Flippases and vesicle-mediated protein transport

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Vol.14 No.12 December 2004

Flippases and vesicle-mediated protein transport Todd R. Graham Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634, USA

The best-understood mechanisms for generating transport vesicles in the secretory and endocytic pathways involve the localized assembly of cytosolic coat proteins such as clathrin, coat protein complex (COP)I and COPII onto membranes. These coat proteins can deform membranes by themselves, but accessory proteins might help to generate the tight curvature needed to form a vesicle. Enzymes that pump phospholipid from one leaflet of the bilayer to the other (flippases) can deform membranes by creating an imbalance in the phospholipid number between the two leaflets. Recent studies describe a requirement for the yeast Drs2p family of P-type ATPases in both phospholipid translocation and protein transport in the secretory and endocytic pathways. This indicates that flippases work with coat proteins to form vesicles. Small vesicles and tubular–vesicular structures mediate protein transport between organelles of the secretory and endocytic pathways. For example, coat protein complex (COP)II-coated vesicles bud from the endoplasmic reticulum (ER) to deliver cargo to the Golgi, and COPI-coated vesicles mediate retrograde transport from the Golgi back to the ER. Clathrin associates with organelle-specific adaptor proteins (APs) to form vesicles from the transGolgi network (TGN), endosomes and the plasma membrane, and seems to mediate multiple transport pathways between these compartments [1,2]. The small, GTPbinding protein ADP-ribosylation factor (ARF), and its guanine-nucleotide-exchange factors (GEFs) and GTPase activating proteins, regulate the assembly of COPI, clathrin-adaptor and AP-3 coat proteins on the Golgi and/or endosomes [3]. In addition, genetic studies in yeast have implicated Drs2p in ARF and clathrin-dependent formation of transport vesicles from the TGN [4,5]. Drs2p and its mammalian ortholog ATPase II are founding members of a large subfamily of eukaryotic P-type ATPases that are thought to be aminophospholipid translocases (APLTs, also known as flippases) [6]. Several reviews have described the function of mammalian flippases in establishing membrane asymmetry, regulation of blood clotting and clearance of apoptotic cells [6–10]. In this review, I focus on the biochemical and cell biological functions of the Drs2p family of P-type ATPases in yeast. Corresponding author: Todd R. Graham ([email protected]). Available online 2 November 2004

Flippases and membrane asymmetry A phospholipid molecule in a membrane bilayer can diffuse rapidly throughout one leaflet, but faces a substantial barrier to the translocation, or ‘flipping’, of its polar headgroup through the hydrophobic interior of the membrane to the other leaflet [11]. Consequently, the two leaflets of a membrane can be completely different in phospholipid composition and the plasma membrane of most eukaryotic cells is asymmetric. The extracellular leaflet is made primarily of sphingomyelin and phosphatidylcholine (PtdCho), with phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer) restricted to the cytosolic leaflet [6,10,12,13]. Synthesis of phospholipid occurs primarily on the cytosolic leaflet of the ER and so w50% of the newly made phospholipid must be flipped to the lumenal leaflet to produce a bilayer [14]. The ER membrane contains an undefined protein or proteins that facilitate rapid, bidirectional, energy-independent flip-flop of phospholipid between lumenal and cytosolic leaflets [15–18] to produce a symmetrical ER membrane. Thus, asymmetry must be established as membrane flows through the Golgi, and/or on arrival at the plasma membrane. This asymmetry seems to be regulated by the action of flippase, floppase and scramblase activities (Figure 1), at least in red blood cell membranes in which most studies have been performed [6,10]. APLT Twenty years ago, the Devaux group discovered a plasma membrane flippase activity that is specific for PtdEtn and PtdSer, named APLT, by monitoring the translocation of nitroxide spin-labeled phospholipid derivatives across the plasma membrane [19]. A similar APLT activity that flips spin-labeled PtdSer from the lumenal to the cytosolic leaflet has been found in chromaffin granules [20]. Although several phospholipid derivatives have been used in these experiments [7], typically they contain a short acyl chain in the sn2 position (e.g. six carbons long) with either spin-labeled or fluorescent 7-nitro-2–1,3-benzoxadiazol-4-yl (NBD) reporter attached to the last carbon. The mammalian APLT flips one phospholipid for each ATP hydrolyzed [21] and seems to recognize the headgroup and glycerol backbone of the aminophospholipids rather than the reporter [10]. The APLT defined using labeled phospholipids also seems to translocate unmodified, long-chain PtdSer and PtdEtn [22], but more studies are needed to confirm this.

