Accepted Manuscript Phosphatidylcholine is transferred from chemically-defined liposomes to chloroplasts through proteins of the chloroplast outer envelope membrane Congfei Yin, Mats X. Andersson, Hongsheng Zhang, Henrik Aronsson PII: DOI: Reference:
S0014-5793(14)00860-6 http://dx.doi.org/10.1016/j.febslet.2014.11.044 FEBS 36945
To appear in:
FEBS Letters
Received Date: Revised Date: Accepted Date:
26 September 2014 24 November 2014 24 November 2014
Please cite this article as: Yin, C., Andersson, M.X., Zhang, H., Aronsson, H., Phosphatidylcholine is transferred from chemically-defined liposomes to chloroplasts through proteins of the chloroplast outer envelope membrane, FEBS Letters (2014), doi: http://dx.doi.org/10.1016/j.febslet.2014.11.044
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Phosphatidylcholine is transferred from chemically-defined liposomes to chloroplasts through proteins of the chloroplast outer envelope membrane
Congfei Yin1,2, Mats X Andersson2, Hongsheng Zhang1*, Henrik Aronsson2* 1
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural
University, Nanjing, People's Republic of China 2
Department of Biological and Environmental Sciences, University of Gothenburg, SE-405
30 Gothenburg, Sweden
*For correspondence: Address: Department of Biological and Environmental Sciences, University of Gothenburg, SE-405 30 Gothenburg, Sweden. Phone: +46 31 7864802 Fax: +46 31 7862540 Email: [email protected]
Adress: State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, People's Republic of China. Email: [email protected]
ABSTRACT Chloroplasts maintain their lipid balance through a tight interplay with the endoplasmic reticulum (ER). The outer envelope membrane of chloroplasts contains a large proportion of the phospholipid phospatidylcholine (PC), which is synthesized in the ER and also a possible precursor for thylakoid galactolipids. The mechanism for PC transport from the ER to chloroplasts is not known. Using isolated chloroplasts and liposomes containing radiolabelled PC we investigated non-vesicular transport of PC in vitro. PC uptake in chloroplasts was time- and temperature-dependent, but nucleotide-independent. Increased radius of liposomes stimulated PC uptake, and protease treatment of the chloroplasts impaired PC uptake. This implies that the chloroplast outer envelopes contains an exposed proteinaceous machinery for the uptake of PC from closely apposed membranes.
Keywords: phosphatidylcholine (PC); chloroplast; liposome; lipid uptake
Abbreviations: DAG, diacylglycerol; DGDG, digalactosyldiacylglycerol; ER, endoplasmic reticulum;
MGDG,
monogalactosyldiacylglycerol;
PA,
phosphatic
acid;
PC,
phosphatidylcholine; PE, phosphatidylethanolamine; PLAM, plastid associated membranes; TGD, trigalactosyl diacylglycerol.
Introduction Chloroplasts are vital organelles of plants as they harbour the photosynthesis machinery, nitrogen- and sulphur metabolism as well as produce, among other vital compound classes, amino- and fatty acids. The chloroplast contains an inner membrane system, the thylakoids, being surrounded by an aqueous stroma. The stroma is in turn surrounded by the envelope membrane, divided into an outer and inner envelope membrane. Chloroplast membranes constitute about 70% of the total plant mesophyll cell membranes, and 80% are galactolipids e.g. monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) both being more common in the thylakoid membranes than in the envelope (Dörmann and Benning 2002). In the outer leaflet of the outer envelope membrane, facing the cytosol, the phospholipid phospatidylcholine (PC) is a major constituent (32-44 mol%) (Block et al. 1983; Cline et al. 1981). Chloroplast galactolipids are synthesized in the envelope through a coordinated interplay with the endoplasmic reticulum (ER) e.g. chloroplasts export fatty acyl moieties to the ER and import diacylglycerol backbones that act as galactolipid precursors back from the ER (Benning 2008). In vivo pulse chase studies indicate that PC (detected in envelope/chloroplast) synthesized in the ER could be the immediate precursor for galactolipid synthesis in chloroplasts (Heinz and Roughan 1983; Hellgren et al. 1995; Hellgren and Sandelius 2001; Roughan et al. 1987; Slack et al. 1977). In addition, diacylglycerol (DAG) (Williams et al. 2000), phosphatic acid (PA) (Xu et al. 2005) and lyso-PC (Mongrand et al. 1997) have been suggested to be galactolipid precursors transported from the ER. Since chloroplasts lack capacity to synthesize the head group of PC, and both the outer envelope and the ER contain a significant amount of PC it seems likely that PC is transported from the ER whether or not it is the major precursor for galactolipids. However, the mechanism behind PC transport from ER to the envelope is unclear.
Movement of lipids between cytosolic organelles may use vesicular transport e.g. endocytic or exocytic pathways or non-vesicular transport (Holthuis and Levine 2005). Lipid interchange between ER and chloroplasts has been proposed to occur by contact sites between the plastid envelope and a specialized ER domain coined plastid associated membranes (PLAM) (Andersson et al. 2007; Benning 2009). Trigalactosyl diacylglycerol (TGD) proteins are located in the chloroplast envelope and facilitate lipid transport into chloroplasts from the ER (Awai et al. 2006; Wang et al. 2012; Xu et al. 2003). TGD4 alone makes up a complex in the outer envelope and TGD1, 2 and 3 a complex in the inner envelope (Wang and Benning 2012). TGD proteins are proposed to facilitate transfer of PA from ER to the chloroplast where it is used as a galactolipid precursor after dephosphorylation in the inner envelope (Wang et al. 2012). Recently, it was proposed that the ER and the outer chloroplast envelope can form hemifusions at contact sites to facilitate transfer of lipophilic compounds (Mehrshahi et al. 2013). It has been shown in vitro that radiolabelled phospholipids can be transferred from donor ER to isolated chloroplasts in a time and temperature dependent manner but without need for nucleotides or cytosolic factors (Andersson et al. 2004; Xu et al. 2008). Thus, available evidence suggests that chloroplasts can take up phospholipids from donor membranes, provided these make close contact to the organelle. Yeast mitochondria have been shown to be able to take up phospholipids from chemically defined donor liposomes by means of a protein machinery in their outer membrane (Wu and Voelker 2004). In this case larger liposomes were more efficient donors and this was interpreted as those larger liposomes can contribute a “flatter” contact surface with the mitochondria. In this study we tested if isolated chloroplasts have the ability to take up PC from donor liposomes. We used an in vitro system using radiolabelled PC in liposomes mixed with isolated chloroplasts to test if chloroplast like mitochondria could take up phospholipids form chemically defined donor liposomes. We
show that PC is absorbed by chloroplasts in a time and temperature dependent way, and that the PC uptake rate is related to the size of liposomes. Moreover, the process is independent of exogenous nucleotides, whereas, removal of proteins on the outer envelope by thermolysin impairs it, but which protein(s) responsible remains to be elucidated.
Methods and materials
Plant material and chloroplast preparation Pea (Pisum sativum L. cv Kelvedon Wonder) were sown on soil, covered with vermiculite and grown at 22°C and 12 h light. Isolation of chloroplasts from 10-12 days old peas was performed as previously described (Aronsson et al. 2001). Chlorophyll concentration was measured spectroscopically according to (Lichtenthaler and Wellburn 1983).
