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Is Lipid Translocation Involved During Endo- and Exocytosis?

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Is Lipid Translocation Involved During Endo- and Exocytosis? Biochimie 82 (2000) 497−509 © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400002091/FLA 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 phosphati- dylethanolamine 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 lipo- some 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 redistribu- tion 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 spontane- ous 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].
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