Membranes that make fat: roles of membrane lipids as acyl donors for triglyceride synthesis and organelle function

Triglycerides constitute an inert storage form for fatty acids deposited in lipid droplets and are mobilized to provide metabolic energy or membrane building blocks. The biosynthesis of triglycerides is highly conserved within eukaryotes and normally involves the sequential esterification of activated fatty acids with a glycerol backbone. Some eukaryotes, however, can also use cellular membrane lipids as direct fatty acid donors for triglyceride synthesis. The biological significance of a pathway that generates triglycerides at the expense of organelle membranes has remained elusive. Here we review current knowledge on how cells use membrane lipids as fatty acid donors for triglyceride synthesis and discuss the hypothesis that a primary function of this pathway is to regulate membrane lipid remodeling and organelle function.

Triglycerides constitute an inert storage form for fatty acids deposited in lipid droplets and are mobilized to provide metabolic energy or membrane building blocks.The biosynthesis of triglycerides is highly conserved within eukaryotes and normally involves the sequential esterification of activated fatty acids with a glycerol backbone.Some eukaryotes, however, can also use cellular membrane lipids as direct fatty acid donors for triglyceride synthesis.The biological significance of a pathway that generates triglycerides at the expense of organelle membranes has remained elusive.Here we review current knowledge on how cells use membrane lipids as fatty acid donors for triglyceride synthesis and discuss the hypothesis that a primary function of this pathway is to regulate membrane lipid remodeling and organelle function.
Keywords: endoplasmic reticulum; fat; lipid droplet; membrane; nuclear membrane; phospholipid; triglyceride Lipid droplets (LDs) are ubiquitous organelles dedicated to the storage of nonpolar or "neutral" lipids, such as triglycerides (TGs), steryl, wax, or retinyl esters [1].The storage and breakdown of neutral lipids to and from LDs plays diverse physiological roles in organisms.TGs are an abundant neutral lipid in many cell types and function as storage molecules for fatty acyl (FA) chains.The biochemistry of TG synthesis is a highly conserved process that serves multiple key functions in eukaryotic physiology.It provides a source of energy during times of nutrient scarcity as well as lipid intermediates for the biogenesis of membranes; prevents the disruption of membrane integrity mediated by increased levels of free FA; and restricts the secondary messenger activity of some lipids.The predominant pathway for TG synthesis in eukaryotes involves the esterification of FAs to a diacylglycerol (DG) backbone and takes place predominantly in the ER [2].The FAs destined for storage must be first activated by thioesterification with coenzyme A (CoA) by enzymes called acyl-CoA synthetases.The DG used for TG synthesis is provided by dephosphorylation of phosphatidate (PA), catalyzed by the conserved family of PA phosphatases known as lipins.Activated FAs and DG are then condensed into TG by diacylglycerol O-acyltransferases (DGAT; EC 2.3.1.20)enzymes.This acyl-CoA-dependent pathway is considered the "canonical" way to synthesize TG.Intriguingly, some unicellular eukaryotes and plants have been known to utilize an alternative, acyl-CoA-independent mechanism that uses endogenous membrane lipids as FA donors to make TG (Fig. 1), but its functional significance has been unclear.As a result, this pathway has largely gone under the radar in the field.Biotechnological and evolutionary aspects of this alternative TG biosynthetic pathway have been discussed in other articles [3,4].In this perspective article, we present an overview of how cells use membrane lipids to generate TG and discuss the hypothesis that the role of this pathway is primarily linked to membrane acyl lipid remodeling and organelle function.