Liposome preparation Lipids were extracted from 7-9 days old pea seedlings. They were cut with razor blade, and soaked in 12 ml 33P (2.66*108 kdpm) (as H333PO4, Perkin Elmer) radiolabelled water for 48 h in a fume hood with lights on before lipid extraction. Thereafter total lipids were extracted (Kourtchenko et al. 2007) and a phospholipid fraction prepared by fractionation on a silica column. Total extracted lipids were loaded onto a 500 mg silica column (Supelco). Neutral lipids were eluted with CHCl3:acetone (9:1, by volume), glycolipids with acetone:methanol (9:1, by volume), and total phospholipids with methanol. The procedure of isolation of liposomes was adapted from (Olson et al. 1979) with some modifications as described (Berglund et al. 1999). Total phospholipids isolated from labeled pea seedlings or a mix of commercial 14C-dipalmitoyl phosphatidylcholine ([14C]PC, specific activity 3.6 GBq/mmol, Perkin Elmer) or commercial L-3-phosphatidylethanolamine, 1palmitoyl-2-[1-14C]linoleoyl ([14C]PE, specific activity 2.04 GBq/mmol, Perkin Elmer) corresponding to 50 kdpm per assay with soybean PC and PE (Sigma-Aldrich) (mix 4:1 by weight, 10 µg/assay) dissolved in chloroform were dried under N2. One ml of import buffer was added to the dried lipid film heated to 60°C for about 10 s and vortexed thoroughly. After
four cycles of freeze-thawing liposomes were pressure extruded through double 50, 100, 200, 400 or 1000 nm (as indicated) polycarbonate filters eleven times, and stored at 4°C until use.
Liposome import assay Liposomes corresponding to about 30 kdpm of 14C- or 33P-label were mixed with chloroplasts corresponding to 50 µg of chlorophyll in a total volume of 100 µl of import buffer (0.33 M sorbitol; 50 mM Hepes; KOH pH 8.0). Import assays were performed on ice or 25°C water bath with 150 mol*m-2*s-1 light using different time points. The reaction was stopped by adding 200 µl ice cold isolation buffer. The suspension was transferred to the top of a 1 ml 35% Percoll mixed in isolation buffer, centrifuged 5000 g, 8 min at 4°C, and the pellet being washed 3 times in 1 ml isolation buffer. Finally the pellet was re-suspended in 200 µl isolation buffer and transferred to a scintillation vial containing 1 ml methanol, and 8 ml scintillation cocktail (Ready Protein+, Beckman) was added before and the radioactivity counted in a TriCarb 2900 TR liquid scintillation analyzer. Thermolysin treatment was performed according to (Garcia et al. 2010) with slight modifications. Chloroplasts were incubated in 50 µg/ml thermolysin and 300 µM CaCl2 for 30 min on ice in darkness. Control samples had thermolysin replaced with an equal volume of import buffer. Reactions were terminated by addition of an equal volume of 50 µM EDTA (final concentration 25 µM). The suspension was transferred to the top of a 1 ml “cushion” of 35% (by volume) Percoll™ in isolation buffer, centrifuged at 5000 g, 5 min at 4°C, and the pellet washed twice in 1 ml import buffer, then finally re-suspended in 50 µl import buffer to continue the liposomes import assay. For nucleotide experiments, ATP/GTP was added to the reaction to a final concentration of 0.5 mM or 1 mM (only for ATP). Control samples had ATP/GTP replaced with the same volume of import buffer. Reactions were incubated at 25°C in a water bath with 150 mol*m-
2
*s-1 light for 30 min or 60 min.
Results and discussion
Uptake of PC from liposomes to isolated chloroplasts is time and temperature dependent As ER derived lipid transport to chloroplasts has been suggested to occur at contact sites between the ER and the chloroplast and yeast mitochondria apparently harbors a machinery for the uptake of phospholipids from protein free liposomes, we decided to test if intact plant chloroplasts could take up radiolabelled phospholipids from large unilamellar liposomes. An initial experiment was performed with large (400 nm) unilamellar liposomes (LUVs) prepared
from
phospholipids
isolated
[33P]ortophosphate. LUVs containing
33
from
pea
seedlings
pre-incubated
with
P-labelled lipids were mixed with isolated
chloroplasts isolated from pea seedlings. After incubation the chloroplasts were washed and the amount of radiolabelled transferred to the chloroplasts determined (figure. 1A). There was a clear time dependent incorporation of radiolabel into the chloroplast fraction demonstrated by a more than 3-fold increase in label after 30 min compared to zero time (figure. 1A). Moreover, phospholipid uptake was also temperature dependent; after 30 min at 25°C uptake of phospholipids was approximately double that of samples incubated on ice (figure. 1A). Unfortunately the amount of radiolabel transferred was too low to determine the exact composition of phospholipids transferred. We thus conclude that isolated chloroplasts can take up phospholipids from protein free liposomes in vitro. We next tested if PC, as this lipid is most likely transported from the ER to the outer chloroplast envelope (see above), could be transferred from chemically defined liposomes to isolated chloroplasts. To this end [14C]PC was incorporated into liposomes (400 nm in diameter) made from a mix of soybean PC:PE (4:1). The PC:PE ratio is around 2:1 in ER, but the total amount differ in plants, PC and PE being around 50% and 25%, respectively (Moreau et al. 1998), mammals, PC and PE
representing around 60% and 30%, respectively, and yeast where PC and PE being around 40% and 20%, respectively (Van Meer et al. 2008). Thus, the amount of PE was rather well reflected in the liposomes whereas increased concentration of PC in the liposomes enabled improved detection but also replaced other phospholipids not of investigated in this study. The uptake of the total pool of unlabeled and labeled [14C]PC in chloroplasts was increased from 15.4 pmol at 0 min, to 59.4 pmol at 30 min, and finally 74.8 pmol at 60 min (figure. 1B). Performing the experiment at 25°C allowed substantially more PC taken up compared to incubation on ice (figure. 1B). Uptake of the total PC pool when compared was after 30 min 23.6 pmol on ice versus 59.4 pmol at 25°C, and after 60 min 27.6 pmol on ice versus 74.8 pmol at 25°C. In a separate experiment, liposomes were prepared with radiolabelled PE and transfer of this to isolated chloroplasts was measured. The uptake of PC was 10 fold higher than that of PE e.g. 74.8 pmol PC versus 6.98 pmol PE at 60 min at 25°C (figure. 1B). Thus, PC was clearly taken up from protein free liposomes by isolated chloroplasts in a time and temperature dependent fashion. This thus indicates that there is a lipid import machinery present in chloroplasts that can operate in the absence of any proteins on the donor membrane. The efficiency of PC uptake was clearly time and temperature dependent (figure. 1B). The significantly lower uptake of PE demonstrates selectivity in the lipid import machinery. This result is in accordance with that PE is not found in any significant levels in isolated chloroplasts or envelope membranes. In addition, the very low uptake of PE demonstrates that the result is not due to binding of liposomes to the chloroplasts; there really is transfer of lipids from liposomes to chloroplasts. To test whether nucleotides are needed during PC uptake to chloroplasts in vitro, ATP or GTP was added to the transfer reaction. Reactions performed at 25°C under light condition with additional ATP or GTP (final concentration 0.5 mM) demonstrated slightly lower PC uptake into chloroplasts compared with mock reactions without any additional
nucleotides added (figure. 1C). Furthermore, no increase in PC uptake was observed when the concentration of ATP was increased from 0.5 to 1 mM. Thus, energy in the form of ATP or GTP is not a requirement during PC uptake to chloroplasts. Our results support the notion i.e. even with ATP or GTP employed in the reaction, chloroplasts still cannot absorb more PC. This thus confirms that nucleotides are not necessarily needed for lipid transport to chloroplasts. This is in line with earlier studies of transfer of lipids from ER to chloroplasts (Andersson et al. 2004; Moreau et al. 1991; Xu et al. 2008). Vesicular transport requires energy and reconstituted lipid transport (presumably also vesicular-mediated) between ER and Golgi in plants (Morré et al. 1991; Sturbois et al. 1994) and animals (Moreau and Morré 1991; Moreau et al. 1991; Morré 1998) cells require ATP, but previous studies using purified ER as donor and chloroplast as acceptor showed no ATP dependent transfer of total lipids (Morré et al. 1991). Lipids transport from ER to mitochondria in yeast has been extensively studied by various in vitro approaches (de Kroon et al. 2003; Gaigg et al. 1995; Voelker 2000; 2003), and the general conclusion is that no nucleotides are required for lipid transport from ER to mitochondria. Apparently, there are similarities between lipid transport from ER to mitochondria in yeast and ER to chloroplast lipid transport in higher plants.
