Transport of proteins into chloroplasts. Binding of nuclear-coded chloroplast proteins to the chloroplast envelope

A system has been constructed in vitro for the binding of cytoplasmically synthesized chloroplast proteins to the chloroplast envelope which precedes the uptake into the organelle in vivo. Isolated chloroplast envelopes from young pea or spinach are capable of binding the majority of proteins obtained by translation of poly(A)-containing RNA from greening plants in vitro. Among the bound proteins the precursors to the light-harvesting chlorophyll a/b apoprotein and the small subunit of ribulose-1,5-bisphosphate carboxylase are prominent. Binding is an intrinsic property of the envelope membrane and does not require energy in the form of ATP. Bound proteins remain on the surface of the envelope vesicles and can be digested by protease. Binding is complete within minutes, shows a high affinity of the reactants, and is non-ionic in nature. Protein binding is specific for translation products of poly(A)-containing RNA from greening plants. Precursors to chloroplast protein are bound preferentially as compared to the mature proteins. The specificity is further demonstrated by the low binding of proteins obtained by run-off translation of polysomes. Binding of radioactive labeled proteins is subject to competition by excess unlabeled homologous proteins. Once bound, the proteins are withdrawn from competition indicating a high binding stability. All the properties found for binding of proteins to isolated envelopes are consistent with the concept of the so-called envelope carrier hypothesis.     

Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts.

The chloroplastic envelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and post-translationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments.     

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.     

Detection of small GTP-binding proteins in the outer envelope membrane of pea chloroplasts

We found small GTP-binding proteins in the outer envelope membrane of pea chloroplasts. The proteins in this membrane were separated by SDS-PAGE, transferred to a nitrocellulose filter, and incubated with [alpha-32P]GTP. Three GTP-binding proteins with the molecular weight of 24,000 were found. Binding was prevented by 10(-8)-10(-7) M GTP or by 10(-7) M guanosine 5'-[gamma-thio]triphosphate or GDP; binding was unaffected by 10(-8)-10(-6) M ATP. Thermolysin treatment of intact chloroplasts resulted in the loss of GTP-binding activity, suggesting that these proteins were in the cytosolic side of the outer envelope membrane.     

Emerging roles of the chloroplast outer envelope membrane

The chloroplast is essential for the viability of plants. It is enclosed by a double-membrane envelope that originated from the outer and plasma membranes of a cyanobacterial endosymbiont. Chloroplast biogenesis depends on binary fission and import of nuclear-encoded proteins. Our understanding of the mechanisms and evolutionary origins of these processes has been greatly advanced by recent genetic and biochemical studies on envelope-localized multiprotein machines. Furthermore, the latest studies on outer envelope proteins have provided molecular insights into organelle movement and membrane lipid remodeling, activities that are vital for plant survival under diverse environmental conditions. Ongoing and future research on the chloroplast outer envelope should add to our knowledge of organelle biology and the evolution of eukaryotic cells.     

Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone.

Nine different proteins were imported into isolated pea chloroplasts in vitro. For seven of these [the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), beta-subunit of ATP synthase, glutamine synthetase, the light-harvesting chlorophyll a/b binding protein, chloramphenicol acetyltransferase, and pre-beta-lactamase], a fraction was found to migrate as a stable high-molecular-weight complex during nondenaturing gel electrophoresis. This complex contained the mature forms of the imported proteins and the groEL-related chloroplast chaperonin 60 (previously known as Rubisco subunit binding protein). Thus, the stable association of imported proteins with this molecular chaperone is widespread and not necessarily restricted to Rubisco subunits or to chloroplast proteins. With two of the imported proteins (ferredoxin and superoxide dismutase), such complexes were not observed. It seems likely that, in addition to its proposed role in assembly of Rubisco, the chloroplast chaperonin 60 is involved in the assembly or folding of a wide range of proteins in chloroplasts.     

Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts

We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes.     

Evidence for two-step processing of nuclear-encoded chloroplast proteins during membrane assembly

A plastome (chloroplast genome) mutant of tobacco, lutescens-1, displays abnormal degradation of the chloroplast-encoded polypeptides which form the core complex of photosystem II (PSII). Two nuclear-encoded proteins (present in polymorphic forms), which normally function in the water oxidation process of PSII, accumulate as larger size-class polypeptides in mutant thylakoid membranes. These accumulated proteins are intermediate in size between the full-length primary protein synthesized in the cytoplasm and the proteolytically processed mature polypeptides. Trypsin treatment of unstacked mutant thylakoids and of inside-out vesicle (PSII-enriched) preparations indicated that the intermediate size forms were correctly localized on the inner surface of the thylakoid membrane, but not surface-exposed in the same way as the mature proteins. Only one of the intermediate size-class proteins could be extracted by salt washes. We interpret these data to be consistent with the idea that the two imported proteins that function in the water oxidation step of photosynthesis and are localized in the loculus (the space within the thylakoid vesicles) undergo two-step processing. The second step in proteolytic processing may be related to transport through a second membrane (the first transport step through the chloroplast envelope having been completed); this step may be arrested in the mutant due to the absence of the PSII core complex.     