An alternative pathway to make triglyceride using membrane lipids as acyl donors
In vitro assays using yeast or plant microsome fractions identified an activity esterifying DG with acyl chains of phospholipids yielding TG and a lysophospholipid (Fig. 1) [5,6].The enzyme responsible for this activity, called phospholipid diacylglycerol acyl transferase (PDAT; EC 2.3.1.158,also referred to as "transacylase"), was first cloned in budding yeast Saccharomyces cerevisiae based on its sequence homology to the mammalian lecithin:cholesterol acyltransferase (LCAT; EC 2. 3.1.43).LCAT catalyzes the biochemically analogous acyl-CoA independent transfer of an acyl chain from phosphatidylcholine (PC) to cholesterol to generate cholesteryl esters and lysoPC.Yeast expresses one PDAT, called LCAT-related open reading frame (Lro1).Two PDAT proteins, AtPDAT1 and AtPDAT2, have been described in the cress Arabidopsis thaliana [7], while one has been described in the unicellular green alga Chlamydomonas reinhardtii [8].Similar to Lro1, the activities of AtPDAT1 and CrPDAT1 have been confirmed biochemically [7,8].Lro1, AtPDAT1, and CrPDAT1 represent thus the founding members of the PDAT family in fungi, plants, and algae, respectively, the three major taxonomic groups that are known to express PDAT orthologues.Predicted PDATs are also found in the flagellates Euglenozoa, the fungal-like Oomycetes [9], and diatoms where there is transcriptomic evidence that they may participate in TG synthesis [10].Mammals do not express any predicted PDAT proteins.An acyl-CoA-independent pathway that deacylates PC, however, has been described in mammals.The enzyme lecithin:retinol acyltransferase (LRAT; EC 2.3.1.135)transfers a fatty acyl chain from PC to retinol to generate retinyl esters which, similar to TG, are stored in LDs [11,12].LRAT is anchored onto the membrane via a carboxy-terminal transmembrane domain and, unlike PDATs and LCAT, its catalytic domain is oriented towards the cytoplasmic side of the ER membrane.Finally, it should be mentioned that in yeast, Lro1 has been also reported to generate 1-acylceramides in vitro [13], which are stored in LDs [14].The biological function of this activity has not been investigated so far.
PDATs have a conserved topology and share a number of key features.Their primary structure adopts a type 2 membrane configuration with a short cytoplasmic amino-terminal sequence, a single transmembrane domain, and a luminal carboxy-terminal catalytic domain.PDATs belong to the alpha/beta hydrolase superfamily and contain within their catalytic domain a catalytic triad (Ser-Asp-His), also present in LCAT, which shares 24% identity with Lro1 within its catalytic domain.However, LCAT and LCAT-related proteins, such as lysosomal phospholipase A2 (LPLA2), have lost their transmembrane anchors and have instead signal peptides that direct them as soluble proteins to the blood plasma or lysosomal lumen, respectively [15].
Consistent with the predicted topology of PDATs, full-length Lro1 behaves biochemically as an integral membrane protein [16,17], is glycosylated, and its catalytic triad is located at the ER lumen [16].A version of Lro1 lacking the transmembrane domain retains its PDAT activity in vitro [18] and in vivo [19].However, when expressed as a soluble protein, Lro1 still requires an ER membrane interaction motif in order to exert its function in vivo [19].Notably, this modified soluble Lro1 is active from the cytosolic leaflet of the ER membrane, suggesting that PDATs have the capacity to make TG and induce LD biogenesis from either side of the ER membrane.
A critical regulatory element in many lipase and LCAT enzymes is a flexible "lid" located in proximity over their active site [20].Lids consist of a loop or helix and control access to the enzyme's active site by alternating between an "open" or "closed" conformation.The structure of LCAT has confirmed the presence of a short loop that has a lid-like function [21,22].Although the topology of lids with respect to the catalytic site of lipases is conserved, their specific sequence is not necessarily so [20].Therefore, if and how the activity of PDATs is controlled by similar lidlike domains will need to await the elucidation of their structure, which is currently missing.

Regulation and function of PDATs
PDATs use membrane lipids to generate storage lipids packed in LDs.To understand the physiological significance of this conversion, we need first to ask (a) which membrane lipids PDATs use as substrates, (b) which are the organelles that PDATs associate with and what factors control the targeting and activity of PDATs on them, and (c) what are the physiological conditions that promote PDAT activity.Below we briefly discuss recent work that addresses these questions.

Membrane lipid substrates of PDATs
The basic preferences of the phospholipid classes that act as acyl chain donors for PDATs have been studied.In vitro, Lro1 and AtPDAT1 have a preference for transferring an acyl chain from the sn-2 position of phosphatidylethanolamine (PE) or PC to DG, and in both cases, PE is preferred over PC [6,7].In yeast, there is further in vivo evidence for a preference of PE by Lro1 [23,24].Moreover, under conditions of enhanced PC fatty acyl exchange, Lro1 was not required for the remodeling of PC [25].Another study provided evidence that loss of Lro1 leads to the increase of both PC and PE, suggesting that both of these phospholipids are substrates of Lro1 [26].In addition, lack of Lro1 affects total cellular fatty acid composition with a significant decrease in C16:0 and a more modest increases in C16:1 and 18:1 [27].In plants, PDATs display a preference for PC enriched in unusual fatty acids, which are channeled and stored into TG in seeds [6,28,29].In contrast to the yeast and plant enzymes, CrPDAT shows a broad preference to anionic phospholipids including phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA) [8]; notably, CrPDAT is also active on the galactolipid galactosyldiacylglycerol (MGDG), a major lipid in chloroplast membranes which makes approximately 40% of the total lipid in C. reinhardtii [8].These different acyl donor preferences exhibited by CrPDAT, compared to fungi and plants, may reflect the different membrane lipid composition of this alga, which lacks PC, as well as its possible site of function at the chloroplast membrane (see below).The PDAT from the stramenopile alga Nannochloropsis oceanica (NoPDAT) shows strong preference in vitro to PG and much less to MGDG.Consistently, loss of NoPDAT results in increases of PG but also PE [30].Finally, it should be noted that there is some evidence suggesting that PDATs can also generate TG in the absence of any phospholipidor galactolipidsubstrate: Lro1, AtPDAT1, and CrPDAT all show low levels of DG: DG transacylase activity in vitro, where DG is both the donor and acceptor of the acyl chain [7,8,18].A similar DG transacylase activity has been also described in rat intestinal microsomes [31].Collectively, yeast and plant PDATs appear to be primarily active against PE and PC while algal enzymes display a broader range of substrates, likely reflecting their distinct metabolic and organellar specialization.