Proteins on the outer envelope are needed for efficient PC uptake To investigate if there are proteins on the outer envelope to assist PC uptake, intact chloroplasts were treated with thermolysin. This protease is unable to penetrate the chloroplast outer envelope membrane, and thus only digests proteins exposed at the outer envelope surface of intact chloroplasts (Cline et al. 1984). It was obvious that after chloroplast treatment with thermolysin the lipid uptake efficiency was much lower than for those chloroplasts not treated with protease (figure. 2). This demonstrates that protein(s) protruding
on the surface of the outer envelope play an important role in uptake of PC from liposomes by isolated chloroplasts.
Increased liposome size accelerates PC uptake to chloroplasts We interpret our findings as that there is a machinery in the outer chloroplast envelope aiding in transfer of phospholipids from a donor membrane, provided this membrane is situated close enough to the chloroplast. A donor liposome of larger radius would present a larger flatter surface to the chloroplast and thus in theory allows a more efficient transfer of lipids. This was in fact the case with phospholipid uptake by isolated yeast mitochondria from donor liposomes (Wu and Voelker 2004). To test whether the size of liposomes also affect PC absorption to chloroplasts, liposomes made from [14C]PC in a mixture with soybean PC:PE (ratio 4:1) with different diameters (50 nm up to 1000 nm in diameter) were used for lipid uptake to isolated pea chloroplasts in vitro (figure. 3). This clearly demonstrated that the uptake rate of PC increased with the size of liposomes e.g. chloroplasts took up about 2-fold more PC from 1000 nm liposomes in diameter compared to 50 nm liposomes (figure. 3). Thus, increased size of the liposomes affected lipid uptake into chloroplast positively.
Conclusions We herein proved clear evidence for that isolated chloroplasts can take up the phospholipid PC from chemically defined protein free liposomes. Furthermore, the uptake is dependent on proteins exposed on the outer envelope surface of the chloroplast. In theory, this could be explained by lipids adsorbed from the surface of the liposomes by lipid transport proteins protruding from the chloroplast surface. Alternatively, proteins in the outer envelope could induce hemifusions with the liposomes. We cannot, with this experimental set up completely
discriminate between these two situations and the literature lends some support to both. However, the lack of PE uptake from liposomes demonstrates the liposomes at least not remain attached to the chloroplast to a large extent. Lipid transport seems to be mediated between mitochondria and ER as well as between Golgi and ER by membrane bound lipid transport proteins at contact sites (Holthuis and Levine 2005; Kornmann and Walter 2010). It was also recently shown that there is likely exchange of hydrophobic molecules as hemifusions between the ER and chloroplasts (Mehrshahi et al. 2013). Other questions that could be addressed in future studies using similar methodology as we used are if other lipids are transported, if the transport is unidirectional and if TGD-proteins are involved in the observed transfer of PC from liposomes.
Acknowledgements This work was supported by Olle Engkvist Byggmästare Foundation (to H.A.), and PhD student fellowships from the China Scholarship Council (CSC) and Nanjing Agricultural University (to C.Y.).
Figure legends
Figure 1. Uptake of PC from liposomes to chloroplasts is time and temperature dependent but nucleotide independent. Liposomes containing (A) 33P labeled phospholipids, (B) [14C]PC or [14C]PE were mixed with chloroplasts isolated from pea seedlings and incubated at the indicated temperature for the indicated time. After re-purification of the chloroplasts, the amount of labelled lipid transferred to the chloroplasts was measured. C. Indicated concentrations of ATP and GTP were added to the import reaction where liposomes containing [14C]PC were mixed with chloroplasts isolated from pea seedlings and the amount of labelled PC transferred to the chloroplasts measured. All data presented are representative of two independent experiments, and range derived from three replicates within that specific experiment.
Figure 2. Envelope proteins susceptible to thermolysin facilitate PC uptake to chloroplasts. Liposomes containing [14C]PC were mixed with chloroplasts isolated from pea seedlings and the amount of labelled PC transferred to the chloroplasts measured. Chloroplasts were incubated with thermolysin or mock treated prior to a 60 min liposome import assay on ice or at 25°C. Data presented are representative of two independent experiments, average and range derived from three replicates within that experiment is shown.
Figure 3. Increased liposome diameter accelerates PC uptake to chloroplasts. Liposomes containing [14C]PC of the indicated diameter were mixed with chloroplasts isolated from pea seedlings and the amount of labelled PC transferred to the chloroplasts measured. Data presented are representative of two independent experiments, and range derived from three replicates within that experiment.
