Phospholipid Synthesis and Transport in Mammalian Cells

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1 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi: /tra Review Phospholipid Synthesis and Transport in Mammalian Cells Jean E. Vance Department of Medicine and Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, AB Canada Corresponding author: Jean E. Vance, Abstract Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter-organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria-associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter-organelle phospholipid transport in mammalian cells. Keywords endoplasmic reticulum, inter-organelle transport, lipid transfer proteins, membrane contact sites, mitochondria, mitochondria-associated membranes, phospholipids, plasma membrane Received 4 September 2014, revised and accepted for publication 18 September 2014, uncorrected manuscript published online 22 September 2014, published online 15 October 2014 The most abundant phospholipid in mammalian cells is phosphatidylcholine (PC). A variety of other phospholipids, including phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (SM), cardiolipin (CL), as well as phosphatidylinositol (PI) and its phosphorylated derivatives, are also important membrane constituents (Table 1). In addition to the phospholipids, other lipids such as cholesterol and glycosphingolipids are components of mammalian cell membranes. Yeast contain most of the same phospholipid classes that are present in mammalian cells but lack SM and cholesterol, although other sterols are present. Unlike mammalian cells, in which PC is the major phospholipid, Escherichia coli is devoid of PC, SM, PI and cholesterol, whereas the most abundant phospholipid of this organism is PE. In addition, the outer membrane of this Gram-negative bacterium is composed primarily of lipopolysaccharides rather than phospholipids and contains the unique, complex lipid, lipid A. Although the lipid composition of E. coli membranes is profoundly different from that of mammalian cells, prokaryotic cells such as E. coli areabletoperformmanyofthesamebasicfunctions that operate in mammalian cells. The actions of proteins that are embedded in membranes of eukaryotic cells depend critically on the membrane phospholipid composition (1). Different types of cells and 1

2 Vance Table 1: Lipid composition of a typical nucleated mammalian cell Percentage of total lipids a Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol Phosphatidylserine 5 10 Phosphatidic acid 1 2 Sphingomyelin 5 10 Cardiolipin 2 5 Phosphatidylglycerol <1 Glycosphingolipids 2 5 Cholesterol a Data are averaged from several sources. tissues of mammals have defined phospholipid compositions and major alterations in the phospholipid content of mammalian membranes are not well tolerated. In addition, the different organelles of mammalian cells also have distinct phospholipid compositions although, in general, the differences in lipid composition are quantitative rather than qualitative. Thus, a fundamental biological question regarding the functioning of cells is: how is the unique phospholipid composition of the various organelle membranes established and maintained? Table 2 compares the phospholipid compositions of several subcellular organellesisolatedfromratliver.thevaluesgivenintable2 are approximate because isolation of pure organelles is difficult and is usually confounded by contamination of the enriched organelle fraction by other organelle membranes. In all organelles of mammalian cells, PC is the major phospholipid, comprising 40 50% of total phospholipids. PE is the second most abundant phospholipid in mammalian cells and is particularly enriched in inner membranes of mitochondria ( 35 40% of total phospholipids) compared with other organelles ( 17 25%). The high content of PE in mitochondria is consistent with the hypothesis that mammalian mitochondria originated from a symbiotic relationship between primitive mammalian cells and bacteria. The PS content of mitochondrial inner membranes and lysosomal membranes is much lower than that of the endoplasmic reticulum (ER), nucleus, Golgi and plasma membrane. Cardiolipin (CL), which is required for normal functioning of mitochondria, is present only in mitochondria, particularly in the inner membranes (2); the detection of cardiolipin in non-mitochondrial membranes is probably due to contamination of the isolated membrane fraction by mitochondria. The plasma membrane is highly enriched in SM and cholesterol compared with other organelles (Table 2). Consequently, the molar ratio of cholesterol:phospholipid in the plasma membrane is 0.75 whereas this ratio is <0.1intheERandmitochondria. Multiple glycosphingolipid species are also present in mammalian cells and are highly enriched in the outer leaflet of the plasma membrane compared with other organelles. Thus, although most of the major phospholipid classes are present in all organelle membranes, some phospholipids are more abundant in one organelle than in others (Table 2; reviewed in 3). This unequal distribution of phospholipids among organelles raises the question of how this distribution arises. Several mechanisms might be responsible for establishing the distinct phospholipid compositions of organelle membranes. First, the enzyme that synthesizes the phospholipid that is enriched in an organelle might be localized primarily to that organelle. Second, the enzymes that degrade the enriched phospholipid might be less active in that organelle compared with other organelles. Third, distinct pathways might exist for transport of a specific phospholipid between the organelle that synthesizes that phospholipid and the membrane in which the phospholipid is enriched. In addition to the unequal distribution of phospholipids among organelle membranes, phospholipids are also asymmetrically distributed across the two sides of the membrane bilayer. For example, in the plasma membrane of mammalian cells, PE and PS are normally highly enriched in the inner, compared with the outer, leaflet of the bilayer, whereaspcandsmareenrichedintheouterleaflet(4). This asymmetric transbilayer distribution of phospholipids in the plasma membrane serves important physiological functions. For example, during apoptosis PS becomes exposed on the external leaflet of the plasma membrane thereby targeting the apoptotic cell for engulfment via PS receptors on macrophages (5). Moreover, movement of PS from the cytoplasmic to the external leaflet of the plasma membrane of platelets is required for initiation of the blood clotting cascade (6). The active sites of enzymes that catalyze the terminal reactions of most phospholipid biosynthetic pathways are located on the cytosolic face of the ER (7). Yet, phospholipidsthataremadeonthecytosolicsideofthemembrane 2 Traffic 2015; 16: 1 18

