Is Lipid Translocation Involved During Endo- and Exocytosis?

Is lipid translocation involved during endo- and exocytosis?

Philippe F. Devaux*

Institut de Biologie Physico-Chimique, UPR-CNRS 9052, 13, rue Pierre-et-Marie-Curie, 75005 Paris, France (Received 28 January 2000; accepted 17 March 2000)

Abstract — Stimulation of the aminophospholipid translocase, responsible for the transport of phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaﬂet of the plasma membrane, provokes endocytic-like vesicles in erythrocytes and stimulates endocytosis in K562 cells. In this article arguments are given which support the idea that the active transport of lipids could be the driving force involved in membrane folding during the early step of endocytosis. The model is sustained by experiments on shape changes of pure lipid vesicles triggered by a change in the proportion of inner and outer lipids. It is shown that the formation of microvesicles with a diameter of 100–200 nm caused by the translocation of plasma membrane lipids implies a surface tension in the whole membrane. It is likely that cytoskeleton proteins and inner organelles prevent a real cell from undergoing overall shape changes of the type seen with giant unilamellar vesicles. Another hypothesis put forward in this article is the possible implication of the phospholipid ‘scramblase’ during exocytosis which could favor the unfolding of microvesicles. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientiﬁques et médicales Elsevier SAS aminophospholipid translocase / membrane budding / spontaneous curvature / liposomes / K562 cells

1. Introduction yet whether clathrin polymerizes and then pinches off the membrane to form the buds or if polymerization takes During the last 10–15 years, a large number of proteins place around a pre-formed bud. An alternative explanation have been recognized as playing central roles during proposed for membrane budding during endocytosis is the endo-exocytosis phenomena and more generally during formation of local invaginations named caveola formed by vesicle traffic: clathrin, NSF, ARF, dynamin, caveolin, self association of the hydrophobic protein caveolin with annexins and GTP-binding proteins, have been implicated specific lipids [6]. To what extent is ATP necessary for in the processes of membrane vesicularization (also called budding is still debated [7]. In summary, the physical budding) or during fission, targeting and fusion of origin of the local membrane curvature is still unexplained vesicles. A few reconstitution experiments with a limited in spite of the correlation between the presence of specific number of proteins have reproduced, if not quantitatively, proteins and the formation of coated pits. Concerning at least qualitatively some of the important steps [1, 2] and exocytosis, it is believed that the translocation of lipids from these observations, generalized models were pro- which a priori is necessary after the fusion of micro- posed. However, the main emphasis of earlier research has vesicles to the relatively flat plasma membrane can take been to show which proteins are required, rather than to place spontaneously, yet it is known that lipid flip-flop dissect the molecular mechanisms involved. Membrane requires several hours which is quite incompatible with folding which is a prerequisite for vesicle formation, is the time scale of a continuous endocytotic process. often assumed to be explained by the binding of clathrin In general, the role of lipids has been largely over- or of coat proteins. Yet, endocytosis can exist without looked in these cell biology investigations. Only recently, clathrin or without clathrin polymerization [3, 4]. Further- in experiments with reconstituted vesicles to which coat more, increasing the amount of polymerized clathrin does proteins were attached, has the importance of lipids been not systematically increase the number of coated pits at realized. In fact, even pure liposomes enable one to mimic the plasma membrane [5]. In fact, it has not been proven some fundamental steps of biological membrane traffic. To those who believe that the representation of a biologi- * Correspondence and reprints: [email protected] cal membrane by a mere lipid bilayer is irrelevant, it Abbreviations: PC, phosphatidylcholine; PS, phosphatidylserine; should be recalled that membrane budding, fission and PE, phosphatidylethanolamine; L-PC, lyso-phosphatidylcholine; fusion refer primarily to the status of the lipid bilayer. PG, phosphatidylglycerol; GUV, giant unilamellar vesicle; LUV, large unilamellar vesicle; SUV, sonicated unilamellar vesicle; Thus, a detailed analysis of the physical constraints NMR, nuclear magnetic resonance; MDR, multi drug resistance associated with folding and fusion of a pure lipid bilayer protein is of primary importance. Of course, investigating liposome behavior would be sterile for biology if one does not 498 Devaux keep in mind the numerous specificities of biological In a biological membrane, local deformations can be membranes. Not only the lipid composition of all eukar- achieved by applying external forces for example by the yotic membranes is complicated, i.e., involves mixtures of contraction of cytoskeleton proteins. In a liposome