References Andersson MX, Goksör M, Sandelius AS (2007) Optical manipulation reveals strong attracting forces at membrane contact sites between endoplasmic reticulum and chloroplasts. Journal of Biological Chemistry 282: 1170-1174 Andersson MX, Kjellberg JM, Sandelius AS (2004) The involvement of cytosolic lipases in converting phosphatidyl choline to substrate for galactolipid synthesis in the chloroplast envelope. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1684: 46-53 Aronsson H, Sundqvist C, Timko MP, Dahlin C (2001) The importance of the C-terminal region and Cys residues for the membrane association of the NADPH: protochlorophyllide oxidoreductase in pea. FEBS Lett 502: 11-15 Awai K, Xu C, Tamot B, Benning C (2006) A phosphatidic acid-binding protein of the chloroplast inner envelope membrane involved in lipid trafficking. Proceedings of the National Academy of Sciences 103: 10817-10822 Benning C (2008) A role for lipid trafficking in chloroplast biogenesis. Progress in lipid research 47: 381-389 Benning C (2009) Mechanisms of lipid transport involved in organelle biogenesis in plant cells. Annual Review of Cell and Developmental 25: 71-91 Berglund AH, Nilsson R, Liljenberg C (1999) Permeability of large unilamellar digalactosyldiacylglycerol vesicles for protons and glucose–influence of α-tocopherol, β-carotene, zeaxanthin and cholesterol. Plant Physiology and Biochemistry 37: 179-186 Block MA, Dorne A-J, Joyard J, Douce R (1983) Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. II. Biochemical characterization. Journal of Biological Chemistry 258: 13281-13286 Cline K, Andrews J, Mersey B, Newcomb EH, Keegstra K (1981) Separation and characterization of inner and outer envelope membranes of pea chloroplasts. Proceedings of the National Academy of Sciences 78: 35953599 Cline K, Werner-Washburne M, Andrews J, Keegstra K (1984) Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. Plant Physiol 75: 675-678 de Kroon AI, Koorengevel MC, Vromans TA, de Kruijff B (2003) Continuous equilibration of phosphatidylcholine and its precursors between endoplasmic reticulum and mitochondria in yeast. Mol Biol Cell 14: 2142-2150 Dörmann P, Benning C (2002) Galactolipids rule in seed plants. Trends in plant science 7: 112-118 Gaigg B, Simbeni R, Hrastnik C, Paltauf F, Daum G (1995) Characterization of a microsomal subfraction associated with mitochondria of the yeast, Saccharomyces cerevisiae. Involvement in synthesis and import of phospholipids into mitochondria. Biochimica et Biophysica Acta (BBA)-Biomembranes 1234: 214-220 Garcia C, Khan NZ, Nannmark U, Aronsson H (2010) The chloroplast protein CPSAR1, dually localized in the stroma and the inner envelope membrane, is involved in thylakoid biogenesis. Plant Journal 63: 73-85 Heinz E, Roughan PG (1983) Similarities and differences in lipid metabolism of chloroplasts isolated from 18: 3 and 16: 3 plants. Plant Physiol 72: 273-279 Hellgren LI, Carlsson AS, Selldén G, Sandelius AS (1995) In situ leaf lipid metabolism in garden pea (pisum sativum L.) exposed to moderately enhanced level of ozone. J Exp Bot 46: 221-230 Hellgren LI, Sandelius AS (2001) Age-dependent variation in membrane lipid synthesis in leaves of garden pea (Pisum sativum L.). J Exp Bot 52: 2275-2282 Holthuis JC, Levine TP (2005) Lipid traffic: floppy drives and a superhighway. Nature reviews molecular cell biology 6: 209-220 Kornmann B, Walter P (2010) ERMES-mediated ER-mitochondria contacts: molecular hubs for the regulation of mitochondrial biology. J Cell Sci 123: 1389-1393 Kourtchenko O, Andersson MX, Hamberg M, Brunnström Å, Göbel C, McPhail KL, Gerwick WH, Feussner I, Ellerström M (2007) Oxo-phytodienoic acid-containing galactolipids in Arabidopsis: jasmonate signaling dependence. Plant Physiol 145: 1658-1669 Lichtenthaler H, Wellburn A (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochemical Society Transactions 11: 591-592 Mehrshahi P, Stefano G, Andaloro JM, Brandizzi F, Froehlich JE, DellaPenna D (2013) Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts. Proceedings of the National Academy of Sciences 110: 12126-12131 Mongrand S, Bessoule J, Cassagne C (1997) A re-examination in vivo of the phosphatidylcholine–galactolipid metabolic relationship during plant lipid biosynthesis. Biochemical Journal 327: 853-858 Moreau P, Bessoule J, Mongrand S, Testet E, Vincent P, Cassagne C (1998) Lipid trafficking in plant cells. Progress in lipid research 37: 371-391 Moreau P, Morré DJ (1991) Cell-free transfer of membrane lipids. Evidence for lipid processing. Journal of
Biological Chemistry 266: 4329-4333 Moreau P, Rodriguez M, Cassagne C, Morré D, Morré D (1991) Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver. Journal of Biological Chemistry 266: 43224328 Morré D, Penel C, Morré DM, Sandelius AS, Moreau P, Andersson B (1991) Cell-free transfer and sorting of membrane lipids in spinach. Protoplasma 160: 49-64 Morré DJ (1998) Cell-free analysis of Golgi apparatus membrane traffic in rat liver. Histochemistry and cell biology 109: 487-504 Olson F, Hunt C, Szoka F, Vail W, Papahadjopoulos D (1979) Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochimica et Biophysica Acta (BBA)Biomembranes 557: 9-23 Roughan PG, Thompson Jr GA, Cho SH (1987) Metabolism of exogenous long-chain fatty acids by spinach leaves. Archives of biochemistry and biophysics 259: 481-496 Slack CR, Roughan PG, Balasingham N (1977) Labelling studies in vivo on the metabolism of the acyl and glycerol moieties of the glycerolipids in the developing maize leaf. Biochemical Journal 162: 289-296 Sturbois B, Moreau P, Maneta-Peyret L, Morré D, Cassagne C (1994) Cell-free transfer of phospholipids between the endoplasmic reticulum and the Golgi apparatus of leek seedlings. Biochimica et Biophysica Acta (BBA)-Biomembranes 1189: 31-37 Van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nature reviews molecular cell biology 9: 112-124 Wang Z, Benning C (2012) Chloroplast lipid synthesis and lipid trafficking through ER-plastid membrane contact sites. Biochemical Society Transactions 40: 457-463 Wang Z, Xu C, Benning C (2012) TGD4 involved in endoplasmic reticulum-to-chloroplast lipid trafficking is a phosphatidic acid binding protein. Plant Journal 70: 614-623 Williams J, Imperial V, Khan M, Hodson J (2000) The role of phosphatidylcholine in fatty acid exchange and desaturation in Brassica napus L. leaves. Biochemical Journal 349: 127-133 Voelker DR (2000) Interorganelle transport of aminoglycerophospholipids. Biochimica et Biophysica Acta (BBA)Molecular and Cell Biology of Lipids 1486: 97-107 Voelker DR (2003) New perspectives on the regulation of intermembrane glycerophospholipid traffic. Journal of lipid research 44: 441-449 Wu W-I, Voelker DR (2004) Reconstitution of phosphatidylserine transport from chemically defined donor membranes to phosphatidylserine decarboxylase 2 implicates specific lipid domains in the process. Journal of Biological Chemistry 279: 6635-6642 Xu C, Fan J, Cornish AJ, Benning C (2008) Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein. Plant Cell 20: 2190-2204 Xu C, Fan J, Froehlich JE, Awai K, Benning C (2005) Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis. Plant Cell 17: 3094-3110 Xu C, Fan J, Riekhof W, Froehlich JE, Benning C (2003) A permease‐like protein involved in ER to thylakoid lipid transfer in Arabidopsis. The EMBO journal 22: 2370-2379
Figure 1
Figure 2
Figure 3
Bullet points:
Chloroplasts take up PC from unilamellar liposomes in a time and temperature dependent fashion
ATP and GTP are dispensable for in vitro PC uptake into chloroplasts