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PS

PE

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PI

PC or Sph

Scramblase Ca2+

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Extracellular or lumenal leaflet

ER flippase

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Figure 1. Proteins thought to mediate the transbilayer movement of phospholipids and regulate membrane asymmetry. ATP-dependent flippases catalyze the translocation of phosphatidylethanolamine (PE) and phosphatidylserine (PS) from either the extracellular or lumenal leaflet to the cytosolic leaflet, whereas floppases mediate a reverse reaction that is kinetically slower and has no apparent headgroup specificity. The net result of these activities is an asymmetrical membrane. Scramblase, which is activated by an influx of Ca2C, enables the phospholipids to flow down their concentration gradient to produce a symmetrical membrane. The endoplasmic reticulum (ER) membrane also contains an energy-independent flippase that enables bi-directional movement of phospholipids during membrane synthesis, which results in a symmetrical membrane. ATP-dependent flippases seem to be P-type ATPases in the Drs2p–ATPase II family, and the floppase activity is thought to be catalyzed by ATP-binding cassette (ABC) transporters. The identities of scramblase and the ER flippase are uncertain [6,10]. Abbreviations: PC, phosphatidylcholine; PI, phosphatidylinositol; Sph, sphingosine.

Identification of the APLT has been hampered by the lack of specific inhibitors and difficulties reconstituting this activity with a demonstrably pure enzyme. However, the biochemical properties of APLT correlate well with the plasma membrane Mg2C-ATPase [23] and the ATPase II purified from chromaffin granules [24,25]. The gene that encodes ATPase II has been cloned and its sequence is extremely similar to that of Drs2p from Saccharomyces (46% identity), and both proteins show weaker similarity to many P-type ATPases, which comprise a family of ion and heavy metal transporters that are integral to the membrane [26]. From several genome-sequencing projects, it is apparent that Drs2p and ATPase II are founding members of a distinct subfamily of P-type ATPases that includes five yeast and 14 human members, each of which are considered potential APLTs [26–28] (Table 1).

The yeast Drs2p family of potential phospholipid translocases (PLTs) The discovery of a yeast homolog of ATPase II provided the opportunity to apply genetic approaches to determine whether Drs2p and, by extension, ATPase II are APLTs. As in mammalian cells, yeast expose little PtdEtn and PtdSer on the outer leaflet of the plasma membrane [29,30] and can translocate NBD–PtdSer and NBD–PtdEtn across this membrane [26,29,31,32]. Table 1. Yeast and human phospholipid translocasesa Saccharomyces cerevisiae Drs2p, Dnf3p Dnf1p, Dnf2p Neo1p

a

Homo sapiens

Refs

ATP8A1 (ATPase II), ATP8A2 ATP8B1 (FIC1), ATP8B2, ATP8B3 ATP8B4 ATP9A, ATP9B ATP10A, ATP10C (mouse pfatp), ATP10D ATP11A, ATP11B, ATP11C

[27,28]

The human subgroups are based on phylogenetic analyses in the referenced papers. A BLAST ‘best hit’ comparison indicates that Drs2p and Dnf3p are most closely related to the human ATP8A pair, that Dn1p and Dnf2p are most closely related to the ATP8B group and that Neo1p is most closely related to the ATP9 pair.

