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ScienceDirect Lipid trafficking at endoplasmic reticulum–chloroplast membrane contact sites Maryse A Block and Juliette Jouhet Glycerolipid synthesis in plant cells is characterized by an intense trafficking of lipids between the endoplasmic reticulum (ER) and chloroplasts. Initially, fatty acids are synthesized within chloroplasts and are exported to the ER where they are used to build up phospholipids and triacylglycerol. Ultimately, derivatives of these phospholipids return to chloroplasts to form galactolipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, the main and essential lipids of photosynthetic membranes. Lipid trafficking was proposed to transit through membrane contact sites (MCSs) connecting both organelles. Here, we review recent insights into ER– chloroplast MCSs and lipid trafficking between chloroplasts and the ER. Address Laboratoire de Physiologie Cellulaire et Ve´ge´tale, Unite´ Mixte Recherche 5168, Centre National Recherche Scientifique, Universite´ de GrenobleAlpes, Institut National de la Recherche Agronomique, Commissariat a` l’Energie Atomique et Energies Alternatives, Institut de Recherches en Technologies et Sciences pour le Vivant, 17 Avenue des Martyrs, F38054 Grenoble, France Corresponding author: Block, Maryse A ([email protected], [email protected])

Current Opinion in Cell Biology 2015, 35:21–29 This review comes from a themed issue on Cell organelles Edited by Maya Schuldiner and Wei Guo

http://dx.doi.org/10.1016/j.ceb.2015.03.004 0955-0674/# 2015 Elsevier Ltd. All rights reserved.

Introduction The chloroplast, like mitochondrion, is a semiautonomous organelle. It contains an extensive membrane system, the thylakoids, and has a unique lipid composition, where the two galactoglycerolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are essential. Through photosynthesis it achieves the conversion of light energy into chemical energy and produces a diversity of compounds crucial for plant development [1,2]. The chloroplast is the main site of synthesis of fatty acids (FA) which are exported to the endoplasmic reticulum (ER) for phospholipid and triacylglycerol (TAG) synthesis [3–5]. On the other hand, diacylglycerol (DAG), the substrate for galactolipid synthesis which occurs in the chloroplast www.sciencedirect.com

envelope [5–8], is for a large part generated from phospholipids formed in the ER [9,10]. FA and glycerolipid trafficking between chloroplasts and the ER is therefore very active. However, lipids cannot move freely through the cytosol due to their weak water solubility and except for the import of a limited number of glycosylated proteins from the Golgi, chloroplasts are mostly unconnected with the endomembrane vesicular trafficking network [11]. A few components of the lipid transfer machineries have been identified but most of the transport mechanisms remain to be characterized. The observation of numerous membrane contact sites (MCSs) between chloroplasts and the ER [12,13] lead to the hypothesis that these MCSs could play a role in lipid trafficking [12,14]. We will outline recent results on MCSs and lipid trafficking between chloroplasts and the ER in order to deduce some guidelines for future studies of plant lipid biosynthesis.

General features of the chloroplast–ER membrane contact sites The ER has a reticulated structure with some cisternae that line up with chloroplasts [13]. By following fluorescent proteins expressed either in the ER or in chloroplasts, it has been observed that some branch end-points of the ER locate at the chloroplast surface [15] and that some thin tubular formations called stromules (stromafilled tubules) extend from the chloroplast envelope and align along ER tubules, branching accordingly with the ER [16,17] (Figure 1a). Rapid dynamic behavior of stromules suggests a stretch of the chloroplast envelope relying on membrane tethering sites with the ER [17]. Physical association between the ER and chloroplast surface has been demonstrated on isolated Arabidopsis thaliana chloroplasts by optical manipulation with laser tweezers [14]. Some ER fragments cannot detach from chloroplasts up to a pulling force of 400 pN, a magnitude relevant with protein–protein interactions. These ER– chloroplast membrane connections have been released from purified chloroplasts by incubation at a lowered pH and have been identified with ER-associated enzyme activities [14]. They were called plastid associated membranes (PLAM). They differ from the chloroplast outer envelope membrane (OEM) by their low galactolipid content and presence of phosphatidylethanolamine (PE) and from the ER by their low glycosylceramide and sterol contents. PE and phosphatidylcholine (PC) are by far the main glycerolipids of PLAM and are equally present [14]. The protein profile of PLAM is also different from the ER and chloroplast envelope [14]. Interestingly, PLAM contains PC synthase but not Current Opinion in Cell Biology 2015, 35:21–29