3 Mammalian Phospholipid Synthesis and Trafficking Table 2: Phospholipid composition of rat liver organelles (% total phospholipids) a Mitochondria Lipid ER Inner Outer Lysosomes Nuclei Golgi Plasma membrane PC PE SM PI PS CL Other 5 <1 < Chol/PL molar ratio Approximate phospholipid content is given as % total lipid phosphorus. Chol, cholesterol. a Data are averaged from several sources. are present on both leaflets of the bilayer. Numerous studies with model membranes have demonstrated that the transbilayer movement of phospholipids such as PS, PC and PE is energetically unfavorable and does not occur spontaneously. Thus, proteins such as flippases, floppases and scramblases are required to establish and maintain the transbilayer asymmetry of phospholipids in organelle membranes. Several of these proteins have now been identifiedandwillbediscussedinthearticlebygrahamand coworkers (8) in this Thematic Series. The following discussion provides an overview of the pathways and organelles involved in the biosynthesis of the major membrane phospholipids in mammalian cells. In addition, mechanisms that have been proposed for the inter-organelle trafficking and assembly of phospholipids into organelle membranes will be considered. Phospholipid Biosynthetic Pathways in Mammalian Cells Biosynthesis of the phospholipid precursors diacylglycerol and CDP-diacylglycerol The synthesis of all mammalian phospholipids requires the acquisition of a diacylglycerol unit that is contributed by either diacylglycerol per se or CDP-diacylglycerol (Figures 1 3). These two phospholipid precursors are generated from phosphatidic acid (Figure 1). First, 1- acylglycerol-3-phosphate (also called lyso-phosphatidic acid) is made either from glycerol-3-phosphate via glycerol -3-phosphate acyltransferase or from the acylation of dihydroxyacetone phosphate and reduction of 1-acyl-dihydroxyacetone phosphate to 1-acylglycerol-3-P (Figure 1). Membranes of the ER and mitochondria contain distinct isoforms of glycerol-3-phosphate acyltransferase (reviewed in 9, 10). The 1-acylglycerol-3-phosphate is then converted into phosphatidic acid by acyltransferase activities (Figure 1) that are primarily associated with the ER and are also present in mitochondrial outer membranes. Subsequently, diacylglycerol is generated from phosphatidic acid by the action of phosphatidic acid phosphatase-1 (reviewed in 11), a cytosolic enzyme that becomes activated upon binding to ER membranes. Alternatively, CDP-diacylglycerol synthase, an enzyme that is associated primarily with the ER, and has also been detected on mitochondrial membranes, catalyzes a reaction between CTP and phosphatidic acid leading to formation of CDP-diacylglycerol (Figure 1). In other eukaryoticcellssuchasyeast,parallelpathwaysareutilized for synthesis of phosphatidic acid, diacylglycerol and CDP-diacylglycerol[reviewedinthearticlebyTamura and coworkers (12) in this Thematic Series]. Biosynthesis of phosphatidylcholine All nucleated mammalian cells make PC via the CDP-choline pathway, also known as the Kennedy pathway (13,14) (Figure 2). First, extracellular choline is imported into the cell and rapidly phosphorylated to phosphocholine by the cytosolic enzyme choline kinase. This kinase activity is encoded by two distinct genes (reviewedin15).thesecondreactionofthispathway for PC biosynthesis is catalyzed by CTP:phosphocholine cytidylyltransferase which generates CDP-choline. Under most metabolic conditions, production of CDP-choline is Traffic 2015; 16:

4 Vance Figure 1: Biosynthesis of the phospholipid precursors, diacylglycerol and CDP-diacylglycerol in mammalian cells. CDP-diacylglycerol (CDP-DG) and diacylglycerol are formed from phosphatidic acid by the action of CDP-DG synthase (CDP-DGS) and phosphatidic acid phosphatase- 1 (PAP-1), respectively. Phosphatidic acid is produced from both glycerol-3-phosphate and dihydroxyacetone phosphate. Glycerol-3-phosphate acyltransferase (GPAT) acylates glycerol- 3-P to 1-acylglycerol-3-P [also called lyso-phosphatidic acid (lyso-pa)] which is subsequently further acylated to phosphatidic acid. In the second pathway for phosphatidic acid synthesis, dihydroxyacetone-p is acylated to 1-acyl-dihydroxyacetone-P which is sequentially converted to lyso-pa and then phosphatidic acid. Enzymatic activities indicated in blue are present in both ER and mitochondrial membranes, whereas the enzyme indicated in red (PAP-1) is cytosolic and becomes activated upon binding to ER membranes. the rate-limiting reaction for PC biosynthesis (reviewed in 16). CTP:phosphocholine cytidylyltransferase exists as two isoforms, α and β, bothofwhichareactivated upon binding to membranes (17; reviewed in 15). The α, butnottheβ, isoform contains a nuclear localization sequence. Thus, the α isoform is located primarily in the nucleus and is also present in the cytoplasm, whereas the β isoform is extranuclear. The biological explanation for why CDP-choline is made in the nucleus is not clear because the final reaction of the CDP-choline pathway for PC synthesis is catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase, an integral membrane protein localized primarily to the ER; this enzyme transfers diacylglycerol to CDP-choline with production of PC (Figure 2). Figure 2: Legend on next page. In an alternative pathway for PC synthesis, PE is converted to PC by three sequential methylation reactions, all of which are catalyzed by PE N-methyltransferase (reviewed in 18, 19), an enzyme that is embedded in ER membranes. Hepatocytes are the only type of mammalian cells in which this reaction produces significant amounts of PC ( 30% of total). Parallel pathways are utilized for PC synthesis in yeast but in this organism, under most culture conditions, themajorityofpcismadebypemethylation.amajor difference in the methylation pathway between yeast and mammalian cells is that two distinct methyltransferases are required for conversion of PE to PC in yeast, whereas in mammalian cells a single methyltransferase performs all three methylation reactions (20). Another choline-containing lipid, the phosphosphingolipid sphingomyelin, is highly enriched in the plasma membrane compared with other organelle membranes of mammalian cells (Table 2). SM is synthesized from two precursors ceramideandpc thataremadeintheer and are transported to the Golgi for SM synthesis; the majority of SM is made by SM synthase-1 in the Golgi apparatus (reviewed in 21). Small quantities of SM are also made via a second synthase, SM synthase-2, which is present in the plasma membrane. In addition, a closely 4 Traffic 2015; 16: 1 18