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Surprisingly, NBD–PtdCho is also translocated efficiently into yeast, which suggests that yeast translocase(s) have broader substrate specificity (a PLT rather than an APLT) [32–34]. How NBD–PtdCho translocase activity relates to membrane asymmetry is unknown. Strains carrying a disruption of DRS2 (drs2D) are viable and initial studies differed on whether translocation of NBD–PtdSer across the drs2D plasma membrane is impaired significantly [26,35–37]. In retrospect, these studies were complicated by functional redundancy between Drs2p and other potential PLTs (Table 1), and the role of Drs2p in protein transport. DRS2 and the DNF genes form part of an essential group, any one member of which can support viability, which indicates a common biochemical function for these proteins [38]. Drs2p and Dnf3p co-localize in late Golgi membranes whereas Dnf1p and Dnf2p localize to the plasma membrane and internal organelles (Golgi and/or endosomal) [4,29,38]. Dnf1p and, to a lesser extent, Dnf2p localize to sites of polarized growth (small buds and mother-bud neck during cytokinesis) and seem to cycle actively between the endocytic and exocytic pathways [38,39]. Based on their localization, Dnf1p and Dnf2p are the most likely candidates for the plasma membrane PLT. Consistently, the energy-dependent translocation of NBD–PtdEtn, NBD–PtdSer and NBD–PtdCho across this membrane is abolished in dnf1 and dnf2 double mutants (dnf1,2D), and these cells expose endogenous PtdEtn on the cell surface [29]. Loss of Drs2p function perturbs exocytosis [5,38] and seems to deplete the cell surface of Dnf1p (and possibly Dnf2p) partially [39]. The extent of Dnf1p depletion from the drs2D cell surface might vary with strain background and growth conditions, which might cause the disparate data on NBD–PtdSer translocation from different laboratories. Thus, Drs2p does not seem to contribute directly to PLT activity in the plasma membrane of wild-type cells; however, it is positioned to contribute to plasma membrane asymmetry as membrane flows from the TGN to the plasma

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membrane. In support of this possibility, drs2D also exposes PtdEtn on the outer leaflet of the plasma membrane, and the drs2D dnf1,2D triple mutant exposes more PtdEtn than dnf1,2D cells [29]. These data support the proposed PLT activity of the Drs2p and Dnf proteins, but do not rule out the possibility that they regulate either the activity or localization of other proteins that are directly responsible for NBD–phospholipid translocation and membrane asymmetry. Strong evidence that ATPases in the Drs2p family are PLTs comes from the recent demonstration that Drs2p is directly responsible for an APLT activity in TGN membranes in yeast [40]. To assess the contribution of Drs2p to this activity, TGN membranes were purified from strains that are deficient for the Dnf proteins and that express either wild-type or a temperature-sensitive form of Drs2p (either DRS2 dnf1,2,3D or drs2-ts dnf1,2,3D). Growth of these cells at the permissive temperature alleviates a problem associated with deleting DRS2: loss of Drs2p function in vivo causes mislocalization of resident TGN proteins making the drs2D TGN membranes deficient in multiple proteins relative to wild-type controls. The ability to inactivate the Drs2p-ts protein after membrane isolation enabled comparison of APLT activity in the same membranes before and after inactivation of a single protein. These experiments indicate that Drs2p function is required for translocation of NBD–PtdSer from the lumenal to the cytosolic leaflet of the TGN [40]. ATPdependent translocation of NBD–PtdEtn or NBD–PtdCho was undetectable in either membrane preparation, indicating that Drs2p does not catalyze these activities. However, as described later, PtdSer is not the only substrate for Drs2p and it is possible that these assays are insufficiently sensitive to detect NBD–PtdEtn translocase activity catalyzed by Drs2p. This possibility is consistent with the loss of PtdEtn asymmetry of the drs2D plasma membrane. The Drs2p-coupled NBD–PtdSer translocase requires hydrolyzable ATP and Mg2C, but no other specific ion cofactor, which indicates that Drs2p is a Mg2C-dependent ATPase that directly flips NBD–PtdSer across the membrane [40]. However, it is possible that ATPases of the Drs2p family are coupled to lipid translocation through an ion intermediate. In this model, the P-type ATPase pumps an ion across the membrane to establish a concentration gradient, which subsequently drives phospholipid translocation through an undefined symporter. In fact, a proton gradient seems to be necessary for translocation of NBD–PtdCho and NBD–PtdEtn across the plasma membrane [34]. However, this gradient is established by the plasma membrane HC-ATPase (Pma1p) rather than Dnf1p and Dnf2p, and so how the proton gradient contributes to phospholipid translocation or asymmetry is unknown. Disruption of the putative symporter should also perturb phospholipid translocation and phenotypically mimic the drs2 and dnf mutants. Intriguingly, mutations in members of an essential gene family (LEM3, CDC50 and YNR048) that encodes integral membrane proteins (Figure 2) produce phenotypes that are comparable to dnf1,2 and drs2 mutations [39,41,42]. www.sciencedirect.com

