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.     

Analysis of membrane protein complexes by blue native PAGE

Blue native polyacryamide gel electrophoresis is a special case of native electrophoresis for high resolution separation of enzymatically active protein complexes from tissue homogenates and cell fractions. The method is powerful between 10 and 10,000 kDa. Also membrane protein complexes are separated well after solubilization of complexes with mild neutral detergents. The separation principle relies on binding of Coomassie blue G250 which provides negative charges to the surface of the protein. During migration to the anode, protein complexes are separated according to molecular mass and/or size and high resolution is obtained by the decreasing pore size of a polyacrylamide gradient gel. The principles of 2-dimensional blue native sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented here together with a practical step-by-step guide to performing the method in the laboratory.     

Solubilization of membrane protein complexes for blue native PAGE

Blue native PAGE is an electrophoretic technique for high-resolution separation of membrane proteins. The method has been proven especially useful for investigation of native protein complexes enabling a characterization of potential protein-protein interactions in the context of functional proteomics. Blue native PAGE is easy to realise, results are reproducible and a high number of protocols are available. However, care should be taken during solubilization of protein complexes to achieve significant results in BN-PAGE analysis. Solubilization of membranes and proteins is not only influenced by detergent-lipid and detergent-protein interactions but also by lipid-lipid, lipid-protein and protein-protein interactions. Interactions have been investigated experimentally and theoretically. But, in practice, the experimental results do not always mirror the theoretical basis and therefore optimal solubilization conditions for each membrane and membrane protein complex should be investigated individually to tap the full potential of BN-PAGE analysis.     

Protein translocon at the Arabidopsis outer chloroplast membrane.

Chloroplasts are organelles essential for the photoautotrophic growth of plants. Their biogenesis from undifferentiated proplastids is triggered by light and requires the import of hundreds of different precursor proteins from the cytoplasm. Cleavable N-terminal transit sequences target the precursors to the chloroplast where translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes enable their import. In pea, the Toc complex is trimeric consisting of two surface-exposed GTP-binding proteins (Toc159 and Toc34) involved in precursor recognition and Toc75 forming an aequeous protein-conducting channel. Completion of the Arabidopsis genome has revealed an unexpected complexity of predicted components of the Toc complex in this plant model organism: four genes encode homologs of Toc159, two encode homologs of Toc34, but only one encodes a likely functional homolog of Toc75. The availability of the genomic sequence data and powerful molecular genetic techniques in Arabidopsis set the stage to unravel the mechanisms of chloroplast protein import in unprecedented depth.     

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.     

The preprotein conducting channel at the inner envelope membrane of plastids

The preprotein translocation at the inner envelope membrane of chloroplasts so far involves five proteins: Tic110, Tic55, Tic40, Tic22 and Tic20. The molecular function of these proteins has not yet been established. Here, we demonstrate that Tic110 constitutes a central part of the preprotein translocation pore. Dependent on the presence of intact Tic110, radiolabelled preprotein specifically interacts with isolated inner envelope vesicles as well as with purified, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of beta-sheets, a structure typically found in pore proteins. In planar lipid bilayers, recombinant Tic110 forms a cation-selective high-conductance channel with a calculated inner pore opening of 1.7 nm. Purified transit peptide causes strong flickering and a voltage-dependent block of the channel. Moreover, at the inner envelope membrane, a peptide-sensitive channel is described that shows properties basically identical to the channel formed by recombinant Tic110. We conclude that Tic110 has a distinct preprotein binding site and functions as a preprotein translocation pore at the inner envelope membrane.     

Regulation of two GTPases Toc159 and Toc34 in the translocon of the outer envelope of chloroplasts

The GTPases Toc159 and Toc34 of the translocon of the outer envelope of chloroplasts (TOC) are involved in recognition and transfer of precursor proteins at the cytosolic face of the organelle. Both proteins engage multiple interactions within the translocon during the translocation process, including dimeric states of their G-domains. The units of the Toc34 homodimer are involved in the recognition of the transit peptide representing the translocation signal of precursor proteins. This substrate recognition is part of the regulation of the GTPase cycle of Toc34. The Toc159 monomer and the Toc34 homodimer recognize the transit peptide of the small subunit of Rubisco at the N- and at the C-terminal region, respectively. Analysis of the transit peptide interaction by crosslinking shows that the heterodimer between both G-domains binds pSSU most efficiently. While substrate recognition by Toc34 homodimer was shown to regulate nucleotide exchange, we provide evidence that the high activation energy of the GTPase Toc159 is lowered by substrate recognition. The nucleotide affinity of Toc34_G homodimer and Toc159_G monomer are distinct, Toc34_G homodimer recognizes GDP and Toc159_G GTP with highest affinity. Moreover, the analysis of the nucleotide association rates of the monomeric and dimeric receptor units suggests that the heterodimer has an arrangement distinct from the homodimer of Toc34. Based on the biochemical parameters determined we propose a model for the order of events at the cytosolic side of TOC. The molecular processes described by this hypothesis range from transit peptide recognition to perception of the substrate by the translocation channel.     

