On the molecular mechanism of flippase- and scramblase-mediated phospholipid transport

    On the molecular mechanism of flippase- and scramblase-mediated phospholipid transport C´edric Montigny, Joseph Lyons, Philippe Champeil, Poul Nissen, Guillaume Lenoir PII: DOI: Reference:

S1388-1981(15)00242-5 doi: 10.1016/j.bbalip.2015.12.020 BBAMCB 57870

To appear in:

BBA - Molecular and Cell Biology of Lipids

Received date: Revised date: Accepted date:

2 October 2015 20 December 2015 28 December 2015

Please cite this article as: C´edric Montigny, Joseph Lyons, Philippe Champeil, Poul Nissen, Guillaume Lenoir, On the molecular mechanism of flippase- and scramblasemediated phospholipid transport, BBA - Molecular and Cell Biology of Lipids (2015), doi: 10.1016/j.bbalip.2015.12.020

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ACCEPTED MANUSCRIPT On the molecular mechanism of phospholipid transport

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Cédric Montigny1, Joseph Lyons2, Philippe Champeil1, Poul Nissen2, Guillaume Lenoir1,* 1

Corresponding author. E-mail address: [email protected]

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Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France 2 DANDRITE, Nordic-EMBL Partnership for molecular Medicine, and PUMPkin, Danish National Research Foundation, Aarhus University, Department of Molecular Biology and Genetics, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark

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Phospholipid flippases are key regulators of transbilayer lipid asymmetry in eukaryotic cell membranes, critical to many trafficking and signaling pathways. P4-ATPases, in particular, are responsible for the uphill transport of phospholipids from the exoplasmic to the cytosolic leaflet of the plasma membrane, as well as membranes of the late secretory/endocytic pathways, thereby establishing transbilayer asymmetry. Recent studies combining cell biology and biochemical approaches have improved our understanding of the path taken by lipids through P4-ATPases. Additionally, identification of several protein families catalyzing phospholipid ‘scrambling’, i.e. disruption of phospholipid asymmetry through energy-independent bi-directional phospholipid transport, as well as the recent report of the structure of such a scramblase, opens the way to a deeper characterization of their mechanism of action. Here, we discuss the molecular nature of the mechanism by which lipids may ‘flip’ across membranes, with an emphasis on active lipid transport catalyzed by P4-ATPases. 1. Introduction

Ongoing lipidomics initiatives have revealed that eukaryotic cells contain thousands of different lipid structures [1,2]. This diversity reflects the numerous different functions attributed to cell lipids. In addition to their well-known structural role in cell compartmentalization by membranes, lipids are used for energy storage, as important regulators of membrane protein function, and are directly involved in membrane trafficking or in organizing subdomains in biological membranes, the so-called lipid rafts. In membranes of the late secretory pathway, this range of functions is accompanied by an asymmetric transbilayer distribution of lipids: whereas most lipids are symmetrically distributed across the endoplasmic reticulum (ER) membrane, the trans-Golgi network (TGN), plasma and endo/lysosomal membranes display an asymmetric distribution, with sphingomyelin and glycosphingolipids mainly in the non-cytosolic leaflet and the aminophospholipids (APLs) phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEth) primarily restricted to the cytosolic leaflet [3,4]. The prominent role played by PtdSer sidedness is for instance illustrated by its ability to recruit the signaling protein K-Ras, which in turn promotes cell proliferation through activation of the mitogen-activated protein kinase cascade [5].

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Conversely, cell-surface exposure of PtdSer may trigger recognition of apoptotic cells by macrophages or activation of the blood coagulation cascade [6,7]. Selective enrichment of lipids in one of the two leaflets of the membrane bilayer is probably the result of various mechanisms, with integral membrane transport proteins being major contributors to this process. Members of two unrelated protein families have been shown to mediate ATP-dependent unidirectional phospholipid transport across membranes, against a concentration gradient, namely the ATP-binding cassette (ABC) transporters and the P-type ATPases (Fig. 1A) [8,9]. The corresponding members of the ABC family generally catalyze the transport of lipids from the cytosolic to the exoplasmic leaflet of membranes (‘flop’). An exception to this rule is ABCA4, an ABC transporter found in photoreceptor cells that actively transports N-retinylidene-PtdEth and PtdEth from the lumen to the cytoplasmic leaflet of disc membranes [10]. Conversely, P-type ATPases from the P4 subfamily (referred to hereafter as P4-ATPases) catalyze the transport of phospholipids from the exoplasmic to the cytoplasmic leaflet of membranes (‘flip’), thereby allowing APLs to be confined intracellularly in normal quiescent cells [11–13]. Such transporters may be named ‘flippases’ and ‘floppases’ for active transport of lipids from the exoplasmic to the cytosolic leaflet (inward), or opposite (outward), respectively (Fig. 1A). The long-sought after proteins responsible for dissipation of lipid asymmetry are the scramblases, which catalyze an ATP-independent and bidirectional movement (‘flip-flop’) of a broad range of lipid classes across eukaryotic plasma membranes [14]. The identity of scramblases has remained obscure until several recent reports suggested that Xk-family protein members are necessary for caspase-dependent PtdSer scrambling [15,16] and that members of the TMEM16 family support Ca2+-dependent phospholipid scrambling (Fig. 1A) [17]. There are at least fourteen P4-ATPases encoded for in mammals, with deficiencies for a subset of them associated with pathological conditions (see e.g. [18–21]). For example, ATP8A1 and ATP8A2 are associated with neurological disorders in mice and humans, respectively. Mutations in the most extensively studied P4-ATPase, ATP8B1, have been directly correlated to progressive familial intrahepatic cholestasis, a severe liver disease that progresses toward liver failure and death before adulthood [21]. Other human P4-ATPases might be important, as in mouse the ortholog of human ATP10A has been implicated in lipid metabolism, obesity, and type 2 diabetes [22]. In yeast, most of the five P4-ATPases are linked to vesicle-mediated protein transport [23]. Previously, it was shown that the plasma membrane-localized Dnf1p and Dnf2p are required for endocytosis at low temperature [24], and that the TGN-resident flippase Drs2p is required for budding of post-Golgi exocytic vesicles [25], the formation of clathrin-coated vesicles [26], and bidirectional transport between the TGN and early endosomes [27]. Collectively, these findings suggest that flippases play a critical role in vesicle biogenesis at late secretory membranes. To explain the membrane trafficking defects that occur after inactivation of P4-ATPases in yeast, one hypothesis is that the mere imbalance in phospholipid number caused by P4-ATPasecatalyzed lipid translocation is sufficient to cause the membrane bending required for vesicle formation [28–31]. Alternatively, P4-ATPases might serve as a platform for the recruitment of proteins more directly involved in membrane trafficking. In view of the importance of lipids in cell physiology, there has been a growing interest in the mechanism by which, at a molecular level, they are transported. So far, P-type ATPases of most subfamilies have been shown to translocate cations, and within the whole family, the fair conservation of key residues involved in catalysis as well as the predicted similar domain organization suggest a common transport mechanism [32]. Therefore, a classical conundrum referred to as the ‘giant substrate’ problem is posed by the nature of the

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substrate transported by P4-ATPases [33]; how have P4-ATPases evolved to provide a pathway of sufficient size to allow for translocation of lipids instead of ions? In this review, we first summarize and discuss recent insights into the molecular mechanism of lipid transport catalyzed by P4-ATPases. Focus will be on the possible path taken by lipid molecules into the protein structure and will be discussed in light of the structural and functional data accumulated in the past decades for cation-transporting P-type ATPases. We also consider the mechanism of phospholipid scrambling, based on the recent crystallographic structure of an ATP-independent flippase [34]. 2. Structure and transport mechanism of P-type ATPases 2.1 Overall structure of P-type ATPases

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P-type ATPases are integral membrane proteins that mainly transport cations, but also lipids, across membranes and against a steep concentration gradient. Transport is achieved thanks to the hydrolysis of ATP. P-type ATPases are found in all kingdoms of life [35] and sequence alignments of P-type ATPases resulted in defining five subfamilies (dubbed P1, P2, P3, P4, and P5) reflecting different substrate specificities. Several reviews describing these five subfamilies have been previously published (see e.g. [36–40]). Briefly, ATPases from the P1 subfamily are mainly monovalent or divalent metal transporters (Cu+, Ag+, Cu2+, Zn2+, Co2+, Pb2+ and Cd2+). The P2 subfamily includes the most thoroughly studied P-type ATPases: the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) which transports two Ca2+ from the cytosol to the ER lumen per cycle in exchange for two to three H+ in the opposite direction, and the Na+/K+-ATPase in the plasma membrane which expels three Na+ ions out of the cell in exchange for two imported K+ ions per cycle. P2-ATPases also include the gastric H+/K+- ATPase which is closely related to the Na+/K+ATPase, and Ca2+-calmodulin activated plasma membrane calcium ATPases. P-type ATPases from the P3 subfamily comprise H+-ATPases, which energize the plant and fungal plasma membrane by creating a large electrochemical H+ gradient. P5-ATPases are orphan transporters whose substrates have not yet been identified. P4-ATPases have been shown to be phospholipid transporters. The presence of several conserved motifs among all P-type ATPases argues for a common catalytic mechanism between the various subfamilies. Moreover, a number of highresolution structures of members from different subfamilies (P1, P2, and P3) reveals a conserved architecture (Fig. 1B, [41–47]). The transmembrane domain is composed of 6 to 12 -helices, with a central membrane core of 6 transmembrane helices (M1 to M6) [36]. The cytoplasmic portion of these proteins is organized into three subdomains (N, nucleotide binding; P, phosphorylation; A, actuator). C- or N-terminal extensions may exist and serve various regulatory purposes, for instance in plasma membrane Ca2+-ATPases, proton pumps, or metal pumps. The N-domain (Fig. 1B, red) contains strictly conserved residues that are essential for Mg-ATP binding. A hallmark of P-type ATPases is their transient autophosphorylation during the transport cycle. The phosphorylated aspartate residue is located in the P-domain (Fig. 1B, blue), within the conserved DKTG sequence. The P-domain sits on top of M4 and M5, which carry several residues critical for binding of the transported ions. For a transport cycle to be completed, the enzyme needs to dephosphorylate. This function is fulfilled by the A-domain (Fig. 1B, yellow), which contains a glutamate residue critical for hydrolysis of the phosphorylated intermediate. The A-domain is connected to M1 and M2 through two long and flexible linkers. Some P-type ATPases are dependent on ancillary subunits to fulfill their transport function. For instance, phospholamban and sarcolipin are homologous, small single-span