Envelope proteins susceptible to thermolysin facilitate PC uptake to chloroplasts
Increased liposome radius accelerates PC uptake to chloroplasts
S0014-5793(14)00860-6 http://dx.doi.org/10.1016/j.febslet.2014.11.044 FEBS 36945
To appear in:
FEBS Letters
Received Date: Revised Date: Accepted Date:
26 September 2014 24 November 2014 24 November 2014
Please cite this article as: Yin, C., Andersson, M.X., Zhang, H., Aronsson, H., Phosphatidylcholine is transferred from chemically-defined liposomes to chloroplasts through proteins of the chloroplast outer envelope membrane, FEBS Letters (2014), doi: http://dx.doi.org/10.1016/j.febslet.2014.11.044
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Phosphatidylcholine is transferred from chemically-defined liposomes to chloroplasts through proteins of the chloroplast outer envelope membrane
Congfei Yin1,2, Mats X Andersson2, Hongsheng Zhang1*, Henrik Aronsson2* 1
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural
University, Nanjing, People's Republic of China 2
Department of Biological and Environmental Sciences, University of Gothenburg, SE-405
30 Gothenburg, Sweden
*For correspondence: Address: Department of Biological and Environmental Sciences, University of Gothenburg, SE-405 30 Gothenburg, Sweden. Phone: +46 31 7864802 Fax: +46 31 7862540 Email: [email protected]
Adress: State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, People's Republic of China. Email: [email protected]
ABSTRACT Chloroplasts maintain their lipid balance through a tight interplay with the endoplasmic reticulum (ER). The outer envelope membrane of chloroplasts contains a large proportion of the phospholipid phospatidylcholine (PC), which is synthesized in the ER and also a possible precursor for thylakoid galactolipids. The mechanism for PC transport from the ER to chloroplasts is not known. Using isolated chloroplasts and liposomes containing radiolabelled PC we investigated non-vesicular transport of PC in vitro. PC uptake in chloroplasts was time- and temperature-dependent, but nucleotide-independent. Increased radius of liposomes stimulated PC uptake, and protease treatment of the chloroplasts impaired PC uptake. This implies that the chloroplast outer envelopes contains an exposed proteinaceous machinery for the uptake of PC from closely apposed membranes.
Keywords: phosphatidylcholine (PC); chloroplast; liposome; lipid uptake
Abbreviations: DAG, diacylglycerol; DGDG, digalactosyldiacylglycerol; ER, endoplasmic reticulum;
MGDG,
monogalactosyldiacylglycerol;
PA,
phosphatic
acid;
PC,
phosphatidylcholine; PE, phosphatidylethanolamine; PLAM, plastid associated membranes; TGD, trigalactosyl diacylglycerol.
Introduction Chloroplasts are vital organelles of plants as they harbour the photosynthesis machinery, nitrogen- and sulphur metabolism as well as produce, among other vital compound classes, amino- and fatty acids. The chloroplast contains an inner membrane system, the thylakoids, being surrounded by an aqueous stroma. The stroma is in turn surrounded by the envelope membrane, divided into an outer and inner envelope membrane. Chloroplast membranes constitute about 70% of the total plant mesophyll cell membranes, and 80% are galactolipids e.g. monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) both being more common in the thylakoid membranes than in the envelope (Dörmann and Benning 2002). In the outer leaflet of the outer envelope membrane, facing the cytosol, the phospholipid phospatidylcholine (PC) is a major constituent (32-44 mol%) (Block et al. 1983; Cline et al. 1981). Chloroplast galactolipids are synthesized in the envelope through a coordinated interplay with the endoplasmic reticulum (ER) e.g. chloroplasts export fatty acyl moieties to the ER and import diacylglycerol backbones that act as galactolipid precursors back from the ER (Benning 2008). In vivo pulse chase studies indicate that PC (detected in envelope/chloroplast) synthesized in the ER could be the immediate precursor for galactolipid synthesis in chloroplasts (Heinz and Roughan 1983; Hellgren et al. 1995; Hellgren and Sandelius 2001; Roughan et al. 1987; Slack et al. 1977). In addition, diacylglycerol (DAG) (Williams et al. 2000), phosphatic acid (PA) (Xu et al. 2005) and lyso-PC (Mongrand et al. 1997) have been suggested to be galactolipid precursors transported from the ER. Since chloroplasts lack capacity to synthesize the head group of PC, and both the outer envelope and the ER contain a significant amount of PC it seems likely that PC is transported from the ER whether or not it is the major precursor for galactolipids. However, the mechanism behind PC transport from ER to the envelope is unclear.
Movement of lipids between cytosolic organelles may use vesicular transport e.g. endocytic or exocytic pathways or non-vesicular transport (Holthuis and Levine 2005). Lipid interchange between ER and chloroplasts has been proposed to occur by contact sites between the plastid envelope and a specialized ER domain coined plastid associated membranes (PLAM) (Andersson et al. 2007; Benning 2009). Trigalactosyl diacylglycerol (TGD) proteins are located in the chloroplast envelope and facilitate lipid transport into chloroplasts from the ER (Awai et al. 2006; Wang et al. 2012; Xu et al. 2003). TGD4 alone makes up a complex in the outer envelope and TGD1, 2 and 3 a complex in the inner envelope (Wang and Benning 2012). TGD proteins are proposed to facilitate transfer of PA from ER to the chloroplast where it is used as a galactolipid precursor after dephosphorylation in the inner envelope (Wang et al. 2012). Recently, it was proposed that the ER and the outer chloroplast envelope can form hemifusions at contact sites to facilitate transfer of lipophilic compounds (Mehrshahi et al. 2013). It has been shown in vitro that radiolabelled phospholipids can be transferred from donor ER to isolated chloroplasts in a time and temperature dependent manner but without need for nucleotides or cytosolic factors (Andersson et al. 2004; Xu et al. 2008). Thus, available evidence suggests that chloroplasts can take up phospholipids from donor membranes, provided these make close contact to the organelle. Yeast mitochondria have been shown to be able to take up phospholipids from chemically defined donor liposomes by means of a protein machinery in their outer membrane (Wu and Voelker 2004). In this case larger liposomes were more efficient donors and this was interpreted as those larger liposomes can contribute a “flatter” contact surface with the mitochondria. In this study we tested if isolated chloroplasts have the ability to take up PC from donor liposomes. We used an in vitro system using radiolabelled PC in liposomes mixed with isolated chloroplasts to test if chloroplast like mitochondria could take up phospholipids form chemically defined donor liposomes. We
show that PC is absorbed by chloroplasts in a time and temperature dependent way, and that the PC uptake rate is related to the size of liposomes. Moreover, the process is independent of exogenous nucleotides, whereas, removal of proteins on the outer envelope by thermolysin impairs it, but which protein(s) responsible remains to be elucidated.
Methods and materials
Plant material and chloroplast preparation Pea (Pisum sativum L. cv Kelvedon Wonder) were sown on soil, covered with vermiculite and grown at 22°C and 12 h light. Isolation of chloroplasts from 10-12 days old peas was performed as previously described (Aronsson et al. 2001). Chlorophyll concentration was measured spectroscopically according to (Lichtenthaler and Wellburn 1983).