5 Mammalian Phospholipid Synthesis and Trafficking relatedenzyme,smsynthase-r,ispresentintheerand produces small amounts of the ethanolamine analog of SM: ceramide phosphoethanolamine (22). Yeast do not make SM. Biosynthesis of phosphatidylserine and phosphatidylethanolamine PS is synthesized in mammalian cells by two distinct PS synthases, PS synthase-1 and PS synthase-2 Figure 2: Biosynthetic pathways for PC, PE and PS in mammalian cells. PS is synthesized by a base-exchange reaction from either PC [via PS synthase-1 (PSS-1)] or PE [via PS synthase-2 (PSS-2)]; these enzymatic reactions occur in elements of the ER. PS is transported to mitochondria and decarboxylated to PE by PS decarboxylase (PSD), an enzyme restricted to mitochondrial inner membranes. PE is also made in the ER by the CDP-ethanolamine pathway. Ethanolamine is first phosphorylated to phosphoethanolamine by ethanolamine kinase (EK), a cytosolic protein. CTP-phosphoethanolamine cytidylyltransferase (ET), another cytosolic enzyme, then converts P-ethanolamine and CTP to CDP-ethanolamine which combines with diacylglycerol (DG) to generate PE via the ER integral membrane protein CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EPT). PC is synthesized by a parallel series of reactions that comprise the CDP-choline pathway. Choline is phosphorylated by the cytosolic enzyme choline kinase (CK) to phosphocholine which is converted to CDP-choline by CTP-phosphocholine cytidylyltransferase (CT); CT is mainly located in the nucleus and is also present in cytosol and in association with microsomal membranes. In the final step of this pathway, the integral ER membrane protein, CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT), catalyzes the formation of PC from CDP-choline and DG. In an alternative pathway for PC synthesis, PE is converted to PC by three successive methylation reactions catalyzed by the ER/MAM enzyme PE methyltransferase (PEMT); hepatocytes are the only mammalian cells in which this reaction produces significant amounts of PC. SM, which is highly enriched in the plasma membrane, is synthesized from PC and ceramide in the Golgi by the enzyme SM synthase-1 (SMS1). Enzymes of ER membranes are indicated in blue, whereas cytosolic enzymes are in red. CT, primarily a nuclear protein, is highlighted in purple. The mitochondrial enzyme, PSD, is shown in turquoise, and SMS1,aGolgi protein, is in brown. Figure 3: Biosynthesis of phospholipids from CDPdiacylglycerol (CDP-DG) in mammalian cells. PIismade from CDP-DG and myo-inositol via PI synthase (PIS), an enzyme of ER membranes. PI is the precursor of multiple phosphorylated PI derivatives (PIPs) that play key roles in cell signaling events. Phosphatidylglycerol phosphate (PGP) is synthesized from glycerol-3-p and CDP-DG by PGP synthase (PGPS), an enzyme that is located primarily in the ER, with lower activity in mitochondria. Subsequently, PGP phosphatase (PGP-Pase) dephosphorylates PGP to produce PG. For the synthesis of CL, also known as diphosphatidylglycerol, PG combines with a second molecule of CDP-DG in a reaction catalyzed by cardiolipin synthase (CLS), an enzyme restricted to mitochondrial inner membranes. The nascent CL is remodeled to mature CL by a phospholipase and transacylations of monolyso-cl to CL in a cascade of reactions, some of which occur on the MAM. Another phospholipid that is derived from PG is lyso(bis-phosphatidic acid) (LBPA), also called bis(monoacylglycerol)phosphate. This phospholipid is highly enriched in intra-lumenal membranes of late endosomes/lysosomes. LBPA has a complex biosynthetic pathway that involves deacylation/reacylation reactions, and a unique stereochemistry in which the glycerol-1-phospho-1 -glycerol backbone is esterified at the sn-3 and sn-3 positions by acyl groups. Enzymes localized to the ER are shown in red; enzymes present in both mitochondria and ER are in blue, and the mitochondrial enzyme, CLS, is shown in turquoise. Traffic 2015; 16:

6 Vance (Figure 2), that are enriched in a specific domain of the ER called mitochondria-associated membranes (MAM) (Inter-Organelle Phospholipid Transport via Membrane Contact Sites section) (23). These synthases catalyze parallel base-exchange reactions in which serine replaces the choline or ethanolamine head group of PC (PS synthase-1) and PE (PS synthase-2), respectively (Figure 2) (reviewed in 3). In contrast, in yeast, PS is made by a completely different enzymatic reaction in which CDP-diacylglycerol reacts with serine. The CDP-diacylglycerol pathway for PS synthesis has not been detected in mammalian cells. PE is made in mammalian cells by two major biosynthetic pathways that operate in spatially separated organelles: the ER and mitochondria (reviewed in 3, 14). In the CDP-ethanolamine pathway, the final step of which occurs on ER membranes, the reactions parallel those of the CDP-choline pathway for PC synthesis (13). As depicted in Figure 2, ethanolamine is phosphorylated to phosphoethanolamine by the cytosolic enzyme ethanolamine kinase. Subsequently, CTP reacts with phosphoethanolamine to produce CDP-ethanolamine in a reaction catalyzed by the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase. In the final step of PE synthesis by this pathway, the integral ER membrane protein CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase converts CDP-ethanolamine and diacylglycerol to PE. In other