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membrane proteins that regulate Ca2+ transport by binding to SERCA1a and SERCA2a. Recently, the structures of rabbit SERCA1a in complex with sarcolipin or phospholamban were reported. The structures revealed that both sarcolipin and phospholamban bind to SERCA1a through a groove between transmembrane helices M2, M6, and M9, in the vicinity of the Ca2+ entry pathway (Fig. 1B; [43,48,49]). Furthermore, it is suggested that sarcolipin specifically binds to a state preceding the binding of Ca2+, thereby modulating the apparent affinity of the pump for Ca2+ ions [50]. Apart from SERCA, the Na+/K+-ATPase catalytic subunit is also associated with auxiliary subunits, namely the -subunit and in some cases members of the FXYD family, e.g. the renal -subunit (Fig. 1B), which are both singlespanning transmembrane proteins. The -subunit is indispensable for function of the Na+/K+ATPase since it is required for export of the catalytic subunit from the ER [51,52]. The subunit has also been shown to alter the K+ affinity of the pump [53]. Because each of the FXYD proteins has a different tissue distribution, they are believed to modulate the kinetic properties of the Na+/K+-ATPase in a tissue-specific manner [54]. 2.2 Transport mechanism

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In view of their predicted similar catalytic mechanism and three-dimensional structure, the wealth of information gained over the last years with respect to the catalytic cycle of iontransporting P2-ATPases may serve as a guide for understanding lipid transport by P4ATPases. Because the Ca2+-transporting ATPase SERCA1a is one of the best-characterized P2-ATPase, its catalytic mechanism will first be described in the following section (Fig. 2A). The Ca2+ transport cycle in SERCA1a starts with binding of Ca2+ to a conformation called ‘E1’, which has high-affinity binding sites in the membrane domain accessible from the cytosolic side (step (i), Fig. 2A). Two Ca2+ ions are transported per ATP hydrolyzed. The changes due to Ca2+ binding to the membrane sites are transmitted to the P-domain via the long M4 and M5 transmembrane spans, allowing subsequent phosphorylation of the conserved aspartate, with bound Mg2+ minimizing the repulsion due to negative charges carried by both ATP and the aspartate (step (ii), Fig. 2A). The resulting intermediate, E1P, is high-energy because it can back-react with ADP to form ATP, and has Ca2+ occluded in the membrane domain, i.e. access to either side of the membrane is transiently closed. Hydrolysis of the ATP molecule disrupts the bridge formed between the N- and P- domains and allows the N-domain to move away from the P domain. The vacancy left by the N-domain is filled by the A-domain, which, after a nearly 90° rotation, places a conserved motif (TGES in P2ATPases and DGET in P4-ATPases) above the phosphorylation site, giving rise to the E2P form (step (iii), Fig. 2A). Rotation of the A-domain is coupled to movements of the first four membrane spans (M1 to M4), through extension of the flexible linkers connecting the Adomain to M1 and M2. Those conformational changes in the membrane domain open up the ion-exit pathway leading to Ca2+ dissociation. The Ca2+-free E2P intermediate is of lowenergy as it is insensitive to ADP, and it exhibits high affinity for the protons to be countertransported (2-3 H+). H+ binding induces conformational changes that are transmitted to the A-domain, allowing nucleophilic attack of the aspartyl-phosphate group. Hydrolysis of the phosphorylated aspartic acid drives the enzyme back to the E2 conformation, which will have to release H+ before binding new Ca2+ ions (steps (iv)-(i), Fig. 2A). This E2 intermediate has high affinity for vanadate, a classical inhibitor of P-type ATPases [55]. Although the catalytic cycle of P4-ATPases has been worked out in much less detail, some progress has been made in recent years. First of all, P4-ATPases become transiently phosphorylated during their catalytic cycle, as revealed by using [-32P]ATP [56–59]. This technique has several appealing features. The phosphoenzyme formed has the advantage of specifically revealing the P4-ATPase considered, as few other proteins form an acid-stable

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phosphoenzyme at the low ATP concentrations used. As a result, it is possible to monitor P4ATPase function in a crude membrane extract without the need of any purification step. In contrast, measurement of turnover rates in crude membranes is often hampered by the high amount of ATP hydrolyzing contaminants. Furthermore, this assay allows dissection of the partial reactions of the catalytic cycle, e.g. phosphorylation and dephosphorylation steps. Purified bovine ATP8A2 and yeast Drs2p require Mg2+ for autophosphorylation by ATP [58,60] and display submicromolar apparent affinities for Mg-ATP, similar to that found for P2-ATPases [58,61]. Similarities between ion transport and phospholipid transport also reside in the fact that the ATP8A2 phosphoenzyme has been found to exist in both ADP-sensitive (E1P) and ADP-insensitive (E2P) forms (Fig. 2B) [58]. Common features between P4- and P2-ATPases are also exemplified by sensitivity to inhibitors such as vanadate [58–60,62,63] and metal fluoride compounds that mimic the phosphate group [59,61]. Assuming that P4ATPases and P2-ATPases have similar reaction schemes, binding of phospholipid to the P4ATPase phosphoenzyme should, by analogy with proton binding to SERCA, stimulate dephosphorylation of the pump (Fig. 2B). Phospholipid-induced dephosphorylation has indeed been observed for several P4-ATPases, including ATP8A1, Drs2p and ATP8A2 [58,59,61,62]. In support of a similar catalytic cycle between P2- and P4-ATPases, vanadate has been found to bind ATP8A2 with a much higher affinity in the presence than in the absence of PtdSer [58], suggesting that PtdSer displaces the E1 – E2 equilibrium toward E2, as classically observed when K+ or H+ bind to the transport sites on Na+/K+-ATPase or Ca2+ATPase, respectively. Collectively, these data argue in favor of a similar catalytic mechanism between P2- and P4-ATPases. Note, however, that in cation-transporting P-type ATPases, binding of the ion being translocated from the cytosolic side to the exoplasmic side of the membrane is a prerequisite for subsequent phosphorylation, whereas no particular species has been identified that is required to activate phosphorylation of ATP8A2 and Drs2p by ATP, suggesting that phospholipids are the only species transported by these enzymes [58,59].

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3. Transbilayer asymmetry in eukaryotic membranes 3.1 Cellular role of lipid asymmetry

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As early 1972, when Singer and Nicolson proposed their fluid mosaic model for cell membranes [64], Bretscher suggested that the erythrocyte plasma membrane displays an asymmetric distribution of lipids, with the cytosolic leaflet enriched in PtdSer and PtdEth, and the exoplasmic leaflet enriched in phosphatidylcholine (PtdCho) and sphingolipids [4,65–67]. Lipid asymmetry is not restricted to the plasma membrane of eukaryotic cells as organelles of the late secretory pathway also display an asymmetric distribution of their constitutive lipids [68]. Conversely, it is generally believed that lipids in the ER membrane are distributed symmetrically. However, new genetically encoded probes have revealed that PtdSer is predominantly located in the luminal leaflet of the ER [69]. Beyond this specific transbilayer lipid distribution, the lipid composition may vary between organelles, conferring specific features to the corresponding membranes and thereby defining ‘territories’ [70,71]. The low concentration of cholesterol and sphingolipids in the ER membrane makes it fluid as opposed to membranes of the late secretory pathway, where cholesterol and sphingolipids concentrations are greatly increased [72]. Transbilayer lipid asymmetry serves critical cell functions. For instance, the enrichment of aminophospholipids, specifically PtdEth, in the cytosolic leaflet of the plasma membrane and on the surface of exocytic and endocytic vesicles may help to keep these membranes in a fusion-competent state [73]. Plasma membrane lipid distribution is also important for activation of the yeast mating pheromone pathway [74]. Furthermore, the

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specific distribution of PtdSer is important in many aspects of cell physiology, as its negatively charged headgroup is the target of C2 domain-containing proteins involved in key processes like protein phosphorylation and membrane fusion [75,76]. Also, the contribution of PtdSer (especially that of the inner leaflet of the plasma membrane) to the negative surface charge of membranes, affects both recruitment of soluble proteins via their polybasic motifs and the function of transmembrane proteins [5,77–80].