Liposome preparation Lipids were extracted from 7-9 days old pea seedlings. They were cut with razor blade, and soaked in 12 ml 33P (2.66*108 kdpm) (as H333PO4, Perkin Elmer) radiolabelled water for 48 h in a fume hood with lights on before lipid extraction. Thereafter total lipids were extracted (Kourtchenko et al. 2007) and a phospholipid fraction prepared by fractionation on a silica column. Total extracted lipids were loaded onto a 500 mg silica column (Supelco). Neutral lipids were eluted with CHCl3:acetone (9:1, by volume), glycolipids with acetone:methanol (9:1, by volume), and total phospholipids with methanol. The procedure of isolation of liposomes was adapted from (Olson et al. 1979) with some modifications as described (Berglund et al. 1999). Total phospholipids isolated from labeled pea seedlings or a mix of commercial 14C-dipalmitoyl phosphatidylcholine ([14C]PC, specific activity 3.6 GBq/mmol, Perkin Elmer) or commercial L-3-phosphatidylethanolamine, 1palmitoyl-2-[1-14C]linoleoyl ([14C]PE, specific activity 2.04 GBq/mmol, Perkin Elmer) corresponding to 50 kdpm per assay with soybean PC and PE (Sigma-Aldrich) (mix 4:1 by weight, 10 µg/assay) dissolved in chloroform were dried under N2. One ml of import buffer was added to the dried lipid film heated to 60°C for about 10 s and vortexed thoroughly. After
four cycles of freeze-thawing liposomes were pressure extruded through double 50, 100, 200, 400 or 1000 nm (as indicated) polycarbonate filters eleven times, and stored at 4°C until use.
Liposome import assay Liposomes corresponding to about 30 kdpm of 14C- or 33P-label were mixed with chloroplasts corresponding to 50 µg of chlorophyll in a total volume of 100 µl of import buffer (0.33 M sorbitol; 50 mM Hepes; KOH pH 8.0). Import assays were performed on ice or 25°C water bath with 150 mol*m-2*s-1 light using different time points. The reaction was stopped by adding 200 µl ice cold isolation buffer. The suspension was transferred to the top of a 1 ml 35% Percoll mixed in isolation buffer, centrifuged 5000 g, 8 min at 4°C, and the pellet being washed 3 times in 1 ml isolation buffer. Finally the pellet was re-suspended in 200 µl isolation buffer and transferred to a scintillation vial containing 1 ml methanol, and 8 ml scintillation cocktail (Ready Protein+, Beckman) was added before and the radioactivity counted in a TriCarb 2900 TR liquid scintillation analyzer. Thermolysin treatment was performed according to (Garcia et al. 2010) with slight modifications. Chloroplasts were incubated in 50 µg/ml thermolysin and 300 µM CaCl2 for 30 min on ice in darkness. Control samples had thermolysin replaced with an equal volume of import buffer. Reactions were terminated by addition of an equal volume of 50 µM EDTA (final concentration 25 µM). The suspension was transferred to the top of a 1 ml “cushion” of 35% (by volume) Percoll™ in isolation buffer, centrifuged at 5000 g, 5 min at 4°C, and the pellet washed twice in 1 ml import buffer, then finally re-suspended in 50 µl import buffer to continue the liposomes import assay. For nucleotide experiments, ATP/GTP was added to the reaction to a final concentration of 0.5 mM or 1 mM (only for ATP). Control samples had ATP/GTP replaced with the same volume of import buffer. Reactions were incubated at 25°C in a water bath with 150 mol*m-
2
*s-1 light for 30 min or 60 min.
Results and discussion
Uptake of PC from liposomes to isolated chloroplasts is time and temperature dependent As ER derived lipid transport to chloroplasts has been suggested to occur at contact sites between the ER and the chloroplast and yeast mitochondria apparently harbors a machinery for the uptake of phospholipids from protein free liposomes, we decided to test if intact plant chloroplasts could take up radiolabelled phospholipids from large unilamellar liposomes. An initial experiment was performed with large (400 nm) unilamellar liposomes (LUVs) prepared
from
phospholipids
isolated
[33P]ortophosphate. LUVs containing
33
from
pea
seedlings
pre-incubated
with
P-labelled lipids were mixed with isolated
chloroplasts isolated from pea seedlings. After incubation the chloroplasts were washed and the amount of radiolabelled transferred to the chloroplasts determined (figure. 1A). There was a clear time dependent incorporation of radiolabel into the chloroplast fraction demonstrated by a more than 3-fold increase in label after 30 min compared to zero time (figure. 1A). Moreover, phospholipid uptake was also temperature dependent; after 30 min at 25°C uptake of phospholipids was approximately double that of samples incubated on ice (figure. 1A). Unfortunately the amount of radiolabel transferred was too low to determine the exact composition of phospholipids transferred. We thus conclude that isolated chloroplasts can take up phospholipids from protein free liposomes in vitro. We next tested if PC, as this lipid is most likely transported from the ER to the outer chloroplast envelope (see above), could be transferred from chemically defined liposomes to isolated chloroplasts. To this end [14C]PC was incorporated into liposomes (400 nm in diameter) made from a mix of soybean PC:PE (4:1). The PC:PE ratio is around 2:1 in ER, but the total amount differ in plants, PC and PE being around 50% and 25%, respectively (Moreau et al. 1998), mammals, PC and PE
representing around 60% and 30%, respectively, and yeast where PC and PE being around 40% and 20%, respectively (Van Meer et al. 2008). Thus, the amount of PE was rather well reflected in the liposomes whereas increased concentration of PC in the liposomes enabled improved detection but also replaced other phospholipids not of investigated in this study. The uptake of the total pool of unlabeled and labeled [14C]PC in chloroplasts was increased from 15.4 pmol at 0 min, to 59.4 pmol at 30 min, and finally 74.8 pmol at 60 min (figure. 1B). Performing the experiment at 25°C allowed substantially more PC taken up compared to incubation on ice (figure. 1B). Uptake of the total PC pool when compared was after 30 min 23.6 pmol on ice versus 59.4 pmol at 25°C, and after 60 min 27.6 pmol on ice versus 74.8 pmol at 25°C. In a separate experiment, liposomes were prepared with radiolabelled PE and transfer of this to isolated chloroplasts was measured. The uptake of PC was 10 fold higher than that of PE e.g. 74.8 pmol PC versus 6.98 pmol PE at 60 min at 25°C (figure. 1B). Thus, PC was clearly taken up from protein free liposomes by isolated chloroplasts in a time and temperature dependent fashion. This thus indicates that there is a lipid import machinery present in chloroplasts that can operate in the absence of any proteins on the donor membrane. The efficiency of PC uptake was clearly time and temperature dependent (figure. 1B). The significantly lower uptake of PE demonstrates selectivity in the lipid import machinery. This result is in accordance with that PE is not found in any significant levels in isolated chloroplasts or envelope membranes. In addition, the very low uptake of PE demonstrates that the result is not due to binding of liposomes to the chloroplasts; there really is transfer of lipids from liposomes to chloroplasts. To test whether nucleotides are needed during PC uptake to chloroplasts in vitro, ATP or GTP was added to the transfer reaction. Reactions performed at 25°C under light condition with additional ATP or GTP (final concentration 0.5 mM) demonstrated slightly lower PC uptake into chloroplasts compared with mock reactions without any additional
nucleotides added (figure. 1C). Furthermore, no increase in PC uptake was observed when the concentration of ATP was increased from 0.5 to 1 mM. Thus, energy in the form of ATP or GTP is not a requirement during PC uptake to chloroplasts. Our results support the notion i.e. even with ATP or GTP employed in the reaction, chloroplasts still cannot absorb more PC. This thus confirms that nucleotides are not necessarily needed for lipid transport to chloroplasts. This is in line with earlier studies of transfer of lipids from ER to chloroplasts (Andersson et al. 2004; Moreau et al. 1991; Xu et al. 2008). Vesicular transport requires energy and reconstituted lipid transport (presumably also vesicular-mediated) between ER and Golgi in plants (Morré et al. 1991; Sturbois et al. 1994) and animals (Moreau and Morré 1991; Moreau et al. 1991; Morré 1998) cells require ATP, but previous studies using purified ER as donor and chloroplast as acceptor showed no ATP dependent transfer of total lipids (Morré et al. 1991). Lipids transport from ER to mitochondria in yeast has been extensively studied by various in vitro approaches (de Kroon et al. 2003; Gaigg et al. 1995; Voelker 2000; 2003), and the general conclusion is that no nucleotides are required for lipid transport from ER to mitochondria. Apparently, there are similarities between lipid transport from ER to mitochondria in yeast and ER to chloroplast lipid transport in higher plants.