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The Lem3–Cdc50 family LEM3 (also known as ROS3) was identified in two unbiased, genetic screens for mutants that either aberrantly expose endogenous PtdEtn on the outer leaflet of the plasma membrane or are resistant to a toxic PtdCho analogue. Disruption of LEM3 causes a defect in the translocation of NBD–PtdCho and NBD–PtdEtn across the plasma membrane, but, surprisingly, NBD–PtdSer translocation is unaffected [41,42]. CDC50 was recovered initially in a screen for cold-sensitive, cell-division-cycle mutants, but how cdc50 causes a cold-sensitive block in the G1 to S transition is unknown [43]. These proteins have no sequence features, such as an ATPase domain or similarity to other transporters, that indicate how Lem3p controls the energy-dependent translocation of NBD–PtdCho and PtdEtn across the plasma membrane. However, recent work from the Tanaka group indicates that Lem3p–Dnf1p and Cdc50p–Drs2p form complexes that facilitates exit of these proteins from the ER. Thus, Lem3p and Cdc50p seem to be chaperones that are responsible for the proper localization of Drs2p–Dnf family ATPases, rather than translocases or symporters themselves [39]. However, these observations do not rule out the possibility that Lem3p and Cdc50p also have a more direct role in either coupling phospholipid translocation to an energy source (either ATP hydrolysis by Drs2p–Dnf or a proton gradient) or determining substrate specificity once the protein complex arrives at the proper membrane. Requirements for Drs2p family P-type ATPases in protein transport A role for Drs2p in protein transport was suggested initially from genetic interactions with ARF and clathrin, and subsequent studies have implicated Drs2p in a clathrin-dependent pathway exiting the TGN [4,5,44]. The TGN produces several kinds of vesicles that are destined for the vacuole, late endosomes, early endosomes and the plasma membrane (Figure 3). In yeast, two classes of exocytic vesicles, dense and light, which carry different cargo, mediate polarized secretion to the bud [45]. Unexpectedly, Drs2p, clathrin and a dynamin-related protein (Vps1p) are required to form the dense class of exocytic vesicles that carry the cargo protein invertase [5,46,47]. The drs2, clathrin heavy chain (chc1) and vps1 mutants also mislocalize the TGN resident protein Kex2p to the plasma membrane, probably by disrupting the normal trafficking of Kex2p to the early endosome. Thus, it seems that Drs2p and clathrin are responsible for producing vesicles containing Kex2p (targeted to the early endosome) in addition to the dense vesicles containing invertase (Figure 3). There is debate over whether the dense exocytic vesicles bud from either the TGN or late endosome. The latter possibility was proposed because blocking protein transport through the late endosome also perturbs the formation of dense exocytic vesicles. A caveat to both these models is that the trafficking pathways between the TGN, plasma membrane and endosomes might be so interdependent that is impossible to perturb one pathway without indirectly affecting the others. Nonetheless, most available data indicate that Drs2p