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3.2 Energy-dependent inward-directed phospholipid transport by P4-ATPases versus energy-independent phospholipid transport

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Most lipid species are synthesized at the cytosolic leaflet of the ER, and newly synthesized lipids must be transported to the exoplasmic leaflet to allow proper membrane expansion. However, although lipids diffuse quickly within the two-dimensional membrane plane, their spontaneous transfer across the ER membrane, coined ‘flip-flop’, is usually slow and depends on the size and chemical nature of the lipid headgroup (from minutes for cholesterol to several hours for glycerophospholipids; [72]). Therefore, it was proposed that protein catalysts facilitate the flip-flop process in biogenic membranes [81]. Such catalysts appear not to require ATP for lipid flip-flop and to catalyze a transport reaction that is essentially independent of the nature of the lipid headgroup. These catalysts in the ER were originally dubbed ‘flippases’, although the term ‘flip-floppase’ might be less confusing today. Thus far, their identity in the ER has not yet been revealed [82]. In addition, the mere insertion of membrane-spanning peptides in model membranes may trigger a fast flip-flop, suggesting that this process, in ER membranes, does not necessarily depend on a particular protein and could be an inherent property of fluid membranes. Importantly, this peptidedependent acceleration of spontaneous flip-flop is strongly inhibited by the presence of cholesterol [83], suggesting that such spontaneous process may not be relevant for the plasma membrane, or more generally for membranes of the late secretory pathway. Those membranes are more rigid and because of this more impermeable to phospholipids polar headgroups, thus preventing a fast spontaneous flip-flop. For lipid asymmetry to be established and maintained in eukaryotes, cells are equipped with flippases and floppases that catalyze an inward-directed or outward-directed lipid transport, respectively. To drive this transport against a concentration gradient, the flippases and floppases require energy derived from ATP consumption. In 1984, Devaux and Seigneuret identified a flippase activity in the erythrocyte plasma membrane [84]. By using spin-labeled phospholipids to study the transbilayer distribution of PtdCho, PtdSer and PtdEth, the authors observed a Mg-ATP-dependent rapid transfer of PtdSer and PtdEth (but not of PtdCho) toward the cytosolic leaflet. They proposed that an ATP-dependent membrane protein transports PtdSer and PtdEth from the exoplasmic to the cytosolic leaflet of the erythrocyte plasma membrane, the so-called aminophospholipid translocase. Shortly after, a comparable flippase activity was detected in bovine chromaffin granules [85]. Purification of the membrane fraction responsible for this activity and sequence analysis of the corresponding protein showed that it is encoded by the ATP8A1 gene [86]. The ATP8A1 gene product and its ortholog in S. cerevisiae, Drs2p, contain the well-conserved motifs that are key signatures in P-type ATPases. Those two proteins thus became the founding members of a new subfamily of P-type ATPases specifically involved in lipid transport. 4. P4-ATPases as inward-directed phospholipid transporters 4.1 P4-ATPases and Cdc50 proteins, key regulators of phospholipid asymmetry

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Following the discovery of the identity of the chromaffin granule ATPase in mammalian cells responsible for transbilayer aminophospholipid transport [85,86], pioneering work in yeast unveiled a prominent role for P4-ATPases in phospholipid transport. There are five P4-ATPase members in yeast, including Dnf1p and Dnf2p, which reside at the plasma membrane, Neo1p in the endosomes, and Drs2p and Dnf3p in the TGN [25,87,88]. Deletion of Dnf1p and Dnf2p inhibits ATP-dependent transport of fluorescent (NBD-labeled) analogs of PtdCho and PtdEth [24,89], while removal of Drs2p and Dnf3p abolishes NBDPtdSer/NBD-PtdEth and NBD-PtdCho/NBD-PtdEth transport, respectively [90,68]. Moreover, those two TGN-localized P4-ATPases do contribute to steady-state phospholipid asymmetry as chemical labeling of the cytosolic leaflet of post-Golgi secretory vesicles, with a compound reacting with the primary amine in the headgroup of PtdEth, revealed that PtdEth sidedness requires both Drs2p and Dnf3p [68]. Neo1p is essential and cannot be removed. With respect to mammalian P4-ATPases, the picture is somewhat less clear. Early biochemical studies revealed the existence of a ~120-kDa ATPase in human red blood cells and in bovine brain chromaffin granules that was activated by aminophospholipids and inhibited by orthovanadate [91–93]; as mentioned above, the ATPase contained in these fractions was later shown to catalyze the transport of spin-labeled PtdSer and PtdEth [84,85,94]. But although the gene encoding the lipid transporter in chromaffin granules has been cloned and identified as ATP8A1 [86], ATP11C has only been recently identified as a major aminophospholipid flippase in the erythrocyte plasma membrane [95]. Using UPS-1 cells defective in PtdSer translocation, the related plasma membrane-localized P4-ATPase ATP8B1 has been suggested to promote flipping of PtdSer [96]. The defect in the nonendocytic uptake of NBD-PS in these UPS-1 defective cells has now been attributed to a mutation in the ATP11C gene [97]. In contrast, in Caco-2 cells, depletion of ATP8B1 by RNAi did not lead to any PtdSer transport inhibition [98]. This apparent discrepancy might simply reflect the fact that ATP8B1-mediated PtdSder transport represents only a minor fraction of total PtdSer transport in the plasma membrane of Caco-2 cells. In another study ATP8B1 was suggested to be a cardiolipin importer, with cardiolipin binding to ATP8B1 via a positively charged sequence located in the ATP-hydrolyzing domain [99]. The relevance for localization of this cardiolipin-binding domain in the aqueous cytoplasm remains to be elucidated [100]. Re-examination of ATP8B1 substrate specificity recently showed that ATP8B1 preferentially catalyzes flipping of NBD-PtdCho, and not NBD-PtdSer, suggesting that PtdSer is indeed not a bona fide substrate of ATP8B1 [101]. The substrate specificity of other P4-ATPases localized to the plasma membrane has recently been investigated: ATP8B1, ATP8B2, and ATP10A exhibit a preference for NBD-PtdCho whereas ATP11A and ATP11C catalyze NBD-PtdSer and NBD-PtdEth transport [101,102]. Furthermore, homologous enzymes in A. thaliana and C. elegans have also been shown to transport NBD-labeled lipids [103–106]. Definitive evidence of the involvement of P4-ATPases in the transport of NBD-lipids has come from the purification and reconstitution in proteoliposomes of yeast Drs2p and bovine ATP8A2 [60,107]. Flippase activity was assayed by incorporating NBD-lipids in proteoliposomes prior to monitoring the leaflet distribution of NBD-lipids using the membrane-impermeable fluorescence quencher dithionite. The extent of NBD-lipid flipping is given by the difference between ATP-treated and ATP-untreated proteoliposomes. In both cases, treatment of proteoliposomes with ATP results in a modest (~5-10 %) but significant increase in NBD-PtdSer accessibility to dithionite. In addition, both Drs2p and ATP8A2 exhibit a strong preference for NBD-PtdSer over other NBD-lipids [60,107]. It should be stressed that P4-ATPases do not act alone: reminiscent of the association observed between the Na+/K+-ATPase and its  and  subunits or between the SR Ca2+ATPase and sarcolipin, P4-ATPases have been found to form heteromeric complexes with

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members of the Cdc50 protein family. In yeast, the Cdc50 family consists of 3 members, namely Cdc50p, Lem3p and Crf1p [108], which associate with Drs2p, Dnf1p/Dnf2p and Dnf3p, respectively. The human genome also contains three genes encoding CDC50 proteins: CDC50A, CDC50B and CDC50C. Cdc50 proteins are ~40-50 kDa proteins predicted to contain two transmembrane spans connected by a large N-glycosylated ectodomain [59,109] and with the N- and C-termini located toward the cytosol. Furthermore, conserved disulfide bridges stabilize the ectodomain [110]. N-glycosylation of the ectodomain is required for stable expression of P4-ATPases [109,111] but does not seem to be critical for either binding to the P4-ATPase or for its flippase activity [59,111]. In contrast, disruption of one out of the two disulfide bridges in yeast Cdc50p or Lem3p significantly impairs its interaction with the P4-ATPase and alters P4-ATPase catalyzed phospholipid transport [110]. It is now clear that the association between P4-ATPases and Cdc50 proteins is of primary importance. In a number of cells or organisms (including yeast, mammalian cells, plants, and worms), Cdc50 proteins and P4-ATPases display a mutual dependence for their exit from the ER [57,96,102,109,112–116]. Of note, the yeast P4-ATPase Neo1p and its closest mammalian homolog ATP9B localize to the endosomes and the TGN, respectively, in a Cdc50 proteinindependent manner [112,117]. In this case, the N-terminal cytoplasmic region of ATP9B has been attributed as being a TGN signal sequence [117]. Beyond their critical function in P4-ATPase maturation and sorting, Cdc50 proteins are proposed to play an intimate role in the transport cycle catalyzed by P4-ATPases [56,57,103,111,118]. CDC50A is the preferred binding partner of ATP8A2. Exchanging the N-terminal portion of CDC50A preceding the first transmembrane span with that of CDC50B did not prevent ATP8A2 to interact with CDC50A but substantially decreased its NBD-PS flipping activity after purification of the complex, supporting a role for CDC50 proteins in P4-ATPase-mediated phospholipid transport [111]. A pivotal role of the cytosolic N-terminus of the Leishmania Cdc50 subunit LiRos3 in miltefosine uptake (a substrate of LiMT, a Leishmania infantum P4-ATPase) was also inferred from site-directed mutagenesis of strongly conserved residues [119]. In another study, Takahashi et al investigated the contribution of Cdc50p to the in vivo function of Drs2p [118]. Using a genetic screen, the authors identified mutated alleles of CDC50 that could still interact with Drs2p and promote its localization to TGN membranes but which were defective in the formation of transport vesicles from early endosomes to TGN, suggesting that Cdc50p plays a pivotal role in Drs2p lipid transport activity. Deletion of CDC50 also causes a substantial reduction in Drs2p expression, and overexpression of Drs2p without Cdc50p leads to a P4-ATPase unable to be phosphorylated [59]. Co-expressing Drs2p and Lem3p in a cdc50 background rescued Drs2p expression and resulted in the purification of a Drs2p/Lem3p complex in amounts similar to that obtained for the physiological Drs2p/Cdc50p complex. However, the ability of the Drs2p/Lem3p complex to undergo phosphorylation from [-32P]ATP was essentially abolished, arguing for a direct and specific contribution of Cdc50p in the Drs2p transport cycle [56]. It should however be emphasized that given the importance of Cdc50 proteins as P4-ATPase chaperones, it remains difficult to dissociate their role in early trafficking steps from a role in catalytic function per se. Hence, we will have to adopt new approaches to understand precisely the role of Cdc50 proteins in P4-ATPase-catalyzed lipid transport. 4.2 Molecular mechanism of P4-ATPase-dependent phospholipid transport In the past few years, landmark papers aiming at deciphering the molecular mechanism by which phospholipids are translocated through P4-ATPases have been published. Random and site-directed mutagenesis approaches allowed the identification of P4-ATPase residues critical for lipid binding. The paragraphs below describe the two main

