FEBS Letters 584 (2010) 17791786

journal homepage: www.FEBSLetters.org

Review

Transbilayer (ip-op) lipid motion and lipid scrambling in membranes

F.-Xabier Contreras a, Lissete Snchez-Magraner b, Alicia Alonso c, Flix M. Goi c,*
a
Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany
b
Department of Biotechnology, University of Verona, 37134 Verona, Italy
c
Unidad de Biofsica (Centro Mixto CSIC-UPV/EHU), Departamento de Bioqumica, Universidad del Pas Vasco, P.O. Box 644, 48080 Bilbao, Spain

a r t i c l e i n f o a b s t r a c t

Article history: This paper reviews the current knowledge on the various mechanisms for transbilayer, or ip-op,
Received 19 October 2009 lipid motion in model and cell membranes, enzyme-assisted lipid transfer by ippases, oppases
Accepted 18 December 2009 and scramblases is briey discussed, while non-catalyzed lipid ip-op is reviewed in more detail.
Available online 30 December 2009
Transbilayer lipid motion may occur as a result of the insertion of foreign molecules (detergents, lip-
Edited by Wilhelm Just
ids, or even proteins) in one of the membrane leaets. It may also be the result of the enzymatic gen-
eration of lipids, e.g. diacylglycerol or ceramide, at one side of the membrane. Transbilayer motion
rates decrease in the order diacylglycerol  ceramide  phospholipids. Ceramide, but not diacyl-
Keywords:
Transmembrane lipid motion
glycerol, can induce transbilayer motion of other lipids, and bilayer scrambling. Transbilayer lipid
Lipid ip-op diffusion and bilayer scrambling are dened as two conceptually and mechanistically different pro-
Bilayer scrambling cesses. The mechanism of scrambling appears to be related to local instabilities caused by the non-
Ceramide lamellar ceramide molecule, or by other molecules that exhibit a relatively slow ip-op rate, when
Diacylglycerol asymmetrically inserted or generated in one of the monolayers in a cell or model membrane.
2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction amine (PE) and phosphatidylserine (PS) are exclusively present in

As in all living structures, the molecules forming eukaryotic cell
membranes are in a non-equilibrium state. This is mainly due to a
continuous intracellular trafcking of molecules that control cell
survival. In the last decades, a great deal of information has been
gathered concerning membrane protein synthesis, transport, mat-
uration and distribution into the target membrane [1]. However,
the means by which membrane lipids are transported to their nal
destination after synthesis in the endoplasmic reticulum (ER) is
still to be claried. Cellular membranes are composed of a double
layer of lipids in which proteins are embedded. At least some bio-
logical membranes, and particularly the plasma membrane (PM),
are known to be asymmetric and composed of lipids that are het-
erogeneously distributed in the different organelles. This specic
membrane lipid composition requires the transbilayer movement
of lipids from one leaet to the other. Phospholipid synthesis takes
place almost exclusively in the cytoplasmic leaet of the ER, which
leads to an asymmetry in lipid composition. In order to maintain
the balance of lipid molecules in the ER, about one-half of the new-
ly synthesized lipids are transferred to the other leaet [2].
The PM is characterized by a strict lipid asymmetry in which
phosphatidylcholine (PC) and sphingomyelin (SM) are mainly
localized in the extracellular leaet, whereas phosphatidylethanol-
* Corresponding author. Fax: +34 94 601 33 60.
E-mail address: felix.goni@ehu.es (F.M. Goi).
the cytoplasmic leaet. Maintenance of the lipid asymmetry in
the PM is an energy-dependent process that is important for a vari-
ety of cell processes [3]. For example, PS exposure on the cell sur-
face leaet after certain stimuli has been associated with apoptosis
and senescence [4,5]. It is thought that the recognition of exposed
PS by macrophages is necessary for the engulfment of apoptotic
cells [6]. It is also reported that PS exposure is observed in vascular
cells in tumors but not in normal cells [7].
Transbilayer lipid motion has been dubbed ip-op and it is
unfortunate that this rather childish term has stayed in the scientic
literature, together with the ippase and oppase terms that desig-
nate lipid translocating enzymes. Maintenance of membrane
asymmetry in the resting state cannot be mediated by passive lipid
ip-op alone, since spontaneous translocation of phospholipids
across the bilayer has been postulated to be extremely slow
(10 15 s 1), on average a single lipid ip-op event every 24 h
[8,9]. The slow pace of this translocation is due to the high energy
barrier imposed by the resistance to the passage of polar head groups
through the hydrophobic core but also to the additional resistance
imposed by the increased lateral tension developed in the receiving
monolayer upon insertion of the new phospholipids [10]. Therefore,
asymmetric membranes cannot be maintained by passive ip-ops.
Establishment and maintenance of lipid asymmetry involves several
different membrane proteins. These lipid translocators include ATP-
dependent ippases and oppases [10,11] and energy-independent
scramblases [11]. The energy-dependent translocators have been