eukaryotes, such as yeast, equivalent reactions and enzymes are used for PE synthesis by the CDP-ethanolamine pathway. The other major pathway for PE synthesis in mammalian cells utilizes PS decarboxylase, an enzyme that is restricted to mitochondrial inner membranes and decarboxylates PS to PE (24; reviewed in 3) (Figure 4). Small amounts of PE are also made in mammalian cells by a base-exchange reaction via PS synthase-2 in the ER (23). In addition, some PE is made by the acylation of lyso-pe, a reaction that occurs primarily on the MAM in yeast (25). Thus, the majority of PE is synthesized in mammalian cells in two spatially distinct organelles: the mitochondria and the ER. Thus,poolsofPEappeartobecompartmentalizedonthe basis of their biosynthetic origin. Indeed, PE in mitochondrial membranes is primarily made in situ in mitochondria from PS decarboxylation, rather than being imported from the ER (26,27). The requirement of PE synthesis by each of the two major pathways in mammalian cells was highlighted by the finding that elimination of PS decarboxylase activity in mice is incompatible with life, despite operation of the CDP-ethanolamine pathway (28). Furthermore, reduction of PS decarboxylase activity in mammalian cells severely impairs cell growth, as well as mitochondrial morphology and function, even when PE is produced from CDP-ethanolamine (29). Reciprocally, elimination of the CDP-ethanolamine pathway in mice, by disruption of the gene encoding CTP:phosphoethanolamine cytidylyltransferase, is also embryonic lethal despite PE being synthesized via PS decarboxylase (30). These findings indicate that the pools of PE made in the ER and mitochondriaarefunctionallydistinct,andthatpemadeinthe ER is not efficiently imported into mitochondria. Importantly, because the PS precursor of PE synthesis via the PS decarboxylation pathway is synthesized in the MAM, the PS must be imported from the MAM to mitochondrial inner membranes. The mechanism by which newly made PS is transported from MAM to the site of PS decarboxylase in mitochondria will be discussed in detail in Transfer of PS from the ER to Mitochondria section (Figure 4). In mammalian cells, only a single PS decarboxylase gene that encodes a mitochondrial protein has been identified. In contrast, yeast express two distinct PS decarboxylases: one (Psd1) that functions in mitochondria, like the mammalian enzyme, and the other PS decarboxylase (Psd2) is located in the Golgi/vacuole (31,32). The transport and decarboxylation of PS in yeast will be discussed in the article by Voelker and coworkers (33) in this Thematic Series. Biosynthesis of phospholipids from CDP-diacylglycerol In mammalian cells, CDP-diacylglycerol provides the diacylglycerol unit for the synthesis of PI (Figure 3) and its numerous phosphorylated derivatives that are involved in cell signaling events. PI is made by PI synthase in the ER frommyo-inositol and CDP-diacylglycerol. For the synthesis of phosphatidylglycerol (PG) and two derivatives of PG (Figure 3), first, PG-phosphate is made from glycerol-3-phosphate and CDP-diacylglycerol in a reaction catalyzed by PG-P synthase; this enzymatic activity is associated with both ER and mitochondrial membranes. Subsequently, PG-P is dephosphorylated to PG by PG-P phosphatase, which also appears to be active in both the ER and mitochondria. Two less abundant 6 Traffic 2015; 16: 1 18

7 Mammalian Phospholipid Synthesis and Trafficking stereochemistry of this phospholipid is unique in that the glycerol-1-phospho-1 -glycerol backbone is acylated at the sn-3 and sn-3 positions. The biosynthetic pathway for lyso(bis-phosphatidic acid) is complex and involves an isomerization reaction as well as deacylation/reacylation steps (37). Little is known about either the enzymes that convert PG to lyso(bis-phosphatidic acid) or the lipid traffickingpathwaysrequiredforitssynthesis. Mechanisms Proposed for Inter-Organelle Phospholipid Transport Figure 4: Trafficking of PS and PE between the ER and mitochondria in mammalian cells. PS is synthesized by two PS synthases (PSS-1 and PSS-2) in MAM, a domain of the ER that is closely juxtaposed to mitochondria. The MAM have been proposed to form contact sites with mitochondrial outer membranes and mediate the import of newly made PS into mitochondria for decarboxylation to PE via PS decarboxylase (PSD). In contrast, PE made in the ER from ethanolamine (etn) via the CDP-ethanolamine pathway is inefficiently transported (indicated by dotted line) to mitochondria. In yeast, lyso-pe (LPE) is acylated to PE in the MAM by the acyltransferase Ale1; a parallel pathway for PE synthesis is proposed to exist in mammalian cells and PE made from LPE is exported from MAM to mitochondria. PE made in both the MAM and the ER is exported to other organelles by unknown mechanisms (indicated by?). Abbreviations for enzyme names are shown in red. phospholipids cardiolipin (CL; also called diphosphatidylglycerol) and lyso(bis-phosphatidic acid) [also called bis(monoacylglycerol)phosphate] are generated from PG (Figure 3). For CL synthesis, PG combines with a second molecule of CDP-diacylglycerol in a reaction catalyzed by CL synthase, an enzyme that is restricted to mitochondrial inner membranes (reviewed in 34, 35). The third member of the PG family, lyso(bis-phosphatidic acid), is highly enriched in membranes of the multivesicular bodies that are present within the lumen of late endosomes/lysosomes (36). The General mechanistic considerations A common theme that has emerged from identification of the subcellular sites of phospholipid biosynthesis is that the final step in the biosynthetic pathways for most phospholipids including PC, PS, PI, as well as some PE and PG in mammalian cells occurs on ER membranes. Yet, membranes of all mammalian organelles contain different proportions of each of these phospholipids. Thus, transport processes must exist for delivery of these phospholipids from the ER to the other organelles. In addition, SM is highly enriched in the plasma membrane compared with other organelles (Table 2), yet the enzyme that synthesizes SM from ceramide and PC operates primarily in the Golgi. Consequently, ceramide and PC must be transported from the ER to the Golgi for SM synthesis (38). On the other hand, the machinery