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Vol.14 No.12 December 2004

the vacuole in the AP-3 pathway, whereas Dnf1p and Dnf2p have redundant functions in an early endosome to TGN pathway [38]. The latter pathway seems to be mediated by AP-1 and clathrin [48]. Interestingly, the dnf1,2D mutant exhibits a cold-sensitive defect in the internalization step of endocytosis that is exacerbated by additional deletion of DRS2 [29,38]. Neo1p is essential for viability and is required for an ARF–COPI-dependent retrograde pathway from the Golgi to the ER [49] (Figure 3). Strikingly, proteins of the Drs2p family are implicated in every trafficking pathway in which ARF is thought to function in producing coated vesicles.

ATPase C N N

C Cytosolic

Lumenal Drs2 Neo1 Dnf1 Dnf2 Dnf3

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Potential mechanisms for Drs2p function in vesicle formation Several lines of evidence indicate that phospholipid translocation by Drs2p is required for vesicle formation. Inactivation of drs2-ts by temperature-shift rapidly inactivates NBD–PtdSer translocation in vitro and dense vesicle formation in vivo [5,40]. In addition, an ATPasedead form of Drs2p (drs2-D560N) blocks formation of these exocytic vesicles [5], indicating that the ability of Drs2p to flip PtdSer to the cytosolic leaflet of the TGN is required to support vesicle formation. Surprisingly, this is not the case: even though PtdSer composes w20% of Golgi phospholipid in wild-type cells, cho1D mutants, which are PtdSer-deficient, transport and process proteins normally in the secretory pathway. Moreover, PtdSer-deficient cells require Drs2p to produce dense vesicles. Therefore, PtdSer is not an obligatory substrate for the function of Drs2p in vesicle formation [40].

Cdc50 Lem3 Ynr048w TRENDS in Cell Biology

Figure 2. Predicted topologies of the Drs2p family of P-type ATPases and their potential chaperones. Interaction of Cdc50p with Drs2p and of Lem3p with Dnf1p is required for the export of these proteins from the endoplasmic reticulum.

functions at the TGN to facilitate the formation of clathrin-coated vesicles from this compartment [4,5,38]. The requirement for Drs2p in forming exocytic vesicles has prompted investigation of other members of the Drs2p/Neo1p family, to determine if they are also involved in protein transport. Functional overlap between Drs2p and Dnf proteins also occurs at the level of protein transport. For example, Drs2p and Dnf1p have interchangeable roles for delivering alkaline phosphatase to

Golgi complex

ER

Pma1p (light)

drs2∆ Invertase (dense) Kex2p

neo1-ts ALP Vacuole

drs2∆ dnf1∆

CPY

dnf1∆ dnf2∆

dnf1∆ dnf2∆ < 20˚

Early endosome

CPY Late endosome

Coat proteins: Cargo proteins:

COPII ALP

CPY

COPI Kex2p

AP-3 Invertase

Clathrin Pma1p TRENDS in Cell Biology

Figure 3. Vesicle-mediated protein-transport pathways that require P-type ATPases of the Drs2p family. Pathways that are perturbed by mutations in the DRS2 family genes (red text) are indicated by ‘T-bars’. Emphasis is placed on transport pathways exiting the trans-Golgi network (TGN; right-hand cisterna of Golgi complex), which are defined by the cargo proteins indicated (blue text), the vesicle density (light or dense) or the coat proteins. The roles of clathrin in the yeast system are still uncertain but pathways that are perturbed by clathrin mutations are indicated with a clathrin coat (black). In yeast, all trafficking pathways in which ADP-ribosylation factor (ARF)-dependent coat proteins are implicated (COPI, AP-3 and clathrin) require one or more Drs2p family ATPases. Abbreviations: ALP, alkaline phosphatase; COP, coat protein complex; CPY, carboxypeptidase Y; ER, endoplasmic reticulum. www.sciencedirect.com