a many different lipids, the proportion of which seems to be protrusion forming a long tether can be formed by sucking strictly regulated, but in addition the plasma membrane of the membrane with a micropipette or by pulling it with eukaryotic cells has an asymmetrical transmembrane dis- optical tweezers. Alternatively, the shape can be changed tribution of phospholipids which is difficult, if not impos- in a more subtle manner by progressive modification of sible, to reproduce with model membrane [8]. Another the ratio between inner and outer areas: Ai and Ao. For important characteristic of in vivo vesicularization is the example, if the two opposing monolayers react differently actual size of the vesicles involved in membrane traffic. to their environment or are selectively modified by addi- Sufficient attention has not been paid to this point in tion or depletion of lipids, they eventually have different ∆ 7 previous investigations on shape change of liposomes by areas ( A=Ai-Ao 0), then within the framework of the physicists attempting to mimic endo- and/or exo-cytosis in so-called bilayer couple hypothesis [16], the membrane giant vesicles. bends. The major issue that I will address in this article is the As indicated originally by Helfrich, the bending energy following: is membrane bending and unfolding during of a liposome can be written in the following way [17]: endo-exocytosis caused by the transmembrane redistribution of lipids by the aminophospholipid translocase which kc 2 E = ͐ ͑ C + C − 2C ͒ dA c 1 2 0 is a ubiquitous lipid transporter present in the plasma 2 A membrane of eukaryotic cells? I shall first try to shed some light on physical constraints which cannot be Here, the Gaussian curvature term is omitted. As long as ignored when trying to understand how membranes in- vesicle fission (or fusion) has not taken place, we deal vaginate. In view of our knowledge on lipid-protein with vesicles of spherical topology and the Gaussian interactions this will bring up some suggestions on pos- curvature term can be omitted. kc has the dimension of an sible mechanisms involved at the early stage of endocy- energy and is of the order of a few times the thermal tosis and the late stage of exocytosis, in particular the energy (kBT). The integral extends over the whole area A putative role of ‘flippases’ and ‘scramblases’. The possible of the lipid vesicle. C1 and C2 are the local curvatures (see involvement of the aminophospholipid translocase was figure 1). In practice for a closed vesicle the two leaflets presented several years ago by myself as a working have a slightly different area. Under the condition that the hypothesis [9, 10]. Recent studies have given experimen- two leaflets are everywhere separated by the same spacing ∆ tal support to this idea [11–14]. h which is of the order of 6 nm, A is related to the local curvature by the following relation which is correct to order h/R [18]: 2. Membrane budding DA = h ͐ dA͑ C + C ͒ 1 2 Let us consider first a protein-free giant unilamellar vesicle containing one or several types of phospholipids. C0 is the spontaneous curvatjure that can be defined as the Unlike a soap bubble, the surface tension of a liposome in curvature that the membrane would take in a relaxed state, the absence of osmotic pressure, is very low and the i.e., not constrained by the necessity to form a closed membrane undergoes visible surface undulations. How- liposome in order to avoid exposing the hydrophobic lipid ever, it would be an erroneous conclusion to infer that a 7 chains to water. C0 0 can be due to an asymmetry in the bilayer is easy to deform. The budding of a synaptic-like lipid composition or lipid environment or simply to a microvesicle out of the surface of a giant liposome difference in the number of lipids present at each interface. imposes locally a high curvature which for a bilayer must In Helfrich’s formulation C0 includes both the local and be associated with a difference in the area of the inner and non-local tendency to bend. A more explicit formalism outer monolayers. If lipids were free to diffuse from one was proposed recently in the laboratory of Wortis [18]. In side of a membrane to the other, invaginations and this model, the elastic energy has two separate terms, budding would happen as a manifestation of thermal hence the difference in area of the two leaflets is ac- fluctuations as it happens with surfactant films. But in a counted for by a term corresponding to a new elastic lipid bilayer, in general it costs a lot of energy for a stretching energy: phospholipid to traverse the membrane: t1/2 of spontaneous phosphatydylcholine flip-flop is of the order of several k E = c ͑ C + C − 2C ͒2 dA + j͑ p/2 Ah2 ͒͑DA − DA ͒2 hours or even days at physiological temperatures [15]. c 2 ͐ 1 2 0 o Furthermore, the surface compressibility of a lipid mono- A ∆ ∆ layer is low, so thermal fluctuations only give rise to A is the actual difference in area, whereas Ao is the surface undulations. difference in area that would exist in a relaxed state. j is Is lipid translocation involved during endo- and exocytosis? 499

