The chloroplastic protein import machinery contains a Rieske-type iron-sulfur cluster and a mononuclear iron-binding protein.

Transport of precursor proteins across the chloroplastic envelope membranes requires the interaction of protein translocons localized in both the outer and inner envelope membranes. Analysis by blue native gel electrophoresis revealed that the translocon of the inner envelope membranes consisted of at least six proteins with molecular weights of 36, 45, 52, 60, 100 and 110 kDa, respectively. Tic110 and ClpC, identified as components of the protein import apparatus of the inner envelope membrane, were prominent constituents of this complex. The amino acid sequence of the 52 kDa protein, deduced from the cDNA, contains a predicted Rieske-type iron-sulfur cluster and a mononuclear iron-binding site. Diethylpyrocarbonate, a Rieske-type protein-modifying reagent, inhibits the translocation of precursor protein across the inner envelope membrane, whereas binding of the precursor to the outer envelope membrane is still possible. In another independent experimental approach, the 52 kDa protein could be co-purified with a trapped precursor protein in association with the chloroplast protein translocon subunits Toc86, Toc75, Toc34 and Tic110. Together, these results strongly suggest that the 52 kDa protein, named Tic55 due to its calculated molecular weight, is a member of the chloroplastic inner envelope protein translocon.     

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.     

Organization in photosynthetic membranes of purple bacteria in vivo: The role of carotenoids

Photosynthesis in purple bacteria is performed by pigment–protein complexes that are closely packed within specialized intracytoplasmic membranes. Here we report on the influence of carotenoid composition on the organization of RC–LH1 pigment–protein complexes in intact membranes and cells of Rhodobacter sphaeroides. Mostly dimeric RC–LH1 complexes could be isolated from strains expressing native brown carotenoids when grown under illuminated/anaerobic conditions, or from strains expressing green carotenoids when grown under either illuminated/anaerobic or dark/semiaerobic conditions. However, mostly monomeric RC–LH1 complexes were isolated from strains expressing the native photoprotective red carotenoid spheroidenone, which is synthesized during phototrophic growth in the presence of oxygen. Despite this marked difference, linear dichroism (LD) and light-minus-dark LD spectra of oriented intact intracytoplasmic membranes indicated that RC–LH1 complexes are always assembled in ordered arrays, irrespective of variations in the relative amounts of isolated dimeric and monomeric RC–LH1 complexes. We propose that part of the photoprotective response to the presence of oxygen mediated by synthesis of spheroidenone may be a switch of the structure of the RC–LH1 complex from dimers to monomers, but that these monomers are still organized into the photosynthetic membrane in ordered arrays. When levels of the dimeric RC–LH1 complex were very high, and in the absence of LH2, LD and ?LD spectra from intact cells indicated an ordered arrangement of RC–LH1 complexes. Such a degree of ordering implies the presence of highly elongated, tubular membranes with dimensions requiring orientation along the length of the cell and in a proportion larger than previously observed.     

Comparison of extracellular ATP and UTP signalling in rat renal mesangial cells. No indications for the involvement of separate purino- and pyrimidino-ceptors.

The visible c.d. spectrum of wild-type Rhodospirillum rubrum shows positive bands [Dratz, Schultz & Sauer (1966) Brookhaven Symp. Biol. 19, 303-318] that are largely due to the B880 antenna pigments, bacteriochlorophyll a and carotenoids. The bacteriochlorophyll c.d. band was absent from the spectrum of R. rubrum G9, a mutant unable to synthesize coloured carotenoids, and could be partly restored by adding extracted carotenoids to freeze-dried membrane vesicles isolated from that mutant. Therefore it seems to arise from either bacteriochlorophyll-carotenoid interactions or bacteriochlorophyll-protein interactions that are induced by the carotenoid. The more complex carotenoid c.d. band had different shapes in native and reconstituted carotenoid-containing membranes. Such differences suggest that the optical activity of the carotenoid in the B880 antenna arises from both non-degenerate and degenerate interactions.     

Envelope Permeability to Possible Precursors of Carotenoid Biosynthesis during Chloroplast-Chromoplast Transformation

During the transformation of chloroplasts to chromoplasts in Capsicum annuum L., the permeability of the envelope membranes to possible precursors of carotenoid biosynthesis (acetate, mevalonate, citrate) was tested. The plastids were isolated by gel filtration, and the uptake of labeled compounds was measured by a filtering centrifugation technique, using silicone oil. The different ripening stages were characterized by the ratio of chlorophyll to carotenoid content. The chloroplast membranes were shown to be impermeable to all of the metabolites tested. During the transformation there was a sharp increase of membrane permeability. In the mature chromoplast, the permeability to mevalonate and acetate again decreased to about 20% of the maximum value and reached zero for citrate. The results give evidence that during the transformation of chloroplasts to chromoplasts, precursors for carotenoid biosynthesis are translocated from extraplastidic sites into the plastids, there being possibly incorporated into carotenoids.     