Proteins on the outer envelope are needed for efficient PC uptake To investigate if there are proteins on the outer envelope to assist PC uptake, intact chloroplasts were treated with thermolysin. This protease is unable to penetrate the chloroplast outer envelope membrane, and thus only digests proteins exposed at the outer envelope surface of intact chloroplasts (Cline et al. 1984). It was obvious that after chloroplast treatment with thermolysin the lipid uptake efficiency was much lower than for those chloroplasts not treated with protease (figure. 2). This demonstrates that protein(s) protruding
on the surface of the outer envelope play an important role in uptake of PC from liposomes by isolated chloroplasts.
Increased liposome size accelerates PC uptake to chloroplasts We interpret our findings as that there is a machinery in the outer chloroplast envelope aiding in transfer of phospholipids from a donor membrane, provided this membrane is situated close enough to the chloroplast. A donor liposome of larger radius would present a larger flatter surface to the chloroplast and thus in theory allows a more efficient transfer of lipids. This was in fact the case with phospholipid uptake by isolated yeast mitochondria from donor liposomes (Wu and Voelker 2004). To test whether the size of liposomes also affect PC absorption to chloroplasts, liposomes made from [14C]PC in a mixture with soybean PC:PE (ratio 4:1) with different diameters (50 nm up to 1000 nm in diameter) were used for lipid uptake to isolated pea chloroplasts in vitro (figure. 3). This clearly demonstrated that the uptake rate of PC increased with the size of liposomes e.g. chloroplasts took up about 2-fold more PC from 1000 nm liposomes in diameter compared to 50 nm liposomes (figure. 3). Thus, increased size of the liposomes affected lipid uptake into chloroplast positively.
Conclusions We herein proved clear evidence for that isolated chloroplasts can take up the phospholipid PC from chemically defined protein free liposomes. Furthermore, the uptake is dependent on proteins exposed on the outer envelope surface of the chloroplast. In theory, this could be explained by lipids adsorbed from the surface of the liposomes by lipid transport proteins protruding from the chloroplast surface. Alternatively, proteins in the outer envelope could induce hemifusions with the liposomes. We cannot, with this experimental set up completely
discriminate between these two situations and the literature lends some support to both. However, the lack of PE uptake from liposomes demonstrates the liposomes at least not remain attached to the chloroplast to a large extent. Lipid transport seems to be mediated between mitochondria and ER as well as between Golgi and ER by membrane bound lipid transport proteins at contact sites (Holthuis and Levine 2005; Kornmann and Walter 2010). It was also recently shown that there is likely exchange of hydrophobic molecules as hemifusions between the ER and chloroplasts (Mehrshahi et al. 2013). Other questions that could be addressed in future studies using similar methodology as we used are if other lipids are transported, if the transport is unidirectional and if TGD-proteins are involved in the observed transfer of PC from liposomes.
Acknowledgements This work was supported by Olle Engkvist Byggmästare Foundation (to H.A.), and PhD student fellowships from the China Scholarship Council (CSC) and Nanjing Agricultural University (to C.Y.).
Figure legends
Figure 1. Uptake of PC from liposomes to chloroplasts is time and temperature dependent but nucleotide independent. Liposomes containing (A) 33P labeled phospholipids, (B) [14C]PC or [14C]PE were mixed with chloroplasts isolated from pea seedlings and incubated at the indicated temperature for the indicated time. After re-purification of the chloroplasts, the amount of labelled lipid transferred to the chloroplasts was measured. C. Indicated concentrations of ATP and GTP were added to the import reaction where liposomes containing [14C]PC were mixed with chloroplasts isolated from pea seedlings and the amount of labelled PC transferred to the chloroplasts measured. All data presented are representative of two independent experiments, and range derived from three replicates within that specific experiment.
Figure 2. Envelope proteins susceptible to thermolysin facilitate PC uptake to chloroplasts. Liposomes containing [14C]PC were mixed with chloroplasts isolated from pea seedlings and the amount of labelled PC transferred to the chloroplasts measured. Chloroplasts were incubated with thermolysin or mock treated prior to a 60 min liposome import assay on ice or at 25°C. Data presented are representative of two independent experiments, average and range derived from three replicates within that experiment is shown.
Figure 3. Increased liposome diameter accelerates PC uptake to chloroplasts. Liposomes containing [14C]PC of the indicated diameter were mixed with chloroplasts isolated from pea seedlings and the amount of labelled PC transferred to the chloroplasts measured. Data presented are representative of two independent experiments, and range derived from three replicates within that experiment.