Toc12, a novel subunit of the intermembrane space preprotein translocon of chloroplasts

Translocation of proteins across membranes is essential for the biogenesis of each cell and is achieved by proteinaceous complexes. We analyzed the translocation complex of the intermembrane space from chloroplasts and identified a 12-kDa protein associated with the Toc machinery. Toc12 is an outer envelope protein exposing a soluble domain into the intermembrane space. Toc12 contains a J-domain and stimulates the ATPase activity of DnaK. The conformational stability and the ability to stimulate Hsp70 are dependent on a disulfide bridge within the loop region of the J-domain, suggesting a redox-regulated activation of the chaperone. Toc12 is associated with Toc64 and Tic22. Its J-domain recruits the Hsp70 of outer envelope membrane to the intermembrane space translocon and facilitates its interaction to the preprotein.     

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.     

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.     

Organization of the native ribosome–translocon complex at the mammalian endoplasmic reticulum membrane

BackgroundIn eukaryotic cells, many proteins have to be transported across or inserted into the endoplasmic reticulum membrane during their biogenesis on the ribosome. This process is facilitated by the protein translocon, a highly dynamic multi-subunit membrane protein complex.Scope of reviewThe aim of this review is to summarize the current structural knowledge about protein translocon components in mammals.Major conclusionsVarious structural biology approaches have been used in synergy to characterize the translocon in recent years. X-ray crystallography and cryoelectron microscopy single particle analysis have yielded highly detailed insights into the structure and functional mechanism of the protein-conducting channel Sec61, which constitutes the functional core of the translocon. Cryoelectron tomography and subtomogram analysis have advanced our understanding of the overall structure, molecular organization and compositional heterogeneity of the translocon in a native membrane environment. Tomography densities at subnanometer resolution revealed an intricate network of interactions between the ribosome, Sec61 and accessory translocon components that assist in protein transport, membrane insertion and maturation.General significanceThe protein translocon is a gateway for approximately one third of all synthesized proteins and numerous human diseases are associated with malfunctioning of its components. Thus, detailed insights into the structure and molecular organization of the translocon will not only advance our understanding of membrane protein biogenesis in general, but they can potentially pave the way for novel therapeutic approaches against human diseases.     

Characterization of rat TOM40, a central component of the preprotein translocase of the mitochondrial outer membrane

We cloned a 38-kDa rat mitochondrial outer membrane protein (OM38) with structural homology to the central component of preprotein translocase of the fungal mitochondrial outer membrane, Tom40. Although it has no predictable alpha-helical transmembrane segments, OM38 is resistant to alkaline carbonate extraction and is inaccessible to proteases and polyclonal antibodies added from outside the mitochondria, suggesting that it is embedded in the membrane, probably in a beta-barrel structure, as has been similarly speculated for fungal Tom40. Immunoprecipitation demonstrated that OM38 is associated with the major import receptors rTOM20 and rTOM22, and several other unidentified components with molecular masses of 5-10 kDa in digitonin-solubilized membrane: OM10, OM7.5, and OM5. Blue native polyacrylamide gel electrophoresis revealed that OM38 is a component of a approximately 400-kDa complex, firmly associating with rTOM22 and loosely associating with rTOM20. The preprotein in transit to the matrix interacted with the TOM complex containing OM38, and immunodepletion of OM38 resulted in the loss of preprotein import activity of the detergent-solubilized and reconstituted outer membrane vesicles. Taken together, these results indicate that OM38 is a structural and functional homolog of fungal Tom40 and functions as a component of the preprotein import machinery of the rat mitochondrial outer membrane.     

