Analysis of Acyl Fluxes through Multiple Pathways of Triacylglycerol Synthesis in Developing Soybean Embryos

The reactions leading to triacylglycerol (TAG) synthesis in oilseeds have been well characterized. However, quantitative analyses of acyl group and glycerol backbone fluxes that comprise extraplastidic phospholipid and TAG synthesis, including acyl editing and phosphatidylcholine-diacylglycerol interconversion, are lacking. To investigate these fluxes, we rapidly labeled developing soybean (Glycine max) embryos with [¹⁴C]acetate and [¹⁴C]glycerol. Cultured intact embryos that mimic in planta growth were used. The initial kinetics of newly synthesized acyl chain and glycerol backbone incorporation into phosphatidylcholine (PC), 1,2-sn-diacylglycerol (DAG), and TAG were analyzed along with their initial labeled molecular species and positional distributions. Almost 60% of the newly synthesized fatty acids first enter glycerolipids through PC acyl editing, largely at the sn-2 position. This flux, mostly of oleate, was over three times the flux of nascent [¹⁴C]fatty acids incorporated into the sn-1 and sn-2 positions of DAG through glycerol-3-phosphate acylation. Furthermore, the total flux for PC acyl editing, which includes both nascent and preexisting fatty acids, was estimated to be 1.5 to 5 times the flux of fatty acid synthesis. Thus, recycled acyl groups (16:0, 18:1, 18:2, and 18:3) in the acyl-coenzyme A pool provide most of the acyl chains for de novo glycerol-3-phosphate acylation. Our results also show kinetically distinct DAG pools. DAG used for TAG synthesis is mostly derived from PC, whereas de novo synthesized DAG is mostly used for PC synthesis. In addition, two kinetically distinct sn-3 acylations of DAG were observed, providing TAG molecular species enriched in saturated or polyunsaturated fatty acids.     

Biosynthesis and desaturation of prokaryotic galactolipids in leaves and isolated chloroplasts from spinach

Mono- and digalactosyldiacylglycerol (MGDG and DGDG) were isolated from the leaves of sixteen 16:3 plants. In all of these plant species, the sn-2 position of MGDG was more enriched in C₁₆ fatty acids than sn-2 of DGDG. The molar ratios of prokaryotic MGDG to prokaryotic DGDG ranged from 4 to 10. This suggests that 16:3 plants synthesize more prokaryotic MGDG than prokaryotic DGDG. In the 16:3 plant Spinacia oleracea L. (spinach), the formation of prokaryotic galactolipids was studied both in vivo and in vitro. In intact spinach leaves as well as in chloroplasts isolated from these leaves, radioactivity from [1-¹⁴C]acetate accumulated 10 times faster in MGDG than in DGDG. After 2 hours of incorporation, most labeled galactolipids from leaves and all labeled galactolipids from isolated chloroplasts were in the prokaryotic configuration. Both in vivo and in vitro, the desaturation of labeled palmitate and oleate to trienoic fatty acids was higher in MGDG than in DGDG. In leaves, palmitate at the sn-2 position was desaturated in MGDG but not in DGDG. In isolated chloroplasts, palmitate at sn-2 similarly was desaturated only in MGDG, but palmitate and oleate at the sn-1 position were desaturated in MGDG as well as in DGDG. Apparently, palmitate desaturase reacts with sn-1 palmitate in either galactolipid, but does not react with the sn-2 fatty acid of DGDG. These results demonstrate that isolated spinach chloroplasts can synthesize and desaturate prokaryotic MGDG and DGDG. The finally accumulating molecular species, MGDG(18:3/16:3) and DGDG(18:3/16:0), are made by the chloroplasts in proportions similar to those found in leaves.     

