The Biochemical Machinery of Plastid Envelope Membranes

Abstract

Plastids are semiautonomous organelles with a wide structural and functional diversity and unique biochemical pathways. As such, they are able to transcribe and translate the information present in their own genome but are strongly dependent on imported proteins that are encoded in the nuclear genome and translated in the cytoplasm. Plastids are present in every plant cell, with very few exceptions (such as the highly specialized male sexual cells), and their structural and functional diversity reflects their role in different cell types. According to their developmental stage, we distinguish them as juvenile (proplastids), differentiating, mature, and senescent. Meristematic cells contain proplastids, which ensure the continuity of plastids from generation to generation and are capable of considerable structural and metabolic plasticity to develop into various types of plastids that remain interconvertible. When leaves are grown in darkness, proplastids differentiate into etioplasts, which can be converted into chloroplasts under illumination. The metabolism of these various types of plastids is linked to the function of the tissue in which they are found. For instance, whereas the chief function of illuminated leaves is the assimilation of CO2 by chloroplasts, root plastids are mainly involved in the assimilation of inorganic nitrogen. Amyloplasts, which contain large starch grains, behave as storage reservoirs in stems, roots, and tubers. Chromoplasts synthesize large amounts of carotenoids and are present in petals, fruits, and even roots. The interconversions between these different plastids are accompanied by dramatic changes, including the development or regression of internal membrane systems (e.g. thylakoids and prolamellar bodies) and the acquisition of specific enzymatic equipment reflecting specialized metabolism. However, at all stages of these transformations, the two limiting envelope membranes remain apparently unchanged. Located at the interface between plastids and the surrounding cytosol, the envelope is a key structure for the integration of plastid metabolism within the cell. Because plastids are semiautonomous organelles, a tight coordination between plastidial development and cell differentiation is required. Envelope membranes are an essential checkpoint between the expression of plastidial and nuclear genomes, for example, as the site for the specific recognition and transport of the precursor plastid proteins synthesized on cytosolic ribosomes. Plastid membranes contain an astonishing variety of specific lipids, including polar lipids (e.g. galactolipids, phospholipids, and SLs), pigments (e.g. carotenoids and chlorophylls), and prenylquinones (e.g. plastoquinone and tocopherols). This diversity requires complex metabolic pathways that are closely associated with envelope membranes. Functions of plastid envelope membranes. Plastids rely mostly on the nucleus for their development, and the coordination between the expression of plastid and nuclear genes requires an exchange of information between the nucleus and the organelle. Envelope membranes at the border between plastids and the cytosol play a role in this coordination at least at two levels, by interacting with the plastid translation and transcription apparatus, and through the import of nuclear-encoded proteins. In plastids cpDNA exists as large protein-DNA complexes called nucleoids, which are associated with the translation machinery. Although the replication and transcription of cpDNA are probably regulated within the plastid nucleoids, little is known about how this is accomplished or about nucleoid structure. The morphology of plastid nucleoids is subject to dynamic changes during plastid development (Sato et al., 1997). Proplastids contain a single, centrally located nucleoid. In developing plastids nucleoids move to the periphery, apparently associated with envelope membranes, and are extensively replicated. Conversely, in mature chloroplasts plastid nucleoids are dispersed within the organelle as small particles associated with thylakoids. As leaves undergo senescence, the number of plastid nucleoids and the copy number of cpDNA decrease. The inner envelope membrane of developing plastids contains a DNA-binding protein, PEND (plastid envelopeDNA), which binds to several specific regions of cpDNA (Sato et al., 1993). The cDNA for PEND protein was cloned and the corresponding protein was purified and characterized. This protein contains a bZIP domain, a sextuplet repeat region, and a putative membrane-spanning region (Sato et al., 1998). Its expression is restricted to the early stages of plastid development; the