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4.2.1 Two-gate mechanism Based on the fact that yeast Dnf1p and Drs2p exhibit different substrate specificities, with Dnf1p specifically recognizing PtdCho and PtdEth and Drs2p preferentially recognizing PtdSer, Graham and colleagues generated various chimeras by exchanging transmembrane segments from Drs2p into Dnf1p. By swapping M3+4 and the intervening loop (L3/4) from Drs2p into Dnf1p, the latter gains specificity toward NBD-PtdSer accompanied by a reduced activity toward NBD-PtdCho and NBD-PtdEth [120]. Further dissection of the importance of the segment encompassing M3 and M4 indicated that exchanging M3 from Drs2p into Dnf1p merely reduces NBD-PtdCho and NBD-PtdEth transport whereas substitution of M4 resulted in a large increase in NBD-PtdSer activity of Dnf1p, suggesting that M4 contains residues that allow for NBD-PtdSer selection and that M3 contributes residues important for NBDPtdCho selection. Strikingly, mutation of a single residue on the cytoplasmic side of M4 of Dnf1p, Y618, to phenylalanine, had greatest influence on conferring PtdSer specificity (Fig. 3A). Y618 is part of a PISLY motif in Dnf1p (the so-called ‘P+4’ residue, named after the presence of the strongly conserved proline residue) corresponding in P2-ATPases to the conserved PE309GLP312 sequence (SERCA numbering) and to PISLF511 in Drs2p. The reciprocal substitution (F511Y) in Drs2p gives rise to a partial loss of PtdSer asymmetry in yeast plasma membrane. However, when swapping F511 for a leucine, the equivalent residue in the PtdSer-specific P4-ATPase ATP8A2, loss of PtdSer asymmetry was also observed, suggesting that several residues contribute to the formation of the PtdSer-binding pocket (see also [121]). Importantly, the specificity for PtdSer of the Dnf1p Y618F variant is not the result of an altered interaction with Cdc50 proteins as PtdSer transport was strictly dependent on the expression of Lem3p and not on Cdc50p [120]. By random mutagenesis, Graham and colleagues identified additional key residues involved in PtdSer and PtdCho selection. Replacing G230 and A231 in M1 of Dnf1p with the corresponding residues in Drs2p (G230Q/A231Q) conferred specificity toward NBD-PtdSer (Fig. 3A and 3B). Similarly, mutation of N550 on the cytosolic side of M3 to various other residues and the mutations V621A and E622V on the cytosolic side of M4 confer to Dnf1p specificity toward NBD-PtdSer (Fig. 3A and 3B). It is however surprising that aminoacids at positions 550, 621, and 622 are identical in both Dnf1p and Drs2p [122]. Following the same experimental approach, I234, F235, P240, G241, all located on the L1/2 loop, as well as I545 on the cytosolic side of M3 and I590 in the L3/4 loop were found to be critical for NBD-PtdCho transport (Fig. 3A and 3B; [122]). Moreover, by screening M3 and L3/4 mutants for resistance to edelfosine, a toxic PC analog [123], these authors identified a residue, F587, whose mutation significantly decreased the ability of Dnf1p to transport edelfosine and NBD-PtdCho [120]. Although no significant role in substrate selection could be attributed to putative transmembrane segments 5 to 10, this might be due to improper folding or diminished expression, as the Dnf1p chimera bearing the corresponding M5-10 segments of Drs2p were retained in the ER [120]. From these studies, it appears that M3, L1/2, and L3/4 in Dnf1p mostly contain sequences that recognize PtdCho whereas M1, M2, and M4 contain sequences that are critical for PtdSer transport activity. Altogether, these data suggest a peripheral path for the flip of phospholipids from the exoplasmic to cytoplasmic leaflet of membranes, in a groove delineated by M1, M3, and M4, along the protein/lipid interface (Fig. 3B; [120–122]). They seem to suggest that the phospholipid molecule could be first selected on the exoplasmic side of the membrane by a cluster of residues forming an ‘entry gate’. After translocation, a

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second cluster, the ‘exit gate’, would select the phospholipid to be delivered on the cytosolic leaflet (Fig. 3B). Residues conferring to Dnf1p the ability to transport di-acylated PtdSer were also identified, namely F213, T254, D258 and N550. All these residues being clustered at the cytosolic interface (Fig. 3B), it seems that distinguishing di-acylated lipids from lysolipids is a faculty of the exit gate [121]. Interestingly, residues in SERCA corresponding to some of these residues, i.e. T254, D258, and N550, together with the ‘P+4’ residue in M4 (Y618 in Dnf1p or F511 in Drs2p), are in proximity of a binding pocket for a phospholipid molecule identified in several E2 structures of SERCA1a [124,125], suggesting that this primordial lipid-binding site in P2-ATPases might have evolved into a site important for phospholipid translocation in P4-ATPases [32]. As those exit gate residues seem to discriminate di-acylated lipids from lysolipids, it seems likely that the ester linkage between the fatty acyl chain and the glycerol backbone is part of the lipid recognition pocket in P4-ATPases. In other words, although the polar headgroup of the lipid is the only part of the lipid molecule that ‘really needs to find’ a hydrophilic cavity inside the protein structure, the first atoms of the fatty acyl chains might also be recognized.

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4.2.2 Hydrophobic gate mechanism As M4 proved to be a critical element in the transduction mechanism of cationtransporting ATPases, the importance of residues in M4 was investigated in the purified ATP8A2/CDC50A flippase complex. M4 is subjected to movements along an axis normal to the plane of the membrane during the E1P to E2P and E2 to E1 transitions. Mutation of I364 in the P4-conserved PISL motif (PI364SL in bovine ATP8A2, PI508SL in Drs2p; the corresponding glutamate residue in SERCA was previously shown to coordinate Ca2+ at site II) to phenylalanine, glutamate, or methionine (the latter mutation being found in a family suffering from a neurological disorder) results in a decreased apparent affinity of ATP8A2 for PtdSer, as revealed by ATPase activity measurements or by PtdSer-induced dephosphorylation [126]. In contrast, I364A and I364S substitutions result in higher apparent affinities for PtdSer, possibly revealing a stabilization of the lipid bound E2 state (‘E2.PL’, Fig. 2B) by these mutations, suggesting that the size of the side-chain is important for phospholipid selection. These findings highlight that I364 seems to play a pivotal role during the phospholipid transport process. Furthermore, alanine-scanning mutagenesis revealed a critical role for N359, as its mutation to alanine resulted in a variant with strongly reduced maximal velocity as well as decreased apparent affinity for PtdSer [126]. Homology modeling, based on multiple sequence alignments and high-resolution structures of P2ATPases, revealed the presence of a large groove bordered by M1, M2, M4, and M6 helices (Fig. 3A and 3C) as well as a cluster of hydrophobic residues surrounding I364. Mutational analysis of those hydrophobic residues indicated that some of the variants displayed phenotypes similar to some of the I364 variants, and that they form altogether a ‘hydrophobic gate’ (Fig. 3C; [126]). Up and down movements of M4 during the catalytic cycle, and hence of the hydrophobic gate, would drive transport of the phospholipid headgroup [126]. Within this framework, N359 might potentially be crucial for binding the phospholipid headgroup. 4.2.3 Is there a ‘giant substrate’ problem? The two models described above argue for lipid transport pathways outside of the canonical cation-transport pathway as observed for instance in Na+/K+- and Ca2+-ATPase high-resolution structures. However, a number of mutations having a strong impact on the function of P4-ATPases target residues whose homologs in cation-transporting P-ATPases are also critical for ion transport.