0014-5793/$36.00 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2009.12.049
1780 F.-X. Contreras et al. / FEBS Letters 584 (2010) 17791786
described as lipid selective, while scramblases are non-selective,
inducing non-specic transbilayer movement across the membrane
[11]. We intend to briey review the proteins catalyzing ip-op,
and deal in more detail with the less understood phenomenon of
transbilayer lipid motion induced by molecules that were not origi-
nally present in the membrane.
2. Flippases and oppases
Flippases and oppases are ATP-dependent transbilayer lipid
translocators. According to an extensively used, though not univer-
sal, nomenclature they catalyze lipid transfer towards the inward
monolayer (ippase) or towards the outward monolayer (oppas-
es). Some authors however use the name oppase to denote any
enzyme catalyzing transbilayer lipid motion, in some cases even
in the absence of ATP requirements.
Flippases and oppases are required to equalize the number of
lipids at both sides of the membrane when a new membrane/orga-
nelle is being generated, lipid synthesis taking place only on one
side of the bilayer. Thus ippases and oppases help make the
membrane more symmetric. Moreover in some cases they operate
in the opposite way, generating lipid asymmetry, the latter giving
rise in turn to lipid vesiculation. In general, this is a eld in which
many of even the basic aspects remain unknown [12]. Most pre-
dicted ippases have not yet been characterized at the molecular
level, and the few that have been identied by genetic methods
have not been biochemically characterized [13].
An important breakthrough in this area has been the involve-
ment of the so-called P4-ATPases (type 4 P-type ATPases) in the
phospholipid translocation from the exoplasmic to the cytoplasmic
leaet of cell membranes (most other P-type ATPases catalyze cat-
ion transport across membranes). The b-subunit of P4-ATPases
shows similarities with the b and c subunits of Na+, K+-ATPases
[12]. Members of the P4-ATPase group have been identied in Sac-
charomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana
and also in humans. As many as 14 P4-ATPases have been found
in our species [14]. ATP8B1 is a phosphatidylserine translocase that
is probably the most extensively studied mammalian P4-ATPase.
This enzyme contributes to the maintenance of asymmetry in plas-
ma membranes. Mutations in the ATP8B1 gene cause severe liver
disease, however the cellular function and biochemical activity of
most mammalian P4 ATPases remains to be claried. It should be
noted though that no P4-ATPases have been identied in the ER,
where most membrane lipids are synthesized thus the prime locus
for transbilayer lipid motion to occur. The subject of P4-ATPases
and their role as ippases in health and disease has been recently
reviewed by Folmer et al. [15].
A particular case of lipid ipping in the endoplasmic reticu-
lum is that of the glycosyldolichols. Dolichols are a group of a
very long-chain unsaturated isoprene lipids. Protein N-glycosyla-
tion occurs in the lumen of the ER, and the glycosyl units are
provided by glycosyldolichols synthesized on the cytoplasmic
face or ER. Of course ipping of these glycolipids must occur,
but the mechanism is unknown, except that specic ippases
are required [16].
Lipid ippases are also involved in a series of mechanisms that
lead to changes in the membrane physical properties. This aspect
of the enzymes has been reviewed by Devaux et al. [10]. Phospho-
lipid transfer may lead to local or extended cell shape changes
through bending of the lipid bilayer. Bending occurs through the
inbalance of lipids between the two monolayers, and sometimes
tension may be released through vesiculation, i.e. the budding of
new, small vesicles. The newly-formed vesicles may in turn be in-
volved in physiological events such as lipid and protein trafc.
Muthusamy et al. [14] have recently described the involvement
of yeast P4-ATPases in transport vesicle budding.
Floppases catalyze the exofacially oriented transport of lipids
in membranes. Even less is known of this group of enzymes than
of the above-mentioned ippases. The oppases known to date
are all members of the ABC (ATP-binding cassette) superfamily,
a group of proteins that were originally related to multidrug
resistance. The fact that they are rather non-specic transloca-
tors of largely non-polar molecules explains their oppase
activity [11,17]. In fact the best characterized oppase is the
multidrug resistance protein ABCB1, or P-glycoprotein, but even
in this case it is doubtful that it translocates natural
long-chain lipids, as opposed to uorescent short-chain ana-
logues [18].
3. Scramblases: the human PLSCR1
Phospholipid scramblases (PLSCRs) constitute a group of
homologous ATP-independent bidirectional lipid translocators that
are conserved in all eukaryotic organisms, from yeasts to humans.
