Strategies to identify transport systems in plants.

Since the first molecular structures of plant transporters were discovered over a decade ago, considerable advances have been made in the study of plant membrane transport, but we still do not understand transport regulation. The genes encoding the transport systems in the various cell membranes are still to be identified, as are the physiological roles of most transport systems. A wide variety of complementary strategies are now available to study transport systems in plants, including forward and reverse genetics, proteomics, and in silico exploitation of the huge amount of information contained in the completely known genomic sequence of Arabidopsis.

Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana.

In Arabidopsis, monogalactosyldiacylglycerol (MGDG) is synthesized by a multigenic family of MGDG synthases consisting of two types of enzymes differing in their N-terminal portion: type A (atMGD1) and type B (atMGD2 and atMGD3). The present paper compares type B isoforms with the enzymes of type A that are known to sit in the inner membrane of plastid envelope. The occurrence of types A and B in 16:3 and 18:3 plants shows that both types are not specialized isoforms for the prokaryotic and eukaryotic glycerolipid biosynthetic pathways. Type A atMGD1 gene is abundantly expressed in green tissues and along plant development and encodes the most active enzyme. Its mature polypeptide is immunodetected in the envelope of chloroplasts from Arabidopsis leaves after cleavage of its transit peptide. atMGD1 is therefore likely devoted to the massive production of MGDG required to expand the inner envelope membrane and build up the thylakoids network. Transient expression of green fluorescent protein fusions in Arabidopsis leaves and in vitro import experiments show that type B precursors are targeted to plastids, owing to a different mechanism. Noncanonical addressing peptides, whose processing could not be assessed, are involved in the targeting of type B precursors, possibly to the outer envelope membrane where they might contribute to membrane expansion. Expression of type B enzymes was higher in nongreen tissues, i.e., in inflorescence (atMGD2) and roots (atMGD3), where they conceivably influence the eukaryotic structure prominence in MGDG. In addition, their expression of type B enzymes is enhanced under phosphate deprivation.

Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins.

As a complementary approach to genome projects, proteomic analyses have been set up to identify new gene products. One of the major challenges in proteomics concerns membrane proteins, especially the minor ones. A procedure based on the differential extraction of membrane proteins in chloroform/methanol mixtures, was tested on the two different chloroplast membrane systems: envolope and thylakoid membranes. Combining the use of classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses, this procedure enabled identification of hydrophobic proteins. The propensity of hydrophobic proteins to partition in chloroform/methanol mixtures was directly correlated with the number of amino acid residues/number of putative transmembrane regions (Res/TM ratio). Regardless of the particular case of some lipid-interacting proteins, chloroform/methanol extractions allowed enrichment of hydrophobic proteins and exclusion of hydrophilic proteins from both membrane systems, thus demonstrating the versatility of the procedure.

Sulfolipid is a potential candidate for annexin binding to the outer surface of chloroplast.

Using a subcellular-specific proteomic approach, we have identified by protein microsequencing, a putative 35-kDa annexin from among the chloroplast envelope polypeptides. To confirm this identification, we demonstrate that (a) a 35-kDa protein, identified as annexin by antibody cross-reactivity, co-purifies with Percoll-purified chloroplasts and their envelope membranes when extracted in the presence of Ca(2+) and (b) the native spinach annexin protein binds to chloroplast-specific lipids in a Ca(2+)-dependent manner. The binding of the spinach annexin to these glycerolipids occurs at similar Ca(2+) concentrations as those, which promote the interaction of annexins to phospholipids in other membranes. Among chloroplast glycerolipids known to be accessible on the cytosolic face (outer leaflet) of the outer envelope membrane, sulfolipid, and probably phosphatidylinositol, would be the sole candidates for a putative Ca(2+)-dependent interaction of annexin with the chloroplast surface.

The multigenic family of monogalactosyl diacylglycerol synthases.

Because the synthesis of monogalactosyldiacylglycerol (MGDG) is unique to plants, identified as an important marker of the plastid envelope, involved in a key step of plastid biogenesis and is the most abundant lipid on earth, MGDG synthase activity was extensively analysed at the biochemical and physiological levels. In the present paper, we present our current knowledge on the MGDG synthase's function, structure and topology in envelope membranes, and discuss possible roles in plant cell glycerolipid metabolism. The recent discovery of a multigenic family of MGDG synthases raised the possibility that multiple isoenzymes might carry out MGDG synthesis in various tissues and developmental stages.

Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG.

MGDG synthase, the enzyme that catalyzes the synthesis of the major chloroplast membrane lipid monogalactosyldiacylglycerol (MGDG), is encoded by a multigenic family. We have analyzed the biochemical properties, subcellular localization and membrane topology of a spinach chloroplast MGDG synthase, a representative member of the type A family from Spinacia oleracea (soMGD A), using a recombinant protein that was functionally overexpressed in Escherichia coli and specific polyclonal antibodies. We demonstrated that soMGD A could catalyze the synthesis of both 'prokaryotic' and 'eukaryotic' MGDG molecular species in vitro, with a selectivity for diacylglycerol similar to that of purified chloroplast envelope MGDG synthase activity. Furthermore, soMGD A was shown to be sensitive to chemical reagents (dithiothreitol, N-ethylmaleimide and o-phenanthroline) known to affect MGDG synthesis by the partially purified enzyme, as well as in isolated chloroplast envelope membranes. In spinach chloroplasts, soMGD A was localized by Western blot analysis in the inner envelope membrane. Topological studies demonstrated that soMGD A is a monotopic enzyme, embedded within one leaflet of the inner envelope membrane from spinach chloroplasts, a structure which may involve amphipathic alpha helices. We further demonstrated that in vitro, soMGD A precursor is imported and processed to its correct mature form in intact chloroplasts. These results show that soMGD A corresponds to a mature polypeptide of approximately 45 kDa. In addition, inactivation kinetics after gamma-ray irradiation strongly suggest that both native chloroplast envelope MGDG synthase and recombinant soMGD A have a functional molecular mass of 95-100 kDa, indicating that they are probably active as homodimers made of two 45-kDa subunits. This study suggests that, in spite of the growing evidence that MGDG synthesis is catalyzed by a multigenic family of enzymes, in spinach leaves both prokaryotic and eukaryotic MGDG syntheses could be attributable to a unique dimeric enzyme, provided that diacylglycerol is transported from the outer membrane to the inner membrane of the chloroplast envelope.

Technical Advance: Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins.

Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane alpha-helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking.

Do plastid envelope membranes play a role in the expression of the plastid genome?

A unique biochemical machinery is present within the two envelope membranes surrounding plastids (Joyard et al., Plant Physiol. 118 (1998) 715-723) that reflects the stage of development of the plastid and the specific metabolic requirements of the various tissues. Envelope membranes are the site for the synthesis and metabolism of specific lipids. They are also the site of transport of metabolites, proteins and information between plastids and surrounding cellular compartments. For instance, a complex machinery for the import of nuclear-encoded plastid proteins is rapidly being elucidated. The functional studies of plastid envelope membranes result in the characterization of an increasing number of envelope proteins with unexpected functions. For instance, recent experiments have demonstrated that envelope membranes bind specifically to plastid genetic systems, the nucleoids surrounded by plastid ribosomes. At early stages of plastid differentiation, the inner envelope membrane contains a unique protein (named PEND protein) that binds specifically to plastid DNA. This tight connection suggests that the PEND protein is at least involved in partitioning the plastid DNA to daughter plastids during division. The PEND protein can also provide a physical support for replication and transcription. In addition, factors involved in the control of plastid protein synthesis can become associated to envelope membranes. This was shown for a protein homologous to the E. coli ribosome recycling factor and for the stabilizing factors of some specific chloroplast mRNAs encoding thylakoid membrane proteins. In fact, the envelope membranes together with the plastid DNA are the two essential constituents of plastids that confer identity to plastids and their interactions are becoming uncovered through molecular as well as cytological studies. In this review, we will focus on these recent observations (which are consistent with the endosymbiotic origin of plastids) and we discuss possible roles for the plastid envelope in the expression of plastid genome.

Plant ribosome recycling factor homologue is a chloroplastic protein and is bactericidal in escherichia coli carrying temperature-sensitive ribosome recycling factor.

We have isolated a protein, mature RRFHCP, from chloroplasts of spinach (Spinacia oleracea L.) that shows 46% sequence identity and 66% sequence homology with ribosome recycling factor (RRF) of Escherichia coli. RRF recycles ribosomes through disassembly of the posttermination complex. From the cDNA analysis and from the amino-terminal sequencing of the isolated protein, the mature RRFHCP was deduced to have a Mr of 21,838 with 193 aa. It lacks the 78-aa chloroplast targeting sequence encoded by the RRFHCP cDNA sequence. The RRFHCP synthesized in vitro was imported into isolated chloroplasts with simultaneous conversion to the mature RRFHCP. Transcription of the gene coding for RRFHCP was not dependent on light, yet it was limited mostly to photosynthetic tissues in which only one transcript size was detected. Mature RRFHCP exerted a bactericidal effect on E. coli carrying temperature-sensitive RRF at the permissive temperature whereas wild-type E. coli was not affected.