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Among those residues, K873 in M5 of ATP8A2 corresponds to S767 in SERCA, a residue adjacent to N768. The latter has previously been shown to interact with Ca2+ at site I [41]. K873 has been swapped for several other aminoacids in ATP8A2, namely for an alanine, an asparagine, an arginine, or a glutamate [58]. K873A and K873E mutations are more deleterious with respect to sensitivity to PtdSer stimulated dephosphorylation than K873R, suggesting that the positive charge of the side-chain is important [58]. For instance, K873A exhibits a very slow dephosphorylation and overall turnover rate as a result of a decreased apparent affinity for PtdSer. Mutation of N874, equivalent to N768 in SERCA, gives rise to a milder phenotype with respect to reduction in apparent affinity for PtdSer [58]. Aminoacids in the vicinity of N768 in SERCA have been shown to coordinate Ca2+ ions. In addition, this part of the polypeptide chain is essential for transduction events between the cytosolic and the membrane domains in P-type ATPases. Therefore, the phenotypes associated with mutations of K873 and N874 might simply be the result of defective PtdSer binding but also of interrupted transmission between the cytosolic and membrane parts. Along the same lines, the equivalent in Na+/K+-ATPase of the above-mentioned I359 in ATP8A2 is a valine contributing to K+ binding through its backbone oxygen [126]. I364, within the PISL sequence of ATP8A2 transmembrane helix M4, is equivalent to E309 in the PEGL sequence of SERCA, a residue that contributes to Ca2+ binding at site II [41,127]. In M4 of Dnf1p, Y618 is the ‘P+4’ residue equivalent to P312 in SERCA, and mutation of P312 to alanine or glycine leads to a strong reduction in the E1P  E2P transition rate [128]. In P4ATPases, the conformational change associated with the E1P  E2P transition is believed to be critical for binding of the lipid molecule. Baldridge & Graham extended their screen for residues involved in substrate selection to transmembrane segment M6. Swapping I1235 (close to T1230, corresponding to D800 in SERCA) in Dnf1p for the corresponding residue in Drs2p (I1235F) resulted in a marked preference of the I1235F variant for PtdSer. Although the authors favor the hypothesis that this mutation is unlikely to exert its effect through perturbation of the binding of the phospholipid substrate in the canonical site, M6 does contribute several Ca2+-binding residues in SERCA, among which D800 is involved in Ca2+ binding at both sites I and II. It would probably be of great interest to investigate the functional consequences of mutating Drs2p residue F1059 (equivalent to I1235) on its ability to transport PtdSer. Although the ‘two-gate’ and the ‘hydrophobic gate’ models have led to different proposals regarding the transport path taken by phospholipids [121,126], we tend to think that the collected data might be reconciled within a single proposal. With respect to the two-gate mechanism, most of the alleles recovered after random mutagenesis and involved in substrate recognition are located either on the cytoplasmic side or on the exoplasmic side of Dnf1p structure. One might argue here that the identified residues are certainly important for phospholipid selection, either at the cytoplasmic or at the exoplasmic face, but that this does not provide any information about the internal pathway taken by the phospholipid. Intriguingly, the residues suggested to be part of the cytoplasmic exit gate define a region that coincides with the Ca2+ entry path in SERCA: it is widely accepted, thanks to the identification of a hydrated pathway in the protein, that in their journey from the cytoplasm to the Ca2+-binding sites, Ca2+ ions follow a path located between M1, M2, and M4 [129–131]. Moreover, a similar path has been defined for Na+ entry in Na+/K+-ATPase [132]. Thus, residues of the proposed lipid exit gate are strongly reminiscent of the canonical cation-entry pathway in P2-ATPases. Finally, Ca2+ binding at site II in SERCA does occur in a region close to that proposed for lipid binding in ATP8A2, i.e. in the vicinity of the ‘P+1’ residue in M4 (E309 in SERCA and I364 in bovine ATP8A2). As the single Ca2+-binding site in secretory-pathway Ca2+-ATPase (SPCA) and plasma membrane Ca2+-ATPase (PMCA)

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pumps structurally corresponds to site II of SERCA [133], this site indeed seems to be the most efficiently conserved one. Having this in mind, we might speculate that phospholipids initially selected by residues located at the entry gate, follow a path delineated by M1, M2, M4, and M6 (as suggested by the ‘hydrophobic gate’ model) before their release from the protein into the cytosolic leaflet. Such a model for phospholipid movement would also be easier to reconcile with the alternating-access model which states that transport proteins switch conformations to present the substrate binding site to alternate sides of the membrane, with interspersed occluded states, without ever fully opening a channel from one side to the other [134]. In the specific case of a primary active transporter like P4-ATPases, it is somewhat difficult to envision how a lipid transport path located on a peripheral site of the protein would be structurally competent to prevent backflow of phospholipids down their (electro)chemical gradients. Although such a peripheral lipid transport pathway has been proposed as a solution to the so-called ‘giant substrate’ problem, it is worth noting that binding sites for very large inhibitors, like cyclopiazonic acid and thapsigargin derivatives in SERCA and the cardiotonic steroid ouabain in Na+/K+-ATPase, are found deeply buried in the membrane domain (Fig. 4; [44,135–137]). This suggests that there is substantial room between transmembrane helices, for movement and for adapting to the binding of large molecules, including phospholipids. What could then be the role of Cdc50 proteins in P4-ATPase-catalyzed lipid transport? Based on the likely involvement of Cdc50 subunits in P4-ATPase activity, it is tempting to speculate that the subunits contribute transmembrane helices that are potentially involved in the lipid-transport pathway. However, this hypothesis is challenged by the fact that Cdc50 subunits are not critical determinants of the substrate specificity of P4-ATPases [104]. By using Drs2p mutants stuck in the E1P or E2P conformations, Cdc50p has been found to interact more strongly with the E2P intermediate, i.e. at the point where the enzyme is supposed to get loaded with the phospholipid ligand [56]. Rather than providing transmembrane helices that would participate in forming the substrate-binding site, one might envision that binding of Cdc50p gives rise to conformational changes that alter the affinity of the transporter for its lipid substrate, by analogy with the ability of the Na+/K+-ATPase subunit to alter the affinity for K+ of the catalytic subunit. Gaining information on the binding interface between Drs2p and Cdc50p would greatly improve our understanding of the role of Cdc50 proteins. 4.3 Regulation of P4-ATPases Insights into the molecular mechanism of lipid transport and the link between this activity and vesicle-mediated protein transport in the late secretory pathway also emerged from the identification of regulatory mechanisms of P4-ATPase activity. Several regulatory modes have been unveiled in the recent years, some of which are reminiscent of autoregulation of cation-transporting P-type ATPases. 4.3.1 Regulation by lipids Lipids are important regulators of P-type ATPases, as they are at some level for probably all membrane proteins. General effects of the aliphatic chain length and of the fluidity of bulk lipids have been widely investigated and it has been shown that the physical properties of the lipid phase could influence greatly the activity of the SERCA1a Ca2+ATPase [138,139]. Such effects are not related to a direct and specific interaction of a lipid with the pump but mainly result from a lipid mismatch around the transmembrane domain [140,141], which potentially impacts the dynamics of the transporter. Specific interactions of lipids or lysolipids have also been shown to modulate the activity of several P-type ATPases

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and the growing number of high-resolution structures of P-type ATPases has indeed revealed the presence of tightly bound lipids to the Na+/K+ pump [141] and SERCA1a (see [125] and references herein). For the specific case of P4-ATPases, lipids do obviously alter the transport activity, as they may in fact be the transported species. As discussed above, the lipids transported from the exoplasmic leaflet to the cytosolic leaflet of the membrane will for instance stimulate dephosphorylation of the E2P phosphoenzyme. However, specific but nevertheless nonsubstrate lipids have also been found to regulate the activity of P4-ATPases. A synthetic gene array indicated a strong genetic interaction between drs2 and pik1, which encodes a phosphatidylinositol 4-kinase involved in PtdIns(4)P synthesis at the TGN, suggesting a link between phospholipid transport and phosphoinositide metabolism [142]. Depletion of PtdIns(4)P in TGN membranes associated with Pik1p inactivation was subsequently shown to inhibit NBD-PtdSer transport [143]. PtdIns(4)P binds to a stretch of positively charged residues located in the cytosolic C-terminal tail of Drs2p (Fig. 5A), and phosphorylation/dephosphorylation assays on membrane-embedded or purified Drs2p/Cdc50p complex indicated that PtdIns(4)P exerts its stimulating effect by increasing the rate of dephosphorylation [59,61]. Note that PtdSer remains mandatory for PtdIns(4)P to exert its stimulating role on the dephosphorylation step [59,61].