Organelle distribution of PDATs
Compared to DGATs, there is limited information on the cellular distribution of PDATs and their regulation.Most in vitro assays have used microsomes as a source for the enzyme activity suggesting an association of PDATs with the endoplasmic reticulum (ER).Consistently, in yeast, Lro1 is found at the ER but relocates, in a cell-cycle and glucose-dependent manner, to a specialized inner nuclear membrane (INM) subdomain which associates with the nucleolus [9].This INM subdomain is known to expand in response to excess phospholipid synthesis, resulting in nuclear expansion [32].It was proposed that Lro1-mediated membrane remodeling or turnover at the INM could regulate nuclear expansion, which is important during the closed mitosis of yeast cells [9].How the relocation of Lro1 from the ER to the INM, or its PDAT activity at these compartments, is regulated, is not known.Within its cytosolic N-domain, Lro1 contains a nucleolar targeting sequence, but its mutation only partly inhibits Lro1 targeting to the nucleolar INM subdomain, pointing to the presence of additional targeting determinants [9].Loss of the ER-associated protein degradation factor Ubx2 mislocalizes Lro1 from the nuclear membrane and decreases TG levels, suggesting a link between ER homeostasis and Lro1 movement [17].In A. thaliana, both PDATs have predicted C-terminal ER retrieval motifs, implying that they localize to the ER [7].The reported protective requirement of AtPDAT1 during heat or cold stress is consistent with the need for remodeling of membrane phospholipid acyl chains under these conditions [33,34].However, it is unclear whether AtPDAT1 changes its subcellular distribution during stress.Similarly to plants, algae generate acyl lipids and DGs both inside the chloroplast and in the cytoplasm.Various pathways have been proposed to mediate the accumulation of chloroplast-derived TGs in the cytoplasm [35].Interestingly, algal PDATs seem to associate more with plastid membranes.In N. oceanica, NoP-DAT localizes at the outer chloroplast membrane that associates with the ER [30].In C. reinhardtii, CrPDAT contains a predicted chloroplast transit peptide [8] and proteomics analysis identified the protein at the chloroplast [36].Loss of CrPDAT results in a rise of molecular species of the chloroplast galactolipid MGDG and a concomitant decrease in TG.Together with the fact that CrPDAT shows in vitro activity on MGDG, these data support the model that CrPDAT degrades chloroplast membranes to generate TG.Interestingly, this contribution of CrPDAT in TG synthesis takes place under favorable and nutrient-replete conditions.Whether chloroplasts undergo constitutive remodeling by CrPDAT or whether this is a regulated process during the exponential phase of growth, is not known.A more modest requirement of CrPDAT in TG synthesis was also recorded at the early stages of nitrogen starvation, which however waned at later stages.

Regulation of PDAT activity in vivo
Several studies have documented the genetic redundancy and compensation that exists between PDATs and DGATs in fungi and plants.In budding yeast, the single deletion of LRO1 or DGA1 has little or no effect on cell fitness.However, their combined removal effectively abolishes all TG, makes cells highly sensitive to unsaturated fatty acids, and disrupts re-entry to log phase growth [27,[37][38][39].Such genetic redundancy was also described in fission yeast Schizosaccharomyces pombe [40].Similarly, while the single A. thaliana pdat1 and dgat1 mutants show no or a 30% decrease in seed oil content, respectively, the pdat1 dgat1 double mutant display a 70-80% decrease and severe defects in pollen and seed development [41,42].Therefore, TG can be stored in LDs and subsequently mobilized to perform its physiological roles irrespective of the source of its acyl donors.However, this does not necessarily mean that PDAT and DGAT only perform overlapping functions in wild-type yeasts and plants.In fact, there is evidence that the relative contribution of PDATs to cellular TG synthesis depends on the growth stage and is distinct to that of DGATs in wildtype cells.In yeast, Lro1 contributes more to the cellular TG pool during the exponential phase of growth while Dga1 predominates during the stationary phase when yeast cells produce the majority of their TG [37].However, in the absence of Dga1, Lro1 is still able to generate TG and LDs in stationary phase when it localizes at the INM subdomain [9].As mentioned earlier, CrPDAT shows a similar pattern as yeast Lro1, while NoPDAT was shown to generate more TG under nutrient limitation [30].In plants, the relative contribution of the PDAT and DGAT activities to TG synthesis in seeds varied significantly depending on the species [43].These observations suggest that PDATs are likely to perform distinct roles from DGATs in lipid metabolism and cell physiology.