Molecular characterization of the PEND protein, a novel bZIP protein present in the envelope membrane that is the site of nucleoid replication in developing plastids.

Plastid nucleoids are known to bind to the envelope membrane in developing chloroplasts. Here, plastid DNA is extensively replicated. We previously detected a DNA binding protein in the inner envelope membranes of developing plastids in pea and named it PEND (for plastid envelope DNA binding) protein. In this study, we report on the structure and molecular characterization of a cDNA for the PEND protein. As a result of screening cDNA libraries in lambdagt11 with one of the target sequences of the PEND protein as a probe, we obtained a clone (PD2) for a novel DNA binding protein consisting of 633 amino acid residues. Analysis of the N-terminal sequence of the purified PEND protein indicated that the transit peptide is just 16 residues long. The PEND protein was detected specifically in the plastid envelope membrane of young unopened leaf buds by immunoblot analysis. The PEND protein consists of a basic region plus zipper region, an unprecedented sextuple repeat region, and a putative membrane-spanning region. The basic region with a zipper region seems to have diverged from that of other plant transcription factors. In addition, the PEND protein could be a distant homolog of the trans-Golgi network integral membrane proteins. The PEND protein is therefore a novel type of DNA binding protein that binds to the membrane as an intrinsic membrane protein.

Cloning and functional expression of AtCOQ3, the Arabidopsis homologue of the yeast COQ3 gene, encoding a methyltransferase from plant mitochondria involved in ubiquinone biosynthesis.

A mutant of Saccharomyces cerevisiae deleted for the COQ3 gene was constructed. COQ3 encodes a 3,4-dihydroxy-5-hexaprenylbenzoate (DHHB) methyltransferase that catalyses the fourth step in the biosynthesis of ubiquinone from p-hydroxybenzoic acid. A full length cDNA encoding a homologue of DHHB-methyltransferase was cloned from an Arabidopsis thaliana cDNA library by functional complementation of a yeast coq3 deletion mutant. The Arabidopsis thaliana cDNA (AtCOQ3) was able to restore the respiration ability and ubiquinone synthesis of the mutant. The product of the 1372 bp cDNA contained 322 amino acids and had a molecular mass of 35,360 Da. The predicted amino acid sequence contained all consensus regions for S-adenosyl methionine methyltransferases and presented 26% identity with Saccharomyces cerevisiae DHHB-methyltransferase and 38% identity with the rat protein, as well as with a bacterial (Escherichia coli and Salmonella typhimurium) methyltransferase encoded by the UBIG gene. Southern analysis showed that the Arabidopsis thaliana enzyme was encoded by a single nuclear gene. The NH2-terminal part of the cDNA product contained features consistent with a putative mitochondrial transit sequence. The cDNA in Escherichia coli was overexpressed and antibodies were raised against the recombinant protein. Western blot analysis of Arabidopsis thaliana and pea protein extracts indicated that the AtCOQ3 gene product is localized within mitochondrial membranes. This result suggests that at least this step of ubiquinone synthesis takes place in mitochondria.

Redox chains in chloroplast envelope membranes: spectroscopic evidence for the presence of electron carriers, including iron-sulfur centers.

We have shown that envelope membranes from spinach chloroplasts contain (i) semiquinone and flavosemiquinone radicals, (ii) a series of iron-containing electron-transfer centers, and (iii) flavins (mostly FAD) loosely associated with proteins. In contrast, we were unable to detect any cytochrome in spinach chloroplast envelope membranes. In addition to a high spin [1Fe]3+ type protein associated with an EPR signal at g = 4.3, we observed two iron-sulfur centers, a [4Fe-4S]1+ and a [2Fe-2S]1+, associated with features, respectively, at g = 1.921 and g = 1.935, which were detected after reduction by NADPH and NADH, respectively. The [4Fe-4S] center, but not the [2Fe-2S] center, was also reduced by dithionite or 5-deazaflavin/oxalate. An unusual Fe-S center, named X, associated with a signal at g = 2.057, was also detected, which was reduced by dithionite but not by NADH or NADPH. Extremely fast spin-relaxation rates of flavin- and quinone-free radicals suggest their close proximity to the [4Fe-4S] cluster or the high-spin [1Fe]3+ center. Envelope membranes probably contain enzymatic activities involved in the formation and reduction of semiquinone radicals (quinol oxidase, NADPH-quinone, and NADPH-semiquinone reductases). The physiological significance of our results is discussed with respect to (i) the presence of desaturase activities in envelope membranes and (ii) the mechanisms involved in the export of protons to the cytosol, which partially regulate the stromal pH during photosynthesis. The characterization of such a wide variety of electron carriers in envelope membranes opens new fields of research on the functions of this membrane system within the plant cell.