References Andersson MX, Goksör M, Sandelius AS (2007) Optical manipulation reveals strong attracting forces at membrane contact sites between endoplasmic reticulum and chloroplasts. Journal of Biological Chemistry 282: 1170-1174 Andersson MX, Kjellberg JM, Sandelius AS (2004) The involvement of cytosolic lipases in converting phosphatidyl choline to substrate for galactolipid synthesis in the chloroplast envelope. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1684: 46-53 Aronsson H, Sundqvist C, Timko MP, Dahlin C (2001) The importance of the C-terminal region and Cys residues for the membrane association of the NADPH: protochlorophyllide oxidoreductase in pea. FEBS Lett 502: 11-15 Awai K, Xu C, Tamot B, Benning C (2006) A phosphatidic acid-binding protein of the chloroplast inner envelope membrane involved in lipid trafficking. Proceedings of the National Academy of Sciences 103: 10817-10822 Benning C (2008) A role for lipid trafficking in chloroplast biogenesis. Progress in lipid research 47: 381-389 Benning C (2009) Mechanisms of lipid transport involved in organelle biogenesis in plant cells. Annual Review of Cell and Developmental 25: 71-91 Berglund AH, Nilsson R, Liljenberg C (1999) Permeability of large unilamellar digalactosyldiacylglycerol vesicles for protons and glucose–influence of α-tocopherol, β-carotene, zeaxanthin and cholesterol. Plant Physiology and Biochemistry 37: 179-186 Block MA, Dorne A-J, Joyard J, Douce R (1983) Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. II. Biochemical characterization. Journal of Biological Chemistry 258: 13281-13286 Cline K, Andrews J, Mersey B, Newcomb EH, Keegstra K (1981) Separation and characterization of inner and outer envelope membranes of pea chloroplasts. Proceedings of the National Academy of Sciences 78: 35953599 Cline K, Werner-Washburne M, Andrews J, Keegstra K (1984) Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. Plant Physiol 75: 675-678 de Kroon AI, Koorengevel MC, Vromans TA, de Kruijff B (2003) Continuous equilibration of phosphatidylcholine and its precursors between endoplasmic reticulum and mitochondria in yeast. Mol Biol Cell 14: 2142-2150 Dörmann P, Benning C (2002) Galactolipids rule in seed plants. Trends in plant science 7: 112-118 Gaigg B, Simbeni R, Hrastnik C, Paltauf F, Daum G (1995) Characterization of a microsomal subfraction associated with mitochondria of the yeast, Saccharomyces cerevisiae. Involvement in synthesis and import of phospholipids into mitochondria. Biochimica et Biophysica Acta (BBA)-Biomembranes 1234: 214-220 Garcia C, Khan NZ, Nannmark U, Aronsson H (2010) The chloroplast protein CPSAR1, dually localized in the stroma and the inner envelope membrane, is involved in thylakoid biogenesis. Plant Journal 63: 73-85 Heinz E, Roughan PG (1983) Similarities and differences in lipid metabolism of chloroplasts isolated from 18: 3 and 16: 3 plants. Plant Physiol 72: 273-279 Hellgren LI, Carlsson AS, Selldén G, Sandelius AS (1995) In situ leaf lipid metabolism in garden pea (pisum sativum L.) exposed to moderately enhanced level of ozone. J Exp Bot 46: 221-230 Hellgren LI, Sandelius AS (2001) Age-dependent variation in membrane lipid synthesis in leaves of garden pea (Pisum sativum L.). J Exp Bot 52: 2275-2282 Holthuis JC, Levine TP (2005) Lipid traffic: floppy drives and a superhighway. Nature reviews molecular cell biology 6: 209-220 Kornmann B, Walter P (2010) ERMES-mediated ER-mitochondria contacts: molecular hubs for the regulation of mitochondrial biology. J Cell Sci 123: 1389-1393 Kourtchenko O, Andersson MX, Hamberg M, Brunnström Å, Göbel C, McPhail KL, Gerwick WH, Feussner I, Ellerström M (2007) Oxo-phytodienoic acid-containing galactolipids in Arabidopsis: jasmonate signaling dependence. Plant Physiol 145: 1658-1669 Lichtenthaler H, Wellburn A (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochemical Society Transactions 11: 591-592 Mehrshahi P, Stefano G, Andaloro JM, Brandizzi F, Froehlich JE, DellaPenna D (2013) Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts. Proceedings of the National Academy of Sciences 110: 12126-12131 Mongrand S, Bessoule J, Cassagne C (1997) A re-examination in vivo of the phosphatidylcholine–galactolipid metabolic relationship during plant lipid biosynthesis. Biochemical Journal 327: 853-858 Moreau P, Bessoule J, Mongrand S, Testet E, Vincent P, Cassagne C (1998) Lipid trafficking in plant cells. Progress in lipid research 37: 371-391 Moreau P, Morré DJ (1991) Cell-free transfer of membrane lipids. Evidence for lipid processing. Journal of
Biological Chemistry 266: 4329-4333 Moreau P, Rodriguez M, Cassagne C, Morré D, Morré D (1991) Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver. Journal of Biological Chemistry 266: 43224328 Morré D, Penel C, Morré DM, Sandelius AS, Moreau P, Andersson B (1991) Cell-free transfer and sorting of membrane lipids in spinach. Protoplasma 160: 49-64 Morré DJ (1998) Cell-free analysis of Golgi apparatus membrane traffic in rat liver. Histochemistry and cell biology 109: 487-504 Olson F, Hunt C, Szoka F, Vail W, Papahadjopoulos D (1979) Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochimica et Biophysica Acta (BBA)Biomembranes 557: 9-23 Roughan PG, Thompson Jr GA, Cho SH (1987) Metabolism of exogenous long-chain fatty acids by spinach leaves. Archives of biochemistry and biophysics 259: 481-496 Slack CR, Roughan PG, Balasingham N (1977) Labelling studies in vivo on the metabolism of the acyl and glycerol moieties of the glycerolipids in the developing maize leaf. Biochemical Journal 162: 289-296 Sturbois B, Moreau P, Maneta-Peyret L, Morré D, Cassagne C (1994) Cell-free transfer of phospholipids between the endoplasmic reticulum and the Golgi apparatus of leek seedlings. Biochimica et Biophysica Acta (BBA)-Biomembranes 1189: 31-37 Van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nature reviews molecular cell biology 9: 112-124 Wang Z, Benning C (2012) Chloroplast lipid synthesis and lipid trafficking through ER-plastid membrane contact sites. Biochemical Society Transactions 40: 457-463 Wang Z, Xu C, Benning C (2012) TGD4 involved in endoplasmic reticulum-to-chloroplast lipid trafficking is a phosphatidic acid binding protein. Plant Journal 70: 614-623 Williams J, Imperial V, Khan M, Hodson J (2000) The role of phosphatidylcholine in fatty acid exchange and desaturation in Brassica napus L. leaves. Biochemical Journal 349: 127-133 Voelker DR (2000) Interorganelle transport of aminoglycerophospholipids. Biochimica et Biophysica Acta (BBA)Molecular and Cell Biology of Lipids 1486: 97-107 Voelker DR (2003) New perspectives on the regulation of intermembrane glycerophospholipid traffic. Journal of lipid research 44: 441-449 Wu W-I, Voelker DR (2004) Reconstitution of phosphatidylserine transport from chemically defined donor membranes to phosphatidylserine decarboxylase 2 implicates specific lipid domains in the process. Journal of Biological Chemistry 279: 6635-6642 Xu C, Fan J, Cornish AJ, Benning C (2008) Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein. Plant Cell 20: 2190-2204 Xu C, Fan J, Froehlich JE, Awai K, Benning C (2005) Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis. Plant Cell 17: 3094-3110 Xu C, Fan J, Riekhof W, Froehlich JE, Benning C (2003) A permease‐like protein involved in ER to thylakoid lipid transfer in Arabidopsis. The EMBO journal 22: 2370-2379
Figure 1
Figure 2
Figure 3
Bullet points:
Chloroplasts take up PC from unilamellar liposomes in a time and temperature dependent fashion
ATP and GTP are dispensable for in vitro PC uptake into chloroplasts
Envelope proteins susceptible to thermolysin facilitate PC uptake to chloroplasts
Increased liposome radius accelerates PC uptake to chloroplasts
