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CELLULAR & MOLECULAR BIOLOGY LETTERS
Volume 10, (2005) pp 101 – 121
http://www.cmbl.org.pl
Received 6 July 2004
Accepted 2 December 2004
*Corresponding author, e-mail: gummadi@iitm.ac.in
Abbreviations used: PL – phospholipid; PC – phosphatidylcholine; PE –
phosphatidylethanolamine; PS – phosphatidylserine; ER – endoplasmic reticulum; APT
– aminophospholipid translocase; GPI – glycosylphosphatidylinositol; diC
4
PC –
dibutyrylphosphatidylcholine; BSA – bovine serum albumin; NEM – N-ethylmaleimide;
BMV – Bacillus megaterium vesicles; IIMV – inverted inner membrane vesicles; TNBS
–
trinitrobenzene sulfonic acid; PLA
2
– phospholipase A
2
; DPPC –
dipalmitoylphosphatidylcholine; DEPC – diethylpyrocarbonate; SL – spin labeled; PI –
phosphatidylinositol; PG – phosphatidylglycerol.
THE MYSTERY OF PHOSPHOLIPID FLIP-FLOP IN BIOGENIC
MEMBRANES
SATHYANARAYANA N. GUMMADI* and KRISHNA S. KUMAR
Department of Biotechnology, Indian Institute of Technology-Madras, Chennai
600 036, India
Abstract: Phospholipid flip-flop is required for bilayer assembly and the
maintenance of biogenic (self-synthesizing) membranes such as the eukaryotic
endoplasmic reticulum and the bacterial cytoplasmic membrane. Due to the
membrane topology of phospholipid biosynthesis, newly synthesized
phospholipids are initially located in the cytoplasmic leaflet of biogenic
membranes and must be translocated to the exoplasmic leaflet to give uniform
bilayer growth. It is clear from many studies that phospholipid flip-flop in
biogenic membranes occurs very rapidly, within a period of a few minutes.
These studies also reveal that phospholipid translocation in biogenic membranes
occurs bi-directionally, independently of the phospholipid head group, via a
facilitated diffusion process in the absence of metabolic energy input, and that
this type of transport requires specific membrane proteins. These translocators
have been termed biogenic membrane flippases, and they differ from metabolic
energy-dependent transporters (ABC transporters and MDR proteins). No
biogenic membrane flippases have been characterized. This review briefly
discusses the importance of biogenic membrane flippases, the various assay
methods used for measuring the rate of phospholipid flip-flop, and the progress
that has been made towards identifying these proteins.
Key Words: Phospholipids, Biogenic Membranes, Flippases, Bilayer Assembly,
Endoplasmic Reticulum, Bacterial Cytoplasmic Membranes
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INTRODUCTION
Cellular membranes, which are involved in various biochemical functions
including the transport and signalling mechanisms, are made of lipid bilayers.
Seventy-eight years ago, Gorter and Grendel put forward the idea that biological
membranes are based on a lipid bilayer [1]. Since then, important advances have
been made in the understanding of membrane structure, phospholipid (PL)
asymmetry, the organization of membrane embedded molecules and the
biosynthesis of membrane components. Despite the technological advancement
in molecular and cellular biology, the mechanism by which the assembly of the
PL bilayer of cellular membranes occurs is not clearly known, and
understanding the processes of membrane assembly remains a major challenge
in molecular cell biological research.
Fig. 1. Phospholipid flip-flop in biogenic membranes. (a) Phosphatidylcholine synthesis
on the cytoplasmic leaflet of the eukaryotic ER and the translocation of PC to the
opposite leaflet for membrane growth. (b) The synthesis of phosphatidylethanolamine
and phosphatidylserine on the cytoplasmic side of the bacterial inner membrane and its
flip-flop to the opposite leaflet. The double-headed arrows represent the rapid
translocation of phospholipids between the two leaflets of the membrane by flippase (a
membrane protein) without the requirement of metabolic energy. The question marks are
by way of illustrating that the biogenic membrane flippase has not yet been identified.
ER: endoplasmic reticulum; BCM: bacterial cytoplasmic membrane.