for CL biosynthesis is presentonlyinmitochondria,andclisrestrictedtothis organelle. Nevertheless, the CL precursor PG is made in both the ER and mitochondria. Thus, some PG is probably imported into mitochondria from the ER. However, thefractionofpgthatismadeinmitochondriaversus the ER, to be used for CL synthesis, is not clear. Furthermore, the PE of mitochondrial membranes is preferentially synthesized in mitochondria by the decarboxylation of PS, whereas PE made in the ER by the CDP-ethanolamine pathway has only limited access to mitochondria (26). Clearly, therefore, phospholipids do not equilibrate freely among organelle membranes. Instead, specific pathways must exist for inter-organelle phospholipid trafficking. Most phospholipids are poorly soluble in aqueous solutions. It has been generally considered, therefore, that the spontaneous diffusion of phospholipid monomers through the cytosol, from one membrane to another, does Traffic 2015; 16:

8 Vance not occur to any significant extent. However, hydrophilic molecular species of PS are more readily transported to mitochondria and decarboxylated to PE than are more hydrophobic PE species. Moreover, the half-time for transferofpsandpebetweentheerandmitochondriaisup to 10 h for some molecular species (27,39). In addition, the rate of spontaneous diffusion of di-myristoyl-pc between vesicles was increased >100-fold by inclusion of 30 mol % of di-myristoyl-pe in the donor membranes (40). These observationscouldbeconsistentwithsuchadiffusional mechanism. Consequently, the spontaneous diffusion of some phospholipids between organelles cannot be excluded, particularly because the presence of proteins in the membrane might increase the rate of monomer diffusion. Indeed, the half-time of PC transfer between plasma lipoproteins can be as little as 1.5 h (41), perhaps because of the high degree of curvature of the phospholipid monolayer of the lipoprotein particles. Nevertheless, the trafficking of phospholipids from a donor organelle to an acceptor organelle is likely to be achieved primarily by non-diffusional mechanisms. Although the targeting of proteins to defined organelles is, in many cases, mediated by specific targeting motifs within the protein for example, the C-terminal SKL motif directs proteins to peroxisomes lipids lack specific targeting motifs. Instead, the most frequently proposed mechanisms for inter-organelle lipid trafficking are transport via: (i) soluble transport proteins, (ii) vesicles and (iii) close contact between donor and acceptor membranes. The remainder of this article will focus on mechanisms of inter-organelle phospholipid transport in mammalian cells. Inter-organelle phospholipid transport via cytosolic lipid transfer proteins Intuitively, an efficient and specific mechanism for transport of a phospholipid molecule, such as PC, from its site of synthesis in the ER to another organelle, such as the plasma membrane, would be a shuttle process. In such a process, a cytosolic protein would specifically extract a PC monomer from the ER, transport the PC across the cytosol and deliver the PC to the target organelle. Such a mechanism could, theoretically, impart specificity to the transport process and could account for the defined phospholipid compositions of organelles. In vitro reconstitution experiments showed that transport of radiolabeled PC from isolated rat microsomes to isolated mitochondria was stimulated by cytosol (42). Furthermore, in intact mammalian cells newly synthesized [ 14 C]PC was transported rapidly (within 5min) from the ER to the plasma membrane and mitochondria. In contrast, PE was transported from the ER to mitochondria far more slowly (t 1/2 2 h) (43). Subsequently, several types of phospholipid transport proteins were isolated from the cytosol of mammalian cells, including the PC-specific transfer protein (44), the non-specific lipid transfer protein (45) and the PI/PC transfer proteins (46). Each of these abundant proteins was purified and extensively characterized. Hydrophobic pockets, of a size that could accommodate a lipid monomer, were revealed in each of these proteins by 3D structural analysis (reviewed in 47). Further in vitro PC transfer experiments in the 1970s demonstrated that the purified mammalian PC-specific transfer protein accelerated the movement of PC between membranes (44). Interestingly, this transfer protein contains a START motif that is also present in several other proteins, such as MLN64 and the ceramide transfer protein, CERT (38), that have been implicated in lipid metabolism/transport. Thus, the PC transfer protein was proposed to mediate PC transport from the ER to other organelles that were deficient in PC. In addition, the high concentration of the PC transfer protein in liver and lung suggested that this protein mediates PC transport for bile secretion from the liver, and for surfactant secretion from the lung. Nevertheless, despite the ability of the PC transfer protein to promote PC transport between membranes in vitro, the secretion of bile and surfactant was not compromised in mice that lacked this protein. Nor did the animals suffer from respiratory distress. Furthermore, the membrane phospholipid composition was not significantly altered (48). However, more recent studies have demonstrated that PC transfer protein-deficient mice are protectedfromariseinhepaticglucoseproductioninduced by a high-fat diet, although the mechanism underlying this effect has not been elucidated (49). Thus, the cytosolic PC transfer protein does not appear to play a major role in inter-organelle transport of the bulk of PC, and the biological function of this protein remains unknown. The non-specific lipid transfer protein (also called sterol carrier protein-2) binds and exchanges several lipids, 8 Traffic 2015; 16: 1 18