qualitative approach of the present review, I shall continue to use Helfrich’s formulation throughout, and consider that C0 reflects either local spontaneous curvature or the area difference term. For a rigorous and more quantitative development of this section the separation between local and non-local spontaneous curvature would be necessary. If a liposome is formed by gentle swelling of a lipid film in water, in general both leaflets are identical in ≈ composition and density of lipids, hence: C0 0. How- ever, the necessity to close the bilayer in order to avoid exposing hydrophobic surfaces forces the bilayer to bend ≈ and the total energy is minimize for C1 and C2 0, i.e., for an average vesicle radius, < R >, very large. In practice, giant vesicles are formed with a radius of several microns, that is exceeding by far the thickness of the bilayer and corresponding to a low average curvature. Theoreticians have predicted shapes of liposomes for different values of C0 by minimization of Helfrich’s formula or of formula derived from this formula [18–21]. Phase diagrams of vesicle shapes were drawn from these computations and typical shapes corresponding to the budding of a small vesicle from a ‘mother vesicle’ of larger size can be recognized. Figure 2 shows some

Figure 1. Principal radii of curvature of a bilayer of thickness h. Locally any continuous surface can be approximated by an hyperboloid (or a paraboloid) which can be written: 2 2 Z =−1 ͩ x + y ͪ. z is the direction of the normal to the 2 R1 R2 surface; the axes x and y are chosen so as to obtain the symmetrical quadratic form of the surface equation. This defines = 1 = 1 the two principal axes. By definition C1 and C2 are R1 R2 the radii of curvature: they correspond to local parameters. See the text for the definition of the spontaneous curvature C0. a new elastic modulus having also the dimension of an energy. Then C0, the spontaneous curvature, reflects solely the local curvature constraints due for example to local binding of proteins or ions or to the chemical nature of the Figure 2. Schematic ‘phase diagram’ of vesicle shapes. This ‘phase’ diagram shows the shapes of lowest bending energy for phospholipids which are usually asymmetrically distri- ∆ given reduced volume v and equilibrium differential area ao. buted in a biological membrane [8, 10]. In principle, the ∆ κ ao in this diagram is a dimension-less or scaled area difference elastic modulus is different from kc. However, it has the ∆ which is effectively proportional to Ao defined in the text. With same order of magnitude. For example, in the case of the exception of the shapes indicated with a dashed contour, the homogeneous phosphatidylcholine vesicles Miao et al. shapes calculated have an axis of rotational symmetry along the α ≈ [18] have reported that the ratio = j/kc 1. Thus, for the vertical (reproduced from [32]). 500 Devaux predicted shapes. Such shape changes in giant vesicles were observed by several groups who found ways to manipulate ∆A. The contraction or expansion of one of the two leaflets of a liposome or of a biological membrane, which I shall call a C0 modification, can be achieved by chemical modifications in situ resulting, for example, from phospholipase or sphingomyelinase degradation [7, 22–24]. Phosphorylation of phosphonositides on the cytosolic interface of an erythrocyte has been reported to be involved in shape changes in the case of red cells [25]. C0 modification can be triggered by ions and/or proteins interacting with specific lipids of one of the leaflets, causing the formation of rigid domains or ‘rafts’ with a condensed area and therefore leading to asymmetrical membranes [6]. Alternatively, C0 can be modified by the insertion or depletion of lipids from one leaflet. A non-physiological way to enrich the lipid composition of one bilayer leaflet Figure 3. Shape transformation of a unilamellar giant vesicle consist in adding amphiphilic molecules such as lyso- containing egg lecithin and egg phosphatidylglycerol with a phosphatidylcholine (L-PC) to pre-formed giant lipo- molar ratio of 99:1. The shape change is triggered by raising the somes or to erythrocytes. L-PC molecules penetrate the external pH from 6 to 9. The time scale for total shape outer leaflet where they remain and expand selectively the transformation is approximately 10 s. The bar corresponds to external surface since the flip-flop rate of L-PC is ex- 10 µm. Vesicles are observed with an optical microscope with tremely slow even in phosphatidylcholine liposomes [26]. Nomarski configuration (reproduced from [28]). In vivo a net translocation of phospholipids can be achieved by the ‘phospholipid flippases’ which are able to catalyze the translocation of phospholipids or to transport pretation being that even a very small differential thermal phospholipids at the expenses of ATP consumption. expansion of the two monolayers can induce a change of Modulation of the transmembrane asymmetry of certain C0 [30, 31]. lipids, such as phosphatidic acid or phosphatidylglycerol, can be achieved in liposomes if a transmembrane pH gradient is applied [27]. These various techniques which 3. The scaling effect enables one to modulate the spontaneous curvature of a membrane by the formation of asymmetrical bilayers How much asymmetrical has the membrane to be in induce membrane invaginations. order to induce a detectable