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4.3.2 Regulation by cellular protein partners Beyond Cdc50 proteins, genetic and biochemical approaches have identified additional P4-ATPase binding partners, mostly in the yeast S. cerevisiae. Combining formaldehyde cross-linking with tandem-affinity purification and analysis of the isolated complexes by mass spectrometry, several Drs2p binding partners were identified, including Sac1p. Sac1p is a member of the synaptojanin family of lipid phosphatases, which acts as a negative regulator of vesicle-mediated protein transport at the TGN by decreasing the local pool of PtdIns(4)P. PtdIns(4)P is primarily found in TGN membranes and its phosphorylated headgroup helps recruiting proteins involved in vesicle-mediated protein sorting, e.g. AP-1, the major TGN clathrin adaptor [144,145]. Moreover, a two-hybrid screen identified Gea2p, a guanine nucleotide exchange factor (GEF) specific for the small GTP-binding protein Arf, as interacting with a sequence of Drs2p located in its C-terminal extension (Fig. 5A) [146]. These GEF proteins catalyze the exchange of GDP for GTP in Arf proteins and activated GTP-bound Arf in turn recruits vesicle coat proteins to membranes for subsequent budding of transport vesicles [147]. Drs2p was later found to be an effector of Gea2p since addition of the Sec7 domain of Gea2p to isolated TGN membranes stimulated the NBD-PtdSer flippase activity of Drs2p [143]. Interestingly, addition of both Gea2p and PtdIns(4)P to TGN membranes promotes synergistic activation of Drs2p [143], although stimulation by Gea2p of ATP hydrolysis by Drs2p could not be observed with purified and reconstituted Drs2p [148], suggesting that Drs2p activation by Gea2p requires an additional cellular factor. Drs2p as a critical player in membrane trafficking events was further illustrated in yeast by its interaction with the Arf-like protein Arl1p, which contributes to membrane trafficking events at the TGN [149]. Tsai et al. demonstrated a direct interaction between Arl1p and the Drs2p N-terminal extension (Fig. 5A), and that this interaction stimulates Drs2p flippase activity in purified TGN membranes. Interestingly, stimulation of NBD-PtdSer flippase activity of Drs2p by Arl1p is proposed to create a lipid environment suitable for targeting the GTPase-activating protein (GAP) Gcs1p to the TGN: membrane curvature induced by Drs2p flippase activity would allow recruitment of the Drs2p effector Gcs1p through its lipid packing sensor motif and Gcs1p would subsequently inactivate Arl1p by exchanging GTP for GDP [150–153]. These studies therefore identify a feedback regulation mechanism and highlight a critical role of P4-ATPases in coordinating events that control

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4.3.3 Auto-inhibition Because both PtdIns(4)P and Gea2p bind to the cytosolic C-terminus of Drs2p (Fig. 5A) and activate its flippase activity in a synergistic manner, the C-terminal tail of Drs2p might contain an auto-inhibitory domain. This hypothesis was confirmed by expressing a variant of Drs2p for which a TEV cleavage site had been inserted close to the end of predicted transmembrane segment M10 [148]. Cleavage by the TEV protease removed the PtdIns(4)P binding site together with the rest of the C-terminus. Whereas the full-length Drs2p may be activated ~6-fold by PtdIns(4)P with respect to ATPase activity and NBD-PtdSer transport, the truncated version of Drs2p displays an increased basal activity (measured in the absence of PtdIns(4)P) which is no longer stimulated by PtdIns(4)P [148]. This suggested that the Cterminal tail of Drs2p contains an auto-inhibitory domain that can be displaced by addition of PtdIns(4)P. In terms of regulation, the P4-ATPase Drs2p shares striking similarities with other members of the P-type ATPase family, namely with plasma membrane H+-ATPases from plants and fungi (P3 subfamily) and with plasma membrane Ca2+-ATPases (PMCA; P2b subfamily). For example, PMCA is subjected to multiple regulation mechanisms [154], the best-understood being that by Ca2+-calmodulin (Ca2+-CaM), which interacts with high-affinity with the PMCA C-terminal tail (in mammals) or N-terminal tail (in plants) [155–157]. Ca2+CaM binding substantially reduces the Km for Ca2+ by disrupting the intramolecular interaction between the CaM-binding domain and the main body of the pump, thus relieving auto-inhibition. A recent study combining structural data of a plant PMCA regulatory domain/CaM complex with biochemical, biophysical, and modeling data shows that two CaM molecules actually bind to the regulatory domain and that a highly conserved cleft, located at the interface between A, N, and P domains is exposed in the E2 conformation. The regulatory domain of PMCA is proposed to adjust into this conserved cleft thereby restricting motions of the cytoplasmic domains and auto-inhibiting the Ca2+ pump [157]. In fact, another mode of activation of PMCA is its selective cleavage by calpain, which removes the C-terminal tail of the pump including the auto-inhibitory CaM-binding domain [158]. Another common feature arises from the sensitivity of both Drs2p and PMCA to acidic phospholipids, e.g. PtdSer and PtdIns(4,5)P2 in this case, which activate PMCA by lowering the Km for Ca2+ [159]. Two binding sites have been identified, one in the A domain and the other one in the CaM-binding domain [160,161]. In addition, acidic phospholipids PtdIns and PtdSer stimulate Ca2+-ATPase activity by accelerating dephosphorylation of the pump [162]. The interaction of acidic phospholipids with the cytosolic portion of the pump could induce structural rearrangements affecting different steps of the catalytic cycle. As for plant H+-ATPases, synergistic regulation by both N- and C-extensions has recently been suggested [163]. By analogy with PMCA, a model for Drs2p/Cdc50p auto-inhibition may be proposed (Fig. 5B). When PtdIns(4)P levels are low in TGN membranes, thanks for instance to the phosphatase activity of Sac1p, the C-terminus of Drs2p interacts tightly with the cytosolic part of the ATPase, thereby preventing completion of the catalytic cycle even though PtdSer is bound at the transport sites. Because PtdIns(4)P has previously been shown to stimulate the rate of Drs2p/Cdc50p dephosphorylation [59,61] (i.e. the E2P  E2 transition, see Fig. 2), one might further speculate that the Drs2p C-terminus prevents proper positioning of the Adomain with respect to the phosphorylated aspartate. Increased PtdIns(4)P levels, together with binding of Gea2p [143], would disrupt the interaction of the C-terminus of Drs2p with the soluble domain and relieve the ATPase from its auto-inhibited state.

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The results by Zhou et al. [148] raise new questions of how auto-inhibition is precisely achieved for Drs2p. What is the necessary and sufficient region of the C-terminus that is inhibitory? Does auto-inhibition involve interactions between the C-terminus and the cytosolic domains? Is the N-terminus also involved in regulation of the transport cycle, as observed for the plant plasma membrane H+-ATPase [163]? What are the consequences of deleting the C-terminus of Drs2p for interaction with Cdc50p? We might also wonder whether, as it is for instance the case for PMCA, removal of the C-terminal extension by cellular proteases could regulate the activity of Drs2p. If such a mechanism exists, cleavage by such proteases is an irreversible process, which is also linked to specific control mechanisms.

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4.3.4 Proteolysis As mentioned earlier, the appearance of PtdSer in the external leaflet of the plasma membrane is a key event in apoptosis [7]. ATP-independent phospholipid scramblases are responsible for cell surface exposure of PtdSer. Previous work reported that in apoptotic Tcells, an aminophospholipid-specific flippase is downregulated concomitantly to the activation of the nonspecific scramblase [164,165]. However, the identity of the flippase and the mechanism of inactivation remained unclear until recently. The inactivated flippase has been identified as the ATP11C/CDC50A complex, which catalyzes inward-directed NBD-PtdSer transport in various cell lines [101,166]. Inactivation of ATP11C and/or CDC50A results in inhibition of NBD-PtdSer transport at the plasma membrane, and to some extent PtdEth, but not PtdCho. As to the mechanism of its inactivation, it has been found after treatment of mouse W3 cells with Fas ligand (FasL), which induces apoptosis, that this inactivation is associated with proteolysis of ATP11C, as a result of caspase cleavage at three different sites located in the nucleotide-binding domain of ATP11C [166]. In fact, caspase-resistant ATP11C could rescue NBD-PtdSer transport in ATP11C-deficient cells, and did not allow PtdSer exposure upon FasL treatment, suggesting that activation of the phospholipid scramblase is not sufficient: the flippase must be inactivated by caspase for the cell to enter the apoptotic program [166].

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5. Bidirectional and unspecific phospholipid transport catalyzed by eukaryotic scramblases The regulated disruption of phospholipid asymmetry at the plasma membrane is associated with fundamental physiological processes. As a classical example, ATPindependent scramblases are responsible for the cell-surface exposure of PtdSer, the key ‘eatme’ signal in apoptotic cell clearance, by facilitating its transbilayer movement from the cytosolic leaflet to the external leaflet of the plasma membrane. 5.1 A large spectrum of different proteins catalyze bidirectional lipid transport The identity of the phospholipid scramblases enabling platelets to promote blood clotting, or apoptotic cells to be recognized by macrophages, remained elusive for decades. However, recent advances identified Xk-related family members as prime candidates for PtdSer exposure in apoptotic cells [15,16,167]. One of these proteins, Xkr8, mediates PtdSer exposure and engulfment by macrophages in response to apoptotic stimuli when expressed in a variety of cell lines [15]. Xkr8-mediated PtdSer exposure is specific to apoptotic events as it is controlled by caspase-3, which promotes activation of Xkr8 by cleavage in its C-terminal tail. Other members of the Xkr family have now been shown to support caspase-activated phospholipid scrambling during apoptosis [16]. This mechanism of PtdSer exposure in apoptotic cells seems to be evolutionarily conserved as in Caenorhabitis elegans, the protein

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CED-8, the only homolog of Xkr8, is also involved in PtdSer exposure and in a caspasedependent manner [15,167]. However, as Xkr proteins have never been purified and tested for phospholipid translocation in a reconstituted system, it remains to be determined whether they are genuine scramblases or whether they control the activity of another protein responsible for phospholipid scrambling. The same group also identified a candidate protein for Ca2+-activated PtdSer exposure in blood platelets, namely TMEM16F [17]. TMEM16F is part of the TMEM16 or ‘anoctamin’ family, which comprises membrane proteins exclusively expressed in eukaryotic organisms [168]. The various members of this family perform different functions, as TMEM16A forms a Ca2+-activated chloride channel [169–171] while some other members are now proposed to be Ca2+-activated cation channels [172], phospholipid scramblases [34] or dual-function proteins with both scrambling and ion transport activities [173]. How these closely related proteins perform such different functions is currently under intense debate [174]. Although the mammalian TMEM16F protein has not yet been purified in a functional manner, two fungal homologues from Aspergillus fumigatus (afTMEM16) and Nectria haematococca (nhTMEM16) have now been purified and reconstituted, offering the opportunity to assay the ability of pure proteins to scramble lipids in a chemically-defined environment [34,173]. From these studies, both afTMEM16 and nhTMEM16 have the capacity to scramble lipids in proteoliposomes at high rates. Interestingly, phospholipid scrambling in eukaryotic membranes is not restricted to the two above-mentioned protein families as recent reports indicate that, unexpectedly, G-protein coupled receptors, including rhodopsin, the 2-adrenergic receptors as well as A2A receptors are also phospholipid scramblases with broad specificity [175,176]. The physiological relevance of such an activity, which seems to be constitutive, remains to be determined.

