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.     

A novel, bipartite transit peptide targets OEP75 to the outer membrane of the chloroplastic envelope.

OEP75 is an outer envelope membrane component of the chloroplastic protein import apparatus and is synthesized in the cytoplasm as a higher molecular weight precursor (prOEP75). During its own import, prOEP75 is processed first to an intermediate (iOEP75) and subsequently to the mature form (mOEP75). Experiments conducted with stromal extracts indicated that iOEP75 was generated from prOEP75 by the activity of the stromal processing peptidase. The specific processing site was determined and used to divide the prOEP75 transit peptide into N- and C-terminal domains. To determine the targeting functions of the two domains of the transit peptide and of the mature region of prOEP75, we created a deletion mutant construct from prOEP75 and chimeric constructs between domains of prOEP75 and the precursor to a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Analysis of these constructs by in vitro chloroplastic protein import assays revealed that the transit peptide of prOEP75 is bipartite in that the N- and C-terminal portions contain chloroplastic and intraorganellar targeting information, respectively.     

Protein translocation into and across the chloroplastic envelope membranes

Post-translational protein import into chloroplasts follows a common route characterised by the need for nucleoside-triphosphates at various steps and two distinct protein import machineries at the outer and inner envelope membrane, respectively. Several subunits of these complexes have been elucidated. In contrast, protein translocation into the chloroplastic outer envelope uses distinct and various but poorly characterised insertion pathways. A topological framework for single-membrane spanning proteins of the chloroplastic outer envelope is presented.     

A rectifying ATP-regulated solute channel in the chloroplastic outer envelope from pea.

Phosphorylated carbohydrates are the main photoassimilated export products from chloroplasts that support the energy household and metabolism of the plant cell. Channels formed by the chloroplastic outer envelope protein OEP21 selectively facilitate the translocation of triosephosphate, 3-phosphoglycerate and phosphate, central intermediates in the source-sink relationship between the chloroplast and the cytosol. The anion selectivity and asymmetric transport properties of OEP21 are modulated by the ratio between ATP and triosephosphates, 3-phosphoglycerate and phosphate in the intermembrane space. Conditions that lead to export of triosephosphate from chloroplasts, i.e. photosynthesis, result in outward-rectifying OEP21 channels, while a high ATP to triosephosphate ratio, e.g. dark metabolism, leads to inward-rectifying OEP21 channels with a less pronounced anion selectivity. We conclude that solute exchange between plastids and cytosol can already be regulated at the level of the organellar outer membrane.     

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.     

Information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the maize Bt1-encoded protein.

Based on the protein sequence deduced from a cDNA clone, it has been proposed that the maize bt1 locus encodes an amyloplast membrane metabolite translocator protein (Sullivan, T. D., Strelow, L. I., Illingworth, C. A., Phillips, R. L., and Nelson, O. E., Jr. (1991) Plant Cell 3, 1337-1348). The present work provides further evidence for this hypothesis by showing that the gene product of Bt1 could be imported into chloroplasts in vitro and processed to lower molecular weight mature proteins. More importantly, the imported mature proteins were localized to the inner envelope membrane, where metabolite translocators are located in plastids. In addition, the location of information for targeting to the inner membrane was investigated by constructing and analyzing the import of chimeric precursor proteins. A chimeric protein with the transit peptide of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase fused to the mature region of the Bt1-encoded protein was targeted to the inner envelope membrane of chloroplasts. Moreover, a chimeric protein with the transit peptide of the Bt1-encoded protein fused to the mature protein of the light-harvesting chlorophyll a/b binding protein was targeted to the thylakoid. These results indicate that the transit peptide of the Bt1-encoded protein functions primarily as a stromal targeting sequence. The information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the protein.     

Isolation of a carotenoid-containing sub-membrane particle from the chloroplastic envelope outer membrane of pea (Pisum sativum).

Chloroplastic envelope membranes isolated from pea (Pisum sativum) leaves are rich in carotenoids, containing approximately 2 micrograms of carotenoid mg-1 protein. We report here that envelopes can be surfactant-solubilized while maintaining association of carotenoids with protein components of the membrane. Treatment of isolated chloroplastic envelope membranes with 0.5% Deriphat 160 (N-lauryl-beta-imminodipropionate) causes general solubilization but preserves an envelope sub-membrane fragment which is fractionated by centrifugation in a sucrose gradient and by chromatography on a column of DEAE-Sephacel. The isolated submembrane complex contained five major proteins with M(r) values equivalent to 75,000, 36,000, 34,000 17,500, and 14,500. Spectroscopic and chromatographic analyses revealed that the complex contains violaxanthin and at least one other carotenoid. Carotenoid content of the fractionated complex was estimated as 4.8 micrograms mg-1 protein. Immunoblot analysis reveals that the constituent proteins of this complex are derived from the chloroplastic outer envelope membrane. These data suggest that at least some of the carotenoids of the chloroplastic envelope may be organized by apoproteins.     