22 Cell organelles

Figure 1

(a)

(b)

Chloroplast (red) - ER (green) 2

ER Chloroplast (red) - BnCLIP1-eGFP (green) 2

Cytosol

e

St

lop

ve

ro

En

m

ul

ER (red) - BnCLIP1-eGFP (green)

e

Chloroplast

Stroma

2

Thylakoids Reproduced from Tan et al., 2011 [18]

(c)

1: FAT 2: FAX 3: LACS 4: PLA 5: LPCAT 6: ACBP 7: ABCP Lumen

TAG

7 ER

4

5

3

5

: Unknown

6

PC

6

?

Cytosol

4

Lyso-PC

5 3

OEM

4 Acyl-CoA

IMS 2

FA

IEM 1 Stroma

(d)

Chloroplast

Acyl-ACP

1: FAD2 2: TGD4 3: TGD1-3 4: MGD1 5: PAP 6: DGD1 7: FAD6-8 Lumen

PC 1

ER

Lyso-PC Cytosol

?

PA

2

OEM

Acyl-CoA

6

DAG IMS 7

4

3

3 3 5

MGDG

IEM Chloroplast

DGDG

Current Opinion in Cell Biology

Membrane contact sites between chloroplast and endoplasmic reticulum. (a) Schematic position of the MCSs reported between chloroplast and ER. MCSs occur along the ER at different sites on the chloroplast surface, either directly with the chloroplast body or along the highly dynamic chloroplast stromules (stroma-filled tubules). The contact surface can extend to a wide area along an ER branch. Both membranes of the chloroplast envelope are present in the MCSs. In the case of stromules extension, initial contact may preferentially locate on the two beaks corresponding to the extreme ends of the chloroplast ovoid form. (b) Punctate localization of the lipase BnCLIP1 in overlap areas between

Current Opinion in Cell Biology 2015, 35:21–29

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Lipid trafficking between ER and chloroplasts Block and Jouhet 23

MGDG synthase. In tobacco leaves, a chloroplast lipase/ acylhydrolase BnCLIP1 has been detected by confocal imaging at MCSs between the ER and chloroplasts [18] (Figure 1b). This lipase has a discrete localization on the OEM at the junction between the ER and plastids [19]. Presence of PC synthase and lipase at ER–chloroplast MCSs supports a possible role in ER–chloroplast lipid trafficking.

Export of fatty acids from chloroplasts to the ER In higher plants, 16 carbon-FA and 18 carbon-FA are synthesized in the chloroplast stroma as thioesters of acyl carrier protein (16:0-ACP and 18:0-ACP) and 18:0-ACP is monodesaturated into 18:1-ACP [3–5]. FAs are then exported from chloroplasts and converted into acyl-CoAs before insertion into phospholipids and TAG in the ER. According to the model of Fullekrug and co [20], acyl activation, for instance by acyl-CoA synthases, could represent a driving force for FA uptake at MCSs. Indeed, a combination of acyl-ACP thioesterases and acyl-CoA synthases located respectively in the inner and outer side of the chloroplast envelope contributes to FA export [21–23] (Figure 1c and Table 1). The OEM long chain acyl-CoA synthase LACS9 and the ER-located LACS1 and LACS8 are important for TAG formation [24]. However, since the triple lacs1, lacs8, lacs9 KO mutants are only slightly defective in export of acyl groups, some other proteins should also contribute to FA export. Several acyl-ACP synthetases are also present in the chloroplast envelope [25–27]. They are positioned on