Cell Biology of Prokaryotic Organelles

Mounting evidence in recent years has challenged the dogma that prokaryotes are simple and undefined cells devoid of an organized subcellular architecture. In fact, proteins once thought to be the purely eukaryotic inventions, including relatives of actin and tubulin control prokaryotic cell shape, DNA segregation, and cytokinesis. Similarly, compartmentalization, commonly noted as a distinguishing feature of eukaryotic cells, is also prevalent in the prokaryotic world in the form of protein-bounded and lipid-bounded organelles. In this article we highlight some of these prokaryotic organelles and discuss the current knowledge on their ultrastructure and the molecular mechanisms of their biogenesis and maintenance.The emergence of eukaryotes in a world dominated by prokaryotes is one of the defining moments in the evolution of modern day organisms. Although it is clear that the central metabolic and information processing machineries of eukaryotes and prokaryotes share a common ancestry, the origins of the complex eukaryotic cell plan remain mysterious. Eukaryotic cells are typified by the presence of intracellular organelles that compartmentalize essential biochemical reactions whereas their prokaryotic counterparts generally lack such sophisticated subspecialization of the cytoplasmic space. In most cases, this textbook categorization of eukaryotes and prokaryotes holds true. However, decades of research have shown that a number of unique and diverse organelles can be found in the prokaryotic world raising the possibility that the ability to form organelles may have existed before the divergence of eukaryotes from prokaryotes (Shively 2006).Skeptical readers might wonder if a prokaryotic structure can really be defined as an organelle. Here we categorize any compartment bounded by a biological membrane with a dedicated biochemical function as an organelle. This simple and broad definition presents cells, be they eukaryotes or prokaryotes, with a similar set of challenges that need to be addressed to successfully build an intracellular compartment. First, an organism needs to mold a cellular membrane into a desired shape and size. Next, the compartment must be populated with the proper set of proteins that carry out the activity of the organelle. Finally, the cell must ensure the proper localization, maintenance and segregation of these compartments across the cell cycle. Eukaryotic cells perform these difficult mechanistic steps using dedicated molecular pathways. Thus, if connections exist between prokaryotic and eukaryotic organelles it seems likely that relatives of these molecules may be involved in the biogenesis and maintenance of prokaryotic organelles as well.Prokaryotic organelles can be generally divided into two major groups based on the composition of the membrane layer surrounding them. First are the cellular structures bounded by a nonunit membrane such a protein shell or a lipid monolayer (Shively 2006). Well-known examples of these compartments include lipid bodies, polyhydroxy butyrate granules, carboxysomes, and gas vacuoles. The second class consists of those organelles that are surrounded by a lipid-bilayer membrane, an arrangement that is reminiscent of the canonical organelles of the eukaryotic endomembrane system. Therefore, this article is dedicated to a detailed exploration of three prokaryotic lipid-bilayer bounded organelle systems: the magnetosomes of magnetotactic bacteria, photosynthetic membranes, and the internal membrane structures of the Planctomycetes. In each case, we present the most recent findings on the ultrastructure of these organelles and highlight the molecular mechanisms that control their formation, dynamics, and segregation. We also highlight some protein-bounded compartments to present the reader with a more complete view of prokaryotic compartmentalization.     

Plastid biogenesis, between light and shadows

Plastids are cellular organelles which originated when a photosynthetic prokaryote was engulfed by the eukaryotic ancestor of green and red algae and land plants. Plastids have diversified in plants from their original function as chloroplasts to fulfil a variety of other roles in metabolite biosynthesis and in storage, or purely to facilitate their own transmission, according to the cell type that harbours them. Therefore cellular development and plastid biogenesis pathways must be closely intertwined. Cell biological, biochemical, and genetic approaches have generated a large body of knowledge on a variety of plastid biogenesis processes. A brief overview of the components and functions of the plastid genetic machinery, the plastid division apparatus, and protein import to and targeting inside the organelle is presented here. However, key areas in which our knowledge is still surprisingly limited remain, and these are also discussed. Chloroplast-defective mutants suggest that a substantial number of important plastid biogenesis proteins are still unknown. Very little is known about how different plastid types differentiate, or about what mechanisms co-ordinate cell growth with plastid growth and division, in order to achieve what is, in photosynthetic cells, a largely constant cellular plastid complement. Further, it seems likely that major, separate plastid and chloroplast 'master switches' exist, as indicated by the co-ordinated gene expression of plastid or chloroplast-specific proteins. Recent insights into each of these developing areas are reviewed. Ultimately, this information should allow us to gain a systems-level understanding of the plastid-related elements of the networks of plant cellular development.     