Topology of IEP110, a component of the chloroplastic protein import machinery present in the inner envelope membrane.

Proteins from both the inner and outer envelope membranes are engaged in the recognition and translocation of precursor proteins into chloroplasts. A 110 kDa protein of the chloroplastic inner envelope membrane was identified as a component of the protein import apparatus by two methods. First, this protein was part of a 600 kDa complex generated by cross-linking of precursors trapped in the translocation process. Second, solubilization with detergents of chloroplasts containing trapped precursors resulted in the identification of a complex containing both radiolabeled precursor and IEP110. Trypsin treatment of intact purified chloroplasts was used to study the topology of IEP110. The protease treatment left the inner membrane intact while simultaneously degrading domains of inner envelope proteins exposed to the intermembrane space. About 90 kDa of IEP110 was proteolitically removed, indicating that large portions protrude into the intermembrane space. Hydropathy analysis of the protein sequence deduced from the isolated cDNA clone in addition to Western blot analysis using an antiserum of IEP110 specific to the N-terminal 20 kDa, suggests that the N-terminus serves to anchor the protein in the membrane. We speculate that IEP110 could be involved in the formation of translocation contact sites due to its specific topology.     

Envelope membrane proteins that interact with chloroplastic precursor proteins.

The post-translational transport of cytoplasmically synthesized precursor proteins into chloroplasts requires proteins in the envelope membranes. To identify some of these proteins, label transfer cross-linking was performed using precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase (prSSU) that was blocked at an early stage of the transport process. Two envelope proteins were identified: an 86-kD protein and a 75-kD protein, both present in the outer membrane. Labeling of both proteins required prSSU and could not be accomplished with SSU lacking a transit peptide. Labeling of the 75-kD protein occurred only when low levels of ATP were present, whereas labeling of the 86-kD protein occurred in the absence of exogenous ATP. Although both labeled proteins were identified as proteins of the outer envelope membrane, the labeled form of the 75-kD protein could only be detected in fractions containing mixed envelope membranes. Based on these observations, we propose that prSSU first binds in an ATP-independent fashion to the 86-kD protein. The energy-requiring step is association with the 75-kD protein and assembly of a translocation contact site between the inner and outer membrane of the chloroplastic envelope.     

Protein import into chloroplasts

Most chloroplastic proteins are encoded in the nucleus, synthesized on cytosolic ribosomes and subsequently imported into the organelle. In general, proteins destined for the chloroplast are synthesized as precursor proteins with a cleavable N-terminal presequence that mediates routing to the inside of the chloroplast. These precursor proteins have to be targeted to the correct organellar membrane surface after their release from the ribosome and furthermore they have to be maintained in a conformation suitable for translocation across the two envelope membranes. Recognition and import of most chloroplastic precursor proteins are accomplished by a jointly used translocation apparatus. Different but complementary studies of several groups converged recently in the identification of the outer envelope proteins OEP86, OEP75, OEP70 (a Hsp 70-related protein), OEP34, and of the inner envelope protein IEP110 as components of this translocation machinery. None of these proteins, except for OEP70, shows any homology to components of other protein translocases. The plastid import machinery thus seems to be an original development in evolution. Following translocation into the organelle, chloroplastic proteins are sorted to their suborganellar destination, i.e., the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen. This structural and evolutionary complexity of chloroplasts is reflected by a variety of routing mechanisms by which proteins reach their final location once inside the organelle. This review will focus on recent advances in the identification of components of the chloroplastic protein import machinery, and new insights into the pathways of inter-and intraorganellar sorting.     

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. II. Biochemical characterization

In the previous paper (Block, M. A., Dorne, A.-J., Joyard, J., and Douce, R. (1983) J. Biol. Chem. 258, 13273-13280), we have described a method for the separation of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. The two envelope membranes have a different weight ratio of acyl lipid to protein (2.5-3 for the outer envelope membrane and 0.8-1 for the inner envelope membrane). The two membranes also differ in their polar lipid composition. However, in order to prevent the functioning of the galactolipid:galactolipid galactosyltransferase during the course of envelope membrane separation, we have analyzed the polar lipid composition of each envelope membrane after thermolysin treatment of the intact chloroplasts. The outer envelope membrane is characterized by the presence of high amounts of phosphatidylcholine and digalactosyldiacylglycerol whereas the inner envelope membrane has a polar lipid composition almost identical with that of the thykaloids. No phosphatidylethanolamine or cardiolipin could be detected in either envelope membranes, thus demonstrating that the envelope membranes, and especially the outer membrane, do not resemble extrachloroplastic membranes. No striking differences were found in the fatty acid composition of the polar lipids from either the outer or the inner envelope membrane. The two envelope membranes also differ in their carotenoid composition. Among the different enzymatic activities associated with the chloroplast envelope, we have shown that the Mg2+-dependent ATPase, the UDP-Gal:diacylglycerol galactosyltransferase, the phosphatidic acid phosphatase, and the acyl-CoA thioesterase are associated with the inner envelope from spinach chloroplasts whereas the acyl-CoA synthetase is located on the outer envelope membrane.     