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 and insertion of nuclear-encoded preproteins into the mitochondrial outer membrane

Most mitochondrial proteins are synthesized in the cytosol as preproteins with a cleavable presequence and are delivered to the import receptors on the mitochondria by cytoplasmic import factors. The proteins are then imported to the intramitochondrial compartments by the import systems of the outer and inner membranes, TOM and TIM. Mitochondrial outer membrane proteins are synthesized without a cleavable presequence and most of them contain hydrophobic transmembrane domains, which, in conjunction with the flanking segments, function as the mitochondria import signals. Some of the proteins are inserted into the outer membrane by the TOM machinery; the import signal probably arrests further translocation and is released from the translocation channel to the lipid bilayer. The other proteins are inserted into the membrane by a novel pathway independent of the TOM machinery. This article reviews recent developments in the biogenesis of mitochondrial outer membrane proteins.     

Import of preproteins into the chloroplast inner envelope membrane

The chloroplast inner envelope membrane contains many integral proteins which differ in the number of alpha-helices that anchor the protein into the bilayer. For most of these proteins it is not known which pathway they engage to reach their final localisation within the membrane. In yeast mitochondria, two distinct sorting/insertion pathways have been described for integral inner membrane proteins, involving the Tim22 and Tim23 translocases. These routes involve on the one hand a conservative sorting, on the other hand a stop-transfer pathway. In this study we performed a systematic characterisation of the import behaviour of seven inner envelope proteins representing different numbers of predicted alpha-helices. We investigated their energy dependence, import rate, involvement of components of the chloroplast general import pathway and distribution between soluble and membrane fractions. Our results show the existence of at least two different families of inner envelope proteins that can be classified due to the occurrence of an intermediate processing form. Each of the proteins we investigated seems to use a stop-transfer pathway for insertion into the inner envelope.     

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.     

Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.     

Ribosomes specifically bind to mammalian mitochondria via protease-sensitive proteins on the outer membrane

The interaction of ribosomes with specific components of membranes is one of the central themes to the co-translational targeting and import of proteins. To examine ribosome binding to mammalian mitochondria, we used ribosome-nascent chain complexes (RNCs) to follow the in vitro binding of ribosomes that correspond to the initial targeting stage of proteins. Mitochondria were found to contain a limited number of RNC binding sites on the outer membrane. It required more than twice the amount of non-translating ribosomes to inhibit RNC binding by one-half, indicating that RNCs have a competitive binding advantage. In addition, we found that RNCs bind mainly through the ribosomal component and not the nascent chain. RNCs bind via protease-sensitive proteins on the outer membrane, as well as by protease-insensitive components suggesting that two classes of receptors exist. We also show that binding is sensitive to cation conditions. Nearly all of the binding was inhibited in 0.5 m KCl, indicating that they interact with the membrane primarily through electrostatic interactions. In addition, disruption of RNC structure by removing magnesium causes the complete inhibition of binding under normal binding conditions indicating that it is the intact ribosome that is crucial for binding and not the nascent chain. These findings support the hypothesis that the outer mitochondrial membrane contains receptors specific for ribosomes, which would support the conditions necessary for co-translational import.     

Identification of an Arabidopsis inorganic pyrophosphatase capable of being imported into chloroplasts

An Arabidopsis cDNA coding for a previously uncharacterized isoform of inorganic pyrophosphatase was isolated. It was used to complement an E. coli mutant, demonstrating that it coded for an active enzyme. MgCl(2) was necessary for the protein's activity, whilst NaF inhibited it. The K(m) for pyrophosphate and the pH optimum of the protein was determined. The gene coding for this protein was expressed in all tissues, and its expression in rosette leaves was induced by incubation on metabolizable sugars. In vitro import experiments demonstrated that the protein could be imported into chloroplasts and localized to the stromal compartment.     

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins

Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.     

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.     

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.     

Characterization of the translocon of the outer envelope of chloroplasts

The protein translocon of the outer envelope of chloroplasts (Toc) consists of the core subunits Toc159, Toc75, and Toc34. To investigate the molecular structure, the core complex was purified. This core complex has an apparent molecular mass of approximately 500 kD and a molecular stoichiometry of 1:4:4-5 between Toc159, Toc75, and Toc34. The isolated translocon recognizes both transit sequences and precursor proteins in a GTP-dependent manner, suggesting its functional integrity. The complex is embedded by the lipids phosphatidylcholine and digalactosyldiacylglyceride. Two-dimensional structural analysis by EM revealed roughly circular particles consistent with the formation of a stable core complex. The particles show a diameter of approximately 130 A with a solid ring and a less dense interior structure. A three-dimensional map obtained by random conical tilt reconstruction of electron micrographs suggests that a "finger"-like central region separates four curved translocation channels within one complex.