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.     

Chloroplast outer membrane protein targeting and insertion

Proteins in the chloroplast outer envelope membrane are nuclear encoded and post-translationally targeted to the chloroplast. The targeting and membrane insertion of these proteins is not well understood. Although early work suggested otherwise, the best-studied outer membrane proteins (OMPs) use both proteins within the chloroplast and NTPs for insertion. There have been conflicting reports in the field regarding protein targeting and insertion, which have probably arisen because of differences in experimental methodology and different interpretations of reduction (versus abolition) of integration. This review summarizes what is known to date about the mechanism of chloroplast OMP targeting.     

Protein targeting to the bacterial cytoplasmic membrane.

Proteins that perform their activity within the cytoplasmic membrane or outside this cell boundary must be targeted to the translocation site prior to their insertion and/or translocation. In bacteria, several targeting routes are known; the SecB- and the signal recognition particle-dependent pathways are the best characterized. Recently, evidence for the existence of a third major route, the twin-Arg pathway, was gathered. Proteins that use either one of these three different pathways possess special features that enable their specific interaction with the components of the targeting routes. Such targeting information is often contained in an N-terminal extension, the signal sequence, but can also be found within the mature domain of the targeted protein. Once the nascent chain starts to emerge from the ribosome, competition for the protein between different targeting factors begins. After recognition and binding, the targeting factor delivers the protein to the translocation sites at the cytoplasmic membrane. Only by means of a specific interaction between the targeting component and its receptor is the cargo released for further processing and translocation. This mechanism ensures the high-fidelity targeting of premembrane and membrane proteins to the translocation site.     

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.     

Protein targeting across the three membranes of the Euglena chloroplast envelope.

A system has been developed for the import in vitro of precursor proteins into Euglena chloroplasts, which have three envelope membranes. Preparation of functional chloroplasts with intact envelope membranes has been optimized. Import of the precursor (50 kDa) for the tetrapyrrole biosynthesis enzyme porphobilinogen deaminase (PBGD), and processing to the mature size (40 kDa), occurred at 25 degrees C in the light and the presence of ATP, with an estimated efficiency of 62%. Pretreatment of the chloroplasts with proteases abolished this import, suggesting the involvement of specific protein receptors. The presequence of PBGD was found to be cleaved by Escherichia coli leader peptidase to an intermediate form (46 kDa). A construct in which the first 30 residues of the presequence (presumed to be the region removed by leader peptidase) had been deleted was no longer imported. Neither prePBGD nor the truncated precursor were imported into pea chloroplasts, although both bound to the pea chloroplast envelope. Conversely, a chimeric construct, in which the mature PBGD protein was fused downstream of the transit peptide for pea ferredoxin-NADP reductase, was efficiently imported into pea chloroplasts and processed to the mature size. However, this was not imported into Euglena chloroplasts, although again it bound to them. These results provide preliminary evidence for the possibility of two functional domains within the Euglena PBGD presequence. The implications of these findings with respect to the evolution of Euglena chloroplasts are discussed.     

Protein composition of Rhodopseudomonas sphaeroides outer membrane.

The outer membrane polypeptide profile of Rhodopseudmonas sphaeroides was characterized. Solubilization of the outer membrane at 75 or 100 degrees C as opposed to room temperature resulted in the dissociation of 75-, 72-, and 68-kilodalton (kdal) polypeptide aggregates into 29-, 26.5-, and 21.5-kdal polypeptides, respectively, and a shared 47-kdal subunit. Similarly, an 88.5-kdal polypeptide dissociates into a 45-kdal monomeric form, and the electrophoretic mobility of a 58.5-kdal polypeptide was altered to 83 kdal.Lysozyme treatment of outer membrane fractions altered the 21.5-kdal polypeptide mobility to 23 kdal. The presence of lipid in both the 47-kdal polypeptide and an 8- to 10-kdal polypeptide was demonstrated by lipid staining and [14C]acetate incorporation. The lipid component of the 47-kdal polypeptide was neither lipopolysaccharide nor phospholipid. The 8- to 10-kdal polypeptide may be the equivalent of the Braun lipoprotein. Outer membrane fractions isolated from R. sphaeroides-specific phage RS1-resistant mutants were deficient in several of the high-molecular-weight aggregates involving the 47-kdal polypeptide.     

Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins.