Isolation and characterization of an amyloplast envelope-enriched fraction from immature maize endosperm

Gardner, H. W., Miernyk, J. A., Christianson, D. D. and Khoo, U. 1987. Isolation and characterization of an amyloplast envelope-enriched fraction from immature maize endosperm.
A 10000–100000 g pellet obtained by centrifugation of homogenates from immature (25 days after pollination) de-embryonated maize ( Zea mays L., cv. W64A-normal and a typical hybrid) kernels was further fractionated by sedimentation on discontinuous sucrose density gradients. Particles with the highest carotenoid content (0.68% by weight carotenoids based upon total lipid) sedimented at densities of 1.083-1.106 g ml^-1, coincident with the plastid envelope marker enzyme, galacto-syltransferase (EC 2.4.1.46). Lipids extracted from the carotenoid-rich fraction were mainly digalactosyldiacylglycerols, monogalactosyldiacylglycerols, phosphatidylcholines, phosphatidylinositols and phosphatidylglycerols, in order of molar abundance. With increasing particle density (>1.106 g ml¹) the phospholipid and neutral lipid content increased, and the proportion of carotenoids and galactolipids decreased. Electron micrographs of the carotenoid-rich fraction revealed vesicles ranging in size from < 0.1 to 0.5 um, as well as smaller granular membranes. The carotenoid-rich membrane fraction was progressively more difficult to isolate as the endosperm matured, and freezing the immature endosperm prevented subsequent isolation. The lipid and enzyme composition and ultrastructural characteristics of the isolated fraction suggest that it is composed of amyloplast envelope vesicles.     

Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides

The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N-oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer k_ET > 3 · 10¹² s^?1. The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 Å. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Q_y transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.     

Roles of bacteriochlorophyll and carotenoid synthesis in formation of intracytoplasmic membrane systems and pigment-protein complexes in an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114.

Synthesis of bacteriochlorophyll and carotenoids was inhibited in an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114, by alpha, alpha'-dipyridyl and diphenylamine. Formation of two pigment-protein complexes, reaction center-B870 (RC-B870) and B806, and development of the intracytoplasmic membranes of the cells were studied by spectral analysis and electron microscopy. Inhibition of bacteriochlorophyll synthesis by alpha, alpha'-dipyridyl, which was accompanied by a decrease in carotenoid synthesis, suppressed formation of intracytoplasmic membranes in the cells. Growth under illumination had a similar effect on formation of pigments and membranes. On the other hand, inhibition of carotenoid synthesis by diphenylamine did not suppress either development of the membrane system or bacteriochlorophyll synthesis. Formation of RC-B870 and B806 complexes, however, was differentially affected by blockage of carotenoid synthesis. In the presence of diphenylamine, the B806 complex was formed in a much smaller amount than the RC-B870 complex. These results suggest that, in Erythrobacter sp. strain OCh114, bacteriochlorophyll plays an essential role in intracytoplasmic membrane development, and carotenoids are important for assembly of pigment-protein complexes.     

EVIDENCE FOR A SPECIALIZED LOCALIZATION OF THE CHLOROPLAST ATP‐SYNTHASE SUBUNITS α, β, AND γ IN THE EYESPOT APPARATUS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

The eyespot apparatus (EA) of Chlamydomonas reinhardtii P. A. Dang. consists of two layers of carotenoid‐rich lipid globules subtended by thylakoids. The outermost globule layer is additionally associated with the chloroplast envelope membranes and the plasma membrane. In a recent proteomic approach, we identified 202 proteins from isolated EAs of C. reinhardtii via at least two peptides, including, for example, structural components, signalling‐related proteins, and photosynthetic‐related membrane proteins. Here, we have analyzed the proteins of the EA with regard to their topological distribution using thermolysin to find out whether the arrangement of globules and membranes provides protection mechanisms for some of them. From about 230 protein spots separated on two‐dimensional gels, the majority were degraded by thermolysin. Five major protein spots were protected against the action of this protease. These proteins and some that were degradable were identified by mass spectrometry. Surprisingly, the thermolysin‐resistant proteins represented the α and β subunits of the soluble CF₁ complex of the chloroplast ATP synthase. Degradable proteins included typical membrane proteins like LHCs, demonstrating that thermolysin is not in general sterically prevented by the EA structure from reaching membrane‐associated proteins. A control experiment showed that the CF₁ complex of thylakoids is efficiently degraded by thermolysin. Blue native PAGE of thermolysin‐treated EAs followed by SDS‐PAGE revealed that the α and β subunits are present in conjunction with the γ subunit in a thermolysin‐resistant complex. These results provide strong evidence that a significant proportion of these ATP‐synthase subunits have a specialized localization and function within the EA of C. reinhardtii.     