Specific lipids influence the import capacity of the chloroplast outer envelope precursor protein translocon

Recent studies demonstrated that lipids influence the assembly and efficiency of membrane-embedded macromolecular complexes. Similarly, lipids have been found to influence chloroplast precursor protein binding to the membrane surface and to be associated with the Translocon of the Outer membrane of Chloroplasts (TOC). We used a system based on chloroplast outer envelope vesicles from Pisum sativum to obtain an initial understanding of the influence of lipids on precursor protein translocation across the outer envelope. The ability of the model precursor proteins p(OE33)titin and pSSU to be recognized and translocated in this simplified system was investigated. We demonstrate that transport across the outer membrane can be observed in the absence of the inner envelope translocon. The translocation, however, was significantly slower than that observed for chloroplasts. Enrichment of outer envelope vesicles with different lipids natively found in chloroplast membranes altered the binding and transport behavior. Further, the results obtained using outer envelope vesicles were consistent with the results observed for the reconstituted isolated TOC complex. Based on both approaches we concluded that the lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) increased TOC-mediated binding and import for both precursor proteins. In contrast, enrichment in digalactosyldiacylglycerol (DGDG) improved TOC-mediated binding for pSSU, but decreased import for both precursor proteins. Optimal import occurred only in a narrow concentration range of DGDG.     

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.     

Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts

Precursor forms of chloroplast proteins synthesized in cell-free translation systems can be imported posttranslationally into isolated, intact chloroplasts. Radiochemically pure precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and to the light-harvesting chlorophyll a/b protein have been prepared by in vitro translation of hybrid-selected mRNA and used to study this import process. If chloroplasts are pretreated with the uncoupler nigericin, import does not occur, but the precursors bind to the chloroplast surface. Reincubation of the precursor-chloroplast complex in the presence of ATP results in import of bound precursors. The binding appears to be mediated by proteins of the outer chloroplast envelope membrane because pretreatment of chloroplasts with protease inhibits their ability to bind as well as to import precursors. These results indicate that at least a portion of the observed binding is to functional receptor proteins involved in the import process.     

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.     

Localization of CYP86B1 in the outer envelope of chloroplasts

CYP86B1 was cloned from a cDNA library and the protein expressed in E. coli. The protein gave the expected carbon monoxide difference spectrum. Using in vitro import assays with isolated pea chloroplasts, CYP86B1 was shown to be associated with the outer chloroplastic envelope membrane. This study provides the first direct evidence for a chloroplast-localized cytochrome P450-dependent monooxygenase.     

An alternative strategy for the membrane proteome analysis of the green sulfur bacterium Chlorobium tepidum using blue native PAGE and 2-D PAGE on purified membranes

To avoid the specific problems concerning intrinsic membrane proteins in proteome analysis, an alternative strategy is described that is complementary to previous investigations using 2-D polyacrylamide gel electrophoresis (PAGE) techniques. The strategy involves (a) obtaining purified preparations of the membranes from Chlorobium tepidum by washing with 2 M NaBr, which removed membrane-associated soluble proteins and membrane-associated organelles; (b) separation of membrane protein complexes using 1-D Blue-native polyacrylamide gel electrophoresis (BN-PAGE) after solubilization with n-dodecyl-beta-d-maltoside (DDM); (c) combination of the BN with Tricine-SDS-PAGE; (d) high-throughput mass spectrometric analysis after gel band excision, in-gel digestion, and MALDI target spotting; and (e) protein identification from mixtures of tryptic peptides by peptide mass fingerprinting. Using this approach, we identified 143 different proteins, 70 of which have not been previously reported using 2-D PAGE techniques. Membrane proteins with up to 14 transmembrane helices were found, and this procedure proved to be efficient with proteins within a wide pI range (4.4-11.6). About 54% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, and protein translocation, while for the others, a function is not yet known, indicating the potential of the method for the elucidation of the membrane proteomes in general.     

Oxygen evolution and the permeability of the outer envelope of isolated whole chloroplasts

A rapid oxygraph method of studying the permeability of the envelope of isolated chloroplasts was used. The outer envelope of aqueously isolated whole spinach (Spinacia oleracea L.) chloroplasts in buffer is readily permeable to 3-phosphoglyceric acid, which induces an immediate light dependent oxygen evolution. This light dependent oxygen evolution was completely eliminated by swelling these plastids in an osmotically dilute solution. Exogenous adenosine diphosphate, but not inorganic phosphate, strongly stimulated this oxygen evolution. This indicated that the chloroplast envelope is relatively permeable to adenosine diphosphate.

Oxygen evolution and swelling studies indicated that the chloroplast envelope is relatively impermeable to NADP and to ferredoxin.

A method is described whereby the percent of whole chloroplasts present in a chloroplast preparation may be rapidly estimated.