the inner side of the envelope and contribute to short acyl chain elongation and are thus involved in import of acyl chains into chloroplasts rather than export. In our current understanding, free FAs cross the chloroplast envelope membranes by facilitated diffusion. The recently identified FA transporter called FAX1 present in the inner envelope membrane participates in the transfer [28]. Some additional transporters remain however to be characterized in the envelope proteome [28,29,30] since FAX1 deleted lines are only partially affected in chloroplast export of FAs [28]. On the ER, a protein of the ATP-binding cassette family, ABCA9, plays a role in incorporation of newly-synthesized FAs into an 18:3containing pool of TAG present in seeds [31]. The data suggest a role of ABCA9 in coupling of FA export towards TAG synthesis via PC desaturation. Furthermore, ACBPs (Acyl-CoA-binding proteins), such as ACBP4, ACBP5 and ACBP6, have the ability to bind long-chain acylCoAs and could facilitate transfer of FAs from chloroplasts to the ER through the cytosol to feed glycerolipid synthesis [10,32–34]. Expression of ACBP4 and ACBP5 is light-regulated similarly to plastid FA synthesis, and the Arabidopsis acbp4 knock out lines show reduced levels of membrane glycerolipids. ACBP6 is linked to PC metabolism since under cold treatment Arabidopsis plants overexpressing ACBP6 show lower levels of PC and higher levels of phosphatidic acid (PA) than controls. Evidence suggests that PC also participates in FA transport from the chloroplast to the ER (Figure 1c). Indeed, data of

( Legend Figure 1 continued ) chloroplast and ER. The fluorescent micrograph is extracted from Figure 2 of [18]. GFP fluorescence of the fusion protein BnCLIP1-eGFP is observed by confocal imaging during transient expression in tobacco leaves. Top part: The subcellular location of ER relative to chloroplast. Middle part: The subcellular location of fused BnCLIP1-eGFP relative to chloroplast. Bottom part: The subcellular location of fused BnCLIP1-eGFP relative to ER. GFP fluorescence is shown in green in middle and bottom parts; Chloroplast autofluorescence is shown in red; ER network marked with yellow fluorescence protein (YFP) is shown in green in the top part and in red in the bottom part. Overlay of green and red signals shows in yellow. The 2-numbered arrow head indicates a triple overlaying point between a chloroplast, the ER and BnCLIP1eGFP. (c) A model for fatty acid export from chloroplast to ER transiting through MCSs. Fatty acyls are formed as acyl-ACPs in the chloroplast. They are hydrolyzed in the inner envelope membrane (IEM) by fatty acyl thioesterases (1) FATA or FATB. By active transport driven by FAX transporters (2) such as the recently reported FAX1 in the IEM and possibly combined diffusion through the chloroplast envelope membranes and the intermembrane space (IMS) (several black boxes- unknown- on the scheme), free fatty acids reach the outer envelope membrane (OEM). On the chloroplast surface, fatty acids (FA) are activated to acyl-CoAs by long chain acyl-CoA synthetases (LACS) (3) among which LACS9 very likely takes part. As soon as they are synthesized in chloroplast, FAs are incorporated into PC by a acyl-editing cycle of PC expected to go through LACS, phospholipases A (PLA) (4) and lyso-PC:acyl-CoA acyltransferases (LPCAT) (5). Some PLAs and LPCATs are present on the chloroplast outer surface, some of them observed at punctate spots on the OEM or associated with chloroplast-ER MCSs. Several other PLAs and LPCATs are predicted present in the ER. Transfer of acyl-specific pools of the partially water soluble lyso-PC and acyl-CoA molecules across the cytosol is possibly facilitated by tethering of the ER and the chloroplast envelope by unknown proteins (?). Acyl-CoAs can also be taken in charge by soluble acyl-CoA binding proteins (ACBP) (6) to supply acyltransferases in the ER. ABCA9 (7) looks like a membrane transporter involved in the transfer of FAs across the ER membrane and required for synthesis of a pool of triacylglycerol (TAG). (d) A model for glycerolipid import from ER to chloroplast transiting through MCSs. PC is synthesized in the ER and desaturated by FAD2 (1). Our hypothesis is that a flippase (represented as a first black box on the scheme) generates enrichment of the ER cytoplasmic leaflet in FAD2-desaturated PC creating favorable conditions for transfer of PC to chloroplasts. Membrane curvature and properties are locally adjusted to local lipid composition which participates to formation of MCSs possibly alleviated by tethering proteins (?). Unidentified proteins (second black box) locate at MCSs and generate PC transfer to chloroplasts. Phospholipases A and lyso-PC:acyl-CoA acyltransferases likely participate as well as long chain acyl-CoA synthetases such as LACS4 and LACS9. The transfer likely goes through channeling of acyl-specific pools of lyso-PC and acyl-CoA. Increase of PC content at the chloroplast surface supports PC hydrolysis into DAG and PA by cytosolic phospholipases D and C. TGD proteins assemble in the attachment zone, positioning TGD4 (2) in the OEM and a complex containing TGD1, TGD2, and TGD3 (3) in the IEM. TGD assembly enables translocation of the PC-derived DAG across the chloroplast envelope for galactolipid synthesis. PA activates both TGD and the MGDG synthase, MGD1 (4). MGD1 is positioned on the IEM facing the interspace. PA is then converted to DAG by PAP in the IEM (5) joining the major DAG flux feeding MGD1 for MGDG synthesis. By unidentified mechanisms likely positioned at MCSs between the two envelope membranes (several black boxes), MGDG is transferred to the OEM where DGDG synthesis is catalyzed by DGD1 (6), then DGDG is transferred back to the IEM. Final desaturation of MGDG and DGDG occurs in the IEM through FAD6, FAD7 and FAD8 (7). www.sciencedirect.com