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These experiments do not rule out endogenous PtdSer as a substrate of Drs2p, but they do indicate that Drs2p pumps another substrate across the TGN membrane that has an important role in vesicle budding. As described previously, PtdEtn might be the best candidate because the drs2D mutant exhibits a partial loss of PtdEtn asymmetry at the plasma membrane [29] and PtdEtn stimulates the ATPase activity of bovine ATPase II with 10–20% the efficiency of PtdSer [50]. If PtdEtn and PtdSer are interchangeable substrates, the precise phospholipid species might be less important than the physical displacement of phospholipid from the lumenal leaflet to the cytosolic leaflet is crucial. Importantly, we can rule out the possibility that an asymmetric distribution of PtdSer in the TGN is required for vesicle formation through recruitment of PtdSer-binding effectors to the membrane, but further work is required to determine if asymmetry of either PtdEtn or another lipid is important. It is possible that the relevant substrate for Drs2p is an undefined ion rather than phospholipids. Alternatively, the conformational changes associated with ATP hydrolysis might be crucial for vesicle formation by regulating protein interactions. In this regard, it is important to note that the C-terminal tail of Drs2p binds directly to an ARF–GEF (Gea2p) [51]. The Drs2p–Gea2p interaction is not essential for Drs2p function, but it does contribute and should concentrate the vesicle budding machinery (ARF–GEF, ARF, adaptins and clathrin) at sites of Drs2p-catalyzed ATP hydrolysis [51], which is probably coupled to translocation of phospholipid (Figure 4).

ATP ATP

Drs2p

AP-1

Drs2p

ADP ARF–GTP ARF–GDP

ARF–GEF Clathrin

ATP

TRENDS in Cell Biology

Figure 4. Interaction of Drs2p with Gea2p (ARF–GEF) might concentrate the vesiclebudding machinery at sites of Drs2p-dependent phospholipid translocation. Activated ARF–GTP binds to the membrane and recruits adaptor proteins (e.g. AP-1) to facilitate the assembly of clathrin [57]. Hydrolysis of ATP by Drs2p and, presumably, its ability to pump phospholipid across the membrane is required to support the formation of vesicles in vivo. Phospholipid asymmetry produced by Drs2p might also contribute to either the recruitment or function of coat proteins, although phosphatidylserine, a preferred substrate for Drs2p in vitro, is not required for vesicle formation in vivo. Abbreviations: ARF, ADP-ribosylation factor; GEF, guanine-nucleotide-exchange factor. www.sciencedirect.com