Disruption of the plastid ycf10 open reading frame affects uptake of inorganic carbon in the chloroplast of Chlamydomonas.

The product of the chloroplast ycf10 gene has been localized in the inner chloroplast envelope membrane (Sasaki et al., 1993) and found to display sequence homology with the cyanobacterial CotA product which is altered in mutants defective in CO2 transport and proton extrusion (Katoh et al., 1996a,b). In Chlamydomonas reinhardtii, ycf10, located between the psbI and atpH genes, encodes a putative hydrophobic protein of 500 residues, which is considerably larger than its higher plant homologue because of a long insertion that separates the conserved N and C termini. Using biolistic transformation, we have disrupted ycf10 with the chloroplast aadA expression cassette and examined the phenotype of the homoplasmic transformants. These were found to grow both photoheterotrophically and photoautotrophically under low light, thereby revealing that the Ycf10 product is not essential for the photosynthetic reactions. However, under high light these transformants did not grow photoautotrophically and barely photoheterotrophically. The increased light sensitivity of the transformants appears to result from a limitation in photochemical energy utilization and/or dissipation which correlates with a greatly diminished photosynthetic response to exogenous (CO2 + HCO3-), especially under conditions where the chloroplast inorganic carbon transport system is not induced. Mass spectrometric measurements with either whole cells or isolated chloroplasts from the transformants revealed that the CO2 and HCO3- uptake systems have a reduced affinity for their substrates. The results suggest the existence of a ycf10-dependent system within the plastid envelope which promotes efficient inorganic carbon (Ci) uptake into chloroplasts.

Is E37, a major polypeptide of the inner membrane from plastid envelope, an S-adenosyl methionine-dependent methyltransferase?

Using antibodies raised against E37, one of the major polypeptides of the inner membrane from the chloroplast envelope, it has been demonstrated that a single immunologically related polypeptide was present in total protein extracts from various higher plants (monocots and dicots), in photosynthetic and non-photosynthetic tissues from young spinach plantlets, as well as in the cytoplasmic membrane from the cyanobacteria Synechococcus. This ubiquitous distribution of E37 strongly suggests that this protein plays an envelope-specific function common to all types of plastids. Comparison of tobacco and spinach E37 amino acid sequences deduced from the corresponding cDNA demonstrates that consensus motifs for S-adenosyl methionine-dependent methyltransferases are located in both sequences. This hypothesis was confirmed using a biochemical approach. It was demonstrated that E37, together with two minor spinach chloroplast envelope polypeptides of 32 and 39 kDa, can be specifically photolabeled with [3H]-S-adenosyl methionine upon UV-irradiation. Identification of E37 as a photolabeled polypeptide was established by immunoprecipitation. Furthermore, photolabeling of the three envelope polypeptides was specifically inhibited by very low concentration of S-adenosyl homocysteine, thus providing evidence for the presence within these proteins of S-adenosyl methionine- and S-adenosyl homocysteine-binding sites that were closely associated. Taken as a whole these results strongly suggest that E37 is an ubiquitous plastid envelope protein that probably has an S-adenosyl methionine-dependent methyltransferase activity. The 32 and 39 kDa envelope polypeptides probably have a similar methyltransferase activity.

Envelope Membranes from Spinach Chloroplasts Are a Site of Metabolism of Fatty Acid Hydroperoxides.