shape change or more Addition of less than 1% of lipids to the external leaflet precisely to generate budding of a small lipid vesicle out suffices to modify a GUV with a discoid or obloid shape of a larger one that I shall call the ‘mother vesicle’?In into a eight-shape vesicle formed by a small spherical other words, if Co modification is caused by a transfer of vesicle connected to a larger one; similarly a very small lipids, what proportion of lipids has to be transported from lipid transfer of lipids from the inner to the outer leaflet one leaflet to the other to obtain vesicularization? triggers the formation of a budded vesicle with a typical The result from quantitative experiments with GUVs, diameter of the order of a few µm (see figure 3). In some discussed in the above paragraphs, is rather astonishing: instances the small vesicle is separated from the larger 0.1% of lipids in excess on the external leaflet suffice to vesicle by a thin tether difficult to detect unless fluorescent modify a GUV with a typical size of 20–50 µm. Theore- lipids have been incorporated [28]. But in general, the ticians had even predicted that 0.01% asymmetry would single budded vesicle does not shed off. If more than 1% be sufficient [20]. Simple geometrical arguments allow of L-PC is added, the overall shape of the GUV can take one to predict a scaling effect: namely the fraction of more complicated forms with several connected spheres lipids that has to be reoriented or added selectively (figure 4) . If the external leaflet of a liposome is depleted depends on the vesicle size. The scaling parameter is of a fraction of its lipids, for example by removing L-PC essentially the ratio h/Ro where h is the thickness of the from the outer leaflet with bovine serum albumin, a single membrane and Ro is the radius of the vesicle. Obviously, invagination is formed which resembles membrane in- if the thickness h is close to the radius of the vesicle Ro, vaginations during endocytosis [29]. In Sackmann’s labo- then membrane folding is difficult. If N is the average ratory the same sequences of shape change were observed number of lipids per monolayer, the asymmetry in lipid with GUVs when the temperature was varied, the inter- distribution between both leaflets (δ N/N) must be of the Is lipid translocation involved during endo- and exocytosis? 501

and if one neglects temporarily the lateral compressibility, pure geometrical considerations show that the excess of lipids on the outer layer of a budded vesicle must be between 5 and 10%, meaning that a proportion of phospholipids of the order of 5% may have to be translocated. In the latter case, a significant surface tension is generated by the mismatch between both layers and the process requires more energy than for giant vesicles. Indeed, surface asymmetry not only leads to bending but also to surface tension T. By definition: T = Ka, where K is the lateral compressibility coefficient and α the surface strain which is the relative area variation of one leaflet due to lateral compressibility. In the initial state, the surface tension of both leaflets is practically zero. If one adds new molecules to the outer monolayer, because of the coupling with the inner monolayer which resists to this expansion, a surface tension is generated. Eventually a torque is exerted on the membrane. But it is important to note that bending does not relax completely the tension. Because of the conditions of closure (fixed volume), the membrane cannot bend so as to completely relax the tension. In fact the curvature adopted eventually by the membrane equilibrates the difference in surface constraints between the two leaflets, so that the torque is canceled but not the surface tension. In practice, the addition of 0.1% lipids on the external leaflet of a GUV generates a negligible surface tension. However, an asymmetry of 5–10% between the two leaflets of a GUV or of a LUV generates a significant surface tension. One manifestation of the tension is the inhibition of surface undulations which are partially or totally inhibited by addition of lyso-PC to their external surface of vesicles [29]. Recently the symmetrical surface tension generated in liposomes by the asymmetrical addition of L-PC was demonstrated by Traïkia using NMR 31P-chemical shift as a way to detect the molecular packing at the level of the phospholipid head groups of each monolayer in an experiment involving magic angle spinning of lipid vesicles [26]. The surface tension which is equivalent to an increased lipid packing, manifests itself also by an increased resistance to transmembrane lipid diffusion. Thus, while the formation of a 5 µm vesicle budding out of a 50 µm giant unilamellar vesicle can be Figure 4. Shape transformation of an egg phosphatidylcholine achieved in a few minutes at room temperature when the unilamellar vesicle containing fluorescent dextran (0.1 mM) membrane (containing a PC/PG mixture) is submitted to a after L-PC addition in proximity of the vesicle with a micropi- pH gradient [29], the same overall shape with 100 nm pette. A. t=0.B. t=4min.C. t = 7 min. The bar corresponds LUVs required 60 mn of incubation at 60 °C [32]. The to 10 µm (reproduced from [28]). latter observation reported by the