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Flippases, coupled bilayers and vesicle formation The potential role of flippases in vesicle budding has been discussed for several years with models premised on the bilayer-couple hypothesis of Sheetz and Singer [4,6,7,52]. Based on the affect of different membrane intercalating compounds on the shape of red blood cells, this hypothesis states that changes in the surface area of one leaflet relative to the other induce a conformational change in the bilayer. Because the two leaflets are coupled together physically, lateral expansion of one leaflet will face resistance from the opposite leaflet and lead to membrane bending [52]. Flippase-catalyzed translocation of phospholipid molecules from the lumenal (or extracellular) leaflet to the cytosolic leaflet will increase the surface area of the cytosolic leaflet while decreasing that of the lumenal leaflet. Thus, flippases should induce positive curvature in the membrane (bending towards the cytosol) and, therefore, facilitate generation of tightly curved transport vesicles (Figure 4). In Box 1, I extend this discussion to explain how the bilayer-couple hypothesis and flippases might affect homeostasis of vesicle-mediated protein transport between membrane compartments and nonhomeostatic transport events, such as the consumption of the TGN into transport vesicles. The latter event is predicted from the cisternal maturation model, which suggests that Golgi cisternae are formed de novo at the cis face, mature as they progress down the stack, and are consumed at the trans face [53]. The concepts presented above and in Box 1 indicate that Drs2p, and flippases in general, might participate in vesicle formation by one or more of the following mechanisms. (i) Translocation of phospholipid across the membrane might relax the negative curvature in the TGN membrane to produce a substrate that coat proteins can continually deform into vesicles (Box 1a). There is evidence that flippases relax negative curvature in a membrane. Addition of spin-labeled PtdSer to the outer leaflet of red blood cells induces conformational changes in the membrane (bending away from the cytosol) that are reversed by APLT-dependent translocation of the PtdSer to the cytosolic leaflet [19]. (ii) Rather than simply relaxing negative curvature, flippases might use the energy from ATP hydrolysis to produce positive curvature and prime the membrane for vesicle formation. For example, translocation of spin-labeled PtdSer across the plasma membrane to the cytosolic leaflet enhances endocytosis, presumably by increasing positive curvature in this membrane [54]. For Golgi cisternae, flippaseinduced positive curvature should promote fenestration and tubulation of the membrane (Box 1b), features that are typical of this organelle. In addition, Rambourg and colleagues propose that the maturation of Golgi cisternae from cis to trans and the production of exocytic vesicles is accompanied by the membrane transformation events depicted in Box 1b [55]. Thus, flippases might contribute to a maturation process that prepares the membrane for vesiculation. (iii) Flippases might work directly with the vesicle budding machinery to generate positive curvature at the site of vesicle formation. As described above, the interaction of Drs2p with an ARF–GEF might stimulate the localized assemble of clathrin to ‘capture’ curved

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Box 1. Potential impact of flippases on membrane structure and vesicle formation

membrane at the site of phospholipid translocation (Figure 4). (iv) Last, flippases might concentrate a phospholipid other than PtdSer on the cytosolic leaflet that stimulates the recruitment or assembly of coat proteins. For example, a high concentration of PtdEtn (40%) has been shown to stimulate assembly of welldefined clathrin coated buds on liposomes [56]. Concluding remarks In summary, recent studies strongly support the proposed flippase activity of Drs2p family ATPases and have linked this activity to the formation of transport vesicles in vivo. In fact, these are the only integral membrane proteins implicated in ARF and clathrin-dependent transport pathways. Thus, coat proteins might be sufficient to drive vesicle formation in vitro [56,57] but flippases might be necessary for formation of vesicles with the efficiency required to support protein transport in vivo. In addition, these discoveries provide another connection between phospholipid metabolism and vesicle-mediated www.sciencedirect.com

Lumenal leaflet

Balanced bilayer

Cytosolic leaflet

SNAREs

(a)