They are involved in the calcium-dependent transbilayer move-
ment of phospholipids, which tends to eliminate their asymmetric
distribution across the membranes [19].
In humans, four related PLSCR genes have been identied,
named as hPLSCR1hPLSCR4. The predicted open reading frames
encode proteins with 59% (hPLSCR2), 47% (hPLSCR3) and 46%
(hPLSCR4) homology, respectively, to hPLSCR1 [20]. In the cells
PLSCR1 is located in the plasma membrane, PLSCR2 is found in
the nucleus, and PLSCR3 occurs in the mitochondrial membrane.
While hPLSCR1, 3 and 4 are expressed in a variety of tissues includ-
ing heart, kidney, pancreas, spleen, prostate, colon, the expression
of hPLSCR2 is restricted to testis. hPLSCR4 was not detected in
peripheral blood lymphocytes, and hPLSCR1 and hPLSCR3 were
not detected in the brain [20,21].
The rst described member and prototype of this family is
hPLSCR1, a 37 kDa (318 aa) type II endofacial membrane protein
with a single transmembrane segment near the C-terminus.
hPLSCR1 remains inactive under normal conditions, but increased
cytosolic calcium concentrations (1000-fold the basal level in re-
sponse to cell injury, coagulation, cell activation or apoptosis) acti-
vate the protein and induce a rapid transbilayer movement of PE
and PS from the inner to the outer membrane leaet destroying
the bilayer asymmetry [19,20,22]. PLSCR1 is a palmitoylated pro-
tein that partitions with otillin-1 and EGFR into lipid rafts. EGF
stimulates tyrosine phosphorylation of PLSCR1 and promotes
internalization of the protein [23]. In the absence of palmitoyla-
tion, virtually all of the expressed hPLSCR1 localizes to the nucleus
[20].
hPLSCR1 has a variety of important functional regions [24]. The
N-terminal region (aa 185) contains multiple PXXP and PPXY se-
quences, which may bind proteins containing SH3 and WW do-
mains, respectively [20]. The DNA binding domain (aa 86118)
enables the protein to interact with DNA. In the absence of palmi-
toylation hPLSCR1 is imported into the nucleus in an importin a/b-,
GTP-, and Ran-dependent manner and binds to a genomic DNA
with a higher afnity. This suggests a potential nuclear function
as a transcription factor within the nucleus for the unpalmitoylat-
ed protein. One identied gene target of nuclear hPLSCR1 is the
inositol-1,4,5-trisphosphate receptor type 1 gene (IP3R1), nuclear
hPLSCR1 serving to increase both IP3R1 transcription and protein
expression [25,26]. Recent studies suggest the interaction of
hPLSCR1 with topoisomerase II, a protein involved in numerous
cellular processes and essential for cell division [27].
Another important region is the cystein-palmitoylation motif
(184CCCPCC189). The protein is acylated by thioester linkages to
the sulfhydryl groups of one or more cysteines, and this regulates
its trafcking within the cell. This modication is essential for the
 F.-X. Contreras et al. / FEBS Letters 584 (2010) 17791786
hPLSCR1 scrambling activity providing a membrane anchorage for
the N-terminal region. Hydrolysis of the thioester-bound palmitoyl
groups in hPLSCR1 puried from RBC markedly reduced the scram-
bling activity [28,29], and the unmodied hPLSCR1 expressed and
puried from Escherichia coli had approximately 50% of the activity
observed for the endogenous protein puried from RBC [20]. Nu-
clear localization of hPLSCR1 was also observed upon induction
of its de novo synthesis by interferon a (IFNa). These data suggest
that the post-translational acylation acts as a check-point deter-
mining the localization of the protein in the cell and regulating
its normal function in the nucleus or incorporated to the mem-
brane [28,29]. A recent structural model computed by homology
modeling suggests that palmitoylation may represent the principal
membrane anchorage for hPLSCR1 [30].
The nuclear localization of hPLSCR1 depends on cytosolic fac-
tors and energy. The protein has a non-classical nuclear localiza-
tion signal (NLS) (aa 257266) which helps import the protein
into the nucleus by the importin a/b nuclear chaperones. This
NLS is distinct from the prototypical NLS dened by a cluster of ve
basic residues, in this sequence only three out of nine amino acids
are basic (two lysines separated by two neutral amino acids and
one histidine) and it is enriched in hydrophobic residues [25].
hPLSCR1 interacts with importin-a with a high afnity, importin-
a acting as an adaptor that recognizes and binds the scramblase
NLS sequence after association with importin-b. Then the complex
is translocated through nuclear pores. The crystal structure of
importin-a/scramblase/NLS complex has been determined at
2.2 , the structure suggests that NLS binding is not accompanied
by a signicant conformational change in importin-a, and that its
interaction partially overlaps the classical NLS binding site [31].
The calcium binding motif (aa 273284) with a putative EF-