Starting with an Escherichia coli strain missing the outer membrane lipoprotein, multiple mutants were constructed than in addition to this defect miss the outer membrane proteins II, Ia and Ib, or Ia, Ib, and II. In contrast to all single mutants or strains missing the lipoprotein and polypeptides Ia and Ib, drastic influences on the integrity of the outer membrane and cell morphology were observed in mutants without lipoprotein and protein II. Such strains exhibited spherical morphology. They required increased concentrations of electrolytes for optimal growth, and Mg2+ or Ca2+ were the most efficient. These mutants were sensitive to hydrophobic antibiotics and detergents. Electron microscopy revealed abundant blebbing of the outer membrane, and it could clearly be seen that the murein layer was no longer associated with the outer membrane.     

Protein interactions in the outer membrane of Escherichia coli.

Specific protein interactions in Escherichia coli outer membrane were analyzed using chemical cross-linking with truly cleavable reagents and symmetrical two-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The major outer membrane proteins were shown to form cross-linked complexes. These include multimers of lambda receptor, protein I, II, III and the free form of lipoprotein. Lipoprotein was also found to be cross-linked to proteins II and III. The identity of many of these complexes was verified using appropriate mutants missing the proteins in question. No new protein interactions were detected in the mutants even when three of the major proteins were missing. Proteins II, III and the free form of lipoprotein could also be cross-linked to the peptidoglycan layer of the cell wall.     

A two-state allosteric model for autoinhibition rationalizes WASP signal integration and targeting

Remodeling of the actin cytoskeleton is controlled by signaling pathways that include the Wiskott-Aldrich syndrome protein (WASP). WASP is regulated by autoinhibition, and the intramolecular contacts that inactivate the protein can be relieved through binding to the Rho-family GTPase Cdc42. Here, we show that the allosteric regulation of WASP can be quantitatively described by a two-state equilibrium between an active, largely unfolded conformation that is able to stimulate the Arp2/3 complex, and an inactive, folded conformation. The model is parameterized by the stability of WASP against unfolding and by the Cdc42 affinities of WASP constructs that mimic the unfolded and folded conformations. The model is consistent with NMR spectra of GTPase-bound WASP, and accurately predicts changes of amide hydrogen exchange behavior and Cdc42 affinity as a function of WASP stability. The results provide a thermodynamic rationale for the GTPase-independent recruitment of WASP and other autoinhibited effectors to their sites of activity. They also explain how basal activity is suppressed and confirm that WASP needs to integrate multiple cooperative inputs for maximal activation. Our analysis suggests that, in general, simple modulation of a two-state equilibrium may determine several regulatory functions, allowing the generation of complex signaling behavior in vivo.     

Proteomic study of the Arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional two-dimensional electrophoresis

With the completion of the sequencing of the Arabidopsis genome and with the significant increase in the amount of other plant genome and expressed sequence tags (ESTs) data, plant proteomics is rapidly becoming a very active field. We have pursued a high-throughput mass spectrometry-based proteomics approach to identify and characterize membrane proteins localized to the Arabidopsis thaliana chloroplastic envelope membrane. In this study, chloroplasts were prepared from plate- or soil-grown Arabidopsis plants using a novel isolation procedure, and "mixed" envelopes were subsequently isolated using sucrose step gradients. We applied two alternative methodologies, off-line multidimensional protein identification technology (Off-line MUDPIT) and one-dimensional (1D) gel electrophoresis followed by proteolytic digestion and liquid chromatography coupled with tandem mass spectrometry (Gel-C-MS/MS), to identify envelope membrane proteins. This proteomic study enabled us to identify 392 nonredundant proteins.     

Molecular cloning and sequencing of cDNA for yeast porin, an outer mitochondrial membrane protein: a search for targeting signal in the primary structure.

We have cloned a full-length cDNA for yeast porin, the major outer mitochondrial membrane protein from Saccharomyces cerevisiae, and determined its nucleotide sequence. The primary structure of the protein, deduced from the nucleotide sequence, consisted of 283 amino acid residues and its NH2-terminal sequence, Met-Ser-Pro-Pro-Val-Tyr-Ser, coincided with that determined by Edman degradation for yeast porin, except that the initiator methionine was missing in the mature protein. The deduced sequence had an overall polarity index of 46.3%, a value which falls in the normal range for soluble proteins. An evaluation of hydropathy of the protein indicated that the NH2-terminal one third was relatively hydrophilic and the rest of the molecule was rather hydrophobic. An interesting finding was that the NH2-terminal region of yeast porin (consisting of some 50 amino acid residues) shows structural features that resemble those of the corresponding portion of 70-kd protein, which is also a yeast outer mitochondrial membrane protein. We postulate that this NH2-terminal sequence, like that of 70-kd protein, is required for targeting the porin to the outer mitochondrial membrane.