9 Mammalian Phospholipid Synthesis and Trafficking including all the common phospholipids as well as cholesterol, glycolipids and fatty acids, between membranes in vitro (45). This protein is not only present in cytosol but is also associated with peroxisomes by virtue of its C-terminal peroxisomal targeting sequence (reviewed in 50). Disruption of the gene encoding the non-specific lipid transfer protein in mice induced a 10-fold accumulation of phytanic acid and other branched-chain fatty acids in tissues (51), indicating a role for this protein in import of these fatty acids into peroxisomes for oxidation. However, elimination of the non-specific lipid transfer protein in mice did not markedly alter membrane lipid composition. Thus, neither the peroxisomal nor the cytosolic form of this protein appears to be primarily engaged in the bulk transport of phospholipids or cholesterol between organelle membranes. Consequently, although the non-specific lipid transport protein modulates some aspects of lipid metabolism, its precise physiological functions remain an enigma. Mammals express two isoforms of the PI transfer protein, PITPα and PITPβ. The PI transfer proteins are soluble, cytosolic proteins that bind and transfer PI and PC between membranes in vitro (52). The inter-membrane transfer of PIbytheseproteinsis10 20timesmorerapidthanforPC. Remarkably, the mammalian PI transfer protein is highly homologous to the yeast Sec14 protein that is essential for budding of secretory vesicles from the Golgi (53); in Sec14-deficient yeast the PC content of the Golgi apparatus is increased and protein secretion is impaired. The mammalian PI transfer protein also associates with Golgi membranes, indicating that this protein might play a role in vesicle transport/protein secretion in mammalian cells. Additional studies suggest that mammalian PI transfer protein transfers newly made PI from the ER to the plasma membrane for conversion to the phosphoinositides used in signaling pathways (54); however, confirmation of this function of PI transfer protein is required (reviewed in 55). Nevertheless, mammalian PI transfer proteins do play essential roles that have not yet been completely elucidated. For example, PITPα mice die shortly after birth owing to severe neurodegeneration (56). Moreover, elimination of PITPβ from mice is embryonic lethal (57). However, evidence that mammalian PI transfer proteins act as inter-organelle PC and/or PI transport proteins in vivo is lacking, and many questions remain about their biological functions. In summary, these three soluble, cytosolic phospholipid transfer proteins the PC transfer protein, the non-specific lipid transfer protein and the PI transfer proteins efficiently transfer/exchange phospholipids between membranes in vitro. However, evidence is lacking that any of these proteins catalyzes a net transfer of phospholipids between organelles in living cells. Inter-organelle phospholipid transport via vesicles Vesicles that carry proteins to the plasma membrane consist of a bilayer that contains phospholipids and other lipids.thesevesiclesbudfromtheerandtransporttheir protein cargo through the Golgi stacks via a series of fission and fusion steps. The protein-containing vesicles are then exported from the trans-golgi and fuse with the plasma membrane, either depositing their protein cargo inthemembraneorreleasingthesecretoryproteinsinto the extracellular space. Fusion of these vesicles with the plasma membrane would be expected to deliver large amounts of phospholipids from the ER to the plasma membrane. However, in mammalian fibroblasts the major pathways for transport of PC and cholesterol from the ER to the plasma membrane appear to be distinct from the typical vesicle-mediated pathway of protein secretion (58). For example, PC was pulse-labeled in the ER with [ 3 H]choline and the arrival of [ 3 H]PC at the plasma membrane was assessed by rapid isolation of plasma membranes using cationic beads. The newly made PC reached the plasma membrane with a half-time of 1 2 min, whereas the half-time for secretion of newly synthesized proteins was much longer about 20 min. In contrast to protein secretion, the transport of PC to the plasma membrane was not inhibited when the growth temperature was reduced from 37 Cto15 C, or by energy poisons, or by agents such as cytochalasin that disrupted vesicle transport via the cytoskeleton. Interestingly, the transport of PC to the plasma membrane was also distinct in several ways from that of cholesterol (59). These intriguing observations indicate that the bulk of newly made PC is not transported to theplasmamembranefromtheerbythesamevesiclesthat carry proteins for secretion. Nevertheless, a mechanism of PC transport to the plasma membrane via atypical vesicles that do not transport proteins was not excluded. PE transport from the ER to the plasma membrane has also been investigated in mammalian cells. PE, newly made Traffic 2015; 16:

10 Vance in the ER from CDP-ethanolamine, arrived at the plasma membrane within a few minutes, as assessed by derivatization of surface-exposed PE with trinitrobenzene sulfonic acid (60,61). In contrast, newly synthesized proteins reached the cell surface far more slowly. Energy poisons and cytoskeleton-disrupting agents did not impede PE transport to the plasma membrane, whereas these agents inhibited protein secretion. Although interpretation of thesestudiesissomewhatcomplicatedbecauseonlythepe that reached the outer leaflet of the plasma membrane was derivatized, these observations suggest that transport of the bulk of PE from the ER to the plasma membrane is not mediatedbythesamevesiclesthattransportsecretoryproteins. Additional support for this conclusion was provided by experiments in hepatocytes in which PE transport from its sites of synthesis on the ER (via CDP-ethanolamine) and mitochondria (via PS decarboxylation) to the cell surface of hepatocytes was studied using brefeldin A, a fungal metabolite that disaggregates the Golgi (61). PE that was made in the ER or mitochondria was rapidly transported to the outer leaflet of the plasma membrane. Although protein secretion was profoundly impaired by brefeldin A, by energy depletion, and by cytoskeletal disruption, PE transport to the cell surface was not inhibited. It remains a mystery, however, why the phospholipids in the empty secretory vesicles would not be incorporated into the plasma membrane after release of their protein cargo. It is possible that the membrane isolation procedures used in these studies and/or contamination of the isolated organelles by membranes of other organelles might have led to misleading conclusions. Nevertheless, currently available data indicate that PC and PE are transported from the