hand-like structure is essential for the scrambling activity of
hPLSCR1. This motif is known to bind calcium and other ions
(e.g. Tb+3, La+3, Mn+2, Zn+2, Sr+2, Mg+2). In this motif residues in
positions 1, 3, 5, 7, 9 and 12 contribute to the octahedral coordina-
tion of the calcium ion and substitution at any of these positions
reduces phospholipid scramblase function [32,33]. Calcium bind-
ing induces protein oligomerization, either by cross-linking
hPLSCR1 monomers or by inducing a prominent conformational
change that in turn promotes self-association. It remains to be
determined how the conformational change serves to accelerate
the movement of membrane phospholipids between the inner
and outer leaets.
A recent structural model suggests that the calcium binding
motif is not an EF-hand-like structure [30]. Studies of Sahu et al.
suggest that the calcium binding motif present in scramblase rep-
resents a new class of low-afnity calcium binding motifs, the
scramblase-like calcium binding motif being also present in other
unrelated proteins known to exhibit changes in biochemical
parameters upon binding to calcium or other divalent ions [34].
hPLSCR1 structure is completed by the single predicted trans-
membrane helix near the C-terminus (aa 291309) with the in-
side-to-outside orientation, that makes the hPLSCR1 a type II
membrane protein, and by a nal short exoplasmic tail (aa 310
318) [21,24]. Bateman et al. provide an alternative structural mod-
el in which the transmembrane helix is buried within the core of
the hPLSCR1 structure, suggesting that this is not a transmembrane
helix [30].
In addition to palmitoylation and calcium binding hPLSCR1
undergoes a different post-translational modication that affects
protein function, namely phosphorylation of Tyr and Thr residues,
Tyr phosphorylation by c-Abl and c-Src tyrosine kinases at Tyr 69
and 74 and Thr phosphorylation by PKCd at Thr 161 [21,23].
The actual cellular functions of hPLSCR1 remain largely unre-
solved, and different studies suggest that in addition to its putative
role in mediating transbilayer movement of phospholipids,
1781
hPLSCR1 may also function to regulate processes including signal-
ing, cell differentiation, apoptosis, injury, cell proliferation and
transcription [25,26,3537].
4. Transbilayer lipid movement induced by external agents
External addition of detergents to the plasma membrane of red
blood cells at sub-solubilizing concentrations accelerates the slow
transbilayer motion of uorescent-labeled PC analogs (NBD-PC)
across the membrane [38]. Interestingly neutral, zwitterionic, an-
ionic and cationic detergents containing different alkyl chain
lengths (from C6 to C14) are able to accelerate the transfer of lipids
from the outer to the inner leaet. It is important to mention that
the ability of a detergent to induce this effect on the bilayer is com-
pletely independent of its alkyl chain length. This detergent effect
is likely to be induced by formation of transient hydrophobic struc-
tural defects in the membrane barrier, which can be the result of
perturbation in the interfacial region of the bilayer associated with
detergent insertion. Moreover, the concentration of different deter-
gents required to increase the constant rate of NBD-PC ip is very
similar. Therefore the presence of a small neutral hydroxyl group, a
larger sugar residue or an ionic group linked to detergent alkyl
chain is not essential to obtain this detergent accelerating effect
on the transbilayer lipid motion. The membrane concentrations
of non-ionic detergents (i.e. Triton X-100) needed to induce an ob-
servable ip of NBD-PC is around 10-fold lower than that observed
with other detergents. Reduction in the length of the oxyethylene
chain is correlated with a decrease in effectiveness [38].
Our group has also described that alkanes promote the translo-
cation of lipids between leaets [39a]. Alkanes are often used in cell
biology as vehicles to incorporate hydrophobic molecules into cell
membranes. It is generally assumed that these molecules are inac-
tive when used at low concentrations. However, under standard
experimental conditions, we observed that alkanes induce drastic
changes in the physical state of the membrane. Insertion of alkanes
into the outer leaet is accompanied by a rapid transbilayer motion
of lipids across the membrane. As described for detergents above,
acceleration of lipid ip-op may be induced by the transient for-
mation of defects in the bilayer. Since alkanes stabilize and promote
non-lamellar structures in the membrane, a lipid mismatching de-
fect can be created in the borders between lamellar and non-lamel-
lar structures, facilitating lipid translocation within the bilayer.
Protein insertion into membranes may also cause ip-op lipid
motion. Martin et al. [39b] studied the insertion of the so-called
