Stromal low temperature compartment derived from the inner membrane of the chloroplast envelope

Leaf discs of four dicotyledonous species, when incubated at temperatures of 4 to 18°C (optimum at 12°C) for 30 or 60 minutes, responded by accumulations of membranes in the chloroplast stroma in the space between the inner membrane of the envelope and the thylakoids. The accumulated membranes, here referred to as the low temperature compartment, were frequently continuous with the envelope membrane and exhibited kinetics of formation consistent with a derivation from the envelope. Results were similar for expanding leaves of garden pea (Pisum sativum), soybean (Glycine max), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum). We suggest that the stromal low temperature compartment may be analogous to the compartment induced to form between the transitional endoplasmic reticulum and the Golgi apparatus at low temperatures. The findings provide evidence for the possibility of a vesicular transfer of membrane constituents between the inner membrane of the chloroplast envelope and the thylakoids of mature chloroplasts in expanding leaves.     

Translation of the Chloroplast psbA mRNA Requires the Nuclear-encoded Poly(A)-binding Protein,RB47

A set of nuclear mutants of C. reinhardtii were identified that specifically lack translation of the chloroplast-encoded psbA mRNA, which encodes the photosystem II reaction center polypeptide D1. Two of these mutants are deficient in the 47-kD member (RB47) of the psbA RNA-binding complex, which has previously been identified both genetically and biochemically as a putative translational activator of the chloroplast psbA mRNA. RB47 is a member of the poly(A)-binding protein family, and binds with high affinity and specificity to the 5′ untranslated region of the psbA mRNA. The results presented here confirm RB47''s role as a message-specific translational activator in the chloroplast, and bring together genetic and biochemical data to form a cohesive model for light- activated translational regulation in the chloroplast.     

Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677–1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.     

SOME SURFACE CHARACTERISTICS OF THE CHLOROPLAST ENVELOPE

Pronase, cationic ferritin, and ferritin-conjugated plant lectins were used to study the chloroplast envelope. Negative charges (binding cationic ferritin) are fairly uniformly distributed over the envelope surfaces in contact with the hyaloplasm and are not appreciably altered by mild pronase treatment of isolated plastids. All surfaces of stroma-free thylakoids previously exposed to the stroma uniformly bind cationic ferritin. Ricin_II-ferritin binding to the membranes of the chloroplast envelope indicates that galactolipids are distributed in the outer membrane in such a way that their galactose moieties are exposed on the envelope surface. In addition, the outer surface of the inner membrane (the intermembrane face) contains uniformly distributed galactose which binds ricin_II when this membrane is exposed to the reaction medium. Isolated vesicles of the chloroplast envelope bind ricin_II, while isolated envelope vesicles as well as the envelopes of intact chloroplasts failed to bind concanavalin A. Thylakoid surfaces showed minor binding of ricin_II as well as concanavalin A.     

Chloroplast biogenesis. Cell-free transfer of envelope monogalactosylglycerides to thylakoids.

An ATP- and temperature-dependent transfer of monogalactosylglycerides from the chloroplast envelope to the chloroplast thylakoids was reconstituted in a cell-free system prepared from isolated chloroplasts of garden pea (Pisum sativum) or spinach (Spinacia oleracea). Isolated envelope membranes, in which the label was present exclusively in monogalactosylglycerides, were prepared radiolabeled in vitro with [14C]galactose from UDP-[14C]galactose to label galactolipids as the donor. ATP-dependent transfer of radioactivity from donor to unlabeled acceptor thylakoids, immobilized on nitrocellulose strips, was observed. In some experiments linear transfer for longer than 30 min of incubation was facilitated by the addition of stroma proteins but in other experiments stroma was without effect or inhibitory suggesting no absolute requirements for a soluble protein carrier. Transfer was donor specific. No membrane fraction tested (plasma membrane, tonoplast, endoplasmic reticulum, nuclei, Golgi apparatus, mitochondria or thylakoids) (isolated from tissue radiolabeled in vivo with [14C]acetate) other than chloroplast envelopes demonstrated any significant ability to transfer labeled membrane lipids to immobilized thylakoids. Acceptor specificity, while not absolute, showed a 3-10-fold greater ATP-dependent transfer of labeled galactolipids from chloroplast envelopes to immobilized thylakoids than to other leaf membranes. The results provide independent confirmation of the potential for transfer of galactolipids between chloroplast envelopes and thylakoids suggested previously from ultrastructural studies and of the known location of thylakoid galactolipid biosynthetic activities in the chloroplast envelope.     

Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether-like herbicides.

Plant protoporphyrinogen oxidase is of particular interest since it is the last enzyme of the common branch for chlorophyll and heme biosynthetic pathways. In addition, it is the target enzyme for diphenyl ether-type herbicides, such as acifluorfen. Two distinct methods were used to investigate the localization of this enzyme within Percoll-purified spinach chloroplasts. We first assayed the enzymatic activity by spectrofluorimetry and we analyzed the specific binding of the herbicide acifluorfen, using highly purified chloroplast fractions. The results obtained give clear evidence that chloroplast protoporphyrinogen oxidase activity is membrane-bound and is associated with both chloroplast membranes, i.e. envelope and thylakoids. Protoporphyrinogen oxidase specific activity was 7-8 times higher in envelope membranes than in thylakoids, in good agreement with the number of [3H]acifluorfen binding sites in each membrane system: 21 and 3 pmol/mg protein, respectively, in envelope membranes and thylakoids. On a total activity basis, 25% of protoporphyrinogen oxidase activity were associated with envelope membranes. The presence of protoporphyrinogen oxidase in chloroplast envelope membranes provides further evidence for a role of this membrane system in chlorophyll biosynthesis. In contrast, the physiological significance of the enzyme associated with thylakoids is still unknown, but it is possible that thylakoid protoporphyrinogen oxidase could be involved in heme biosynthesis.     

MFP1 is a thylakoid-associated,nucleoid-binding protein with a coiled-coil structure

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.     

Galactolipid Synthesis in Vicia faba Leaves: III. Site(s) of Galactosyl Transferase Activity

Leaves of Vicia faba were fed ¹⁴CO₂ in light for periods of up to 6 hours. At intervals, leaf samples were homogenized and separated into fractions which contained “broken” and “intact” chloroplasts, and three other high speed centrifugal fractions containing other cell membranes and chloroplast envelopes. Analyses of the radioactive labeling of galactose from the galactolipids in these fractions and in purified chloroplast envelopes indicated that the major site of galactosyl transferase enzyme activity was in the chloroplast envelope. The data suggest that in time much of the radioactive galactolipid was transferred from the envelope to the thylakoid-containing fractions. The major site of galactolipid synthesis appears to be in the envelope but there is some evidence of another site in the thylakoids.     

The chloroplast protein CPSAR1, dually localized in the stroma and the inner envelope membrane,is involved in thylakoid biogenesis

Thylakoid biogenesis is a crucial step for plant development involving the combined action of many cellular actors. CPSAR1 is shown here to be required for the normal organization of mature thylakoid stacks, and ultimately for embryo development. CPSAR1 is a chloroplast protein that has a dual localization in the stroma and the inner envelope membrane, according to microscopy studies and subfractionation analysis. CPSAR1 is close to the Obg nucleotide binding protein subfamily and displays GTPase activity, as demonstrated by in vitro assays. Disruption of the CPSAR1 gene via T‐DNA insertion results in the arrest of embryo development. In addition, transmission electron microscopy analysis indicates that mutant embryos are unable to develop thylakoid membranes, and remain white. Unstacked membrane structures resembling single lamellae accumulate in the stroma, and do not assemble into mature thylakoid stacks. CPSAR1 RNA interference induces partially developed thylakoids leading to pale‐green embryos. Altogether, the presented data demonstrate that CPSAR1 is a protein essential for the formation of normal thylakoid membranes, and suggest a possible involvement in the initiation of vesicles from the inner envelope membrane for the transfer of lipids to the thylakoids.     