Enzymes in envelope membranes from spinach (Spinacia oleracea L.) chloroplasts were found to catalyze the rapid breakdown of fatty acid hydroperoxides. In contrast, no such activities were detected in the stroma or in thylakoids. In preparations of envelope membranes, 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid, 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, or 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid were transformed at almost the same rates (1-2 [mu]mol min-1 mg-1 protein). The products formed were separated by reversed-phase high-pressure liquid chromatography and further characterized by gas chromatography-mass spectrometry. Fatty acid hydroperoxides were cleaved (a) into aldehydes and oxoacid fragments, corresponding to the functioning of a hydroperoxide lyase, (b) into ketols that were spontaneously formed from allene oxide synthesized by a hydroperoxide dehydratase, (c) into hydroxy compounds synthesized enzymatically by a system that has not yet been characterized, and (d) into oxoenes resulting from the hydroperoxidase activity of a lipoxygenase. Chloroplast envelope membranes therefore contain a whole set of enzymes that catalyze the synthesis of a variety of fatty acid derivatives, some of which may act as regulatory molecules. The results presented demonstrate a new role for the plastid envelope within the plant cell.

The catalytic site of monogalactosyldiacylglycerol synthase from spinach chloroplast envelope membranes. Biochemical analysis of the structure and of the metal content.

We have analyzed the structure of the active site of monogalactosyldiacylglycerol (MGDG) synthase from spinach chloroplast envelope. Since purification of this membrane-embedded enzyme yielded such low amounts of protein that analyses of the amino acid sequence were so far impossible, we used indirect strategies. Analyses of the inhibition of MGDG synthase by UDP and of its inactivation by citraconic anhydride first indicated that the enzyme contained two functionally independent and topologically distinct binding sites for each substrate. Whereas MGDG synthase binds both the nucleotidic part of UDP-Gal and the acyl chains of 1,2-diacylglycerol, UDP is a competitive inhibitor relatively to UDP-Gal, while it does not compete with 1,2-diacylglycerol for binding on the enzyme. The UDP-Gal-binding site contains lysine residues, as demonstrated for UDP-Gal-binding sites from all galactosyltransferases studied so far. Radiolabeling of MGDG synthase by sulfur labeling reagent, a 35S-labeled lysine-blocking reagent, confirmed that MGDG synthase was a polypeptide with a low molecular mass (around 20 kDa). The 1,2-diacylglycerol-binding site contains reduced cysteines and at least one metal. The divalent cation(s) associated to apo-MGDG synthase was not unambiguously identified, but the results suggest that it could be zinc. Therefore, MGDG synthase presents some structural features in common with diacylglycerol-manipulating enzymes, such as protein kinase C and 1,2-diacylglycerol kinase, which are characterized by the presence of a ubiquitous Cys6His2 domain involved in zinc coordination in their 1,2-diacylglycerol-binding domains.

The outer membrane protein E24 of spinach chloroplast envelope: cloning of a cDNA and topological insertion of the protein in the membrane.

A cDNA coding for the outer membrane protein E24 of spinach chloroplast envelope has been obtained by screening an expression library of spinach cDNA with polyclonal antibodies. Analysis of the protein sequence and comparison with the thermolysin susceptibility of E24 in the chloroplast in situ suggest that E24 is a transmembrane protein and show that its NH2 terminal end is located on the cytosolic side of chloroplasts.

Comparison of the kinetic properties of MGDG synthase in mixed micelles and in envelope membranes from spinach chloroplast.

We have applied the 'membrane partition' kinetic modelling approach proposed by Heirwegh et al. [(1988) Biochem. J. 254, 101-108] to MGDG synthase in isolated envelope vesicles. Comparison of the kinetic parameters obtained for MGDG synthase assayed in purified envelope membranes and in mixed-micelles demonstrates that the latter are relevant to the situation in envelope membranes and that MGDG synthase has a very high affinity for dilinoleoylglycerol. Our results provide additional evidence for the hypothesis that the high affinity of the envelope MGDG synthase for dilinoleoylglycerol could be responsible for the presence of C18 fatty acids at both the sn-1 and sn-2 position of the glycerol backbone in MGDG.

Localization of ferrochelatase activity within mature pea chloroplasts.

Precise localization within the chloroplast of the membrane-bound enzymes involved in heme and chlorophyll biosynthetic pathways is of central importance for better understanding the regulation of the carbon flow into these two pathways. In this study we examine the localization of ferrochelatase activity within mature pea chloroplasts. Our results provide evidence that chloroplast ferrochelatase is associated only with thylakoid membranes. The presence of ferrochelatase in chloroplast thylakoids emphasizes the role of this membrane system in chloroplast protoheme biosynthesis. Furthermore, these results raise the possibility that heme and chlorophyll biosynthesis are compartmentalized in two distinct membrane systems within mature chloroplasts.