Arachidonic acid pools of rat kidney cell nuclei

We have assessed that nuclear lipids from rat kidney cells are not only membrane components, but they are also found within the nucleus. The most abundant nuclear and endonuclear lipids have a high proportion of unsaturated fatty acids (n-6 series: arachidonic > linoleic), mainly esterified to PtdCho. Nuclear most abundant molecular species are 16:0–20:4, 16:0–18:2, 18:0–20:4, 18:0–18:2, and 16:0–18:1. Arachidonic acid is esterified at the sn-2 position of PtdCho: 16:0–20:4(25%), 18:0–20:4(15%), 18:2–20:4(3%), 18:1–20:4(2%). Exogenous [1-¹⁴C]20:4n-6-CoA is esterified in vitro in GP (glycerophospholipids) > > TAG and DAG. Five PtdCho molecular species were labeled: 16:0–20:4, 18:0–20:4, 18:1–20:4, 18:2–20:4, and 20:4–20:4. In conclusion, these results demonstrated that: (1) there is an important lipid pool within kidney cell nuclei; (2) main nuclear and endonuclear lipid pools were PtdCho molecular species which contained a high proportion of unsaturated fatty acids (20:4n-6 and 18:2n-6) esterified at sn-2 position and 16:0 esterified at sn-1 position; (3) kidney cell nuclei also contained the necessary enzymes to esterify exogenous 20:4n-6-CoA to glycerolipids and to GP; (4) exogenous 20:4n-6-CoA was esterified in five PtdCho molecular species with 20:4n-6 at the sn-2 position, although the most actively synthesized PtdCho contained 20:4n-6 at both the sn-1 and sn-2 positions of the molecule; (5) we can infer that by a remodeling process, the unsaturated fatty acids at the sn-1 position of PtdCho molecular species could be replaced by 16:0 and 18:0, and thus PtdCho would achieve the physiological profile characteristic of the organ.     

Studies on the Substrate and Stereo/Regioselectivity of Adipose Triglyceride Lipase,Hormone-sensitive Lipase,and Diacylglycerol-O-acyltransferases

Adipose triglyceride lipase (ATGL) is rate-limiting for the initial step of triacylglycerol (TAG) hydrolysis, generating diacylglycerol (DAG) and fatty acids. DAG exists in three stereochemical isoforms. Here we show that ATGL exhibits a strong preference for the hydrolysis of long-chain fatty acid esters at the sn-2 position of the glycerol backbone. The selectivity of ATGL broadens to the sn-1 position upon stimulation of the enzyme by its co-activator CGI-58. sn-1,3 DAG is the preferred substrate for the consecutive hydrolysis by hormone-sensitive lipase. Interestingly, diacylglycerol-O-acyltransferase 2, present at the endoplasmic reticulum and on lipid droplets, preferentially esterifies sn-1,3 DAG. This suggests that ATGL and diacylglycerol-O-acyltransferase 2 act coordinately in the hydrolysis/re-esterification cycle of TAGs on lipid droplets. Because ATGL preferentially generates sn-1,3 and sn-2,3, it suggests that TAG-derived DAG cannot directly enter phospholipid synthesis or activate protein kinase C without prior isomerization.     

Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

Background

Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope.

Methodology/Principal Findings

Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein.

Conclusions/Significance

Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA proteins to their proper intracellular locations in general.     

Plastid‐associated polyamines: their role in differentiation,structure, functioning,stress response and senescence

Polyamines are low‐molecular weight biogenic amines. They are a specific group of cell growth and development regulators. In the past decade biochemical, molecular and genetic studies have contributed much to a better understanding of the biological role of polyamines in the plant cell. Substantial evidence has also been added to our understanding of the role of polyamines in plastid development. In developing chloroplasts, polyamines serve as a nitrogen source for protein and chlorophyll synthesis. In chloroplast structure, thylakoid proteins linked to polyamines belong mainly to antenna proteins of light‐harvesting chlorophyll a/b–protein complexes. The fact that LHCII oligomeric forms are much more intensely labelled by polyamines, in comparison to monomeric forms, suggests that polyamines participate in oligomer stabilisation. In plastid metabolism, polyamines modulate effectiveness of photosynthesis. The role of polyamines in mature chloroplasts is also related to the photo‐adaptation of the photosynthetic apparatus to low and high light intensity and its response to environmental stress. The occurrence of polyamines and enzymes participating in their metabolism at every stage of plastid development indicates that polyamines play a role in plastid differentiation, structure, functioning and senescence.     