Current Opinion in Cell Biology 2015, 35:21–29

24 Cell organelles

Table 1 List of the main expected or putative Arabidopsis thaliana proteins playing a role in lipid transfer between plastid and ER Name

TAIR accession number

Function

FATA1 FATA2 FATB

At3g25110 At4g13050 At1g08510

Fatty acid thioesterase Fatty acid thioesterase Fatty acid thioesterase

Chloroplast IEM Chloroplast IEM Chloroplast IEM

LACS1 LACS2 LACS3 LACS4 LACS5 LACS8 LACS9

At2g47240 At1g49430 At1g64400 At4g23850 At4g11030 At2g04350 At1g77850

Long Long Long Long Long Long Long

ER ER ER ER ER ER Chloroplast OEM

FAX1

At3g57280

Fatty acid translocater

Chloroplast IEM

chain chain chain chain chain chain chain

acyl-CoA acyl-CoA acyl-CoA acyl-CoA acyl-CoA acyl-CoA acyl-CoA

Localization

synthetase synthetase synthetase synthetase synthetase synthetase synthetase

ABCA9

At5g61730

Fatty acid translocater

ER

ACBP4 ACBP5

At3g05420 At5g27630

Acyl-CoA binding protein Acyl-CoA binding protein

Cytosol Cytosol

DAD1

At2g44810

Phospholipase A1-like acylhydrolase

Chloroplast

DGL

At1g05800 At2g31690 At4g16820 At1g51440 At1g06800 At2g30550 At2g19690 At2g26560

Phospholipase Phospholipase Phospholipase Phospholipase Phospholipase Phospholipase Phospholipase Phospholipase

Chloroplast Chloroplast Chloroplast Chloroplast Chloroplast Chloroplast ER Cytosol

LPCAT1 LPCAT2

At1g63050 At1g12640

Lyso-PC acyltransferase Lyso-PC acyltransferase

ER ER

NPC5

At3g03540

Phospholipase C

Cytosol

PLDa1 PLDa2 PLDa3 PLDa4 PLDb2 PLDg2 PLDg3 PLDz1

At3g15730 At1g52570 At5g25370 At1g55180 At4g00240 At4g11830 At4g11840 At3g16785

Phospholipase Phospholipase Phospholipase Phospholipase Phospholipase Phospholipase Phospholipase Phospholipase

Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol

PAH1 PAH2 LPPe1 LPPe2 LPPg

At3g09560 At5g42870 At3g50920 At5g66450 At5g03080

Phosphatidic Phosphatidic Phosphatidic Phosphatidic Phosphatidic

TGD1 TGD2 TGD3 TGD4

At1g19800 At3g20320 At1g65410 At3g06960

Lipid Lipid Lipid Lipid

MGD1 MGD2 MGD3

At4g31780 At5g20410 At2g11810

MGDG synthase MGDG synthase MGDG synthase

Chloroplast IEM Chloroplast OEM Chloroplast OEM

DGD1 DGD2

At3g11670 At4g00550

DGDG synthase DGDG synthase

Chloroplast OEM Chloroplast OEM

FAD2 FAD3 FAD6 FAD7 FAD8

At3g12120 At2g29980 At4g30950 At3g11170 At5g05580

Lipid Lipid Lipid Lipid Lipid

ER ER Chloroplast IEM Chloroplast IEM Chloroplast IEM

Current Opinion in Cell Biology 2015, 35:21–29

A1-like A1-like A1-like A1-like A1-like A1-like A2-like A2-like

acylhydrolase acylhydrolase acylhydrolase acylhydrolase acylhydrolase acylhydrolase acylhydrolase acylhydrolase

D D D D D D D D

acid acid acid acid acid

translocater translocater translocater translocater

desaturase desaturase desaturase desaturase desaturase

phosphatase phosphatase phosphatase phosphatase phosphatase

Cytosol Cytosol Chloroplast Chloroplast Chloroplast Chloroplast IEM Chloroplast IEM Chloroplast IEM Choroplast OEM (chloroplast associated — ER?)

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Lipid trafficking between ER and chloroplasts Block and Jouhet 25

kinetic labeling experiments support that preformed PC participates in trafficking of newly synthesized acyl chains from plastids to the ER before their incorporation onto glycerol-3-phosphate to form PA, DAG and phospholipids [35,36,37]. The presence of LACS and lyso-PC-acyltransferase activities on the OEM [38–40] and the strong enrichment in PC of the cytosolic leaflet of the OEM [41] contrasting with other chloroplast membranes which are mostly devoid of PC [42] support this model. In addition, since the velocity of PC labeling is very high, the intermediates in the pathway such as acyl-CoAs, lysoPC and PC are expected to undergo substrate channeling without mixing with the bulk pools [37]. In Arabidopsis, various acylhydrolases were reported in chloroplasts [43]. Interestingly, when expressed as GFP fusion, they display a punctate signal at the surface of chloroplasts, suggesting restricted localization to specific domains of the OEM [43], as BnCLIP1, described in tobacco [18]. Enrichment of lipids and enzymes in specific domains such as the PLAM or the chloroplast-ER MCS could favor FA channeling through glycerolipids, however this remains to be verified.

Import of glycerolipids from the ER to chloroplasts Since PC is de novo synthesized in the ER, its presence in the outer leaflet of the OEM clearly indicates the presence of lipid trafficking, however the trafficking mechanism is still unknown. It is noteworthy that only PC and not PE, another abundant ER phospholipid, is present in the OEM and that both phospholipids are absent from other chloroplast membranes. This location may be related to galactolipid synthesis. MGDG and DGDG are formed by sequential galactosylation of DAG through MGD and DGD enzymes in the chloroplast envelope and DAG is either neosynthesized in the IEM, with a ‘prokaryotic’ fatty acyl signature (with 16-carbon acyls at the sn-2 position), or derived from PC and harboring a ‘eukaryotic’ signature (with 18-carbon acyls at the sn-2 position) [5,44,45] (Figure 1d and Table 1). Since no PC synthesis activity was detected in the chloroplast, transfer of the eukaryotic structure is needed, however the nature of the transported molecules, DAG, PA or PC, is still debated [10,46]. Taking into account two possible entry points for PA and DAG in a system including the chloroplast envelope MGD1 and PAP (Phosphatidic acid phosphatase) enzymes, mathematical simulation of galactolipid synthesis gave calculated concentrations of DAG and PA consistent with in vivo concentrations only if the eukaryotic structure was massively imported in the form of DAG, and not of PA [47]. This suggests that a massive influx of eukaryotic PA in the IEM is unlikely and supports an influx of eukaryotic DAG to the IEM [47]. The composition of PLAM with absence of PA or DAG and presence of PC supports a transfer of PC [14]. Transfer of PC likely comprises a transport of the related and more water soluble molecules, lyso-PC and acyl-CoA, between ER and chloroplast. This is supported by the fact that a www.sciencedirect.com