adenylate cyclase toxin from Bordetella pertussis into lipid vesi-
cles. The toxin (1706 aa residues) targets cell membranes, in which
it breaks down the permeability barrier. The NBD-PC test, as men-
tioned above, was used to demonstrate that as the toxin became
incorporated into the vesicles (and efux of liposomal aqueous
contents took place) extensive ip-op motions occurred in the
bilayer. Similar observations have been made with proapoptotic
Bax-type proteins in liposomes [39c,d]. This may be a general
phenomenon for proteins that are not under native conditions part
of the cell membrane, but instead become incorporated into it
under certain circumstances.
5. Spontaneous transbilayer diffusion of biological lipid second
messengers
Important examples of lipid molecules that undergo a rapid
transbilayer motion, and can even induce ip-op of other lipids,
are the bioactive molecules diacylglycerol and ceramide. Although
their average concentration in membranes is very low, it may in-
crease locally by orders of magnitude as a result of cellular activa-
tion by the appropriate agonist.
1782 F.-X. Contreras et al. / FEBS Letters 584 (2010) 17791786
5.1. Diacylglycerol
Diacylglycerol (DAG), a minor component of cellular membranes,
plays major roles in biological processes. An important second mes-
senger molecule, DAG is involved in cell signal transduction [40]. In
DAG a fatty acyl chain is linked at position sn-2 via an ester linkage,
however at position sn-1 the fatty acid can be linked via ester, ether
or alkenyl ether bonds [41]. DAG can exist in at least 50 different
molecular species, containing saturated, mono-unsaturated, di-
unsaturated or poly-unsaturated fatty acids [42,43]. Combinations
of different fatty acids in position sn-1 and sn-2 create a variety of dif-
ferent DAG species. It is still unclear which DAG species play a most
important role in cell signaling. Various studies link an increase in
the degree of unsaturation with a greater activating effect in the cell
[44,45]. It is also still unclear to what extent the membrane structure
is altered after formation of DAG by phospholipase C. Different bio-
physical studies in model membranes have shown that the presence
of DAG causes a change from lamellar to hexagonal structure in the
bilayer [46,47]. This transition to non-lamellar structure depends on
the amount of DAG in the membrane. In addition to the lamellar to
non-lamellar phase transition of lipids, DAG is also involved in fusion
and ssion processes [48,49].
Pagano and Longmuir explored the ability of uorescent-labeled
DAG to freely diffuse across the membrane [50]. In their studies the
plasma membrane of Chinese hamster broblasts was labeled with
uorescent phosphatidic acid (NBD-PA). Upon dephosphorylation,
the resulting product NBD-DAG was readily distributed into cyto-
plasmic membranes. The labeling of intracellular membranes could
only be due to a transbilayer movement of DAG within the plasma
membrane. Labeling of the cells directly with NBD-DAG or enzy-
matic production of uorescent DAG from NBD-PC upon phospho-
lipase C addition led to a similar intracellular uorescent DAG
distribution. To rule out the involvement of cytosolic proteins that
may directly transport the lipid into the cytosolic membranes and a
possible lateral diffusion of the NBD-DAG along cytoplasmic mem-
branes in contact with or in close proximity of the plasma mem-
brane, these authors observed in articial lipid vesicles, mainly
composed of DOPC, a direct transbilayer movement of NBD-DAG,
but not NBD-PA. These results are in agreement with the transbilay-
er movement of endogenous DAG described previously in human
erythrocytes [51]. Additional studies in vesicles using 13C NMR
spectroscopy and other physical techniques show that the transbi-
layer movement of DAG compared to phospholipids is extremely
fast. Predictions from the chemical shift of the carbonyl 1,2-DAG
signal in PC vesicles containing 20% DAG estimated a DAG transbi-
layer movement rate of 63 s 1 (t1/2  11 ms at 38 C) [52].
Decreasing DAG concentration in the membrane accelerates the ex-
change rate of the molecule between the leaets.
Other studies using chemical tools estimate that the ip-op of
DAG occurs in the range of seconds [53] to 1 min [51], in any case
several orders of magnitude faster than that observed for phospho-
lipids. Compared to other hydrophobic molecules (i.e. alkanes,
detergents or proteins) that induce the scrambling of other lipids
after insertion in the membrane, it should be noted that DAG, which
can freely diffuse through the membrane and change its physical
properties, does not facilitate the transbilayer movement of other
lipids. Studies from our group have shown that increasing DAG levels
in the outer leaet either via phospholipase C treatment or by exter-
nal addition of DAG to preexisting vesicles does not induce transbi-
