenced for atToc75-III (atToc75-III↓) or AtOEP80 (AtOEP80↓), as well as additional studies on a stable, atToc75-III missense allele (toc75-III-3/modifier of altered response to gravity1), and our results indicated that both proteins are important for chloroplast biogenesis at postembryonic stages of development. Moreover, both are important for photosynthetic and nonphotosynthetic development, albeit to different degrees: atToc75-III↓ phenotypes were considerably more severe than those of AtOEP80↓. Qualitative similarity between the atToc75-III↓ and AtOEP80↓ phenotypes may be linked to deficiencies in atToc75-III and other TOC proteins in AtOEP80↓ plants. Detailed analysis of atToc75-III↓ plants, by electron microscopy, immunoblotting, quantitative proteomics, and protein import assays, indicated that these plants are defective in relation to the biogenesis of both photosynthetic and nonphotosynthetic plastids and preproteins, confirming the earlier hypothesis that atToc75-III functions promiscuously in different substrate-specific import pathways.     

Protein import into chloroplasts

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.     

Molecular Characterization and Expression Analysis of Chloroplast Protein Import Components in Tomato (Solanum lycopersicum)

The translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and initial import into the organelle of thousands of nucleus-encoded proteins. These proteins are translated in the cytosol as precursor proteins with cleavable amino-terminal targeting sequences called transit peptides. The majority of the known Toc components that mediate chloroplast protein import were originally identified in pea, and more recently have been studied most extensively in Arabidopsis. With the completion of the tomato genome sequencing project, it is now possible to identify putative homologues of the chloroplast import components in tomato. In the work reported here, the Toc GTPase cDNAs from tomato were identified, cloned and analyzed. The analysis revealed that there are four Toc159 homologues (slToc159-1, -2, -3 and -4) and two Toc34 homologues (slToc34-1 and -2) in tomato, and it was shown that tomato Toc159 and Toc34 homologues share high sequence similarity with the comparable import apparatus components from Arabidopsis and pea. Thus, tomato is a valid model for further study of this system. The expression level of Toc complex components was also investigated in different tissues during tomato development. The two tomato Toc34 homologues are expressed at higher levels in non-photosynthetic tissues, whereas, the expression of two tomato Toc159 homologues, slToc159-1 and slToc159-4, were higher in photosynthetic tissues, and the expression patterns of slToc159-2 was not significantly different in photosynthetic and non-photosynthetic tissues, and slToc159-3 expression was limited to a few select tissues.     

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.     

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.