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5.2 How is phospholipid asymmetry achieved in cells expressing scramblases? We previously mentioned that in some cell types, inhibition of a flippase was necessary for PtdSer exposure in apoptotic cells [166]. This is, in fact, rather unexpected, since one would expect activation of scramblases to be sufficient. Indeed, flip-flop catalyzed by ATP-independent scramblases typically occurs at rates faster than 104 per second (see e.g. [176–179]), while in view of the highest ATP hydrolysis rates reported so far, active transporters like P4-ATPases are believed to reach turnover numbers around 102 per second [58]. Although the relative expression levels of scramblases compared to that of ATP11C is unknown, it is difficult to envision how P4-ATPases could efficiently counteract randomization of lipids by scramblases [166]. In the particular case of ATP11C, the flip-flop rate induced by Xkr8 after its purification and reconstitution in proteoliposomes remains to be determined. In this respect, a surprising result stems from the discovery of constitutive phospholipid scramblases that are nonetheless located in membranes displaying transbilayer phospholipid asymmetry [176]. For instance, rhodopsin is found in the plasma membrane of photoreceptor outer segments. Therefore, there must be a mechanism that makes rhodopsin silent in membranes where asymmetry must be preserved. It is possible that the lipid bilayer composition restricts rhodopsin scramblase activity at the plasma membrane, for example through the high sterol content found in these membranes [180]. 5.3 The specific case of the TMEM16 family As mentioned earlier, fungal homologues of mammalian TMEM16 proteins have now been purified, reconstituted and their biochemical activity provides definitive evidence that they are scramblases. The crystal structure of nhTMEM16 has recently been solved, providing a framework for understanding the molecular mechanism of lipid scrambling [34].

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5.3.1 Structure of Nectria haematococca TMEM16 Based on epitope-tagging of TMEM16G and hydrophobicity plots, TMEM16 proteins were initially proposed to contain eight membrane-spanning segments (hence coined anoctamins) [181,182]. However, in light of the recent crystal structure of nhTMEM16 [34], it appears that nhTMEM16 is a homodimeric protein with each protomer comprising ten transmembrane helices, representing a novel membrane protein fold (Fig. 6A). After purification and reconstitution into proteoliposomes, nhTMEM16 was shown to catalyze a fast movement of NBD-PtdSer and NBD-PtdEth that is stimulated by submicromolar Ca2+ concentrations, a behavior highly similar to that of afTMEM16 [173]. In contrast with afTMEM16, nhTMEM16 does not display any ion channel activity [34]. The dimer interface in the crystal is primarily provided by contacts between the N-terminus of one monomer and the C-terminus of the other, in the cytoplasmic domain, and to a minor extent between residues in the N-terminal part of transmembrane helix M10 (extracellular side, Fig. 6A and 6B). At the dimer interface, there are two pores (the dimer cavities) that merge into a large one at the intracellular half of the membrane (Fig. 6B). Because the residues lining the pore are mainly hydrophobic and aromatic, it is proposed that this cavity is filled with lipids [34].

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5.3.2 Insights into the mechanism of phospholipid scrambling At the opposite side of the membrane dimer interface, there is a narrow crevice, dubbed the ‘subunit cavity’ and contributed by transmembrane helices M3 to M7, that forms along the whole lipid bilayer a structure reminiscent of a ‘spiral staircase’ (Fig. 6C and 6D). In spite of its exposure to the hydrophobic phase of the membrane, this crevice bears hydrophilic residues and is wide enough to accommodate a lipid headgroup, suggesting that it might be the site of ion and lipid conduction through nhTMEM16 and other TMEM16 family members [34], perhaps coupled to horizontal, brownian motions in the membrane. One of the striking features of the nhTMEM16 structure also lies in the identification of a Ca2+-binding site conserved throughout the whole TMEM16 family. The Ca2+-binding site is buried within the transmembrane region of the protein, and some of the acidic residues contributing to Ca2+ binding are part of the helices forming the subunit cavity (Fig. 6C). In support of the subunit cavity providing the ion conduction path, mutational analysis of murine TMEM16F revealed that substituting a glutamine residue facing this cavity for a lysine impaired selectivity and conductance of the channel [172]. Moreover, mutation of residues involved in Ca2+ coordination reduces phospholipid scrambling by nhTMEM16 and alters the ion conduction properties of murine TMEM16A [34]. As some of the Ca2+-binding residues are borne by transmembrane helices forming the subunit cavity, it is tempting to speculate that Ca2+ binding may induce a conformational change that would be transmitted to the nearby subunit cavity. A recent study examined the role of the subunit cavity in phospholipid scrambling, by analyzing a chimera of a non-scrambling TMEM16 protein (TMEM16A) and a scrambling TMEM16 protein (TMEM16F). A small domain in TMEM16F containing the cytosolic portion of transmembrane helices 4 and 5 as well as the intervening loop was found to confer scrambling activity to TMEM16A [183]. This ‘scrambling domain’ turned out to be part of the subunit cavity ‘staircase’, substantiating a role for the cavity in phospholipid scrambling [34]. Collectively, these data suggest that ions and lipids follow the same path through TMEM16 family members and that Ca2+ regulates both functions. In such a case, how can some TMEM16 proteins prevent lipid scrambling while allowing ion current? Noteworthy, a similar path for ions and lipids has recently been questioned. The channel activity of purified and reconstituted afTMEM16 is sensitive to certain lipid compositions whereas its lipid scrambling activity is not, suggesting that separate pathways might co-exist for ion and lipid

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6. Concluding remarks

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Since their discovery as putative phospholipid transporters, P4-ATPases have been under intensive investigation. Their role in NBD-lipid transport is now firmly established, thanks to the purification and reconstitution of select P4-ATPases in a chemically defined environment. Genetics and cell biology approaches have also convincingly demonstrated the key role of P4-ATPases in coordinating membrane trafficking events, although their precise contribution to vesicle-mediated protein transport remains to be elucidated. Additionally, there is increasing evidence showing that Cdc50 proteins are key players in phospholipid translocation. This is for instance illustrated by the fact that Cdc50 proteins still interact with P4-ATPases after their trafficking to the correct membrane, suggesting that they play an intimate role in phospholipid transport beyond their role as a chaperone. From a molecular point of view, though, many important aspects remain to be addressed. Although invaluable tools for studying transbilayer lipid transport, NBD-lipids are not perfect mimics of natural lipids. Due to its relative hydrophilicity, the NBD moiety placed at the end of a fatty acyl chain tends to loop back toward the membrane-water interface [69,184]. Such behavior might explain that while Dnf1p is capable of transporting NBDlipids, it does not seem to support transport of di-acylated lipids [121]. The development of new tools that can probe natural lipid transport will be key to identifying genuine transport substrates of P4-ATPases. Another point of particular importance is to figure out whether P4ATPases transport any ion or molecule against phospholipids, i.e. from the cytosolic to the exoplasmic aspect of membranes. In cation-transporting P2-ATPases, binding of the ion translocated from the inside to the outside is a prerequisite for autophosphorylation. In the case of P4-ATPases, neither Na+, K+, Cl-, Ca2+, Zn2+, Co2+, nor La3+ seemed to affect phosphorylation of the pump, suggesting that at variance with P2-ATPases, ions are not transported by P4-ATPases [58,59]. It remains possible that P4-ATPases exchange certain classes of phospholipids for others. If it were the case, lipid transport by P4-ATPases would not expand the cytoplasmic leaflet at the expense of the exoplasmic one and we would therefore need to revise the model postulating that P4-ATPase-catalyzed phospholipid transport creates a mass imbalance between membrane leaflets. The recent crystal structures of the nhTMEM16 scramblase and of the bacterial ABC transporter PglK (the latter flips lipid-linked oligosaccharides from the cytoplasmic side to the periplasmic side of the plasma membrane), shed new light on the molecular mechanism of transbilayer lipid transport [34,185]. Comparison of those two structures also highlights the likely diversity of mechanisms employed by proteins for transporting lipids across the membrane bilayer. Although the structure by Brunner et al. provides an appealing model for lipid transport by ATP-independent scramblases [34], we might anticipate a rather different mechanism for active and specific transporters like P4-ATPases. Indeed, how can backflow of phospholipids, a critical step for establishing lipid asymmetry, be prevented with a lateral transport groove similar to that proposed for nhTMEM16? Although recent studies provided a great deal of insightful data with respect to the molecular mechanism of lipid transport [120,122,126], no doubt a combination of structural, cellular and biochemical approaches will soon help in the elucidation of the detailed mechanism of lipid translocation.