layer translocation of uorescent-labeled lipids within the
membrane [54].
5.2. Ceramide
Ceramide represents the backbone structure of most sphingoli-
pids and it also exists in free form, at low concentrations, in the cell
membranes. This small sphingolipid is produced in cell mem-
branes via direct degradation of sphingomyelin (SM) or ganglio-
sides. Recently much attention has focused on ceramide as a
potent biological molecule involved in several biochemical and cel-
lular responses to stress including, among others, apoptosis, senes-
cence, cell cycle arrest and differentiation [55,56]. In the last two
decades the role of ceramide in apoptosis has been intensively
studied. Results from different groups clearly show that ceramide
is involved in apoptosis, in that ceramide accumulation regulates
the execution phase of apoptosis involving mitochondria [55].
Recent data suggests that ceramide may act in changing the
physical state of the membrane. Various studies show that forma-
tion of ceramide in the membrane leads to the segregation of this
lipid into Cer-rich domains forming large platforms or rafts in
which different cell surface receptors oligomerize (i.e. Fas, CD40).
Formation of Cer-enriched domains has been intensively studied
in model membranes [57]. These rigid domains can be explained
by the strong inter-molecular interaction between ceramide
molecules, forming a compact membrane structure. London and
co-workers show that formation of Cer-rigid domains induces the
segregation of cholesterol from rafts [58,59]. The authors observed
that ceramide and cholesterol, in liquid-ordered (Lo) mixtures,
compete for SM-rich domains [60]. Additional studies described
the requirement of at least 8 methylene groups in the ceramide
N-acyl chain to segregate sterols from Lo domains (e.g. [61]). This
cholesterol displacement enables the formation of SM/Cer rigid
gel domains [62]. The stability of SM/Cer interactions in domains
has been intensively studied using electron spin resonance (ESR)
[63] and confocal uorescence microscopy [64].
Another dening characteristic of ceramide is the ability to af-
fect general membrane curvature and stability, probably because
of its intrinsic negative curvature, that facilitates formation of
non-lamellar structures [46,65]. It is thought that via the transient
formation of non-bilayer intermediates, ceramide induces vesicle
efux [66], membrane fusion [67] and vesicle budding [68]. Since
ceramide production takes place in the extracellular leaet, via
an exported sphingomyelinase, while proteins that interact with
ceramide are intracellular, several studies have emphasized the
ability of ceramide to diffuse through the membrane. For several
years a controversy has existed concerning the ability of natural
ceramide to freely diffuse across the membrane. Several groups re-
port that ceramide, after formation, is strictly localized for a con-
siderable time in the leaet where it is synthesized [69]. Other
studies postulate that the half-life for spontaneous bilayer transfer
of a BODIPY-Ceramide in PC vesicles is 22 min at 37 C [70]. Com-
pared to other lipid analogs, labeled with the same uorophore, the
spontaneous ceramide transfer across the membrane is faster than
that measured for other lipids containing a more bulky polar
group. Taking into account that the presence of a bulky uores-
cence group, more polar than a fatty acid, can inuence the rate
of transbilayer lipid movement in the membrane, observation of
unlabeled-ceramide diffusion across the membrane has been nec-
essary to understand the inuence of the uorophore on transbi-
layer movement.
Recently, Devauxs group investigated the transmembrane dif-
fusion of different unlabelled ceramide species in giant unilamellar
vesicles (GUVs) [71]. Ceramide was incorporated into the outer
leaet of GUVs and the transbilayer movement of ceramide in
the membrane was followed monitoring the GUV shape changes.
Note that insertion of a small fraction (1%) of exogenous lipid
in one of the membrane lipid monolayers can lead to clearly visible
changes in cell or vesicle shape [72]. Using this technique, the half-
life of ceramide ip-op was found to be 30 s at 37 C, one order of
magnitude lower than that observed with uorescence analogs.
Similar half-lives for ceramide diffusion were observed, using
spin-labeled ceramide analogues, in vesicles s1/2 = 1.1 min at
 F.-X. Contreras et al. / FEBS Letters 584 (2010) 17791786
20 C) and human erythrocyte membranes s1/2 = 1.1 min at 20 C).
Interestingly short-chain ceramide, compared to long-chain cera-
mide, can easily diffuse across erythrocyte membranes.
An additional study in GUVs containing liquid-ordered (Lo) and
liquid-disordered (Ld) domains, after SM to ceramide conversion
in situ by sphingomyelinase (SMase), shows a drastic decrease in
Lo domains followed by an increase in Cer-rich gel domains and
the generation of transitory nanoscale defects [73]. Formation of
structural defects is the result of an increase in the lateral surface
tension induced by the enzymatic activity of SMase in one leaet.
This surface tension, produced by the asymmetric ceramide forma-
tion in one leaet, can in turn induce shape changes and transitory
membrane rupture. Our group has also reported that ceramide for-
mation by SMase activity in one side of the membrane induces the
transbilayer lipid movement of other lipids in vesicle and erythro-
cyte membranes [74]. SMase activity induces the ip-op not only
of uorescence labeled phospholipids (PC, PE, PS), but also of nat-
ural gangliosides (i.e. GM3) containing a bulky polar group. Gangli-
oside diffusion across the membrane was investigated using a
novel method in which vesicles containing neuraminidase trapped
inside the liposomes were labeled on the outer leaet with GM3.
After SMase addition and SM hydrolysis, GM3 ops to the inner
leaet, to be hydrolyzed by neuraminidase (Fig. 1). During this pro-
cedure no neuraminidase efux from the vesicles was observed. In
this experiment ceramide formation (and presumably transbilayer
motion) facilitates the translocation of other lipids.
Fig. 1. Flip-op of gangliosides induced by sphingomyelinase activity in large
unilamellar vesicles. (A) Sphingomyelin hydrolysis by sphingomyelinase. Average
values S.E. (n = 4). (B) GM3 ganglioside hydrolysis by entrapped neuraminidase.
Average values S.E. (n = 5). Vesicle composition was SM:PE:Ch (2:1:1) + 10 mol%
GM3 on the outer leaet only [75].
1783
In our opinion, SMase induces the transbilayer movement of
lipids due, probably, to the intrinsic negative curvature of ceramide
and subsequent ability to promote non-lamellar phase structures
in the membrane. The presence of a non-lamellar lipid such as
Cer in one of the membrane monolayers destabilizes locally the bi-
layer. Cer could even induce transient non-lamellar structures
coexisting in a short time frame with lamellar structures in the
membrane; defects transiently present between both structures
can facilitate the motion of lipids across the leaets leading to a
more symmetric lipid distribution. The transient membrane de-
fects induced by ceramide formation in one leaet have also been
suggested by other investigators [73]. It remains to be claried if
the transient nanoscale pores observed by Devaux and co-workers
mostly at the interface between Lo and Ld domains, in the presence
of Cer-rich domains, and the corresponding lipid packaging defects
between both domains can facilitate the transbilayer movement of
other lipids in the membrane. Unfortunately observation of transi-
tory non-lamellar states or more stable transient pores induced by
ceramide using electron microscopy has not yet been possible.
In a parallel study we investigated the ability of ceramide,
added externally to pre-formed vesicles containing pyrene-la-
belled PC (pyr-PC) in the outer leaet, to promote this kind of lipid
translocation [54,74]. Insertion of long-chain ceramide into the
outer leaet induces the redistribution of pyr-PC into both mono-
layers resulting in an increase in monomer uorescence intensity
and a decrease in the excimer uorescence intensity (Fig. 2). In
the same study we demonstrated that short N-acyl chain cera-
mides (C2 and C6) have a lower ability to trigger ip-op between
monolayers. Short chain ceramides are used in cell biology to mi-
mic some endogenous ceramide effects (i.e. apoptosis, cell differ-
entiation), but are less potent than natural ceramides in
facilitating the lamellar-to-inverse hexagonal phase transition of
lipids [75]. This effect is related to the molecular geometry of
short-chain ceramides. In the latter ceramides a short N-acyl-chain
mismatches with the sphingosine backbone, leading to a smaller
diameter of its hydrophobic moiety section, compared to the diam-
eter of the polar group section. Therefore short-chain ceramides
have a molecular shape that induces positive curvature of the mon-
olayers, forming micelles that oppose the formation of inverted
hexagonal phases in the bilayer [76]. In contrast, long-chain cera-
mide has a shape that favors negative curvature and promotes
the formation of inverted hexagonal phases in the membrane. It
is important to stress that the low ability of short-chain ceramides
Fig. 2. Ceramide-induced transbilayer movement of pyr-PC in LUVs composed of
SM/PE/Ch. The original lipid concentration was 300 lM. The following additions
were made to 1 ml of the LUV suspension at time zero: 30 lM (nal concentration
in buffer) egg ceramide in 2 ll ethanol (curve A), 2 ll ethanol (curve B), 1.6 U
sphingomyelinase (curve C), and none (curve D). The data have been tted to
monoexponential curves of the form y = a (1 e bx)c [54].
1784 F.-X. Contreras et al. / FEBS Letters 584 (2010) 17791786

to promote lipid ip-op in membranes is also connected with

their less potent effect in ceramide-dependent signaling pathways
(i.e. apoptosis or cell differentiation) [77]. Dihydroceramide, the
saturated ceramide counterpart and the precursor of ceramide dur-
ing de novo synthesis, is unable to induce ip-op motion. This
inability is matched by the fact that the absence of the trans-D4
double bonds seems to transform the lipid into a less biologically
active molecule, incapable of reproducing most of the effects ob-
served with the unsaturated ceramide, even though its uptake
and metabolism is similar [77]. To mention but one example, dihy-
droceramide inhibits the apoptotic effect of ceramide in mitochon-
dria [78].

6. Molecular dynamics simulations of lipid ip-op

The mechanism of transmembrane diffusion of lipids in a pro-

tein-free membrane is poorly understood. However, it is assumed
that the diffusion of a lipid in a membrane lacking proteins takes
place as a single molecular event in which nearby lipids play an
important role [79,80]. Perturbation of the membrane physical
properties (i.e. protein insertion, ionic concentration imbalance
across the membrane, enzymes) induces defects in the membrane
that could allow the spontaneous formation of transitory water
pores, which would in turn facilitate the diffusion of lipids through
the hydrophobic membrane core [81,82]. These nanoscale water

Copyright 2007 American Chemical Society 2007

pores are the result of lipid mismatching within the membrane,
and they could be related to the transient structural defects men-
tioned in the previous section. As discussed above, ceramide pro-
duction could induce the formation of such transitory water
pores as a result of the bilayer destabilization caused by the pres-
ence of Cer in one of the monolayers.
Molecular dynamics simulations (MD) aid our understanding of
the mechanism associated with lipid ip-op [83]. MD simulations
that mimic the physiological condition of the cell provide evidence
that formation of defects in the membrane can lead to the genera-
tion of transient water pores. Once the water pore is created, the
spontaneous translocation of lipids within the membrane can oc-
cur (Fig. 3) [84]. Interestingly, the average time required for the
ip-op of a lipid across a water pore was found to be around
60 ns. Together with the high number of ip-op events observed
during the MD, these results suggest that translocation of a lipid
across the bilayer is a very fast process in which water pore forma-
tion is the rate-limiting step. It has also been suggested that the Fig. 3. Pore-mediated lipid ip-op: (A) 0 ps, (B) 43.85 ns, (C) 118.9 ns, (D)
translocation of one lipid mediated by this water pore can promote 122.4 ns, (E) 152.7 ns, (F) 204.65 ns, (G) 208.9 ns, and (H) 215 ns. Lipids (except
for the ip-opped one) are not shown; water is shown in red and white, acyl
the ip-op of other lipids. In order to keep the pore stable, diffu- chains of the ip-opped lipid are shown in yellow, and its choline and phosphate
sion of one lipid to the opposite leaet, at the edge of the pore, has groups are shown in orange and green, respectively. (Taken from Gurtovenko, A.A.
to be substituted by another lipid from the same leaet. In the and Vattulainen, I. (2002) Molecular mechanism for lipid ip-ops. J. Phys. Chem. B.
event that this new lipid reaches the opposite leaet, the substitu- 111, 1355413559).
tion process has to be repeated again. Moreover, from these simu-
lations, the authors observed that the number of ip and op
events were symmetric (similar number of ip and op events single molecule may diffuse to the opposite bilayer spontaneously,
were found). The transient water pores in the simulations by as a result of thermal motion. This may be a relatively frequent
Gurtovenko and Vattulainen [8184] may have their physical cor- event, as with diacylglycerol, or a rare one, as with phospholipids.
relates in the structural defects observed by Lpez-Montero et al. Transmembrane motion of a single lipid molecule may be enzyme-
[73], as suggested by the latter authors. catalyzed. Lipid scrambling however is somewhat of a precipitous
event, which requires the building-up of a certain amount of en-
ergy to occur.
7. Flip-op motion and bilayer scrambling The different effects of DAG and ceramide are rather clarifying
in this respect. Both molecules undergo spontaneous ip-op mo-
An overview of the results discussed above may clarify one as- tion although at widely differing rates, about 1 min 1 for DAG but
pect of transbilayer lipid motion that has remained less clear in the three orders of magnitude slower for ceramide. DAG ip-op is not
past, i.e. the difference between ip-op, or transbilayer motion, accompanied by bilayer scrambling, while both phenomena are de-
and lipid scrambling in bilayers. The difference is both conceptual tected with Cer.
and mechanistic. Conceptually, transbilayer lipid motion refers to One possible explanation, hinted at by Ruiz-Argello et al. [67]
the diffusion of single molecules, while lipid scrambling involves years ago, is that because of the fast exchange DAG does not give
necessarily a population of lipid molecules. Mechanistically, a rise to any signicant membrane asymmetry, thus the process does
 F.-X. Contreras et al. / FEBS Letters 584 (2010) 17791786
not involve other than thermal energy and it is not energically cou-
pled to any other event. Cer on the contrary has a much slower ip-
op rate. When Cer becomes incorporated in one of the membrane
leaets, a certain degree of membrane asymmetry occurs. This
asymmetry is both chemical (ceramide concentration at both sides
of the membrane are different) and mechanical. See [85] for a
recent review on ceramide-induced mechanical stress.
When ceramide, e.g. in organic solvent, is externally added to
a membrane suspension, so that it gets incorporated into the
outer monolayers, insertion of foreign ceramide molecules will
lead to an imbalance of lateral tensions of the inner and outer
monolayers, being higher in the outer monolayer. The same phe-
nomenon, with an opposite sign, occurs when SM molecules in
the outer monolayers are hydrolyzed by a sphingomyelinase giv-
ing rise to ceramide. In the latter case the outer leaet lateral
tension will decrease, because the area per molecule of ceramide
is smaller than that of SM. Moreover, the propensity of ceramide
to aggregate in the form of non-lamellar phases will also help in
destabilizing the bilayer. In any case, the asymmetric distribu-
tion of ceramide, which cannot be relaxed via rapid ip-op, will
generate a mechanical energy of bending, which will ultimately
lead to the local destabilization of the bilayer, and the resulting
lipid scrambling. When ceramide is incorporated in the lipid
mixture prior to liposome preparation, a symmetric distribution
of ceramide molecules in the two monolayers is achieved, and
no scrambling is observed under these conditions [54].
The same mechanism may explain the observed lipid scram-
bling caused by the asymmetric insertion of proteins, or of some
surfactants, e.g. lysophosphatidylcholine, that are known to have
slow ip-op rates. However, in the absence of specic ip-op
rate values, it cannot be ascertained whether all detergents cause
scrambling via the same mechanism. In their case, the spontane-
ous tendency to form lipid-detergent mixed micelles may provide
a specic mechanism for bilayer scrambling to occur.
Acknowledgements
This work was supported by grants from the Spanish Ministerio
de Ciencia e Innovacin No. BFU 2008-01637/BMC (A.A.), BFU
2007-62062 (F.M.G.) and from the Basque Government (IT 461-
07) (F.M.G.). F.X.C. is a postdoctoral fellow from Federation of Euro-
pean Biochemical Societies. L.S.M. is supported by a postdoctoral
grant from the Basque Government.
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