Cullis’group is in accordance with previously published theoretical predic- tions [33, 34]. order of h/Ro for ‘vesicularization’. In the case of a GUV Finally, I would like to emphasize as a conclusion of with a typical diameter of about 50 µm, the experiments this section that the formation of one or several mi- show that the ‘small’ vesicles which are generated by lipid crovesicles with a diameter of about 20 nm is always asymmetry have a size of 5–10 µm. Since the bilayer accompanied by a surface tension. Because of the conti- thickness is around 6 nm, a very small asymmetry, below nuity between a budded vesicle and the rest of the 1%, is sufficient in principle. In the case of a 200 nm LUV liposome this tension must be the same in the whole 502 Devaux vesicle membrane, even in a GUV. In other words, and eventually the surface of the sphere may become although local fluctuations of the surface density in a GUV covered by microvesicles. could give birth to a microvesicle of the size encountered Figure 5 shows an experiment where a large number of in vivo during endocytosis or vesicle shedding, this relatively small vesicles appear at the external surface of vesicle would promptly disappear because of the instabil- a giant vesicle. This figure was obtained with multilamel- ity of a local surface tension. Thus a stable budded vesicle lar vesicles (MLVs) on the surface of which a large excess connected to the rest of the plasma membrane is only of L-PC was added. The inner membranes probably possible if the whole plasma membrane is constrained. prevent the liposome from an overall deformation or collapse. The large excess of lipids on the external leaflet of the external membrane can only form series of small 4. Is microvesicle formation in liposomes and red vesicles which eventually shed off, thereby pealing the cells relevant to endocytosis? external bilayer of this multilamellar vesicle. But even in this example the size of the small vesicles (≈ 1 µm) was Typical diameters of the vesicles involved in much larger than the size of endo-exo-cytic vesicles. The endocytosis-exocytosis, and more generally vesicle traffic above example is reminiscent of the situation obtained within eukaryotic cells, are around 200 nm. They result with red blood cells to which a large excess of L-PC has from local membrane curvature, either of the plasma been added. In the latter case, very small membrane membrane itself or of organelles such as the ER or the protrusions cover the cell surface which seems to shrink Golgi system, the typical size of which is in the range of and form a uniform sphere (spherocyte) if viewed with an one or several microns. Although lipid vesicle experi- optical microscope. In reality, the erythrocyte surface is ments demonstrate a close relationship between vesicle covered by small vesicles with a radius below resolution shape and lipid transmembrane distribution and although of the optical microscope. The cell resists because of the they show that endocytic-like invaginations may be trig- cytoskeleton and eventually lyses if more L-PC is added gered in GUVs by the redistribution of a small proportion (> 5%). of lipids, they do not satisfactorily mimic endocytosis. Figure 6a-f shows in an (over)simplified way the shape Indeed, endocytosis implies the formation of one or transformations that can undergo an obloid GUV several microvesicles from the invaginations of a cell (figure 6a), by adding to the external surface molecules membrane which has a typical radius of several µm. This such as L-PC [28, 29] or by increasing area to volume is not what is observed in giant unilamellar vesicles when ratio by heating [30, 31] Shapes in figure 6b, c are the for example 1% of L-PC is added externally. We have canonical axial symmetrical shapes calculated by theore- mentioned above that the formation of very small spheri- ticians (see figure 2). The radius R of the spherical cal vesicles requires a lipid asymmetry of the order of ‘budded’ vesicle(s) is large. Such transformation does not 5–10% and because these vesicles (or invaginations) resemble in vivo budding. Shapes in figure 6d, e corre- remain at least temporarily connected to the rest of the spond to the formation of relatively small vesicles. They membrane, the same lipid asymmetry must exist all along require first a transformation of the ‘mother vesicle’ into a the cell surface and will cause an important surface sphere. Again, overall this is not what is seen with real tension. The so-called large unilamellar vesicles (LUVs) cells. In the case of figure 6f, which represents a multila- with a diameter of 100–200 nm can sustain surface tension mellar vesicle (MLV), the obloid shape is maintained by without collapse and form budded vesicles or protrusions the inner bilayers. In vivo the cell shape is maintained by with a diameter of about 50 nm or less. On the other hand, the cytoskeleton and by the presence of organelles. a giant vesicle of the size of an eukaryotic cell will not I infer that the activity of a lipid pump transporting form at its surface a series of small vesicles with a lipids from the outer monolayer to the inner monolayer (or diameter which is ≈ 10–3 times the average diameter of a selective fraction of the outer leaflet