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It is a known fact that most of the glycerophospholipid-synthesizing enzymes in
eukaryotic cells are membrane-bound proteins located in the microsomal
fraction. These enzymes have their active sites oriented towards the cytoplasmic
leaflet. For example, cholinephosphotransferase, a membrane-bound enzyme
involved in the synthesis of phosphatidylcholine (PC), is present on the
cytoplasmic face of the endoplasmic reticulum (ER) (Fig. 1A). In the
prokaryotes, phosphatidylethanolamine (PE) is synthesized by two
cytoplasmically disposed enzymes, namely phosphatidylserine synthase and
phosphatidylserine decarboxylase (Fig. 1B). Due to the membrane topology of
PL biosynthesis, newly synthesized phospholipids are initially located in the
cytoplasmic leaflet of biogenic membranes and must be translocated to the
exoplasmic leaflet to permit uniform bilayer growth [2].
Thermodynamic analysis revealed that PL translocation is energetically
unfavorable because of the energy barrier to be overcome in translocating the
polar head group of the phospholipids through the hydrophobic interior of the
bilayer [2-4]. Consequently, PL translocation is slow in artificial liposomal
systems and in certain biomembranes, lasting a few hours to days [5-7].
However, it is clear from many studies that phospholipid flip-flop in the ER and
bacterial cytoplasmic membranes occurs very rapidly, taking a few seconds to
minutes [8-13]. These studies revealed that PL translocation in the ER and in
other biogenic membranes occurs bi-directionally independently of the PL head
group. It was also demonstrated that this transport requires specific membrane
proteins and does not require any metabolic energy.
Based on these results, it was hypothesized that was another class of lipid
translocators that facilitate the transbilayer movement of phospholipids in a
metabolic energy-independent fashion [2, 9, 10, 13]. These translocators are
named biogenic membrane flippases and they differ from metabolic energy-
dependent transporters. For example, eukaryotic plasma membranes are
equipped with special kinds of transporters that translocate phospholipids from
one leaflet to other at the expense of ATP hydrolysis (Fig. 2). In this class of PL
translocators are aminophospholipid translocase (APT), an inward-directed
pump specific for phosphatidylserine (PS) and PE [14-16], and the ABC family
of transporters, including the multi-drug resistance gene family of products,
which are ATP-driven, outward directed pumps [17-19]. Apart from these, a
special class of PL translocators exists, namely “scramblases”, which catalyze
the bi-directional, ATP-independent, Ca
2+
-dependent transport of phospholipids
resulting in their redistribution [20-22]. Even though ABC transporters,
scramblases and APT are attractive candidates for lipid flippases, they are
unlikely candidates for biogenic membrane flippases, as they require metabolic
energy or calcium for the rapid flip-flop of phospholipids. The importance of
these metabolic energy-dependent flippases was discussed in many reviews [2,
16, 22].
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Fig. 2. The transbilayer movement of phospholipids in cellular membranes. Three
classes of lipid translocators have been characterized so far. The gray double-headed
arrows represent bi-directional energy-independent Ca
2+
-dependent PL translocation
(scramblase), the black single-headed arrows represent vectorial, energy-dependent PL
translocation (aminophospholipid and the ABC family of transporters), and the black
double-headed arrows represent bi-directional energy-independent PL flip-flop (biogenic
membrane flippases).
Metabolic energy-dependent (ATP-dependent) lipid translocators are typically
located in the plasma membranes and secretory granule membranes of
eukaryotic cells, whereas biogenic membranes possess the metabolic energy-
independent translocators (Fig. 2). In addition, there is a possibility that
metabolic energy-dependent flippases might be present in biogenic membranes
as a consequence of trafficking through the ER to the plasma membrane.
However, no reports are available on the effect of the presence of these energy
dependent flippases on biogenic membranes during trafficking through the ER to
the plasma membrane. No biogenic membrane flippase have yet been identified.
In this review, the state of knowledge on biogenic membrane flippases is
discussed, including different assays used to measure flippase activity and the
latest progress towards identifying them.