ER to the plasma membrane mainly by a process that is independent of the vesicles that transport secretory proteins and the transport does not require an intact Golgi apparatus. Overall, these observations are consistent with the idea that movement of PC and PE from the ER to the plasma membrane occurs via a non-vesicular process, such as one mediated by an unidentified cytosolic carrier protein or by a membrane contact event between the ER and plasma membrane (62; reviewed in 63, 64). Inotherstudies,thetransportofPCandPEintheopposite direction from the plasma membrane to intracellular organelles was investigated. For example, a fluorescent phospholipid analog, 4-nitrobenzo-2-oxo-1,3-diazole (NBD)-PE (65) or NBD-PC (66), was inserted into the plasma membrane of fibroblasts at low temperature and, when the cells were warmed, the fluorescent phospholipid was internalized into vesicles via both clathrin-dependent and clathrin-independent endocytosis. Subsequently, the fluorescent phospholipid was distributed to internal organelles such as the Golgi, ER and mitochondria via an ATP-dependent process. The kinetics of phospholipid transport from the plasma membrane to intracellular organelles were different from the kinetics of ER-to-plasma membrane transport, suggesting that these two pools of phospholipids do not mix freely. Nevertheless, NBD-labeled phospholipids are significantly more polar than their natural counterparts and are, therefore, more likely to diffuse between membranes within the aqueous phase than are the more hydrophobic phospholipids. However, the molecular details of the mechanism by which phospholipids are translocated from the plasma membrane to other organelles in mammalian cells remain elusive. Recent studies in yeast indicate that a multiprotein complex, ESCRT1, plays a role in the vesicle-mediated transportofpcfromtheplasmamembranetothevacuole (reviewed in 67). The mechanism by which SM is transported from its site of synthesis, primarily the Golgi, to the plasma membrane has also been examined. SM is highly enriched in the plasma membrane compared with other organelles. Thus, an efficient mechanism must exist for SM transport from the Golgi to the plasma membrane. The involvement of vesicles in SM transport to the cell surface was suggested by studiesinfibroblastsinwhichafluorescentceramideanalog was converted to SM. The half-time for the transport of newly synthesized fluorescent SM to the plasma membrane was min, similar to the time required for protein secretion (68,69). Moreover, SM transport to the plasma membrane required GTP and was partially inhibited by monensin that blocks protein trafficking through the Golgi stacks. In other studies, SM transport from the plasma membrane to other organelles was investigated. Fluorescent NBD-SM was inserted into the plasma membrane of fibroblasts and was endocytosed by multiple pathways that required ATP. The endocytosed SM was transported back to the plasma membrane, apparently by a vesicle-mediated pathway that was independent of the Golgi (reviewed 10 Traffic 2015; 16: 1 18

11 Mammalian Phospholipid Synthesis and Trafficking in 70). One caveat for interpretation of this type of experiment, in which fluorescent lipid analogs are used, is that one must consider whether the fluorescent lipid analog, which is more hydrophilic than its natural counterpart, accurately mimics the behavior of the natural lipid. In other studies in rat hepatocytes, natural (i.e. non-fluorescent) SM was radiolabeled with [ 3 H]choline and the arrival of the SM at the cell surface was monitored by degradation of cell surface SM with exogenously added sphingomyelinase. In this system, SM transport to the plasma membrane was inhibited at low temperatures but only slightly reduced by energy poisons, monensin or brefeldin A, despite profound inhibition of protein secretion (71). Thus, the observations with radiolabeled SM are consistent with a process for SM transport to the plasma membrane that does not require the Golgi and is largely independent of the vesicles that carry secretory proteins. Although the mechanism of SM transport to the plasma membrane has not yet been unambiguously established, the combined data suggest that this transport occurs either by a non-vesicular process or by vesicles that are distinct from those used for protein secretion. Overall, studies on the inter-organelle transport of phospholipids in mammalian cells indicate that mechanisms responsible for transport of each lipid class between each pair of organelle membranes might be distinct, and that not a single, unifying mechanism exists for the intracellular transport of all phospholipids. Inter-organelle phospholipid transport via membrane contact sites In the absence of strong evidence that the majority of inter-organelle phospholipid transport is mediated by either vesicles or soluble transfer proteins, alternative mechanisms, such as transport via regions of close juxtaposition between donor and acceptor membranes, are attractive (reviewed in 64, 72, 73). Indeed, evidence is emerging that membrane contact sites between donor and acceptor organelles are formed by multiprotein tetheringcomplexesthatconsistofbothsolubleandintegral membrane proteins, as well as specific lipids such as phosphoinositides. Some of these tethering complexes have been reported to contain proteins that simultaneously bind the two apposing membranes at the contact site. The idea that membrane contact sites enhance inter-organelle lipid transport is attractive because transfer of a hydrophobic phospholipid molecule between organelles could be achieved without the energetically unfavorable movement of the lipid through the aqueous cytosol. Numerous electron microscopy studies have revealed regions of close juxtaposition (contact sites) between the ER and mitochondria (74, 75; reviewed in 73). Indeed, up to 20% of mitochondria in HeLa cells appear to be closely apposed to the ER (75). High-resolution electron microscopy showed that the distance between the ER and mitochondrial outer membranes at these contact sites in mammalian cells is nm (76,77), a separation that could accommodate an inter-organelle protein bridge. The existence of contact sites between the ER and mitochondria in rat liver and fibroblasts was