The molecular biology of plastid division in higher plants

Plastids are essential plant organelles vital for life on earth, responsible not only for photosynthesis but for many fundamental intermediary metabolic reactions. Plastids are not formed de novo but arise by binary fission from pre-existing plastids, and plastid division therefore represents an important process for the maintenance of appropriate plastid populations in plant cells. Plastid division comprises an elaborate pathway of co-ordinated events which include division machinery assembly at the division site, the constriction of envelope membranes, membrane fusion and, ultimately, the separation of the two new organelles. Because of their prokaryotic origin bacterial cell division has been successfully used as a paradigm for plastid division. This has resulted in the identification of the key plastid division components FtsZ, MinD, and MinE, as well as novel proteins with similarities to prokaryotic cell division proteins. Through a combination of approaches involving molecular genetics, cell biology, and biochemistry, it is now becoming clear that these proteins act in concert during plastid division, exhibiting both similarities and differences compared with their bacterial counterparts. Recent efforts in the cloning of the disrupted loci in several of the accumulation and replication of chloroplasts mutants has further revealed that the division of plastids is controlled by a combination of prokaryote-derived and host eukaryote-derived proteins residing not only in the plastid stroma but also in the cytoplasm. Based on the available data to date, a working model is presented showing the protein components involved in plastid division, their subcellular localization, and their protein interaction properties.     

Emerging facets of plastid division regulation

Plastids are complex organelles that are integrated into the plant host cell where they differentiate and divide in tune with plant differentiation and development. In line with their prokaryotic origin, plastid division involves both evolutionary conserved proteins and proteins of eukaryotic origin where the host has acquired control over the process. The plastid division apparatus is spatially separated between the stromal and the cytosolic space but where clear coordination mechanisms exist between the two machineries. Our knowledge of the plastid division process has increased dramatically during the past decade and recent findings have not only shed light on plastid division enzymology and the formation of plastid division complexes but also on the integration of the division process into a multicellular context. This review summarises our current knowledge of plastid division with an emphasis on biochemical features, the functional assembly of protein complexes and regulatory features of the overall process.     

Berberine treatment attenuates the palmitate-mediated inhibition of glucose uptake and consumption through increased 1,2,3-triacyl-sn-glycerol synthesis and accumulation in H9c2 cardiomyocytes

Dysfunction of lipid metabolism and accumulation of 1,2-diacyl-sn-glycerol (DAG) may be a key factor in the development of insulin resistance in type 2 diabetes. Berberine (BBR) is an isoquinoline alkaloid extract that has shown promise as a hypoglycemic agent in the management of diabetes in animal and human studies. However, its mechanism of action is not well understood. To determine the effect of BBR on lipid synthesis and its relationship to insulin resistance in H9c2 cardiomyocytes, we measured neutral lipid and phospholipid synthesis and their relationship to glucose uptake. Compared with controls, BBR treatment stimulated 2-[1,2-³H(N)]deoxy-D-glucose uptake and consumption in palmitate-mediated insulin resistant H9c2 cells. The mechanism was though an increase in protein kinase B (AKT) activity and GLUT-4 glucose transporter expression. DAG accumulated in palmitate-mediated insulin resistant H9c2 cells and treatment with BBR reduced this DAG accumulation and increased accumulation of 1,2,3-triacyl-sn-glycerol (TAG) compared to controls. Treatment of palmitate-mediated insulin resistant H9c2 cells with BBR increased [1,3-³H]glycerol and [1-¹⁴C]glucose incorporation into TAG and reduced their incorporation into DAG compared to control. In addition, BBR treatment of these cells increased [1-¹⁴C]palmitic acid incorporation into TAG and decreased its incorporation into DAG compared to controls. BBR treatment did not alter phosphatidylcholine or phosphatidylethanolamine synthesis. The mechanism for the BBR-mediated decreased precursor incorporation into DAG and increased incorporation into TAG in palmitate-incubated cells was an increase in DAG acyltransferase-2 activity and its expression and a decrease in TAG hydrolysis. Thus, BBR treatment attenuates palmitate-induced reduction in glucose uptake and consumption, in part, through reduction in cellular DAG levels and accumulation of TAG in H9c2 cells.     