IMPORTANCE OF BIOGENIC MEMBRANE FLIPPASES
PL and glycolipid translocation across bilayers was found to be important for a
number of biological processes [2]. For example, as discussed earlier, newly
synthesized phospholipids on the cytoplasmic leaflet of eukaryotic ER
membranes need to flip to the opposite leaflet for rapid bilayer growth [8, 11,
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13]. Also, during lipoprotein assembly, a continuous movement of lipids across
the ER and Golgi membranes is required for packaging PC with triglycerides
[22, 23]. Lipoproteins are critical components of the circulatory lipid-handling
apparatus, and their proper assembly is therefore important in reducing the
deposition of nutritionally acquired and endogeneously synthesized lipids into
plaques that block circulation. Hence, flipping PL across the ER and other
biogenic membranes is an important cellular process. The synthesis of
glycosylphosphatidylinositol (GPI) initially starts on the cytoplasmic side of the
ER, but the lipids must reach the lumenal side for GPI anchoring (Fig. 3) [2, 24-
26]. As indicated in Fig. 3, the synthesis of the GPI anchor has three flipping
steps in the ER; they involve glycolipids and phospholipids. The last step in the
synthesis of the GPI anchor is the flipping of PE from the cytoplasmic side to the
lumenal side, without which the GPI anchor is not synthesized. The absence of
GPI proteins leads to a chronic blood disorder (paroxysmal nocturnal
hemoglobinuria) characterized by fragile red blood cells [27]. GPI proteins form
the protective surface coats of parasitic protozoa such as trypanosomes. These
parasites cause deadly diseases like sleeping sickness and Chagas disease, which
Fig. 3. Different steps involved in the synthesis of the GPI anchor. It has been reported
that during the whole process of GPI anchor synthesis, at least 3 different flipping steps
are involved (indicated by the double-headed arrow): the flipping of (i) GluA-PI-PL, (ii)
dolicholphosphate mannose and (iii) phospahtidylethanolamine from the cytoplasmic
leaflet to the lumen of the endoplasmic reticulum. The double-headed arrows represent
rapid translocation of phospholipids between the two leaflets of the membrane by
flippase (membrane protein) without the requirement of metabolic energy. The question
marks are by way of illustrating that experimental evidence is required for the
involvement of biogenic membrane flippase, whereas experimental evidence is available
for the third flipping step (the flipping of PE) [10].
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in turn lead to heart diseases [28]. These studies showed that PL and glycolipid
flip-flop is fundamental to the processes of intracellular lipid traffic and
metabolism in cell. Apart from this, energy-independent translocation of
glycosphingolipids was observed in the Golgi [26]. These studies clearly
suggested the importance of PL translocation across the ER and other biogenic
membranes. In addition, energy-independent translocation was also observed
during the synthesis of glycolipids in the ER. These translocators will not be
discussed here in detail, since this review is focused on metabolic energy-
independent phospholipid flippases in biogenic membranes.
The asymmetry of PL biosynthesis in biogenic membranes is due to the fact that
PL-synthesizing enzymes are asymmetrically embedded in the membrane
bilayer. The concentration gradient generated by these newly synthesized
phospholipids is degenerated by rapid flip-flop. Flip-flop creates a dynamic
equilibrium PL distribution among the leaflets, thereby creating a different
composition of lipids in them. It was found that PC and sphingomyelin are
primarily located on the lumenal side, whereas aminophospholipids (PS & PE)
are located on the cytoplasmic side of the plasma membrane. However,
transbilayer asymmetry in biogenic membranes such as in the eukaryotic ER is
controversial since no clear picture of asymmetry is available [29]. PL
asymmetry has significant physiological consequences in processes such as
membrane budding and membrane protein function.
BIOCHEMICAL ASSAYS TO MONITOR PHOSPHOLIPID FLIP-FLOP
The development of suitable assays to measure PL translocation is crucial in
identifying biogenic membrane flippases. In the simplest assay format, a labeled
phospholipid or phospholipid analogue is inserted into one of the leaflets of the
PL bilayer, and its transbilayer orientation is evaluated after a suitable time
interval [30]. This transbilayer orientation of the phospholipid can be evaluated
in several ways, including via the derivation of amine-containing headgroups
with membrane-impermeant reagents and accessibility to exogenously applied
phospholipases or PL transfer proteins or PL exchange proteins [30, 31]. Most of
the assays developed so far to measure PL translocation involve the use of PL
analogues tagged with radioactive or fluorescent or spin label probes with a
certain degree of water solubility [2]. However, these PL analogues may not
perfectly mimic the behavior of natural long chain lipids. The different types of
PL-translocation assay currently available and described in the literature are
discussed in the following section.