further indicated by subcellular fractionation studies in which mitochondria were isolated that were tightly associated with elements of the ER (26,78). This ER-like fraction (now called mitochondria-associated membranes, abbreviated as MAM) can be separated from mitochondria by Percoll gradient ultracentrifugation (78). Thus, the association betweenmamandmitochondriaisnottheresultofmembrane fusion but is reversible and is in constant flux in cells. The MAM contain many, but not all, proteins typical of the ER. In addition, the MAM are enriched, compared with the bulk of the ER, in several lipid-biosynthesizing enzymes including the two PS synthases (23,79). The mechanisms by which the MAM become enriched with specific proteins are not known although protein palmitoylation might target some proteins to the MAM (80). Membrane domains similar to the MAM of mammalian cells have also been detected in species ranging from yeast (81,82) to plants (reviewed in 83). It is not clear, however, whether mitochondria that associate with MAM have properties that are distinct from those of mitochondria that do not associate with MAM. Transfer of PS from the ER to mitochondria The observation that sites of close juxtaposition exist between the ER and mitochondria led to the idea that the MAM might mediate the transfer of phospholipids, particularly PS, from the ER to mitochondria. This hypothesis was tested using a transport assay based on the knowledge that PS is synthesized in the ER/MAM and must be imported into mitochondria for conversion to PE via PS Traffic 2015; 16:

12 Vance decarboxylase (26,78,84,85) (Figure 4). In mammalian cells, the transport of newly made PS from the ER/MAM to mitochondrial outer membranes is rate limiting for conversion of PS to PE (84). Experiments with reconstituted organelle systems and permeabilized cells demonstrated that the conversion of newly made PS to PE (the half-time of which is several hours) does not require small vesicles or cytosolic proteins (26,84,85). In addition, PS import into mitochondria in mammalian cells is inhibited by energy poisons (26,86,87). On the other hand, in yeast and in in vitro reconstitution models with isolated mammalian mitochondria and microsomes, PS import into mitochondria is independent of ATP (reviewed in 88). Thus, the step in PS import into mitochondria that require ATP in mammalian cells is lost in the reconstituted organelle system. Consistent with MAM being the primary location of the twopssynthases(23),psimportintomitochondriais enhanced by donor membranes that are enriched in PS (89). A high concentration of PS at sites of close proximity between the MAM and mitochondria might increase the tendency for spontaneous efflux of PS monomers from the MAM to mitochondria, analogous to the idea that excess cholesterol increases the potential for cholesterol efflux from membranes (reviewed in 90). Moreover, in HeLa cells, hydrophilic PS species are preferred over hydrophobic PS species for import into mitochondria (27,39). The idea that contact sites between the ER/MAM and mitochondria mediate PS import into mitochondria is supported by the observation that newly made PS is preferentially channeled into mitochondria for decarboxylation to PE (85). In addition, inhibition of PS decarboxylase causes an accumulation of PS in MAM (26,91). A physical connection between the ER/MAM and mitochondrial outer membranes was more directly demonstrated by experiments in mammalian cells in which donor cells containing radiolabeled PS were disrupted, then mixed with disrupted acceptor cells in which PS decarboxylase was active. The disrupted acceptor cells were able to decarboxylate only the pool of PS that had been made in that population of cells, but did not decarboxylate PS made in disrupted donor cells (87). These novel experiments imply that PS export fromtheer/mamtothesiteofpsdecarboxylaseinmitochondrial inner membranes occurs via a channeling of PS between the membranes that synthesize PS and the membranes that contain PS decarboxylase. Thus, the most likely mechanism for PS import into mitochondria appears to be a close juxtaposition, or physical contact, between elements of the ER (MAM) and mitochondrial outer membranes. Similar experiments on PS import into mitochondria havebeenperformedinyeast[seearticlebyvoelkerand coworkers (33) in this Thematic Series]. These studies have identified specific proteins that promote docking between the ER/MAM and mitochondrial outer membranes. In yeast, two proteins that are required for PS import into mitochondria, and facilitate a physical interaction between the ER and mitochondria, are the ubiquitin ligase, Met30p and the related transcription factor Met4p (82). Interestingly, in mammalian cells, a ubiquitin ligase, MITOL, also regulates the formation of contact sites between the ER and mitochondria (92). Moreover, in yeast a protein complex named ERMES (ER mitochondria encounter structure), which is composed of two mitochondrial outer membrane proteins (Mdm10 and Mdm34), a cytosolic protein (Mdm12) and an ER protein (Mmm1), appears to tether the ER and mitochondria. This complex was reported to be required for the conversion of PS to PE (93). However, the requirement of this complex for PS import into mitochondria has been questioned (94). In addition, because mammalian cells do not appear to contain all of the proteins equivalent to the yeast ERMES complex, further investigation is required to determine whether a similar complex tethers the ER to mitochondria in mammalian cells. Overall, little is known about proteins that could tether the ER to mitochondria and mediate PS transport between these organelles in mammalian cells. The dynamin-related GTPase protein, mitofusin 2 (MFN2), is a protein of mammalian mitochondrial outer membranes that regulates mitochondrial fusion. MFN2 is enriched 14-fold in MAM compared to mitochondria (95) and was proposed to be involved in tethering the ER to mitochondrial outer membranes. However, reduction in MFN2 expression in fibroblasts did not inhibit PS import into mitochondria (27). Thus, the primary function of MFN2 appears to be a regulator of mitochondrial fusion. Whether MFN2 has an additional role in stabilizing the contact sites or in forming 12 Traffic 2015; 16: 1 18