Acknowledgments

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We thank Miriam-Rose Ash for preparation of Figure 5B. This work was supported by the ANR young investigator grant ‘AsymLip’ (GL), by the French Infrastructure for Integrated Structural Biology (FRISBI, ANR-10-INSB-05-01) and by the French national center for scientific research (CNRS). JAL is funded by a Lundbeck postdoctoral fellowship.

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Figure 1: Conservation of P-type ATPase architecture. (A) Transmembrane lipid transporters in eukaryotic cell membranes. Flippases actively transport lipids from the exoplasmic to the cytoplasmic side (inward) of the membrane while floppases catalyze an active transport in the opposite (outward) direction. Scramblases disrupt phospholipid asymmetry by catalyzing a fast, bi-directional, energy-independent, and poorly specific transport. The arrows refer to the direction of lipid transport. (B) The figure shows sarcolipinbound rabbit SR Ca2+-ATPase in the E1 conformation (PDB: 4H1W), - and -subunit-bound pig kidney Na+/K+-ATPase in the E2P conformation (PDB: 4HYT), the Legionella pneumophila Cu+-ATPase in the E2P conformation (PDB: 4BBJ), and the AHA2 H+-ATPase from Arabidopsis thaliana in the E1 conformation (PDB: 3B8C). The phosphorylation domain, nucleotide-binding domain, and actuator domain are colored blue, red, and yellow, respectively. The membrane domain is colored tan. The sarcolipin is colored pink. The - and -subunits of Na+/K+-ATPase are colored pink and light blue, respectively. The approximate boundaries of the lipid bilayer are shown as a grey rectangle.

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Figure 2: Comparison of the catalytic cycle of P2- and P4-ATPases. (A) Key intermediates in the catalytic cycle of SR Ca2+-ATPase SERCA1a. The cycle starts with the release of protons (2-3) from the protein, in exchange with binding of two Ca2+ ions from the cytosol step (i)). The Ca2+-bound ATPase is then phosphorylated from Mg-ATP on a conserved aspartic acid to give an E1~P:ADP intermediate (step (ii)), which is converted to an E2P intermediate after release of ADP and exchange, on the luminal side, of two Ca2+ ions (released in the SR lumen) for 2-3 protons (step (iii)). Hydrolysis of the phosphorylated aspartic acid then drives the enzyme back to the E2 conformation (step (iv)), which will have to release H+ to bind new Ca2+ ions. Progression of the catalytic cycle is associated with individual motions of the cytosolic domains depicted by dashed arrows. Ca2+ ions are represented as dark green spheres and H+ as blue spheres. (B) Putative catalytic cycle of P4ATPases based on that of P2-ATPases. The phospholipid substrate (blue) is equivalent to H+ in SR Ca2+-ATPase. Whether phospholipid transport by P4-ATPases (from the outside to the inside) is coupled to transport of another substrate in the opposite direction is currently unknown (symbolized as a question mark). The N-, P-, and A- domains are shown in red, blue, and yellow, respectively. M1-M2 transmembrane helices are represented as a pink cylinder while M3-M4 and M5-M10 helices are represented as light pink and green cylinders, respectively. ‘F’ in the N-domain refers to F587 in rabbit SR Ca2+-ATPase, a residue that is critical for ATP binding. ‘D’ in the P-domain refers to the invariant phosphorylated aspartate. ‘TGES’ is the motif involved in dephosphorylation of P2-ATPases while DGET is the equivalent in P4-ATPases. Cdc50 subunit is omitted for clarity. Figure 3: Models for lipid transport pathways in P4-ATPases. (A) Proposed topology of the membrane domain of the yeast P4-ATPase Drs2p. Pink circles highlight residues which, when mutated in Dnf1p, interfere with NBD-PC transport. Yellow circles highlight residues which, when mutated in Dnf1p, confer preference for NBD-PS (or endogenous PS) and residues of Drs2p important for NBD- and endogenous PS transport. Orange circles highlight residues which, when mutated in Dnf1p, confer preference for di-acylated PS. The Drs2p residues corresponding to those forming the hydrophobic gate in ATP8A2 are highlighted in green. (B) and (C) Two proposed paths for translocation of phospholipids through P4ATPases. The three-dimensional homology model of yeast Drs2p was built using the ITASSER server [186], choosing the crystal structure of the E2P form of rabbit Ca2+-ATPase SERCA1a (PDB ID 3B9B) as the preferred template. The sequence identity between

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SERCA1a and Drs2p is 16 %. Only the membrane part and the bottom part of the P domain of Drs2p are displayed. (B) ‘Two-gate’ model. Transmembrane helices (M1, M3, and M4) that delineate the putative phospholipid transport pathway are colored in blue. Residues involved in phospholipid selection at the entrance and exit gates are colored according to the same color code as in A) and displayed as spheres. Numbering refers to Dnf1p and Drs2p (in brackets). Transmembrane helices bordering the phospholipid transport pathway are numbered. (C) Hydrophobic gate model. Transmembrane helices that delineate the putative phospholipid transport pathway are colored in blue. Residues forming the ‘hydrophobic gate’ are represented as green spheres. Residue N359, whose mutation decreases the apparent affinity of ATP8A2 for PtdSer, is displayed in yellow and as sphere. In order to better visualize the proposed lipid transport groove, the Drs2p model is displayed with a side view seen from the inside of the bilayer (left) and a bottom view seen from the exoplasmic side (right).

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Figure 4: Structures of SR Ca2+-ATPase and Na+/K+-ATPase in the presence of inhibitors bound to the membrane domain. Only the transmembrane region is shown in tan, viewed from within the membrane (left), the cytoplasm (right, for the SR Ca2+-ATPase), or the extracellular side (right, for the Na+/K+-ATPase). (A) Location of the cyclopiazonic acid binding (CPA) site and of a PtdEth binding site in the SERCA1a E2 conformation (PDB: 2EAU). CPA is shown as red sticks and an incomplete PtdEth molecule is shown as purple sticks. The localization of transmembrane helices M1, M2, M3, M4, M5, and M7 is indicated. (B) Location of the thapsigargin derivative Boc-12ADT in the SERCA1a E2 conformation (PDB: 2BY4). Boc-12ADT is shown as green sticks, with oxygen and nitrogen atoms displayed in red and blue, respectively. The localization of transmembrane helices M1-M5 is indicated. (C) Structure of the Na+/K+-ATPase E2P-ouabain complex (PDB: 4HYT). Ouabain is represented by red sticks. The localization of transmembrane helices M1-M6 is indicated.

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Figure 5: Regulation of the P4-ATPase Drs2p. (A) Schematic representation of yeast Drs2p domain organization. Actuator (A-, yellow), nucleotide binding (N-, red), and phosphorylation (P-, blue) domains as well as transmembrane helices are indicated (grey cylinders). The Arl1p regulatory region and the Gea2p/PI4P regulatory/inhibitory region are shown as green boxes with corresponding interacting sequences. Note that while deletion of the first 160 amino acids of Drs2p completely disrupts interaction with Arl1p, deletion of amino acids 153-160 (DSRNKFNI) already drastically diminishes the interaction [149]. (B) Proposed mechanism for regulation by PtdIns(4)P of Drs2p flippase activity based on salient features of the SR Ca2+-ATPase reaction cycle. In the transition state of dephosphorylation (E2-P), PtdSer (orange) is bound to its transport site, resulting in closure of the luminal entry pathway and occlusion of PtdSer. Extensive interactions between the A-, N- and P- domains result in formation of a compact cytosolic headpiece. In the auto-inhibited conformation, the C-terminal tail of Drs2p prevents correct positioning of the DGET loop, possibly by interacting with the cytosolic headpiece. In the un-inhibited state, PtdIns(4)P (green) in the cytosolic leaflet of TGN membranes allows docking of the DGET loop into the phosphorylation site for subsequent nucleophilic attack. After dephosphorylation (E2), the interactions between the A- and P- domains are weakened, resulting in a more relaxed state. The cytosolic gate opens and PtdSer is exchanged with a currently unidentified species. Figure 6: Lipid transport pathway in the nhTMEM16 lipid scramblase. (A) Overall structure of the nhTMEM16 scramblase (PDB: 4WIS). The nhTMEM16 dimer is represented as ribbon and viewed from within the membrane. Bound Ca2+ ions are represented as green spheres. The membrane boundary, the position of transmembrane helix M10 as well as the

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location of N- and C-termini, are indicated. (B) Same as (A) but viewed from the outside of the cell. The location of the dimer cavity is indicated. (C) Location of the subunit cavity in the overall nhTMEM16 structure. The five transmembrane helices (M3-M7) lining the cavity are colored red orange. The cytosolic domain is displayed in blue whereas the remaining transmembrane domain is displayed in tan. Bound Ca2+ ions are represented as green spheres. (D) Subunit cavity from within the membrane. Only the transmembrane domain is displayed, for clarity. The structure is shown in surface representation with the helices lining the cavity colored red orange and the rest of the transmembrane domain colored tan. A possible pathway for lipid translocation within a narrow crevice is indicated.

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Phospholipid flippases are master regulators of lipid asymmetry in cell membranes P4-ATPases use the energy of ATP to maintain transbilayer lipid asymmetry The possible path taken by phospholipids through P4-ATPases is discussed The problem posed by the size of the transported substrate is reassessed The structure of an ATP-independent flippase sheds new light on the mechanism of lipid transport

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