Putative roles of PDATs in membrane lipid remodeling and organelle function
PDATs generate TG, which can provide metabolic energy at times of need, or FA and other lipid precursors.However, this TG is generated at the expense of membrane lipids, which are deacylated and will therefore concomitantly impact on membrane structure and function.This raises the question of whether the function of PDATs is primarily linked to lipid storage or membrane remodeling.While TG storage is normally a response to nutrient limitation or stress in S. cerevisiae and C. reinhardtii, PDATs appear to be more active in these organisms under favorable, nutrient-rich, growth conditions, which would be consistent with a primary role of PDATs in membrane remodeling.How PDATs impact membrane structure and function will largely depend on the metabolic fate of the lysophospholipid (lysoPL) generated by their activity (Fig. 2).LysoPLs are short-lived intermediates that are normally processed either by (a) reacylation or (b) degradation.

PDATs and membrane phospholipid remodeling
In the first possibility, the PDAT-derived lysoPL can be reacylated by a lysophospholipid acyltransferase (LPLAT) that incorporates a FA with different degree of saturation and/or chain length, altering the physical properties of the membrane (Fig. 2).This deacylation-reacylation pathway is known as the Lands cycle and was first described in rat liver microsomes [44].The Lands cycle involves specific phospholipases A and LPLATs and plays important roles in adjusting the post-synthetic phospholipid molecular species composition of the endomembrane system [45,46].Phospholipid acyl chain remodeling is also crucial for the adaptation of cellular membranes in response to environmental changes, such as the homeoviscous adaptation that maintains membrane fluidity during temperature changes [47], or organelle deformation, such as during membrane curvature [48].By using a PDAT to store the deacylated FA into TG, rather than a phospholipase A, cells could avoid rapid local increases of free FA which could build up during the Lands cycle and cause lipotoxic stress.However, this remains a hypothesis that will need to be tested; with the exception of their involvement in the biosynthesis of polyunsaturated FAs in plants [49], the biological significance of PDATs in membrane lipid remodeling remains still poorly defined.
In yeast, a candidate for acylating the lysophospholipid product of Lro1 is Ale1, a multispanning membrane-bound LPLAT which has a broad range of lysophospholipid substrates [50][51][52][53][54]. Interestingly, a highly conserved histidine residue in Ale1 is oriented towards the ER lumen [55].Along similar lines, the recently described structure of chicken lysophosphatidylcholine acyltransferase (LPCAT) contains a reaction chamber connected to two tunnels accessible from both the cytosolic and luminal sides of the ER [56].These findings raise the possibility that some steps of the Lro1-mediated phospholipid remodeling pathway could take place at the luminal side of the ER.In addition to Ale1, budding yeast expresses other enzymes which may have LPLAT activity and could therefore cooperate with Lro1 [57].In plants some evidence links PDAT and LPLAT activities: A. thaliana overexpressing AtPDAT1 shows increases in both LPCAT and lysophosphatidylethanolamine acyltransferase (LPEAT) activities in microsomal fractions, compared to wild-type plants [34].