Identification and characterization of LPLAT7 as an sn-1-specific lysophospholipid acyltransferase

The main fatty acids at the sn-1 position of phospholipids (PLs) are saturated or monounsaturated fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) and are constantly replaced, like unsaturated fatty acids at the sn-2 position. However, little is known about the molecular mechanism underlying the replacement of fatty acids at the sn-1 position, i.e., the sn-1 remodeling. Previously, we established a method to evaluate the incorporation of fatty acids into the sn-1 position of lysophospholipids (lyso-PLs). Here, we used this method to identify the enzymes capable of incorporating fatty acids into the sn-1 position of lyso-PLs (sn-1 lysophospholipid acyltransferase [LPLAT]). Screenings using siRNA knockdown and recombinant proteins for 14 LPLATs identified LPLAT7/lysophosphatidylglycerol acyltransferase 1 (LPGAT1) as a candidate. In vitro, we found LPLAT7 mainly incorporated several fatty acids into the sn-1 position of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), with weak activities toward other lyso-PLs. Interestingly, however, only C18:0-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were specifically reduced in the LPLAT7-mutant cells and tissues from knockout mice, with a concomitant increase in the level of C16:0- and C18:1-containing PC and PE. Consistent with this, the incorporation of deuterium-labeled C18:0 into PLs dramatically decreased in the mutant cells, while deuterium-labeled C16:0 and C18:1 showed the opposite dynamic. Identifying LPLAT7 as an sn-1 LPLAT facilitates understanding the biological significance of sn-1 fatty acid remodeling of PLs. We also propose to use the new nomenclature, LPLAT7, for LPGAT1 since the newly assigned enzymatic activities are quite different from the LPGAT1s previously reported.     

Glycerolipid synthesis in Chlorella kessleri 11h: I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C₁₆ and C₁₈ acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [¹⁴C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C₁₈-sn-2-C₁₆ (C₁₈/C₁₆) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C₁₈-sn-2-C₁₈ (C₁₈/C₁₈) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C₁₈/C₁₈ species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C₁₈/C₁₈ species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

The role of lipids in plastid protein transport

The elaborate compartmentalization of plant cells requires multiple mechanisms of protein targeting and trafficking. In addition to the organelles found in all eukaryotes, the plant cell contains a semi-autonomous organelle, the plastid. The plastid is not only the most active site of protein transport in the cell, but with its three membranes and three aqueous compartments, it also represents the most topologically complex organelle in the cell. The chloroplast contains both a protein import system in the envelope and multiple protein export systems in the thylakoid. Although significant advances have identified several proteinaceous components of the protein import and export apparatuses, the lipids found within plastid membranes are also emerging as important players in the targeting, insertion, and assembly of proteins in plastid membranes. The apparent affinity of chloroplast transit peptides for chloroplast lipids and the tendency for unsaturated MGDG to adopt a hexagonal II phase organization are discussed as possible mechanisms for initiating the binding and/or translocation of precursors to plastid membranes. Other important roles for lipids in plastid biogenesis are addressed, including the spontaneous insertion of proteins into the outer envelope and thylakoid, the role of cubic lipid structures in targeting and assembly of proteins to the prolamellar body, and the repair process of D1 after photoinhibition. The current progress in the identification of the genes and their associated mutations in galactolipid biosynthesis is discussed. Finally, the potential role of plastid-derived tubules in facilitating macromolecular transport between plastids and other cellular organelles is discussed.     

Activities of acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) in microsomal preparations of developing sunflower and safflower seeds

The last step in triacylglycerols (TAG) biosynthesis in oil seeds, the acylation of diacylglycerols (DAG), is catalysed by two types of enzymes: the acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). The relative contribution of these enzymes in the synthesis of TAG has not yet been defined in any plant tissue. In the presented work, microsomal preparations were obtained from sunflower and safflower seeds at different stages of development and used in DGAT and PDAT enzyme assays. The ratio between PDAT and DGAT activity differed dramatically between the two different species. DGAT activities were measured with two different acyl acceptors and assay methods using two different acyl-CoAs, and in all cases the ratio of PDAT to DGAT activity was significantly higher in safflower than sunflower. The sunflower DGAT, measured by both methods, showed significant higher activity with 18:2-CoA than with 18:1-CoA, whereas the opposite specificity was seen with the safflower enzyme. The specificities of PDAT on the other hand, were similar in both species with 18:2-phosphatidylcholine being a better acyl donor than 18:1-PC and with acyl groups at the sn-2 position utilised about fourfold the rate of the sn-1 position. No DAG:DAG transacylase activity could be detected in the microsomal preparations.     