combination of the two long chain acyl-CoA synthetases, the chloroplast LACS9 and the chloroplast-associated ER LACS4 show a role in ‘eukaryotic’ galactolipid formation [48]. Proximity of membranes in MCSs could influence the channeling of specific pools of acyl-CoA such as 18:2CoA to esterify sn-2 position of lyso-PC. However deletion of LACS9 and LACS4 does not completely abolish transfer, indicating a complementary pathway [48]. A protein complex called TGD is essential for the import of ‘eukaryotic’ galactolipid precursors since the tgd mutants are defective in ‘eukaryotic’ galactolipids [49–54,55]. Differently from the lacs4, lacs9 double mutants, the tgd mutants accumulate trigalactolipids. The TGD complex is formed by a non-classical ABC transporter containing 3 different proteins (TGD1/TGD2/TGD3) in the inner envelope membrane (IEM). In addition, a b-barrel protein TGD4 also has a role in the import pathway. TGD4 is located in the OEM closely related with the ER, which is consistent with a position at ER–chloroplast MCSs. The PC content of chloroplasts is enhanced in several tgd mutants [52], suggesting a position of TGD downstream of LACS. TGD function is related to PA metabolism. Two of the TGD proteins (TGD2 and TGD4) bind PA [50,54,56,57]. The tgd mutants accumulate PA and have enhanced MGD1 activity presumably because PA activates MGD1 [50,54,56,57]. Apparently, the TGD complex is not involved in chloroplast export of FAs [58]. However, a chloroplast–ER bi-directional transit of tocopherol precursors/derivatives through TGD seems possible since a link between tocopherol synthesis in chloroplasts and FAD2 (Fatty Acid Desaturase) desaturation in the ER is altered in tgd mutants [59,60]. Kinetics labeling of lipids and mutant analysis indicated that ‘eukaryotic’ galactolipids are formed from FAD2-desaturated PC before desaturation by FAD3 [6]. FAD2 and FAD3 are ER transmembrane proteins and even though their topology in the membrane is identified, we do not yet know with certainty in which leaflet of the membrane the phospholipid molecule goes through desaturation [5,61–64]. It is believed that PC synthesis occurs on the cytosolic side of the ER. However, in the case of PS, which is supposed to be synthesized on the cytosolic side of the ER, data indicate an enrichment of PS on the lumen side and a downstream transfer of PS to the cytosolic side through the action of a flippase in the Golgi compartment [65–67]. Regulation of lipid presentation at lipid trafficking sites, either by adjustment of lipidtransferring proteins or lipid-metabolizing enzymes, is an important aspect that needs investigation [66]. FAD2 and FAD3 work as homodimers and also as a dimer combination of each protein [68]. Their heteromeric dimerization modulates PC desaturation. Enzyme turnover is temperature sensitive and dependent on ubiquitin–proteasome [69]. Since galactolipid synthesis involves a selection of partially desaturated PC-derived molecules, it would worth to analyze if FAD2/FAD3 interaction is affected when lipid precursors are transferred to chloroplasts. Downstream of the transfer, DAG is converted to galactolipids Current Opinion in Cell Biology 2015, 35:21–29

26 Cell organelles

by MGD1. The location of MGD1 relative to membrane junctions is unknown but surprisingly, MGD1 presents a high binding affinity for MGDG, its product, and a low affinity for DGDG. This suggests a role of these lipids for location of MGD1 in specific membrane domains [70]. Moreover, MGDG induces self-organization of MGD1 into elongated and reticulated nanostructures possibly scaffolding thylakoid biogenesis.