Some unusual features of the ultrastructure of the chloroplasts of the green algal orderCaulerpales and their development

Summary The ultrastructure of developing and mature chloroplasts of members of the green algal orderCaulerpales is described. The mature chloroplasts develop from small starch containing plastids. These small starch containing plastids may also develop into the large amyloplasts characteristic of this order. The thylakoid organizing body (TOB), a system of concentric lamellae found at one end of the plastid, appears to be involved in initial thylakoid membrane synthesis. During early plastid development the first formed thylakoids, the plastid DNA and lipid are closely associated with this body. Many developing plastids also have a number of microfilaments near the chloroplast envelope. These microfilaments extend from the TOB towards the opposite end of the plastid.The size and structure of the mature caulerpalean chloroplast varies greatly between species, as does the size and structure of the TOB. The simplest type of TOB occurs inAvrainvillea erecta and the most complex inCaulerpa cactoides. The membranes of the TOB are connected by crossbridges and they are also connected with the inner chloroplast envelope membrane. The structure of the TOB, its relation to the chloroplast envelope, its association with the thylakoids and its possible functions are described.     

In situ incorporation of Fatty acids into lipids of the outer and inner envelope membranes of pea chloroplasts

When incubated with [1-¹⁴C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, ¹⁴C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg²⁺-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their ¹⁴C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The ¹⁴C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.     

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.     

Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment

Induction of cell proliferation requires a concomitant increase in the synthesis of glycosylated lipids and membrane proteins, which is dependent on ER-Golgi protein transport by CopII-coated vesicles. In this process, retrograde transport of ER resident proteins from the Golgi is crucial to maintain ER integrity, and allows for anterograde transport to continue. We previously showed that expression of the CopI specific SNARE protein Use1 (Unusual SNARE in the ER 1) is tightly regulated by eIF4E-dependent translation initiation of Use1 mRNA. Here we investigate the mechanism that controls Use1 mRNA translation. The 5′UTR of mouse Use1 contains a 156 nt alternatively spliced intron. The non-spliced form is the predominantly translated mRNA. The alternatively spliced sequence contains G-repeats that bind the RNA-binding protein G-rich sequence binding factor 1 (Grsf1) in RNA band shift assays. The presence of these G-repeats rendered translation of reporter constructs dependent on the Grsf1 concentration. Down regulation of either Grsf1 or Use1 abrogated expansion of erythroblasts. The 5′UTR of human Use1 lacks the splice donor site, but contains an additional upstream open reading frame in close proximity of the translation start site. Similar to mouse Use1, also the human 5′UTR contains G-repeats in front of the start codon. In conclusion, Grsf1 controls translation of the SNARE protein Use1, possibly by positioning the 40S ribosomal subunit and associated translation factors in front of the translation start site.     

Import of proteins into chloroplasts. Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates

Many of the thylakoid membrane proteins of plant and algal chloroplasts are synthesized in the cytosol as soluble, higher molecular weight precursors. These precursors are post-translationally imported into chloroplasts, incorporated into the thylakoids, and proteolytically processed to mature size. In the present study, the process by which precursors are incorporated into thylakoids was reconstituted in chloroplast lysates using the precursor to the light-harvesting chlorophyll a/b protein (preLHCP) as a model. PreLHCP inserted into thylakoid membranes, but not envelope membranes, if ATP was present in the reaction mixture. Correct integration into the bilayer was verified by previously documented criteria. Integration could also be reconstituted with purified thylakoid membranes if reaction mixtures were supplemented with a soluble extract of chloroplasts. Several other thylakoid precursor proteins in addition to preLHCP, but no stromal precursor proteins, were incorporated into thylakoids under the described assay conditions. These results suggest that the observed in vitro activity represents in vivo events during the biogenesis of thylakoid proteins.