The function and diversity of plastid protein import pathways: a multilane GTPase highway into plastids

The photosynthetic chloroplast is the hallmark organelle of green plants. During the endosymbiotic evolution of chloroplasts, the vast majority of genes from the original cyanobacterial endosymbiont were transferred to the host cell nucleus. Chloroplast biogenesis therefore requires the import of nucleus-encoded proteins from their site of synthesis in the cytosol. The majority of proteins are imported by the activity of Toc and Tic complexes located within the chloroplast envelope. In addition to chloroplasts, plants have evolved additional, non-photosynthetic plastid types that are essential components of all cells. Recent studies indicate that the biogenesis of various plastid types relies on distinct but homologous Toc-Tic import pathways that have specialized in the import of specific classes of substrates. These different import pathways appear to be necessary to balance the essential physiological role of plastids in cellular metabolism with the demands of cellular differentiation and plant development.     

Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids.     

Metabolism of Unsaturated Monogalactosyldiacylglycerol Molecular Species in Arabidopsis thaliana Reveals Different Sites and Substrates for Linolenic Acid Synthesis

Synthesis of unsaturated monogalactosyldiacylglycerol (MGDG) was examined in a mutant of Arabidopsis thaliana (L.) Heynh. containing reduced levels of hexadecatrienoic (16:3) and linolenic (18:3) acids in leaf lipids. Molecular species composition and labeling kinetics following the incorporation of exogenous [¹⁴C]fatty acids suggest that at least two pathways and multiple substrates are involved in desaturation of linoleic acid (18:2) to 18:3 for production of unsaturated galactolipids. A reduction in 18:3/16:3 MGDG and an increase in 18:2/16:2 MGDG, together with labeling kinetics of these molecular species following the incorporation of exogenous [¹⁴C]12:0 fatty acids, suggests that a chloroplastic pathway for production of 18:3 at the sn-1 position of MGDG utilizes 18:2/16:2 MGDG as a substrate. This chloroplastic (prokaryotic) pathway is deficient in the mutant. When exogenous [¹⁴C]18:1 was supplied, a eukaryotic (cytoplasmic) pathway involving the desaturation of 18:2 to 18:3 on phosphatidylcholine serves as the source of 18:3 for the sn-2 position of MGDG. This eucaryotic pathway predominates in the mutant.     

Diacylglycerol Kinase ? Is Selective for Both Acyl Chains of Phosphatidic Acid or Diacylglycerol

The phosphatidylinositol (PI) cycle mediates many cellular events by controlling the metabolism of many lipid second messengers. Diacylglycerol kinase ϵ (DGKϵ) has an important role in this cycle. DGKϵ is the only DGK isoform to show inhibition by its product phosphatidic acid (PA) as well as substrate specificity for sn-2 arachidonoyl-diacylglycerol (DAG). Here, we show that this inhibition and substrate specificity are both determined by selectivity for a combination of the sn-1 and sn-2 acyl chains of PA or DAG, respectively, preferring the most prevalent acyl chain composition of lipids involved specifically in the PI cycle, 1-stearoyl-2-arachidonoyl. Although the difference in rate for closely related lipid species is small, there is a significant enrichment of 1-stearoyl-2-arachidonoyl PI because of the cyclical nature of PI turnover. We also show that the inhibition of DGKϵ by PA is competitive and that the deletion of the hydrophobic segment and cationic cluster of DGKϵ does not affect its selectivity for the acyl chains of PA or DAG. Thus, this active site not only recognizes the lipid headgroup but also a combination of the two acyl chains in PA or DAG. We propose a mechanism of DGKϵ regulation where its dual acyl chain selectivity is used to negatively regulate its enzymatic activity in a manner that ensures DGKϵ remains committed to the PI turnover cycle. This novel mechanism of enzyme regulation within a signaling pathway could serve as a template for the regulation of enzymes in other pathways in the cell.     

Bactofilins,a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus

The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways.