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 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
During the last 10–15 years, a large number of proteins
have been recognized as playing central roles during
endo-exocytosis phenomena and more generally during
vesicle traffic: clathrin, NSF, ARF, dynamin, caveolin,
annexins and GTP-binding proteins, have been implicated
in the processes of membrane vesicularization (also called
budding) or during fission, targeting and fusion of
vesicles. A few reconstitution experiments with a limited
number of proteins have reproduced, if not quantitatively,
at least qualitatively some of the important steps [1, 2] and
from these observations, generalized models were proposed. However, the main emphasis of earlier research has
been to show which proteins are required, rather than to
dissect the molecular mechanisms involved. Membrane
folding which is a prerequisite for vesicle formation, is
often assumed to be explained by the binding of clathrin
or of coat proteins. Yet, endocytosis can exist without
clathrin or without clathrin polymerization [3, 4]. Furthermore, increasing the amount of polymerized clathrin does
not systematically increase the number of coated pits at
the plasma membrane [5]. In fact, it has not been proven
* Correspondence and reprints: [email protected]
Abbreviations: PC, phosphatidylcholine; PS, phosphatidylserine;
PE, phosphatidylethanolamine; L-PC, lyso-phosphatidylcholine;
PG, phosphatidylglycerol; GUV, giant unilamellar vesicle; LUV,
large unilamellar vesicle; SUV, sonicated unilamellar vesicle;
NMR, nuclear magnetic resonance; MDR, multi drug resistance
protein
yet whether clathrin polymerizes and then pinches off the
membrane to form the buds or if polymerization takes
place around a pre-formed bud. An alternative explanation
proposed for membrane budding during endocytosis is the
formation of local invaginations named caveola formed by
self association of the hydrophobic protein caveolin with
specific lipids [6]. To what extent is ATP necessary for
budding is still debated [7]. In summary, the physical
origin of the local membrane curvature is still unexplained
in spite of the correlation between the presence of specific
proteins and the formation of coated pits. Concerning
exocytosis, it is believed that the translocation of lipids
which a priori is necessary after the fusion of microvesicles to the relatively flat plasma membrane can take
place spontaneously, yet it is known that lipid flip-flop
requires several hours which is quite incompatible with
the time scale of a continuous endocytotic process.
In general, the role of lipids has been largely overlooked in these cell biology investigations. Only recently,
in experiments with reconstituted vesicles to which coat
proteins were attached, has the importance of lipids been
realized. In fact, even pure liposomes enable one to mimic
some fundamental steps of biological membrane traffic.
To those who believe that the representation of a biological membrane by a mere lipid bilayer is irrelevant, it
should be recalled that membrane budding, fission and
fusion refer primarily to the status of the lipid bilayer.
Thus, a detailed analysis of the physical constraints
associated with folding and fusion of a pure lipid bilayer
is of primary importance. Of course, investigating liposome behavior would be sterile for biology if one does not
498
keep in mind the numerous specificities of biological
membranes. Not only the lipid composition of all eukaryotic membranes is complicated, i.e., involves mixtures of
many different lipids, the proportion of which seems to be
strictly regulated, but in addition the plasma membrane of
eukaryotic cells has an asymmetrical transmembrane distribution of phospholipids which is difficult, if not impossible, to reproduce with model membrane [8]. Another
important characteristic of in vivo vesicularization is the
actual size of the vesicles involved in membrane traffic.
Sufficient attention has not been paid to this point in
previous investigations on shape change of liposomes by
physicists attempting to mimic endo- and/or exo-cytosis in
giant vesicles.
The major issue that I will address in this article is the
following: is membrane bending and unfolding during
endo-exocytosis caused by the transmembrane redistribution of lipids by the aminophospholipid translocase which
is a ubiquitous lipid transporter present in the plasma
membrane of eukaryotic cells? I shall first try to shed
some light on physical constraints which cannot be
ignored when trying to understand how membranes invaginate. In view of our knowledge on lipid-protein
interactions this will bring up some suggestions on possible mechanisms involved at the early stage of endocytosis and the late stage of exocytosis, in particular the
putative role of ‘flippases’ and ‘scramblases’. The possible
involvement of the aminophospholipid translocase was
presented several years ago by myself as a working
hypothesis [9, 10]. Recent studies have given experimental support to this idea [11–14].
Devaux
In a biological membrane, local deformations can be
achieved by applying external forces for example by the
contraction of cytoskeleton proteins. In a liposome a
protrusion forming a long tether can be formed by sucking
the membrane with a micropipette or by pulling it with
optical tweezers. Alternatively, the shape can be changed
in a more subtle manner by progressive modification of
the ratio between inner and outer areas: Ai and Ao. For
example, if the two opposing monolayers react differently
to their environment or are selectively modified by addition or depletion of lipids, they eventually have different
areas (∆A = Ai-Ao 7 0), then within the framework of the
so-called bilayer couple hypothesis [16], the membrane
bends.
As indicated originally by Helfrich, the bending energy
of a liposome can be written in the following way [17]:
Ec =
兰
共 C1 + C2 − 2 C0 兲 dA
2
A
Here, the Gaussian curvature term is omitted. As long as
vesicle fission (or fusion) has not taken place, we deal
with vesicles of spherical topology and the Gaussian
curvature term can be omitted. kc has the dimension of an
energy and is of the order of a few times the thermal
energy (kBT). The integral extends over the whole area A
of the lipid vesicle. C1 and C2 are the local curvatures (see
figure 1). In practice for a closed vesicle the two leaflets
have a slightly different area. Under the condition that the
two leaflets are everywhere separated by the same spacing
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
Let us consider first a protein-free giant unilamellar
vesicle containing one or several types of phospholipids.
Unlike a soap bubble, the surface tension of a liposome in
the absence of osmotic pressure, is very low and the
membrane undergoes visible surface undulations. However, it would be an erroneous conclusion to infer that a
bilayer is easy to deform. The budding of a synaptic-like
microvesicle out of the surface of a giant liposome
imposes locally a high curvature which for a bilayer must
be associated with a difference in the area of the inner and
outer monolayers. If lipids were free to diffuse from one
side of a membrane to the other, invaginations and
budding would happen as a manifestation of thermal
fluctuations as it happens with surfactant films. But in a
lipid bilayer, in general it costs a lot of energy for a
phospholipid to traverse the membrane: t1/2 of spontaneous phosphatydylcholine flip-flop is of the order of several
hours or even days at physiological temperatures [15].
Furthermore, the surface compressibility of a lipid monolayer is low, so thermal fluctuations only give rise to
surface undulations.
kc
2
DA = h
兰
dA共 C1 + C2 兲
C0 is the spontaneous curvatjure that can be defined as the
curvature that the membrane would take in a relaxed state,
i.e., not constrained by the necessity to form a closed
liposome in order to avoid exposing the hydrophobic lipid
chains to water. C0 7 0 can be due to an asymmetry in the
lipid composition or lipid environment or simply to a
difference in the number of lipids present at each interface.
In Helfrich’s formulation C0 includes both the local and
non-local tendency to bend. A more explicit formalism
was proposed recently in the laboratory of Wortis [18]. In
this model, the elastic energy has two separate terms,
hence the difference in area of the two leaflets is accounted for by a term corresponding to a new elastic
stretching energy:
Ec =
kc
2
兰
共 C1 + C2 − 2 C0 兲 dA + j共 p/2 Ah 兲 共 DA − DAo 兲
2
2
2
A
∆A is the actual difference in area, whereas ∆Ao is the
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. However, 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
the two principal axes. By definition C1 = 1 and C2 = 1 are
R1
R2
the radii of curvature: they correspond to local parameters. See
the text for the definition of the spontaneous curvature C0.
冉
冊
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
phospholipids which are usually asymmetrically distributed in a biological membrane [8, 10]. In principle, the
elastic modulus κ is different from kc. However, it has the
same order of magnitude. For example, in the case of
homogeneous phosphatidylcholine vesicles Miao et al.
[18] have reported that the ratio α = j/kc ≈ 1. Thus, for the
Figure 2. Schematic ‘phase diagram’ of vesicle shapes. This
‘phase’ diagram shows the shapes of lowest bending energy for
given reduced volume v and equilibrium differential area ∆ao.
∆ao in this diagram is a dimension-less or scaled area difference
which is effectively proportional to ∆Ao defined in the text. With
the exception of the shapes indicated with a dashed contour, the
shapes calculated have an axis of rotational symmetry along the
vertical (reproduced from [32]).
500
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
consist in adding amphiphilic molecules such as lysophosphatidylcholine (L-PC) to pre-formed giant liposomes or to erythrocytes. L-PC molecules penetrate the
outer leaflet where they remain and expand selectively the
external surface since the flip-flop rate of L-PC is extremely slow even in phosphatidylcholine liposomes [26].
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
phospholipids at the expenses of ATP consumption.
Modulation of the transmembrane asymmetry of certain
lipids, such as phosphatidic acid or phosphatidylglycerol,
can be achieved in liposomes if a transmembrane pH
gradient is applied [27]. These various techniques which
enables one to modulate the spontaneous curvature of a
membrane by the formation of asymmetrical bilayers
induce membrane invaginations.
Addition of less than 1% of lipids to the external leaflet
suffices to modify a GUV with a discoid or obloid shape
into a eight-shape vesicle formed by a small spherical
vesicle connected to a larger one; similarly a very small
lipid transfer of lipids from the inner to the outer leaflet
triggers the formation of a budded vesicle with a typical
diameter of the order of a few µm (see figure 3). In some
instances the small vesicle is separated from the larger
vesicle by a thin tether difficult to detect unless fluorescent
lipids have been incorporated [28]. But in general, the
single budded vesicle does not shed off. If more than 1%
of L-PC is added, the overall shape of the GUV can take
more complicated forms with several connected spheres
(figure 4) . If the external leaflet of a liposome is depleted
of a fraction of its lipids, for example by removing L-PC
from the outer leaflet with bovine serum albumin, a single
invagination is formed which resembles membrane invaginations during endocytosis [29]. In Sackmann’s laboratory the same sequences of shape change were observed
with GUVs when the temperature was varied, the inter-
Devaux
Figure 3. Shape transformation of a unilamellar giant vesicle
containing egg lecithin and egg phosphatidylglycerol with a
molar ratio of 99:1. The shape change is triggered by raising the
external pH from 6 to 9. The time scale for total shape
transformation is approximately 10 s. The bar corresponds to
10 µm. Vesicles are observed with an optical microscope with
Nomarski configuration (reproduced from [28]).
pretation being that even a very small differential thermal
expansion of the two monolayers can induce a change of
C0 [30, 31].
3. The scaling effect
How much asymmetrical has the membrane to be in
order to induce a detectable shape change or more
precisely to generate budding of a small lipid vesicle out
of a larger one that I shall call the ‘mother vesicle’? In
other words, if Co modification is caused by a transfer of
lipids, what proportion of lipids has to be transported from
one leaflet to the other to obtain vesicularization?
The result from quantitative experiments with GUVs,
discussed in the above paragraphs, is rather astonishing:
0.1% of lipids in excess on the external leaflet suffice to
modify a GUV with a typical size of 20–50 µm. Theoreticians had even predicted that 0.01% asymmetry would
be sufficient [20]. Simple geometrical arguments allow
one to predict a scaling effect: namely the fraction of
lipids that has to be reoriented or added selectively
depends on the vesicle size. The scaling parameter is
essentially the ratio h/Ro where h is the thickness of the
membrane and Ro is the radius of the vesicle. Obviously,
if the thickness h is close to the radius of the vesicle Ro,
then membrane folding is difficult. If N is the average
number of lipids per monolayer, the asymmetry in lipid
distribution between both leaflets (δ N/N) must be of the
Is lipid translocation involved during endo- and exocytosis?
Figure 4. Shape transformation of an egg phosphatidylcholine
unilamellar vesicle containing fluorescent dextran (0.1 mM)
after L-PC addition in proximity of the vesicle with a micropipette. A. t = 0. B. t = 4 min. C. t = 7 min. The bar corresponds
to 10 µm (reproduced from [28]).
order of h/Ro for ‘vesicularization’. In the case of a GUV
with a typical diameter of about 50 µm, the experiments
show that the ‘small’ vesicles which are generated by lipid
asymmetry have a size of 5–10 µm. Since the bilayer
thickness is around 6 nm, a very small asymmetry, below
1%, is sufficient in principle. In the case of a 200 nm LUV
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
31
P-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
achieved in a few minutes at room temperature when the
membrane (containing a PC/PG mixture) is submitted to a
pH gradient [29], the same overall shape with 100 nm
LUVs required 60 mn of incubation at 60 °C [32]. The
latter observation reported by the Cullis’group is in
accordance with previously published theoretical predictions [33, 34].
Finally, I would like to emphasize as a conclusion of
this section that the formation of one or several microvesicles with a diameter of about 20 nm is always
accompanied by a surface tension. Because of the continuity between a budded vesicle and the rest of the
liposome this tension must be the same in the whole
502
vesicle membrane, even in a GUV. In other words,
although local fluctuations of the surface density in a GUV
could give birth to a microvesicle of the size encountered
in vivo during endocytosis or vesicle shedding, this
vesicle would promptly disappear because of the instability of a local surface tension. Thus a stable budded vesicle
connected to the rest of the plasma membrane is only
possible if the whole plasma membrane is constrained.
4. Is microvesicle formation in liposomes and red
cells relevant to endocytosis?
Typical diameters of the vesicles involved in
endocytosis-exocytosis, and more generally vesicle traffic
within eukaryotic cells, are around 200 nm. They result
from local membrane curvature, either of the plasma
membrane itself or of organelles such as the ER or the
Golgi system, the typical size of which is in the range of
one or several microns. Although lipid vesicle experiments demonstrate a close relationship between vesicle
shape and lipid transmembrane distribution and although
they show that endocytic-like invaginations may be triggered in GUVs by the redistribution of a small proportion
of lipids, they do not satisfactorily mimic endocytosis.
Indeed, endocytosis implies the formation of one or
several microvesicles from the invaginations of a cell
membrane which has a typical radius of several µm. This
is not what is observed in giant unilamellar vesicles when
for example 1% of L-PC is added externally. We have
mentioned above that the formation of very small spherical vesicles requires a lipid asymmetry of the order of
5–10% and because these vesicles (or invaginations)
remain at least temporarily connected to the rest of the
membrane, the same lipid asymmetry must exist all along
the cell surface and will cause an important surface
tension. The so-called large unilamellar vesicles (LUVs)
with a diameter of 100–200 nm can sustain surface tension
without collapse and form budded vesicles or protrusions
with a diameter of about 50 nm or less. On the other hand,
a giant vesicle of the size of an eukaryotic cell will not
form at its surface a series of small vesicles with a
diameter which is ≈ 10–3 times the average diameter of
this ‘mother vesicle’ without a complete shape change
unless some scaffolding, analogous to the cytsoskeleton
meshwork in a biological cell prevents the transformation
of this large vesicle. Figure 4 shows that if ∆A is
increased by the addition of a large amount of L-PC into
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
microvesicles. Of course, if the giant vesicle is initially a
perfect sphere and if the internal volume remains approximately constant, there is no possibility of any other shape.
In the latter case, the additional surface on the outer layer
will generate a tension that will inhibit surface undulations
Devaux
and eventually the surface of the sphere may become
covered by microvesicles.
Figure 5 shows an experiment where a large number of
relatively small vesicles appear at the external surface of
a giant vesicle. This figure was obtained with multilamellar vesicles (MLVs) on the surface of which a large excess
of L-PC was added. The inner membranes probably
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
vesicles which eventually shed off, thereby pealing the
external bilayer of this multilamellar vesicle. But even in
this example the size of the small vesicles (≈ 1 µm) was
much larger than the size of endo-exo-cytic vesicles. The
above example is reminiscent of the situation obtained
with red blood cells to which a large excess of L-PC has
been added. In the latter case, very small membrane
protrusions cover the cell surface which seems to shrink
and form a uniform sphere (spherocyte) if viewed with an
optical microscope. In reality, the erythrocyte surface is
covered by small vesicles with a radius below resolution
of the optical microscope. The cell resists because of the
cytoskeleton and eventually lyses if more L-PC is added
(> 5%).
Figure 6a-f shows in an (over)simplified way the shape
transformations that can undergo an obloid GUV
(figure 6a), by adding to the external surface molecules
such as L-PC [28, 29] or by increasing area to volume
ratio by heating [30, 31] Shapes in figure 6b, c are the
canonical axial symmetrical shapes calculated by theoreticians (see figure 2). The radius R of the spherical
‘budded’ vesicle(s) is large. Such transformation does not
resemble in vivo budding. Shapes in figure 6d, e correspond to the formation of relatively small vesicles. They
require first a transformation of the ‘mother vesicle’ into a
sphere. Again, overall this is not what is seen with real
cells. In the case of figure 6f, which represents a multilamellar vesicle (MLV), the obloid shape is maintained by
the inner bilayers. In vivo the cell shape is maintained by
the cytoskeleton and by the presence of organelles.
I infer that the activity of a lipid pump transporting
lipids from the outer monolayer to the inner monolayer (or
a selective fraction of the outer leaflet lipids) can induce
invaginations of the plasma membrane and form endocytic vesicles of the size observed in vivo, provided the
cell surface is reinforced by a cytoskeleton. This cytoskeleton and tubulin meshwork is represented schematically in figure 6g,h
5. Experimental observations in favor of the role of
lipid translocation during endocytosis
The aminophospholipid translocase is present in the
plasma membrane of all animal cells [10, 35–37]; recent
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 = 8 s. C. t = 30 s. D. t = 1 min. 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) or by a
cytoskeleton meshwork (g, h). h.
The external surface is depleted.
See text for more comments.
of plant cells [38]. This protein transports phosphatidylserine (PS) and to a lesser extent phosphatidylethanolamine (PE) from the outer to the inner leaflet where these
aminophospholipids are predominantly located. Cytosolic
ATP is necessary and must be hydrolyzed for this transport
to take place. By controlling the level of cytosolic ATP,
which in vivo is normally of the order of 2–3 mM, it is
possible to modify the equilibrium distribution of the
aminophospholipids and in turn to modify erythrocyte
shapes by shifting a fraction of the lipids from the inner to
the outer leaflet or conversely. Indeed, unpaired lipid
transport is accompanied by echinocyte formation while
artificial ATP enrichment leads to stomatocytes. When the
level of ATP is of the order of 5–6 mM endocytic vesicles
form at the cytosolic interface of erythrocytes. They are
characterized by the formation of a few relatively large
Is lipid translocation involved during endo- and exocytosis?
vesicles and can be quantified by monitoring the acetylcholine esterase activity which normally takes place on
the outer surface of red cells [12, 39]. The decrease in
esterase activity is an indication of the amount of surface
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 β chain which permits their rapid
incorporation (from [12]).
505
becoming non-accessible. If phosphatidylcholine (PC) is
added to the outer surface of red cells this vesicularization
process is affected because the plasma membrane would
rather tend to fold in the opposite direction (see figure 7
506
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 transformed 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 measured 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 β chain bearing the nitroxide: these slightly watersoluble 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 endocytosis activity. In 1999, a different technique was used to
measure endocytosis in K562 cells [14]. The internalization of fluoresceinated membrane proteins was either
watched directly or monitored by the decrease of fluorescence which is quenched by the acidity of early endocytic
vesicles. It was found that the addition of 4% PS on the
external side of the plasma membrane resulted in a
five-fold increase in fluorescence quenching in 10 min at
37 °C. Similarly, PE addition stimulated endocytosis but
lyso-PS, which is not transported by the aminophospholipid translocase [10], inhibited the residual endocytosis
(figure 8) .
In a recent investigation aimed at trying to find the
aminophospholipid translocase protein, Marx et al. [43]
tested the translocation of PS in endocytosis-deficient
yeast cells and found very little specific transport of the
aminophospholipid analogues and essentially no ATP
dependence. These results could suggest that the cells
lacking endocytosis activity have also no aminophospho-
Devaux
Figure 8. Enhancement of bulk protein endocytosis in K562
cells after addition to the external surface of phosphatidylserine
(PS) with a short β chain (C5). Bulk membrane proteins were
biotinylated and coupled with FITC streptavidin. The time
course of internalization of the labeled proteins was monitored
by following the fluorescence decrease of fluorescein which is
quenched in the acidic environment of early endocytic vesicles.
A. Protein internalization is observed as a function of percentage
of PS added. B. Initial bulk flow rate of endocytosis as a function
of the percentage of PS added to the estimate plasma membrane
phospholipids (reproduced from [14]).
lipid transporter. However, it is surprising that these cells
seem to have normal lipid asymmetry.
Actually, the doubt concerning the proper identification
of the aminophospholipid translocase is the most severe
limitation in any experimental proof of its direct implication in endocytosis. Although the sequence of an ATPase
with a molecular mass of 170 kDa has been published by
Tang et al. [44] and identified as the aminophospholipid
Is lipid translocation involved during endo- and exocytosis?
507
Figure 9. Schematic model showing 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 ATPdependent
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,
45]. One will probably have to wait until this protein can
be expressed. Recently, a preliminary report was given
about a successful complementation of an aminophospholipid translocase activity in translocase deficient yeast
cells (DRS2) using RNA obtained from plants [38].
6. Exocytosis and lipid scramblase
The same geometrical argument that led us to conclude
that the formation of microvesicles by bending of the
plasma membrane requires the translocation of lipids from
the outer to the inner leaflet, forces us to conclude that
fusion of small vesicles to the plasma membrane, i.e.,
exocytosis, requires the opposing transfer of lipids (see
figure 9). A priori the two processes (endocytosis and
exocytosis) are not exactly symmetrical. Indeed a single
small vesicle, with a high surface tension and an asymmetrical repartition of phospholipids can fuse with the
plasma membrane without perturbing it because the latter
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
slow spontaneous flip-flop of lipids. However, in a living
cell exocytosis is a continuous process that permits the
whole plasma membrane to be renewed within a short
period, and fusion of synaptic vesicles or granules is a
synchronized event which concerns a large number of
vesicles. It is therefore apparent that independently from
the process that enables the two bilayers to come in close
contact and to merge into a single bilayer, it would be
useful for the efficiency of this process that some mechanism accelerates lipid flip-flop in order to facilitate the
randomization of the transmembrane lipid distribution.
Additionally, if this lipid scrambling process brings to the
outer leaflet some aminophospholipids (PS and PE), it
should facilitate later the formation of inward invaginations (i.e., endocytosis) by the aminophospholipid translocase. In this regard, it is quite interesting to note that
most cells do have in their plasma membrane a protein
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
their outer leaflet when they are stimulated, but it is also
functional in all cells of the blood circulation during
apoptosis [46].
In principle this ‘scramblase’ has been purified and
cloned [47]. There is also a rare and very severe disease
called Scott syndrome which was thought to correspond to
a lack of scramblase; at least it is associated with an
impairment of calcium triggered lipid redistribution in
platelets. However the reconstituted vesicles with the
‘purified’ scramblase have a very low scrambling efficiency [48] and this protein is present in the blood cells of
the Scott patients [49]. So the identification of the protein
may yet have to be confirmed.
7. Discussion and conclusions
It has been demonstrated in giant unilamellar vesicles
that a small area increase of one of the two opposing
monolayers triggers budding or invaginations. In various
eukaryotic cells, a positive correlation between aminophospholipid translocase activity and the endocytic activity was demonstrated. There is also strong evidence that
the stimulation of phospholipid translocation from the
outer to the inner monolayer of the plasma membrane in
K562 cells accelerates endocytosis while the addition of
phospholipids that are not transported has an inhibitory
effect. Therefore, one can infer that the active transport of
508
lipids may serve as a driving force for membrane folding
during endocytosis. It is an ATP driven process which acts
on the whole membrane by generating a non-localized
surface tension; this tension is at least partly relaxed by
the formation of membrane budding and invaginations.
The cytoskeleton is necessary to prevent whole cell
deformation but organelles which are present in eukaryotic cells may help also in a way comparable to inner
vesicles in MLVs (figures 5, 6). The localization of these
invaginations has to be random in a large unilamellar
vesicle which has an homogeneous surface, but in a
biological membrane which contains heterogeneous domains, it is quite conceivable that rigid domains or
localized region with a non-zero spontaneous curvature
imposed by the binding of peripheral proteins such as
clathrin, or associated with the clustering of caveolin,
serve as a point of emergence. Note the model of
domain-induced budding of fluid membranes proposed by
Lipowsky is based on a different physical property,
namely the competition between the minimization of the
bending energy and the energy of the line tension between
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
calcium triggered scramblase during exocytosis is more
speculative at this stage than the hypothesis about the role
of the aminophospholipid translocase during endocytosis.
It clearly needs experimental confirmation. One aspect
that I have not discussed yet is the synchronization
between endocytosis and exocytosis. Clearly in this model
the absence of exocytosis should eventually stop endocytosis because there is a need of substrates for the lipid
pump, namely if no PS and/or PE returns to the exoplasmic leaflet, the pump cannot continue to work. Therefore
the system is safe. If, on the other hand, the pump had no
lipid selectivity, it would continue to pump the whole cell
plasma membrane regardless of what happens within the
cell. This would eventually be a suicide activity. In the
framework of the aminophospholipid translocase model
an evolutionary advantage of lipid heterogeneity becomes
therefore apparent.
There are reports of protein catalyzed outward movement for example by MRP proteins that would return PC
to the outer leaflet in erythrocytes [51]. Although the data
in the literature do not allow one to compare quantitatively
the activity of the aminophospholipid translocase and that
of the MRP protein, the very fact that if the level of ATP
is raised in the erythrocyte cytosol, the membrane bends
inwardly suggests that, if there is competition between the
two transports, the inward movement wins. Thus, again
this is a safe situation. There is one example in nature of
cells shedding off pieces of their own plasma membrane.
Indeed, stimulated platelets send in the blood circulation
microvesicles formed by plasma membrane budding.
However, platelets do not recover from this process.
Whether this vesicle shedding is due to a calcium trig-
Devaux
gered pump transporting an excess of lipids outwardly, or
to the activity of cytoskeleton proteins, is a question not
settled yet [52].
In the framework of this model, clathrin binding could
be necessary to determine which part of the membrane
will invaginate rather than clathrin binding being the
driving force. On the other hand the coat proteins identified as proteins involved in budding of the Golgi system
[1] may have a similar effect than the addition of L-PC
added externally to a GUV. Because there is now compelling evidence that lipid flip-flop in inner membranes is
much more rapid than in the plasma membrane which is
rich in cholesterol [53], it is unlikely than the same
mechanism could be used in the ER or Golgi membranes
to generate microvesicles. Of course several mechanisms
of budding may exist within a cell.
As a final conclusion, the models proposed here have
many implications that can be verified. Reconstitution in
giant vesicles of the purified proteins involved in lipid
traffic appears to be the most significant type of experiments to perform in the future to test the above hypothesis.
Acknowledgments
The author thank Drs. E. Farge, H.G. Döbereiner and S. Cribier
for helpful discussions. Work supported by grants from the
Centre National de la Recherche Scientifique (UPR9052) and the
Université Paris 7-Denis Diderot.
References
[1] Spang A., Matsuoka K., Hamamoto S., Schekman R., Orci L.,
Coatomer, ARF1p and nucleotide are required to bud coat protein
complex I-coated vesicles from large synthetic liposomes, Proc.
Natl. Acad. Sci. USA 98 (1998) 11199–11204.
[2] Bremser M., Nickel W., Schweikert M., Ravazzola M., Amherdt
M., Hughes C.A., Söllner T.H., Rothman J.E., Wieland F.T.,
Coupling of coat assembly and vesicle budding to packaging of
putative cargo receptors, Cell 96 (1999) 495–506.
[3] Sandvig K., van Deurs B., Endocytosis without clathrin, Trends
Cell Biol. 4 (1994) 275–277.
[4] Cupers P., Veithen A., Kiss A., Baudhuin P., Courtoy P.J., Clathrin
is not required for bulk phase endocytosis in rat fetal fibroblasts, J.
Cell Biol. 127 (1994) 725–735.
[5] Miller K., Shipman M., Trowbridge I.S., Hopkins C.R., Transferrin receptors promote the formation of clathrin lattices, Cell 65
(1991) 621–632.
[6] Simons K., Ikonen E., Functional rafts in cell membranes, Nature
387 (1997) 569–572.
[7] Zha X., Pierini L.M., Leopold P.H., Skiba P.J., Tabas I., Maxfield
F.R., Sphingomyelinase treatment induces ATP-independent endocytosis, J. Cell Biol. 140 (1998) 39–47.
[8] Op Den Kamp J.A.F., Lipid asymmetry in membranes, Annu. Rev.
Biochem. 48 (1979) 47–71.
[9] Devaux P.F., Zachowski A., Favre E., Fellmann P, Cribier S.,
Geldwerth D., Hervé P., Seigneuret M., Translocation énergie
dépendante des amino-phospholipides dans la membrane des
globules rouges, Biochimie 68 (1986) 383–393.
[10] Devaux P.F., Static and dynamic lipid asymmetry in cell membranes, Biochemistry 30 (1991) 1163–1173.
Is lipid translocation involved during endo- and exocytosis?
[11] Cribier S., Sainte-Marie J., Devaux P.F., Quantitative comparison
between aminophospholipid translocase activity in human erythrocytes and in K562 cells, Biochim. Biophys. Acta 1148 (1993)
85–90.
[12] Müller P., Pomorski T., Herrmann A., Incorporation of phospholipid analogues into the plasma membrane affects ATP-induced
vesiculation of human erythrocyte ghosts, Biochem. Biophys. Res.
Commun. 199 (1994) 881–887.
[13] Farge E., Increased vesicle endocytosis due to an increase in the
plasma membrane phosphatidylserine concentration, Biophys. J.
69 (1995) 2501–2506.
[14] Farge E., Ojcius D.M., Subtil A., Dautry-Varsat A., Enhancement
of endocytosis due to aminophospholipid transport across the
plasma membrane of living cells, Am. J. Physiol. 276 (1999)
C725–C733.
[15] Kornberg R.D., Mc Connell H.M., Inside-outside translocation of
phospholipids in vesicle membranes, Biochemistry 10 (1971)
1111–1120.
[16] Sheetz M., Singer J.S., Biological membranes as bilayer couples.
A molecular mechanism of drug erythrocyte membrane, Proc.
Natl. Acad. Sci. USA 71 (1974) 4457–4461.
[17] Helfrich W., Elastic properties of lipid bilayers: theory and
possible experiments, Z. Naturforsch. 28c (1973) 693–703.
[18] Miao L., Seifert U., Wortis M., Döbereiner H.G., Budding transitions of fluid bilayer vesicles: the effect of area difference
elasticity, Phys. Rev. E. 49 (1994) 5389–5407.
[19] Svetina S., Zeks B., Membrane bending energy and shape determination of phospholipid vesicles and red blood cells, Eur.
Biophys. J. 17 (1989) 101–111.
[20] Berndl J., Käs J., Lipowsky R., Sackmann E., Seiffert U., Shape
transformation of giant vesicles: extreme sensitivity to bilayer
asymmetry, Eur. Phys. Lett. 13 (1990) 659–664.
[21] Lipowsky R., The conformation of membranes, Nature 349 (1991)
475–481.
[22] Allan D., Walkins C.M., Endovesiculation of human erythrocytes
exposed to sphingomyelinase C: a possible explanation for the
enzyme resistant pool of sphingomyelin, Biochem. Biophys. Acta
938 (1988) 403–410.
[23] Callisen T.H., Talmon Y., Direct imaging by cryo-TEM shows
membrane break-up by phospholipase A2 enzymatic activity,
Biochemistry 37 (1998) 10987–10993.
[24] Schmidt A., Wolde M., Thiele C., Fest W., Kratzin H., Podtelejnikov A.V., Wilker W., Huttner W.B., Söling H.D., Endophilin I
mediates synaptic vesicle formation by transfer of arachidonate to
lysophosphatidic acid, Nature 401 (1999) 133–141.
[25] Ferrell J.E., Huestis W.H., Phosphoinositide metabolism and the
morphology of human erythrocytes, J. Cell Biol. 98 (1984)
1992–1998.
[26] Traïkia M., Thèse de doctorat en Biophysique Moléculaire, Université Denis Diderot, Paris, France, 1999.
[27] Hope M., Redelmeier T.E., Wong K.F., Rodrigueza W., Cullis P.R.,
Phospholipid asymmetry in large unilamellar vesicles induced by
transmembrane pH gradients, Biochemistry 28 (1989) 4181–4187.
[28] Mathivet L., Cribier S., Devaux P.F., Shape change and physical
properties of giant phospholipid vesicles prepared in the presence
of an electric field, Biophys. J. 70 (1996) 1112–1121.
[29] Farge E., Devaux P.F., Shape change of giant liposomes induced
by an asymmetric transmembrane distribution of phospholipids,
Biophys. J. 61 (1992) 347–357.
[30] Kas J., Sackman E., Shape transformation and shape stability of
giant phospholipid vesicles in pure water by area-to-volume
changes, Biophys. J. 60 (1991) 825–844.
[31] Döbereiner H.G., Käs J., Noppi D., Sprenger I., Sackmann E.,
Budding and fission of vesicles, Biophys. J. 65 (1993) 1396–1403.
[32] Mui B.L.S., Döbereiner H.G., Madden T.D., Cullis P.R., Influence
of transbilayer area asymmetry on the morphology of large
unilamellar vesicles, Biophys. J. 6 (1995) 930–941.
[33] Farge E., Devaux P.F., Size-dependent response of liposomes to
phospholipid transmembrane redistribution: from shape change to
induced tension, J. Phys. Chem. 97 (1993) 2958–2961.
509
[34] Farge E., Scale-dependent elastic response of closed phospholipid
bilayers to transmembrane molecular pumping activity: a key for
exo-endocytosis physiological process, Il Nuovo Cimento 16D
(1994) 1457–1470.
[35] Devaux P.F., Lipid transmembrane asymmetry and flip-flop in
biological membranes and in lipid bilayers, Curr. Opin Struct.
Biol. 3 (1993) 489–494.
[36] Zachowski A., Phospholipids in animal eukaryotic membranes:
transverse asymmetry and movement, Biochem. J. 294 (1993)
1–14.
[37] Devaux P.F., Zachowski A., Maintenance and consequences of
membrane phospholipid asymmetry, Chem. Phys. Lipids 73
(1994) 107–120.
[38] Palmgren M.G., Harper J.F., Pumping with plant P-type ATPases,
J. Exp. Bot. 50 (1999) 883–893.
[39] Birchmeier W., Lanz J.H., Winterhalter K.H., Conrad M.J., ATPinduced endocytosis in human erythrocyte ghosts, J. Biol. Chem.
254 (1979) 9298–9304.
[40] Seigneuret M., Devaux P.F., ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane;
relation to shape change, Proc. Natl. Acad. Sci. USA 81 (1984)
3751–3755.
[41] Daleke D.L., Huestis W.H., Incorporation and translocation of
aminophospholipids in human erythrocytes, Biochemistry 24
(1985) 5406–5416.
[42] Pomorski T., Müller P., Zimmermann B., Burger K., Devaux P.F.,
Herrmann A., Transbilayer movement of fluorescent and spinlabeled phospholipids in the plasma membrane of human fibroblasts: a quantitative approach, J. Cell Sci. 109 (1996) 687–698.
[43] Marx U., Polawkowski T., Pomorski T., Lang C., Nelson H.,
Nelson N., Herrmann A., Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of
endocytosis-deficient cells does not require the Drs2 protein, Eur.
J. Biochem. 263 (1999) 254–263.
[44] Tang X., Halleck M.S., Schlegel R.A., Williamson P., A subfamily
of P-type ATPase with aminophospholipid transporting activity,
Science 272 (1996) 1495–1497.
[45] Dolis D., Moreau C., Zachowski A., Devaux P.F., Aminophospholipid translocase and proteins involved in transmembrane phospholipid trafic, Biophys. Chem. 68 (1997) 221–231.
[46] Zwaal R.F.A., Schroit A.J., Pathophysiologic implications of
membrane phospholipid asymmetry in blood cells, Blood 89
(1997) 1121–1132.
[47] Zhou Q., Zha J., Stout J.G., Luhm R.A., Wieder T., Sims P.J.,
Molecular cloning of human plasma membrane phospholipid
scramblase, J. Biol. Chem. 272 (1997) 18240–18244.
[48] Bassé F., Stout J.G., Sims P.J., Wiedmer T., Isolation of an
erythrocyte membrane protein that mediates Ca2+ dependent
transbilayer movement of phospholipid, J. Biol. Chem. 271 (1996)
17205–17210.
[49] Stout J.G., Bassé F., Luhm R.A., Weiss H.J., Wiedmer T., Sims
J.P., Scott syndrome erythrocytes contain a membrane protein
capable of mediating Ca2+-dependent transbilayer migration of
membrane phospholipids, J. Clin. Invest. 99 (1997) 2232–2238.
[50] Lipowsky R., Domain-induced budding of fluid membranes,
Biophys. J. 64 (1993) 1133–1138.
[51] Dekkers D.W.C., Comfurius N., Schroit A.J., Bevers E.M., Zwal
F.A., Transbilayer movement of NBD-labeled phospholipids in red
blood cell membranes: outward-directed transport by the multidrug resistance protein (MRP1), Biochemistry 37 (1998)
1433–1437.
[52] Gaffet P., Bettache N., Bienvenüe A., Phosphatidylserine exposure
on the platelet plasma membrane during A23187 induced activation is independent of cytoskeleton reoganisation, Eur. J. Cell Biol.
67 (1995) 336–345.
[53] Gallet F.P., Zachowski A., Julien R., Fellmann P., Devaux P.F.,
Maftah A., Transbilayer movement and distribution of spin-labeled
phospholipids in the inner mitochondrial membrane, Biochim.
Biophys. Acta 1418 (1999) 61–70.