lipids) can induce this ‘mother vesicle’ without a complete shape change invaginations of the plasma membrane and form en- unless some scaffolding, analogous to the cytsoskeleton docytic vesicles of the size observed in vivo, provided the meshwork in a biological cell prevents the transformation cell surface is reinforced by a cytoskeleton. This cyto- of this large vesicle. Figure 4 shows that if ∆Ais skeleton and tubulin meshwork is represented schemati- increased by the addition of a large amount of L-PC into cally in figure 6g,h the external leaflet of a GUV, the shape undergoes a dramatic change that does not correspond to a mere ‘decoration’ of a large obloid vesicle by a series of 5. Experimental observations in favor of the role of microvesicles. Of course, if the giant vesicle is initially a lipid translocation during endocytosis perfect sphere and if the internal volume remains approximately constant, there is no possibility of any other shape. The aminophospholipid translocase is present in the In the latter case, the additional surface on the outer layer plasma membrane of all animal cells [10, 35–37]; recent will generate a tension that will inhibit surface undulations results suggest that it also exists in the plasma membrane Is lipid translocation involved during endo- and exocytosis? 503

Figure 5. Shape transformation observed by phase contrast microscopy of a multilamellar egg phosphatidylcholine vesicle a after addition of L-PC with a micropipette. A. t=0.B. t=8s.C. t=30s.D. t=1min.E. 2 min. The bar corresponds to 20 µm. Note that the scale is different in E (reproduced from [29]). 504 Devaux

Figure 6. Schematic representation of examples of shape transformations of on obloid giant vesicle when a difference in area ∆A between inner and outer leaflet is imposed. When the external area is increased by 5–10% very small microvesicles will bud at the surface of the ‘mother vesicle’ only if the latter has a spherical shape (d, e) or if the overall shape of the ‘mother vesicle’ is protected by inner lipid vesicles (f)orbya cytoskeleton meshwork (g, h). h. The external surface is depleted. See text for more comments. of plant cells [38]. This protein transports phosphati- aminophospholipids and in turn to modify erythrocyte dylserine (PS) and to a lesser extent phosphatidylethanol- shapes by shifting a fraction of the lipids from the inner to amine (PE) from the outer to the inner leaflet where these the outer leaflet or conversely. Indeed, unpaired lipid aminophospholipids are predominantly located. Cytosolic transport is accompanied by echinocyte formation while ATP is necessary and must be hydrolyzed for this transport artificial ATP enrichment leads to stomatocytes. When the to take place. By controlling the level of cytosolic ATP, level of ATP is of the order of 5–6 mM endocytic vesicles which in vivo is normally of the order of 2–3 mM, it is form at the cytosolic interface of erythrocytes. They are possible to modify the equilibrium distribution of the characterized by the formation of a few relatively large Is lipid translocation involved during endo- and exocytosis? 505 vesicles and can be quantified by monitoring the acetyl- becoming non-accessible. If phosphatidylcholine (PC) is choline esterase activity which normally takes place on added to the outer surface of red cells this vesicularization the outer surface of red cells [12, 39]. The decrease in process is affected because the plasma membrane would esterase activity is an indication of the amount of surface rather tend to fold in the opposite direction (see figure 7

Figure 7. Modulation of endocytosis activity in resealed erythrocyte ghosts as determined by the acetycholinesterase activity. The reduction of the esterase activity which is normally exposed on the external surface of the plasma membrane is an indication of the formation of endocytic vesicles. A. Effect of ATP concentration in the resealed ghosts. ATP stimulates the aminophospholipid translocase. B. Inhibition of endocytosis which normally takes place with 5 mM ATP by addition of phosphatidylcholine to the outer leaflet. C. Inhibition followed by stimulation after addition of phosphatidylserine. This lipid is initially incorporated in the outer leaflet and therefore slows down the inward folding of the plasma membrane. The translocation to towards the inner leaflet by the aminophospholipid translocase reverses the situation and favors membrane invagination. The exogenous lipids added have a short chain which permits their rapid incorporation (from [12]). 