Protein import into chloroplasts

Three proteins from the chloroplastic outer envelope membrane and four proteins from the inner envelope membrane have been identified as components of the chloroplastic protein import apparatus. Multiple molecular chaperones and a stromal processing peptidase are also important components of the import machinery. The interactions of these proteins with each other and with the precursors destined for transport into chloroplasts are gradually being described using both biochemical and genetic strategies. Homologs of some transport components have been identified in cyanobacteria suggesting that at least some of import machinery was inherited from the cyanobacterial ancestors that gave rise to chloroplasts.     

Stable association of chloroplastic precursors with protein translocation complexes that contain proteins from both envelope membranes and a stromal Hsp100 molecular chaperone.

Cytoplasmically synthesized precursors interact with translocation components in both the outer and inner envelope membranes during transport into chloroplasts. Using co-immunoprecipitation techniques, with antibodies specific to known translocation components, we identified stable interactions between precursor proteins and their associated membrane translocation components in detergent-solubilized chloroplastic membrane fractions. Antibodies specific to the outer envelope translocation components OEP75 and OEP34, the inner envelope translocation component IEP110 and the stromal Hsp100, ClpC, specifically co-immunoprecipitated precursor proteins under limiting ATP conditions, a stage we have called docking. A portion of these same translocation components was co-immunoprecipitated as a complex, and could also be detected by co-sedimentation through a sucrose density gradient. ClpC was observed only in complexes with those precursors utilizing the general import apparatus, and its interaction with precursor-containing translocation complexes was destabilized by ATP. Finally, ClpC was co-immunoprecipitated with a portion of the translocation components of both outer and inner envelope membranes, even in the absence of added precursors. We discuss possible roles for stromal Hsp100 in protein import and mechanisms of precursor binding in chloroplasts.     

A permease-like protein involved in ER to thylakoid lipid transfer in Arabidopsis

In eukaryotes, enzymes of different subcellular compartments participate in the assembly of membrane lipids. As a consequence, interorganelle lipid transfer is extensive in growing cells. A prominent example is the transfer of membrane lipid precursors between the endoplasmic reticulum (ER) and the photosynthetic thylakoid membranes in plants. Mono- and digalactolipids are typical photosynthetic membrane lipids. In Arabidopsis, they are derived from one of two pathways, either synthesized de novo in the plastid, or precursors are imported from the ER, giving rise to distinct molecular species. Employing a high-throughput robotic screening procedure generating arrays of spot chromatograms, mutants of Arabidopsis were isolated, which accumulated unusual trigalactolipids. In one allelic mutant subclass, trigalactosyldiacylglycerol1, the primary defect caused a disruption in the biosynthesis of ER-derived thylakoid lipids. Secondarily, a processive galactosyltransferase was activated, leading to the accumulation of oligogalactolipids. Mutations in a permease-like protein of the outer chloroplastic envelope are responsible for the primary biochemical defect. It is proposed that this protein is part of a lipid transfer complex.     

Isolation,characterization, and cDNA sequence of a carotenoid binding protein from the silk gland of Bombyx mori larvae

A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas alpha-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the B. mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.     

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.     

Reconstitution of electrogenic function in isolated pigment-protein complexes of Anabaena variabilis

Treatment of Anabaena variabilis membranes with lauryldimethylamine N-oxide yielded two fractions of pigment-protein complexes which were separable by gel filtration on Sepharose 6B. A green fraction was characterized which had a maximum of the chlorophyll long-wave absorption band at 678 nm and a small amount of carotenoid. In this fraction, Photosystem I activity was higher than in another (brownish-green) fraction which had a maximum of the chlorophyll absorption band at 673 nm and which was enriched in carotenoids. Similarly to isolated membranes, proteoliposomes containing pigment-protein complexes took up tetraphenylborate anions and tetraphenylphosphonium cations and were found to be capable of light-dependent membrane potential generation, when associated with a planar phospholipid membrane in the presence of reduced phenazine methosulfate upon illumination. The spatial arrangement of the pigment-protein complexes in the native and artificial membranes is discussed.