ASSAYS BASED ON PHOSPHOLIPID ANALOGUES
Phospholipid flip-flop in the ER
Bishop and Bell (1985) developed an assay using a water-soluble phospholipid
analogue, dibutyrylphosphatidylcholine (diC
4
PC), to study the flippase activity
in rat liver microsomes [8]. This report was the first evidence for the existence of
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protein-facilitated PL translocation across the ER. A predetermined amount of
[
32
P]diC
4
PC was added to rat liver microsomes, and PL translocation was
measured as the redistribution of radioactivity between the extravesicular and
intravesicular space. The results showed that the transport was saturable, bi-
directional and time-dependent, and also dependent on the amount of
microsomes. The authors also reported that this transport led to an apparent
equilibration between the extravesicular space and vesicular lumen. Later,
Kawashima and Bell showed that a specific protein was involved in the
transmembrane movement of lysophosphatidylcholine in liver microsomes [32].
The results clearly indicated that the transport of monoC
4
PC was saturable and
dependent on time and the amount of microsomes. It was found that the
monoC
4
PC
transported
into
the
microsomes
was
degraded
to
glycerophosphorylcholine. In addition, those authors showed that the transport
of glycerophosphorylcholine was also saturable and dependent on time and the
amount of microsomes.
Backer and Dawidowicz reported on the reconstitution of PL flippase activity
from rat liver microsomes into lipid vesicles [9]. Biochemical reconstitution is
one of the powerful techniques available for the purification of integral
membrane proteins. In this method, the membranes are solubilized in a suitable
detergent and supplemented with exogenous phospholipids. This reconstitution
mixture is subjected to dialysis or treated with detergent-adsorbing beads to form
proteoliposomes. Using this technique, the authors reconstituted PC translocase
activity from the ER. It was found that flippase activity was not detected in the
reconstituted vesicles containing a microsomal lipid extract, suggesting that the
translocase activity is protein mediated. The abundance of flippase among the
total microsomal protein was estimated as 0.6% by weight. It was also reported
separately that most glycerophospholipid classes exhibit flip-flop in microsomes,
and that translocation rates differ depending on the type of phospholipid
(analogue), the temperature, and the method used to study the translocation
process [29].
Menon and co-workers used the assay developed by Bishop and Bell using
diC
4
PC as the transporter reporter to further characterize diC
4
PC transport in rat
liver microsomes [12]. The authors tested the PL translocation activity in the ER
by dissolving ER membranes in Triton X-100 and reconstituting them into egg
PC vesicles. Reconstituted proteoliposomes from detergent-solubilized ER
vesicles were reported to be active, whereas protein-free liposomes containing
ER lipids were found to be inactive. Furthermore, they reported that the slow
step in the equilibration of diC
4
PC between the extra- and intravesicular space
was due to the association of PL at the vesicle surface rather than the
transbilayer movement itself. The amplitude of the transport assay (the extent of
diC
4
PC uptake as a percentage of marker space) was found to be strongly
dependent on the protein/PL ratio in proteoliposomes up to 100 mg/mmol.
Beyond this ratio, the diC
4
PC uptake remained unchanged, indicating that at this
ratio, all the vesicles contained at least one flippase.
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The main disadvantage of an assay using a radiolabeled probe to measure the
rate of transport is the time resolution. For this purpose, many researchers used
fluorescent and spin-labeled PL analogues as transport reporters for their assays.
There are several published reports on fluorescence-based assays to measure PL
flip-flop [e.g. 33-36]. A fast bovine serum albumin (BSA) extraction method
combined with fast filtration was used to assay the translocation of short-chain
radio-labeled PE, PS, and PC, and their spin-labeled analogues across rat liver
microsomes [10]. Briefly, a concentrated solution of phospholipids was added to
microsomes, resulting in the incorporation of the lipids into the microsomes. The
labeled lipids from the outer leaflet were extracted with BSA, following which
the microsomes were isolated by manifold fast filtration. The amount of labeled
phospholipid still associated with the microsomes was quantified and considered
as the amount of flip-flop. Such results have been reported as identical for both
spin and radiolabeled probes. The half-time of PL translocation was reported as
25 s for all the glycerophospholipids tested; this is much faster than what was
reported earlier. The process of PL translocation was found to be bi-directional,
and partially protease and N-ethylmaleimide (NEM) sensitive.
Marx and co-workers developed a stopped flow assay with a high resolution to
characterize the rapid transbilayer movement of phospholipids in the ER [37].
The authors used both spin and fluorescent-labeled PL analogues. In this assay,
the extraction of analogues from the outer leaflet of the microsomes to the BSA
was monitored by measuring the change in paramagnetic resonance or
fluorescence. The time resolution of the assay revealed that the respective half-
time for the transbilayer movement of spin and fluorescent-labeled phospholipid
analogues was 3.5 and 9.5 s.
Phospholipid flip-flop in the bacterial cytoplasmic membrane
The bacterial cytoplasmic membrane is a site for the synthesis of phospholipids,
some of which need to be transported to the other leaflet to maintain uniform
bilayer growth. This transport of phospholipids is also mostly protein mediated,
albeit insensitive to NEM. Based on pulse-labeling studies, it was demonstrated
that the translocation of PE in the Gram-positive bacterium Bacillus megaterium
has a half-time of 3 min [38], and that the PL translocation is independent of
metabolic energy [39].
Hrafnsdóttir and co-workers showed the existence of PL flippase activity in the
cytoplasmic membranes of Bacillus megaterium vesicles (BMV) [34]. The
transport was monitored by measuring the increase in fluorescence as the
transport substrate departed the quenching environment of the donor vesicles.
The increase in fluorescence is due to two transport steps: (a) transfer from the
outer leaflet of the donor vesicles to the outer leaflet of the acceptor vesicles;
and (b) translocation across the membrane. In the case of PL translocation,
biphasic fluorescence traces were obtained. The results showed that PL
translocation in BMV occurs rapidly, with a half-time of 30 s.
As for the ER, diC
4
PC translocase activity has also been demonstrated for
Bacillus subtilis membranes [40]. The bacterial cytoplasmic membranes were
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109
solubilized in detergent and the extracts were reconstituted into proteoliposomes.
The flippase activity was measured using diC
4
PC transport across egg PC
vesicles. The half-time for PL translocation was found to be less than 1 min, and
PL translocation was linearly dependent on the protein/PL content in
reconstituted vesicles up to a ratio of 50 mg/mmol. Kubelt and co-workers also
reported on the transbilayer movement of fluorescent-labeled PL in the inverted
inner membrane vesicles (IIMV) of Escherichia coli [41]. PC and PE
translocation across the IIMV was measured using the fluorescence stopped-
flow technique and BSA back extraction. The half-times for PC and PE
translocation were found to be the same, at1 min. PL translocation in the IIMV
was found to be sensitive to protease, indicating that the rapid transbilayer
movement of PLs is protein mediated.
ASSAYS BASED ON NATURALLY OCCURRING PHOSPHOLIPIDS
The assays discussed so far dealt with the use of PL analogues, and in such
studies, the half-times for PL translocation were quite low. However, PL
analogues may not perfectly mimic the natural long-chain phospholipids. Few
reports are available on assays using natural phospholipids as transport reporters.
Nicolson and Mayinger reconstituted ER membrane proteins prepared from a
yeast microsomal detergent extract in proteoliposomes made of endogenous
aminophospholipids [42]. In this assay, the proteoliposomes or liposomes were
labeled with [
14
C]ethanolamine for 30 min at pH 5.7 and room temperature, and
treated with trinitrobenzene sulfonic acid (TNBS) for 10 min at pH 9.0 and 20ºC
to modify the labeled [
14
C]PE on the outer leaflet. It was observed that
[
14
C]ethanolamine was successfully incorporated into liposomes as [
14
C]PE at
pH 5.7, and that the labeling reaction can be separated from translocation of
[
14
C]PE by changing the pH from 5.7 to 7.4. These results clearly indicated that
the TNBS only reacted with PE on the outer leaflet, and the TNBS-resistant pool
of PE was sequestered to the inner leaflet of the liposomes. In addition, it was
found that a pool of [
14
C]PE unavailable for TNBS modification appeared when
experiments were performed with proteoliposomes derived from yeast
microsomes. The amount of [
14
C]PE unavailable for TNBS modification
depended on the protein content in the proteoliposomes. The half-time for PE
translocation was reported as 10 min. [
14
C] PE translocation has also been
reported as protein-mediated based on its sensitivity to protease.