506 Devaux and [12]). Actually, the difference in the shape change scenarios which are triggered by the addition of various phospholipids to the external surface of erythrocytes were noticed already in 1984 by Seigneuret and Devaux [40] and used later by Daleke and Huestis to monitor the activity of the aminophospholipid translocase [41]. Erythrocytes cannot be considered as the best system to study endocytosis since they do not naturally endocytose. It is interesting to point out that concomitantly the aminophospholipid translocase activity (or ‘flippase’ activity) is rather weak in erythrocytes compared to its activity in cells having a high endocytic activity. Cribier and collaborators have compared the translocase activity in some of the cells of the red cell lineage: erythroblasts, reticulocytes and erythrocytes and found that the highest activity was in the cells provided with the highest endocytic activity, namely in K562 cells which are trans- formed cells derived from human erythroblasts [11]. Similarly, Pomorski et al. [42] have compared the translocase activity in human erythrocytes and in human fibroblasts. It was found that the initial rate of PS translocation was two orders of magnitude larger in fibroblasts than in erythrocytes. In 1995, Farge has mea- sured endocytosis in K562 cells by measuring the uptake of spin-labeled PC added externally and which can only be internalized by endocytosis [13]. He has found that the concomitant addition of PS stimulates PC internalization which suggests an enhanced endocytosis activity. This can be explained by the fact that PS is a substrate of the translocase and that increasing PS should allow more rapid turnover. The interpretation of Farge’s experiments raised some questions because of the instability of the short chain bearing the nitroxide: these slightly water- soluble lipids were found to be good substrates for the endogenous phospholipase A2 present in such cells as the K562 cells. Nevertheless, the increase in initial rate of PC uptake is probably associated with an increased endocy- Figure 8. Enhancement of bulk protein endocytosis in K562 tosis activity. In 1999, a different technique was used to cells after addition to the external surface of phosphatidylserine (PS) with a short chain (C5). Bulk membrane proteins were measure endocytosis in K562 cells [14]. The internaliza- biotinylated and coupled with FITC streptavidin. The time tion of fluoresceinated membrane proteins was either course of internalization of the labeled proteins was monitored watched directly or monitored by the decrease of fluores- by following the fluorescence decrease of fluorescein which is cence which is quenched by the acidity of early endocytic quenched in the acidic environment of early endocytic vesicles. vesicles. It was found that the addition of 4% PS on the A. Protein internalization is observed as a function of percentage external side of the plasma membrane resulted in a of PS added. B. Initial bulk flow rate of endocytosis as a function five-fold increase in fluorescence quenching in 10 min at of the percentage of PS added to the estimate plasma membrane 37 °C. Similarly, PE addition stimulated endocytosis but phospholipids (reproduced from [14]). lyso-PS, which is not transported by the aminophospholipid translocase [10], inhibited the residual endocytosis (figure 8) . lipid transporter. However, it is surprising that these cells In a recent investigation aimed at trying to find the seem to have normal lipid asymmetry. aminophospholipid translocase protein, Marx et al. [43] Actually, the doubt concerning the proper identification tested the translocation of PS in endocytosis-deficient of the aminophospholipid translocase is the most severe yeast cells and found very little specific transport of the limitation in any experimental proof of its direct implica- aminophospholipid analogues and essentially no ATP tion in endocytosis. Although the sequence of an ATPase dependence. These results could suggest that the cells with a molecular mass of 170 kDa has been published by lacking endocytosis activity have also no aminophospho- Tang et al. [44] and identified as the aminophospholipid Is lipid translocation involved during endo- and exocytosis? 507

Figure 9. Schematic model show- ing the translocation of phospholipids from the outer to the inner leaflet during endocytosis and conversely from the cytosolic to the external leaflet during exocytosis. This lipid traffic could be caused (or facilitated) by the ATP- dependent aminophospholipid translocase and the calcium dependent scramblase respectively. Note that even if these proteins were not involved, i.e., were not the driving forces; this translocation of lipids has to take place because of the small size of endocytic vesicles. translocase, there is still a controversy in the literature [43, outer leaflet some aminophospholipids (PS and PE), it 45]. One will probably have to wait until this protein can should facilitate later the formation of inward invagina- be expressed. Recently, a preliminary report was given tions (i.e., endocytosis) by the aminophospholipid trans- about a successful complementation of an aminophospho- locase. In this regard, it is quite interesting to note that lipid translocase activity in translocase deficient yeast most cells do have in their plasma membrane a protein cells (DRS2) using RNA obtained from plants [38]. called lipid ‘scramblase’ which is activated by cytosolic calcium. This scramblase plays an important role in specialized cells such as platelets which expose PS on 6. Exocytosis and lipid scramblase their outer leaflet when they are stimulated, but it is also functional in all cells of the blood circulation during The same geometrical argument that led us to conclude apoptosis [46]. that the formation of microvesicles by bending of the In principle this ‘scramblase’ has been purified and plasma membrane requires the translocation of lipids from cloned [47]. There