PEND transcript was detected in leaf buds of 6-d-old pea seedlings but not in older ones. This is consistent with the observation that the PEND protein, as detected by its DNA-binding activity, was maximal in the envelope membranes of young plastids from 5- or 6-d-old pea seedlings (Sato et al., 1993). This suggests that PEND protein is involved in binding plastid nucleoids to the inner envelope membrane, which might affect the processes of replication, segregation, and/or transcription of cpDNA. Another protein, topoisomerase II, which is required for decatenating plastid DNA molecules after division, is also localized in the vicinity of plastid envelope membranes (Marisson and Leech, 1992). Two observations suggest that envelope membranes could be involved in the regulation of chloroplast gene expression. First, a yellow membrane fraction derived from Chlamydomonas reinhardtii chloroplasts (most likely corresponding to envelope membranes) contain the stabilizing factors of some specific chloroplast mRNAs encoding thylakoid membrane proteins (Zerges and Rochaix, 1998). Second,Rolland et al. (1997) characterized a chloroplast protein homologous to a prokaryotic ribosome-recycling factor. In Escherichia colithis factor is essential to the termination of protein synthesis. A small proportion of this protein was found in envelope membranes, suggesting that it could be involved in the regulation of chloroplast mRNA translation. The import of nuclear-encoded plastid proteins across envelope membranes is another major aspect of the coordination of chloroplast and nuclear genome expression. The characterization of putative constituents involved in the envelope-import machinery is presently the most active field in envelope research. The reader is referred to a recent review by Heins et al. (1998) for a detailed presentation of the envelope-translocation apparatus and its components. It is essential to determine whether all of these components actually play a role in vivo, for example, by using genetic approaches. Finally, the evolution of the envelope-import machinery during plastid development remains to be elucidated. The development of fully functional plastids depends on a complex set of envelope enzymes for the biosynthesis of specific lipid constituents of plastid membranes. Characterized by a unique glycerolipid composition (for review, see Douce and Joyard, 1996), membranes from all plastid types differ strikingly from other plant cell membranes: they contain large amounts of SLs and galactolipids (MGDG and DGDG) and few phospholipids, mostly PG. In chloroplasts this phospholipid is unique because it contains a 16:1t(trans-Δ3-hexadecenoic acid) fatty acid at thesn-2 position of the glycerol backbone. PC is present only in the cytosolic leaflet of the outer envelope membrane and phosphatidylethanolamine cannot be detected in highly purified plastid membranes, including the outer envelope membrane. In contrast, these two phospholipids are major constituents of all extraplastidial membranes. The inner envelope membrane and the thylakoids have a very similar glycerolipid composition. Total (outer plus inner) envelope membranes from proplastids, etioplasts, and chloroplasts have an almost identical lipid composition. The lipids in the outer leaflet of the outer envelope membrane appear to have some physiological importance: their specific interaction with transit peptides affects both targeting and translocation of the precursor proteins to plastids (for review, seeHeins et al., 1998). Galactolipids, especially MGDG, are characterized by a very high content of polyunsaturated fatty acids, mostly 18:3 (linolinic acid) and, to a lesser extent, 16:3 (hexadecatrienoic acid). Plant MGDG consists of two main molecular species: the first (and major) one has 18:3 at both the sn-1 and sn-2 positions of the glycerol backbone (18:3/18:3), and the second has 18:3 at thesn-1 position and 16:3 exclusively at the sn-2 position of the glycerol (18:3/16:3). The former structure (C18/C18) is found in all eukaryotic lipids and is therefore called “eukaryotic.” The latter one is similar to that of cyanobacteria glycerolipids (i.e. with C16 fatty acid at the sn-2 position of the glycerol, C18/C16) and is called “prokaryotic.” Some plants, such as pea, contain MGDG with only the eukaryotic structure (18:3/18:3) and are therefore called “18:3” plants. Other plants, such as spinach, contain MGDG with 16:3 fatty acids and are therefore called “16:3” plants. This fatty acid is present only in prokaryotic MGDG; however, 16:3 plants usually contain both prokaryotic and eukaryotic MGDG, whereas a limited number of species (such asAnthriscus cerefolium) contain only prokaryotic MGDG. Most SL and