Recently, a report was published on an assay to measure the translocation of
dipalmitoylphosphatidylcholine (DPPC) in proteoliposomes derived from
detergent extracts of rat liver ER [13, 43]. PL translocation was measured
depending on the ability of phospholipase A
2
(PLA
2
) to hydrolyze phospholipids
located in the outer leaflet of a vesicle membrane bilayer. It was found that PLA
2
rapidly hydrolyzed phospholipids on the outer leaflet of both proteoliposomes
and liposomes, with a half-time of 0.1 min). In the case of proteoliposomes, the
initial rapid hydrolysis was followed by a slower phase indicating the
translocation of phospholipids from the inner to outer leaflet. The amount of
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110
[
3
H] DPPC hydrolysis depended on the protein content in the reconstituted
vesicles up to a ratio of 60 mg/mmol of PL. The half-time for translocation of
[
3
H] DPPC was reported as 3.3 min.
Tab. 1 Summary of different assays used to measure biogenic membrane flippase
activity.
S.No. Transport reporter
Type of mem-
brane used to
measure activity
Source of
flippase
Important results
References
1
[
32
P] diC
4
PC
ER microsomes Rat liver ER
diC
4
PC transport was
inhibited by NEM and
TNBS, and was protease
sensitive. Transport rate is
2.7 nmol/min/mg
8
2
[
32
P] monoC
4
PC
and [
32
P]
glycerolphosphoryl
choline
ER microsomes Rat liver ER
monoC
4
PC-inhibited by
trypsin, NEM and TNBS
glycerolphosphoryl choline
not inhibited by trypsin,
NEM or TNBS
32
3
Brominated PC
(density labeled)
egg
PC:brominated
PC:egg PE
(1:2.5:1.2)
Rat liver ER
Insensitive to protein
modifying reagents. 15
min for complete
translocation of PC
9
4
[
14
C]-PC; (SL)-PC;
(SL)-PS
ER microsomes Rat liver ER Partially
protease
and
NEM sensitive. Half-time:
25 sec
10
5
C
6
NBD-PC;
C
6
NBD-PE;
(SL)-PC;
(SL)-PE
ER microsomes Rat liver ER
Half-time:
C
6
NBD-PC, C
6
NBD-PE:
9.5s
(SL)-PC, (SL)-PE: 3.5s
37
6
[
14
C]-PE
PC:PI:PE:PS
(40:28:12:7)
Yeast ER
Partially protease sensitive.
Half-time: 10 min
42
7
[
3
H]-DPPC
egg PC
Rat liver ER
Trypsin sensitive. Half-
time: 3.3 min
13
8
C
6
NBD-PC;
C
6
NBD-PE and
C
6
NBD-PG
Bacillus
megaterium lipid
vesicles
Bacillus
megaterium
cytoplasmic
membranes
Sensitive to protease,
Insensitive to NEM. Half-
time: 30s
34
9
[
3
H]diC
4
PC
egg PC
Bacillus
subtilis
membranes
Sensitive to protease. Half-
time: 1 min
40
10
C
6
NBD-PC;
C
6
NBD-PE
egg PC
Inverted inner
membrane
vesicles of
Escherichia
coli
Sensitive to protease. Half-
time: 1 min
41
11
[
3
H]diC
4
PC
egg PC
Rat liver ER
Sensitive to protease. Half-
time: 1 min
12
12
[
14
C]PE
Inner membranes
of Escherichia
Inner
membranes of
Escherichia
coli
Insensitive to sulfhydryl
reagents and protease.
Half-time:

"the work of transmembrane phosphatic acid my done by this"