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

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

The outer envelope protein OEP24 from pea chloroplasts can functionally replace the mitochondrial VDAC in yeast

The chloroplastic outer envelope protein OEP24 from pea forms a high-conductance low specificity solute channel as shown by in vitro studies. In order to establish its function also in an in vivo-like system, the gene encoding OEP24 was transformed into a yeast strain which lacks the general mitochondria solute channel porin, also known as voltage-dependent anion channel (VDAC). Transformation of the yeast VDAC(-) strain with the OEP24 gene resulted in the recovery of a phenotype indistinguishable from the wild-type. The OEP24 polypeptide is targeted to the mitochondrial outer membrane in this heterologous system. We conclude that OEP24 forms a solute channel in pea chloroplasts in planta.     

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

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

Solute channels of the outer membrane: from bacteria to chloroplasts

Chloroplasts, unique organelles of plants, originated from endosymbiosis of an ancestor of today's cyanobacteria with a mitochondria-containing host cell. It is assumed that the outer envelope membrane, which delimits the chloroplast from the surrounding cytosol, was thus inherited from its Gram-negative bacterial ancestor. This plastid-specific membrane is thus equipped with elements of prokaryotic and eukaryotic origin. In particular, the membrane-intrinsic outer envelope proteins (OEPs) form solute channels with properties reminiscent of porins and channels in the bacterial outer membrane. OEP channels are characterised by distinct specificities for metabolites and a quite peculiar expression pattern in specialised plant organs and plastids, thus disproving the assumption that the outer envelope is a non-specific molecular sieve. The same is true for the outer membrane of Gram-negative bacteria, which functions as a permeability barrier in addition to the cytoplasmic membrane, and embeds different classes of channel pores. The channels of these prokaryotic prototype proteins, ranging from unspecific porins to specific channels to ligand-gated receptors, are exclusively built of beta-barrels. Although most of the OEP channels are formed by beta-strands as well, phylogeny based on sequence homology alone is not feasible. Thus, the comparison of structural and functional properties of chloroplast outer envelope and bacterial outer membrane channels is required to pinpoint the ancestral OEP 'portrait gallery'.     

Reconstitution of a chloroplast protein import channel.

The chloroplastic outer envelope protein OEP75 with a molecular weight of 75 kDa probably forms the central pore of the protein import machinery of the outer chloroplastic membrane. Patch-clamp analysis shows that heterologously expressed, purified and reconstituted OEP75 constitutes a voltage-gated ion channel with a unit conductance of Lambda = 145pS. Activation of the OEP75 channel in vitro is completely dependent on the magnitude and direction of the voltage gradient. Therefore, movements of protein charges of parts of OEP75 in the membrane electric field are required either for pore formation or its opening. In the presence of precursor protein from only one side of the bilayer, strong flickering and partial closing of the channel was observed, indicating a specific interaction of the precursor with OEP75. The comparatively low ionic conductance of OEP75 is compatible with a rather narrow aqueous pore (dporeapproximately equal to 8-9 A). Provided that protein and ion translocation occur through the same pore, this implies that the environment of the polypeptide during the transit is mainly hydrophilic and that protein translocation requires almost complete unfolding of the precursor.     

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

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

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

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

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

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

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.     

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 24 mg/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 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.     

Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps

Broad-specificity efflux pumps have been implicated in multidrug-resistant strains of Pseudomonas aeruginosa and other Gram-negative bacteria. Most Gram-negative pumps of clinical relevance have three components, an inner membrane transporter, an outer membrane channel protein, and a periplasmic protein, which together coordinate efflux from the cytoplasmic membrane across the outer membrane through an unknown mechanism. The periplasmic efflux proteins (PEPs) and outer membrane efflux proteins (OEPs) are not obviously related to proteins of known structure, and understanding the structure and function of these proteins has been hindered by the difficulty of obtaining reasonable multiple alignments. We present a general strategy for the alignment and structure prediction of protein families with low mutual sequence similarity using the PEP and OEP families as detailed examples. Gibbs sampling, hidden Markov models, and other analysis techniques were used to locate motifs, generate multiple alignments, and assign PEP or OEP function to hypothetical proteins in several species. We also developed an automated procedure which combines multiple alignments with structure prediction algorithms in order to identify conserved structural features in protein families. This process was used to identify a probable alpha-helical hairpin in the PEP family and was applied to the detection of transmembrane beta-strands in OEPs. We also show that all OEPs contain a large tandem duplication, and demonstrate that the OEP family is unlikely to adopt a porin fold, in contrast to previous predictions.     