plastid PG molecules also have a prokaryotic structure. Biosynthesis of MGDG in a spinach chloroplast. The biosynthesis of fatty acids (under the form of 16:0-acyl-carrier protein [ACP] and 18:1-ACP) occurs in the stroma (some are exported from plastids as acyl-CoA derivatives), whereas UDP-Gal andsn-glycerol-3-phosphate are made in the cytosol. Plastid envelope membranes contain the enzymes for the acylation ofsn-glycerol-3-phosphate to lyso-PA and PA, a phospholipid at the branch point between PG and glycolipid biosynthesis. In the spinach type of 16:3 plants, MGDG is made from diacylglycerol (DAG) synthesized in the envelope (prokaryotic pathway) or derived from extraplastidial glycerolipids (probably PC, which is made in the ER). Very little is known about the extraplastidial (eukaryotic) pathway and how/where PC is converted to DAG. In some 16:3 plants (such as A. cerefolium), only the prokaryotic pathway is active. In 18:3 plants such as pea, phosphatidate phosphatase, which converts PA into DAG, is apparently lacking and only the eukaryotic pathway seems to be functional. Very little is known about lipid transfer between membranes, i.e. DAG transfer to the inner envelope membrane, and about the transfer of newly synthesized glycerolipids such as MGDG to the outer envelope membrane and to thylakoids. Two acyltransferases catalyze the biosynthesis of PA containing 18:1 (oleic acid) and 16:0 (palmitic acid) at the sn-1 andsn-2 positions, respectively, of the glycerol moiety, i.e. with the prokaryotic structure. Therefore, the diacylglycerol synthesized from PA by the envelope phosphatidate phosphatase has the same structure. The acyltransferase responsible for lyso-PA biosynthesis is a soluble enzyme and is the only one of these enzymes for which biochemical and molecular data (and Arabidopsis mutants) are available. MGDG synthase has been characterized on a functional basis (for review, see Douce and Joyard, 1996) and on a molecular basis (Shimojima et al., 1997). MGDG synthase competes with SL synthase for diacylglycerol but also discriminates among the different diacylglycerol molecules available in the membrane (Maréchal et al., 1994). Because MGDG synthase has a high affinity for the 18:2 (linoleate)/18:2 diacylglycerol, this eukaryotic substrate can be used by the enzyme in addition to the prokaryotic 18:1/16:0 diacylglycerol synthesized directly within the inner envelope membrane (Maréchal et al., 1994). The kinetic properties of the envelope MGDG synthase could explain the synthesis of prokaryotic and eukaryotic MGDG molecular species. Finally, MGDG synthase is a very minor protein—about 1/1000 of the envelope proteins, which is surprising considering that MGDG is the most abundant membrane lipid on earth. DGDG synthesis is poorly understood. MGDG and DGDG have different fatty acid compositions, and therefore most MGDG molecules cannot be incorporated directly into DGDG. The only envelope enzyme that synthesizes DGDG is a galactolipid:galactolipid galactosyltransferase that is unable to discriminate (at least in vitro) between MGDG molecular species. This enzyme is localized on the cytosolic face of the outer envelope membrane, and this topographic localization raises questions about its physiological role. Analysis of an Arabidopsis mutant (dgd1) showing a reduced DGDG content suggests that the galactolipid:galactolipid galactosyltransferase is not the main route for DGDG synthesis (Dörmann et al., 1995). Chloroplasts from 18:3 plants are apparently devoid of phosphatidate phosphatase activity. Therefore, in these plants the Kornberg-Pricer pathway in the envelope is not functional and the deficiency in C18/C16 diacylglycerol synthesis is apparently compensated for by an increase in lipid synthesis in the extraplastidal compartments and by a transfer of lipids containing a C18/C18 diacylglycerol backbone to chloroplasts. The same situation probably occurs in an Arabidopsis mutant (act1) defective in acylation of glycerol-3-phosphate. In this mutation Arabidopsis, which is normally a 16:3 plant, is converted to an 18:3 plant. However, the mechanisms responsible for this high flexibility of lipid biosynthesis are poorly understood. A phospholipase C (not yet identified) probably generates diacylglycerol from PC, which is synthesized on the ER. Then diacylglycerol would be transferred from the outer to the inner envelope membrane, where MGDG synthesis takes place. The lipid-transfer mechanisms between membranes, from the ER to the envelope, between the two envelope membranes, or from the inner envelope membrane to thylakoids, are almost unknown. In plant cells most if not all 16:0, 18:0, and 18:1 fatty acids are synthesized within the plastid stroma. The