PDATs and membrane phospholipid degradation
The second possibility is that the lysoPL is further deacylated, resulting in the complete degradation of the original lipid substrate of a PDAT (Fig. 2).In one hypothesis, PDATs could exert this function by themselves.In vitro data support the notion that PDATs display also some phospholipase activity [8,18].Currently, however, there is no biochemical evidence that PDATs could deacylate both fatty acyl chains from their phospholipid substrates.An alternative hypothesis could be that the lysoPL generated by PDATs is processed by additional phospholipases.Yeast cells express several phospholipases which could be mediating the turnover of lyso-PC and/or lyso-PE in conjunction with Lro1 [57].Either way, PDATs would be involved in the turnover of organelles facilitating their membrane degradation.It should be noted that phospholipase-dependent membrane degradation was recently reported by the phospholipase A/acyltransferase (PLAAT) family in mammals.PLAAT3 was reported to degrade organelle membranes at the terminal stages of lens development [58].Moreover, engineered versions of PLAAT members can disrupt mitochondria and peroxisomes in vivo [59].Interestingly, PLAATs show significant sequence homology to LRATs that, as mentioned earlier, display phospholipid-dependent acyltransferase activity [60].Whether PDATs have the capacity to degrade membrane lipids and what are the outcomes on the host organelles remains to be seen.Recent genetic approaches in yeast uncovered critical roles for PDATs in certain mutant backgrounds that highlight their link to membrane function.Lro1 becomes essential in a yeast mutant that lacks PC [24].This finding suggests that Lro1 mediates continuous remodeling of the PC-free membrane in order to maintain membrane functionality.Alternatively, the structural need for lysoPE could help stabilize the membrane due to its cylindrical shape [24].There is also evidence of a requirement of Lro1 for adaptation in mutants where lipid metabolism was re-wired by changing the major subcellular sites of PE and PC synthesis [61].Although the relevant mechanisms remain unknown, these observations provide an experimental handle to investigate PDAT function in vivo.

Membrane phospholipids and TG synthesis in mammals
In contrast to yeast, algae, and plants, no clear PDAT activity has yet been identified in humans.However, studies have shown that FAs released from phospholipids by phospholipases can be used in the biogenesis of TG.Indeed, TG synthesis and LD biogenesis depend on phospholipase A2 during stress [62]; and inhibition of mTORC1 (a nutrient and energy sensor that promotes cell growth) induces the accumulation of TG through lysosome-dependent hydrolysis of phospholipids.This increase in TG levels induced by the inhibition of mTORC1 can be blocked by a phospholipase inhibitor [63].Furthermore, an alternative TG synthesis pathway was recently described in humans.In this pathway, endogenous acyl chains are converted into TG by the protein module TMX1-DIESL within the ER.Mechanistically, DIESL mediates the accumulation of LDs upon loss of TMX1, but not during oleic acid loading.Therefore, DGATs and DIESL may generate different pools of TG in response to specific cellular conditions.DIESL likely uses phospholipids as acyl donors, because only DG and PC showed a significant decrease in cells relying exclusively on DIESL for TG synthesis [64].However, it is not clear whether DIESL mediates a direct transfer of FA from phospholipids to DG or if other enzymes are involved.

Conclusions and outlook
Because it generates TG, PDAT activity has been mostly viewed in the context of metabolic energy storage.The subcellular location of PDATs in plastids, the endoplasmic reticulum, and the nucleus raises the possibility of organelle-specific roles in membrane remodeling and/or degradation.Indeed, the targeted distribution of the yeast PDAT Lro1 during the cell cycle to an expandable nuclear membrane domain suggests a dynamic function for these enzymes during organelle biogenesis.
Several aspects of PDAT regulation remain poorly defined.It is likely that PDAT activity and its impact on membranes may be influenced by the coordinated activity of other lipid metabolic enzymes in its site of action.For example, how the subcellular location and activity of LPLATs and PLAs is coordinated with PDATs remains unknown, but such cooperation is likely to occur in order to control the fate of the lysophospholipids generated by PDATs.It is conceivable that PDATs could regulate membranes in different ways depending on their lipid and protein environment.Moreover, unlike LCATswhich are amphitropic enzymes, i.e., partition between an unbound and bound membrane fraction -PDATs are constitutively membrane-bound, which means that their activity must be tightly controlled to prevent deregulated membrane lipid deacylation.In this respect, the availability of DG as the fatty acyl acceptor may be a critical factor for PDAT activity.It is interesting to note that while the recently solved structure of human DGAT1 shows a hydrophobic channel and reaction center embedded deep within the membrane [65], Lro1 retains its ability to generate TG even when its transmembrane domain is removed, suggesting a different chemistry in the two acyltransferase reactions.Moreover, the molecular basis of how the phospholipid class and fatty acyl species preferences of the substrates of PDATs are established is not known.Structural insights into PDATs will be critical to address these questions.From a wider cell biological viewpoint, we expect future research to focus on the nature of the signals that control the activity of PDATs in fungi, algae, and plants, which remain still largely unknown.Finally, with the recent identification of alternative TG biosynthetic pathways in humans, it will be important to define their mechanistic links with PDATs and their possible roles in normal physiology and disease.

Fig. 1 .
Fig. 1.Schematic representation of the acyl-CoA-dependent and independent pathways of TG synthesis.