What can we learn about chloroplast lipid biosynthesis from the characterization of ER– chloroplast membrane contact sites? Some proteins facilitate tethering of adjacent membranes at MCSs. They are often part of complexes involved in lipid trafficking and in other functions such as organelle dynamics, protein import and primary metabolism as reported in mammals and yeasts [71,72]. A search for homologs of these proteins recognizes few of them in Arabidopsis proteomes of chloroplasts and ER. Interestingly, a Mitofusin homolog called FZL (At1g03160) which belongs to the dynamin protein family is associated with thylakoid and envelope membranes as punctate structures and regulates organization of the thylakoid network and chloroplast morphology [73,74]. In mitochondria, the protein import machinery plays a role in phospholipid transfer through a connection of the ERMES (ER — mitochondria encounter structure) and EMC (ER Membrane Protein Complex) membrane tethering complexes with TOM proteins (protein import machinery of the mitochondrial outer membrane) [72]. In addition, several other protein complexes can guide lipids through mitochondrial MCSs [75,76]. Interestingly, in chloroplasts, protein import is affected in galactolipid synthesis mutants, and the protein import machinery makes a junction between both membranes of the envelope [77]. In addition a TIM17/22/23 family protein (protein import machinery of the mitochondrial inner membrane) was detected in the TGD complex [55]. Actin also possibly interacts with the protein import machinery [78]. It may be hypothesized that by bringing membranes together, the cytoskeleton could facilitate lipid transfer along the protein import system. There is substantial evidence that cytoskeleton-related ER movements [79] and chloroplast movements are dependent on the polymerization state of short actin filaments associated with the OEM [80,81]. In addition, two mechanosensitive-ion channel-like proteins MSL2 and MSL3 show a discrete distribution on the chloroplast surface and appear important for stromule dynamics and plastid morphology [82].

the observation that mutants deleted in one step of the tocopherol synthesis pathway in the IEM can be rescued when the complementary enzyme is addressed to the ER, a mechanism based on hemifusion of membranes at ER– chloroplast MCSs was proposed to facilitate interorganellar diffusion of some nonpolar compounds [85]. This model can be viewed as an alternative for trafficking through lipid transporters however it does not account for transfer selectivity. Finally, in several cases, local modification of membrane structure due to interaction of lipids with proteins helps lipid trafficking across membranes [75,86,87]. Local increase of FAD2-desaturated PC at MCSs could be a key switch in ER-to-chloroplast lipid trafficking. Interestingly, polyunsaturated phospholipids were reported to increase the ability of dynamin to deform synthetic membranes [88]. PA also plays an essential role in ER– chloroplast lipid trafficking [57] and lipids forming hexagonal II phase such as PA may modify membrane curvature and modulate enzyme activity [89].

Conclusion and outlook Lipid trafficking between ER and chloroplasts involves a combination of complex lipid modification steps which, in some cases appear to occur at MCSs. The puzzling question is how the system selects lipid molecules for transfer. Coupling of transfer with lipid synthesis and/or with lipid processing appears essential. Several lipid processing enzymes (PC synthase, TGD4, TGD2, BnCLIP1) are detected at ER–chloroplast membrane junctions. Analysis of membrane topology and mechanism of activity of each lipid processing enzyme should help to understand how lipids are released/consumed on each side of the transfer site. Proximity of membranes in MCSs could improve the selectivity of the transfer by channeling specific pools of lipids such as acyl-CoA or lyso-PC and limiting the lipid dilution into the whole membrane and/or cytosol. Enzymes such as acyl-CoA/acyl-ACP synthetases or MGDG synthases may be important for local organization of the membranes and as pulling forces. Upstream, by locally imposing membrane asymmetry, flippases may act as driving forces to induce the transfer. Arabidopsis contains a large family of P4-type ATPases as possible flippases that will need investigation in the future. Thorough biochemical and biophysical characterization of ER–chloroplast contact sites will be essential to identify the missing components and reconstruct the process of lipid trafficking involved in plant lipid biosynthesis.

Acknowledgments Several different lipid classes are transported between the ER and chloroplasts and this traffic should involve a series of membrane transporters for intermembrane lipid transfer. Yet very few candidate lipid transporters are available in the chloroplast envelope proteome [83,84]. Based on Current Opinion in Cell Biology 2015, 35:21–29

The authors are indebted to the Agence Nationale de la Recherche for financial support of the ReGal project (ANR-10-BLAN-1524), the Reglisse Bioadapt project (ANR-13-ADAP-0008), the ChloroMitoLipid ANRJCJC J Jouhet project (ANR-12-JSV2-0001). They thank Fabrice Re´beille´ and Eric Mare´chal for excellent comments of the manuscript and Melissa Conte for English revision. www.sciencedirect.com

Lipid trafficking between ER and chloroplasts Block and Jouhet 27

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