mechanisms involved in fatty acid export from the stroma to the cytosol for phospholipid (e.g. PC and phosphatidylethanolamine) synthesis in the ER have not been analyzed, but the acyl-CoA synthetase localized on the outer envelope membrane is a good candidate for releasing fatty acids into the cytosolic compartment. Fatty acids of the newly synthesized polar lipids are desaturated to form the polyunsaturated molecular species characteristic of plastid glycerolipids. The characterization of Arabidopsis mutants impaired at five loci, which are named fad4 to fad8, sheds new light on chloroplast membrane desaturases (for review, see Miquel and Browse, 1998). The fad4 gene product is responsible for inserting a Δ3-trans double bond into the 16:0 fatty acid esterified to sn-2 of PG, and thefad5 gene product is responsible for the synthesis of Δ7-16:1 on MGDG and possibly on DGDG. The 16(18):1 desaturase is encoded by the fad6 gene, whereas two 16(18):2 desaturase isozymes are encoded by fad7 andfad8. In contrast, very little is known about the exact localization and biochemistry of these enzymes. A plastidial n-6 desaturase was purified from chloroplast envelope membranes and the corresponding cDNA obtained by Schmidt et al. (1994). Reduced Fd (E′0 = −0.4 V) has been proposed as the electron source for O2 reduction to H2O (E′0 = −0.8 V; for review, see Heinz, 1993), but unambiguous evidence for the involvement of reduced Fd is lacking. Since Fd delivers only one electron at a time, the desaturase must sequentially oxidize two reduced Fd molecules and store the first electron before the double bond is formed. This is possible only if electron-transfer chains exist in envelopes. Some putative components were characterized in spinach chloroplast envelope membranes: (a) semiquinone and flavosemiquinone radicals, (b) a series of iron-sulfur electron-transfer centers, and (c) flavins (mostly FAD) loosely associated with proteins (Jäger-Vottero et al., 1997). Therefore, envelope membranes probably contain all of the enzymatic constituents of a redox essential for fatty acid including electron involved in the and reduction of semiquinone and is the most in thylakoids. In contrast, envelope membranes are devoid of but contain and which are in thylakoids (for review, see Douce and Joyard, The major in envelope membranes is whereas and in thylakoids. The major is in envelope membranes and in thylakoids. In envelope membranes molecules such as and probably have and stabilizing to the of membranes under the that within a chloroplast. molecules have very different but all at least in from a precursor synthesized from a pathway et al., 1997). of the biosynthesis of in chloroplasts. Envelope membranes play a key role in the biosynthesis of the main plastid and which is made from from which is made from and is the precursor for and and for the chains of and Envelope membranes are also the site for the biosynthesis of which is made from and of acid which is made from Plastid are also the for several molecules involved in i.e. from from and The of the the of the enzymes or for for envelope, and for thylakoids. Some pathways are not yet located in plastids and are by Plastid constituents are in whereas molecules involved in are by is a by of two It is the precursor for all desaturated and biosynthesis and its regulation are not as characterized in chloroplasts as they are in and (and also are good with which to enzymes (for review, see 1994). evidence exists for the of envelope membranes in biosynthesis. Proplastids or contain and their envelope membranes only membrane in these could be a site of synthesis. In membranes, which are to from the inner envelope membrane, are very active in biosynthesis. Finally, synthase and desaturase and were in envelope membranes from spinach chloroplasts. The gene encoding from has been cloned et al., and contain a whereas contains a In chloroplasts the inner envelope membrane is the site of and synthesis. are synthesized by of acid and a to form which is converted to or and, to or by a series of and is also synthesized in the inner envelope membrane by of acid and to form which is and to form and et al. proposed that plastoquinone and biosynthesis was localized in membranes and that a specific transport was required for plastoquinone and transfer to chloroplasts and However, the characterization of a nuclear-encoded the in biosynthesis and localized in the inner membrane of plant and Joyard, not this have (for review, see Douce and Joyard, 1996) that both of the (i.e. and the are made in envelope membranes However, the addition of to a by is associated with thylakoids. et al. (1998) that activity, which the of was regulated by its The of from the stroma to envelope