Hemifusion intermediate

d coat p r o F an tei AR ns

As they assemble, vesicle coat proteins deform the membrane into a tightly curved bud and select cargo for export. Release of the vesicle is thought to proceed through a hemifission intermediate whereby the inner leaflet of the bilayer fuses first, followed by fusion of the cytosolic leaflet [63]. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated vesicle fusion with an acceptor membrane proceeds in the opposite direction through a similar hemifusion intermediate [64], with presumably no mixing of phospholipids between the two leaflets during either budding or fusion. The flux of vesiclemediated transport between two membrane-bound compartments must be balanced to prevent the growth of one compartment at the expense of the other. This balance of membrane trade is particularly important when considering the two leaflets of the membrane independently, because an important consequence of budding small transport vesicles (e.g. 25-nm radius) is that the surface area of the cytosolic leaflet is w50% more than that of the lumenal leaflet (Figure Ia). Thus, one would predict that excessive, unbalanced budding of vesicles should quickly generate a deficit of cytosolic leaflet in the donor membrane, whereas excessive vesicle fusion should generate a surplus of cytosolic leaflet. A difference in surface area caused by an imbalance in phospholipid number between the two leaflets places a significant stress (or lateral pressure) on the membrane, which can be relieved through conformational changes [52]. An imbalance in phospholipid number produces either positive curvature in a membrane (when the cytosolic:lumenal leaflet ratio is O1) or negative curvature (when the cytosolic:lumenal leaflet ratio is !1). As little as a 1% change in this ratio can have substantial effects on membrane structure [52,65]. Thus, it is likely that the process of vesicle budding progressively inhibits the formation of new vesicles as the cytosolic:lumenal leaflet ratio decreases and negative curvature increases. Conversely, the process of vesicle fusion might promote the budding of new vesicles as positive curvature increases. Therefore, an advantage of using of small vesicles to transport protein and membrane between organelles might be that it facilitates homeostasis between budding and fusion reactions (for example, at the presynaptic membrane). However, not all exchanges of membrane between organelles are likely to be balanced. For example, the transGolgi network (TGN) is apparently consumed into small vesicles and tubular carriers during the cisternal maturation process. Flippases might provide another route to maintain sufficient cytosolic leaflet of the TGN to support vesicle budding by actively flipping phospholipid from the lumenal leaflet to the cytosolic leaflet (Figure Ia). In addition, flippases might generate excess cytosolic leaflet in a Golgi cisterna (cup-shaped structure in Figure Ib), causing it to fenestrate and produce tubular regions. This could provide a surface primed for efficient vesicle budding by coat proteins.

Flippase

5024 nm2 7850 nm2

25nm 20nm

Negative curvature

Hemifission intermediate

Positive curvature

(b) Unfenestrated

Fenestrated

Flippase

Tubular

Flippase

Vesicular

Coat proteins

Increasing cytosolic:lumenal leaflet ratio TRENDS in Cell Biology

Figure I. Potential roles of flippases in deforming membranes to support vesicle budding. (a) Budding of a transport vesicle (with small radius and high positive curvature) extracts more cytosolic leaflet than lumenal leaflet from a membrane and should, therefore, induce negative curvature in the donor organelle. This negative curvature, which should inhibit budding of additional vesicles, can be relaxed through the fusion of an incoming vesicle or by translocation of lipid to the cytosolic leaflet by a flippase. (b) Alternatively, ATP-dependent flippases could generate positive curvature in a donor membrane such as a Golgi cisterna, inducing conformational changes that prime the membrane for vesicle budding by coat proteins.

protein transport. Classical studies on Sec14p demonstrated that regulation of the phospholipid composition of Golgi by this phosphatidylinositol (PtdIns) transfer protein has an essential role in the formation of exocytic vesicles [58,59]. Similarly, the discovery that a PtdIns 3-kinase (Vps34) is required for vacuolar protein sorting provided the first indication that PtdIns phosphates are important in protein transport [60]. Whereas Sec14p and PtdIns kinases seem to either generate or maintain signaling lipids that recruit effector proteins to membranes [61,62], flippases might have a more mechanical role in helping coat proteins shape membranes into transport vesicles. Clearly, additional studies are needed to test the hypotheses described in this article and determine the mechanism by which Drs2p family ATPases function in protein transport. It is important to define the precise substrates that are transported by each Drs2p family ATPase and whether the Cdc50–Lem3p subunits are solely chaperones or whether they contribute directly to

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phospholipid translocation. Reconstitution of the phospholipid translocase activity with purified proteins in liposomes of defined lipid composition should help to resolve these issues. It is also important to determine whether purified, reconstituted flippases induce an ATP-dependent change in the shape of liposomes, as predicted from the bilayer-couple hypothesis, and the influence of this on recruitment and assembly of clathrin buds onto these liposomes. The requirement for Drs2p family ATPases in protein trafficking in mammalian cells should also be explored. For example, ATPase II is a component of dense-core secretory granules in chromaffin cells [26] and one might predict that ATPase II is needed for the formation or maturation of these exocytic vesicles.

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