A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast.

During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.     

New Insights into the Protein Import Machinery of the Chloroplast's Outer Envelope

A large number of plastid localized proteins are post-translationally imported as precursor proteins from the cytosol into the organelle. Recognition and translocation is accomplished by a subset of chloroplast envelope proteins, which were identified by different but complementary methods. The o uter e nvelope p roteins OEP 86, OEP 75, OEP 70 (a heat shock cognate 70 homologue) and OEP 34 are clearly involved in the import event and can be isolated as one functionally active translocation unit. For three of these proteins cDNA clones have been very recently obtained, namely OEP 86, OEP 75 and OEP 34. OEP 86 seems to be a precursor protein receptor which could be regulated by GTP binding and ATP-dependent phosphorylation-dephosphorylation. OEP 75 is part of the translocation pore traversing the membrane in multiple β-sheets. OEP 34 is tightly associated with OEP 75. It represents a new type of GTP-binding protein which possesses endogenous GTPase activity. Multiple GTP binding and hydrolysis cycles as well as protein phosphorylation-dephosphorylation events might, therefore, regulate the interaction of a precursor protein with the translocation machinery of the outer envelope, making it very distinct from the mitochondrial outer membrane system. Further proteins of the inner envelope membrane, namely IEP 97 and IEP 36, have been implied to function in the translocation event. These recent data allow not only identification of the players in the game but also speculation about mechanisms and regulation of translocation.     

Gene duplication, exon gain and neofunctionalization of OEP16-related genes in land plants

OEP16, a channel protein of the outer membrane of chloroplasts, has been implicated in amino acid transport and in the substrate-dependent import of protochlorophyllide oxidoreductase A. Two major clades of OEP16-related sequences were identified in land plants (OEP16-L and OEP16-S), which arose by a gene duplication event predating the divergence of seed plants and bryophytes. Remarkably, in angiosperms, OEP16-S genes evolved by gaining an additional exon that extends an interhelical loop domain in the pore-forming region of the protein. We analysed the sequence, structure and expression of the corresponding Arabidopsis genes (atOEP16-S and atOEP16-L) and demonstrated that following duplication, both genes diverged in terms of expression patterns and coding sequence. AtOEP16-S, which contains multiple G-box ABA-responsive elements (ABREs) in the promoter region, is regulated by ABI3 and ABI5 and is strongly expressed during the maturation phase in seeds and pollen grains, both desiccation-tolerant tissues. In contrast, atOEP-L, which lacks promoter ABREs, is expressed predominantly in leaves, is induced strongly by low-temperature stress and shows weak induction in response to osmotic stress, salicylic acid and exogenous ABA. Our results indicate that gene duplication, exon gain and regulatory sequence evolution each played a role in the divergence of OEP16 homologues in plants.     

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.     

Functional reconstitution of a voltage-gated potassium channel in giant unilamellar vesicles

Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid composition and physical state. In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in experimental settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane composition, tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compositions and buffers containing low (5 mM) or near-physiological (100 mM) salt concentrations. Protein incorporation into GUVs was characterized with quantitative confocal microscopy, and the protein density of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs containing functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability.     

Expression of human BK ion channels in Sf9 cells, their purification using metal affinity chromatography, and functional reconstitution into planar lipid bilayers

This report describes a procedure for purification of large conductance calcium-activated potassium (BK, maxi-K) channels using immobilised metal affinity chromatography (IMAC) under non-denaturing conditions. An amino-terminal histidine fusion tag was added to hSlo, the human BK channel, and expressed in Sf9 insect cells. Following IMAC purification and production of proteoliposomes, protein function was assessed electrophysiologically in planar bilayer lipid membranes. Single channel openings had conductances of 250-300 pS and were inhibited by paxilline, demonstrating that the BK channels remained functional following IMAC purification. This method to obtain functional human ion channels will be useful in assays to screen potential pharmaceuticals.