membranes, where it is is regulated by the increase of into the stroma under illumination. Two other enzymes of the i.e. et al., and et al., are localized in the envelope and by the and of into The import of the precursor for into chloroplasts requires et al., 1995). et al. on the of plant pigments in characterized proposed a pathway for in plastids and characterized a series of enzymes (e.g. and in the inner envelope membrane. Because can its in envelope membranes can be as a of for the of Therefore, enzymes involved in synthesis and are present in the same membrane but at different stages of plastid the of the envelope biochemical machinery during plastid are a of and (for review, see 1993). other plant are not but are synthesized and in to Some are their The biosynthesis of can be as a lipid that is under and that chloroplast envelope membranes synthesize the derived from of polyunsaturated fatty a these highly molecules are into active (a) and corresponding to the of a (b) that were from synthesized by a (c) synthesized by a that has not yet been and from the of a The same metabolism was in envelope membranes from plastids of buds and Joyard, of these was detected in the stroma or in the thylakoids. Envelope membranes therefore play a role in the of active (and especially of the precursor for and this that of biosynthesis of molecules is essential for their The envelope enzymes are for plant development regulation and to Arabidopsis mutants devoid of fatty acids were unable to from 18:3 and as a not and were highly to (for review, see Miquel and Browse, 1998). Plastids are a source of For example, recent observations of reinhardtii mutants suggest that some or its but not or can the pathway the expression of some nuclear genes et al., 1997). In some major plant (e.g. and from synthesized within chloroplasts for review, see et al., 1997). The involvement of envelope membranes to be to how in the regulation of is a product of (e.g. and The biochemical of the and Arabidopsis mutants have been (for review, see et al., 1998). The gene at of Arabidopsis was and characterized in to et al., The mutant in Arabidopsis is impaired in the of from to biosynthesis is a first plastids to and extraplastidial compartments is converted into This suggests that envelope membranes are involved at some in biosynthesis (for review, see et al., 1997). Envelope membranes and the regulation of In addition to the that the cytosolic for that are during the inner envelope membrane contains a that inorganic into chloroplasts This protein is encoded by a chloroplast gene In leaves the chloroplast envelope contains a that for the biosynthesis of acid In chloroplasts a similar is probably involved in the export of for to by cytosolic In leaves the interaction between and metabolism two of the inner envelope membrane, a that into chloroplasts for the synthase essential for the of that from reduction or and a that data are available for several of these in and For et al. (1998) the acid from obtained from a wide diversity of plastids and and encoding types of and have only but highly especially in the membrane-spanning In addition to these envelope membranes contain a series of proteins, and other et al., et al., et al., et al., essential for the functional integration of plastids within Most of these proteins have been characterized in little is known about For example, et al. found that the outer envelope membrane of plastids contains a highly homologous to that of but different from the chloroplast that remains to be Finally, the involvement of envelope membranes in the regulation of metabolism is probably complex et al. (1997) the gene an inner envelope in reinhardtii and the of the with cells or chloroplasts that the affects both CO2 and suggesting the of a that inorganic into chloroplasts for this observation is that is associated with a in the chloroplast envelope involved in of both and CO2 into the chloroplast. However, another is that the on inorganic in the mutant could only be might be involved in regulation and could be associated with of the chloroplast envelope redox (Jäger-Vottero et al., that be involved in and in the export of to the cytosol. not which is but the of CO2 across envelope membranes is and is not to the of CO2 by chloroplasts. The characterization of these proteins in plastid envelope membranes the flexibility of plastid metabolism in reflecting that of the various in which they are found. of envelope membrane from spinach chloroplasts by a the of the of envelope membranes, the that it only a of the envelope (a) envelope proteins with are not in this envelope have and (b) the most envelope proteins not into these from et al. DNA acid a fatty containing and double