is also a rare and very severe disease the outer to the inner leaflet, forces us to conclude that called Scott syndrome which was thought to correspond to fusion of small vesicles to the plasma membrane, i.e., a lack of scramblase; at least it is associated with an exocytosis, requires the opposing transfer of lipids (see impairment of calcium triggered lipid redistribution in figure 9). A priori the two processes (endocytosis and platelets. However the reconstituted vesicles with the exocytosis) are not exactly symmetrical. Indeed a single ‘purified’ scramblase have a very low scrambling effi- small vesicle, with a high surface tension and an asym- ciency [48] and this protein is present in the blood cells of metrical repartition of phospholipids can fuse with the the Scott patients [49]. So the identification of the protein plasma membrane without perturbing it because the latter may yet have to be confirmed. can be considered as a sink of infinite size. The small perturbation brought by fusion of one vesicle is a local perturbation that will eventually be canceled out by the 7. Discussion and conclusions slow spontaneous flip-flop of lipids. However, in a living cell exocytosis is a continuous process that permits the It has been demonstrated in giant unilamellar vesicles whole plasma membrane to be renewed within a short that a small area increase of one of the two opposing period, and fusion of synaptic vesicles or granules is a monolayers triggers budding or invaginations. In various synchronized event which concerns a large number of eukaryotic cells, a positive correlation between amino- vesicles. It is therefore apparent that independently from phospholipid translocase activity and the endocytic activ- the process that enables the two bilayers to come in close ity was demonstrated. There is also strong evidence that contact and to merge into a single bilayer, it would be the stimulation of phospholipid translocation from the useful for the efficiency of this process that some mecha- outer to the inner monolayer of the plasma membrane in nism accelerates lipid flip-flop in order to facilitate the K562 cells accelerates endocytosis while the addition of randomization of the transmembrane lipid distribution. phospholipids that are not transported has an inhibitory Additionally, if this lipid scrambling process brings to the effect. Therefore, one can infer that the active transport of 508 Devaux lipids may serve as a driving force for membrane folding gered pump transporting an excess of lipids outwardly, or during endocytosis. It is an ATP driven process which acts to the activity of cytoskeleton proteins, is a question not on the whole membrane by generating a non-localized settled yet [52]. surface tension; this tension is at least partly relaxed by In the framework of this model, clathrin binding could the formation of membrane budding and invaginations. be necessary to determine which part of the membrane The cytoskeleton is necessary to prevent whole cell will invaginate rather than clathrin binding being the deformation but organelles which are present in eukaryo- driving force. On the other hand the coat proteins identi- tic cells may help also in a way comparable to inner fied as proteins involved in budding of the Golgi system vesicles in MLVs (figures 5, 6). The localization of these [1] may have a similar effect than the addition of L-PC invaginations has to be random in a large unilamellar added externally to a GUV. Because there is now compel- vesicle which has an homogeneous surface, but in a ling evidence that lipid flip-flop in inner membranes is biological membrane which contains heterogeneous do- much more rapid than in the plasma membrane which is mains, it is quite conceivable that rigid domains or rich in cholesterol [53], it is unlikely than the same localized region with a non-zero spontaneous curvature mechanism could be used in the ER or Golgi membranes imposed by the binding of peripheral proteins such as to generate microvesicles. Of course several mechanisms clathrin, or associated with the clustering of caveolin, of budding may exist within a cell. serve as a point of emergence. Note the model of As a final conclusion, the models proposed here have domain-induced budding of fluid membranes proposed by many implications that can be verified. Reconstitution in Lipowsky is based on a different physical property, giant vesicles of the purified proteins involved in lipid namely the competition between the minimization of the traffic appears to be the most significant type of experi- bending energy and the energy of the line tension between ments to perform in the future to test the above hypothesis. two domains [50]. The applicability of the latter model to biological membranes is still very speculative. The scramblase hypothesis which suggests a role of the Acknowledgments calcium triggered scramblase during exocytosis is more speculative at this stage than the hypothesis about the role The author thank Drs. E. Farge, H.G. Döbereiner and S. Cribier of the aminophospholipid translocase during endocytosis. for helpful discussions. Work supported by grants from the It clearly needs experimental confirmation. 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