(Received for publication, June 7, 1995; and in revised form, January 9, 1996)
From the
A relatively rapid transbilayer motion of phospholipids in the
microsomal membrane seems to be required due to their asymmetric
synthesis in the cytoplasmic leaflet. Marked discrepancies exist with
regard to the rate and specificity of this flip-flop process. To
reinvestigate this problem, we have used both spin-labeled and
radioactively labeled long chain phospholipids with a new fast
translocation assay. Identical results were obtained with both types of
probes. Transbilayer motion of glycerophospholipids was found to be
much more rapid than previously reported (half-time less than 25 s) and
to occur identically for phosphatidylcholine, phosphatidylserine, and
phosphatidylethanolamine. Such transport is nonvectorial and leads to a
symmetric transbilayer distribution of phospholipids. In contrast,
transverse diffusion of sphingomyelin was 1 order of magnitude slower.
Phospholipid flip-flop appears to occur by a protein-mediated transport
process displaying saturable and competitive behavior. Proteolysis,
chemical modification, and competition experiments suggest that this
transport process may be related to that previously described in the
endoplasmic reticulum for short-chain phosphatidylcholine (Bishop, W.
R., and Bell, R. M.(1985) Cell 42, 51-60). The
relationship between phospholipid flip-flop and nonbilayer structures
occurring in the endoplasmic reticulum was also investigated by P-NMR. Several conditions were found under which the
P isotropic NMR signal previously attributed to nonbilayer
structures is decreased or abolished, whereas transbilayer diffusion is
unaffected, suggesting that the flip-flop process is independent of
such structures. It is concluded that flip-flop in the endoplasmic
reticulum is mediated by a bidirectional protein transporter with a
high efficiency for glycerophospholipids and a low efficiency for
sphingomyelin. In vivo, the activity of this transporter would
be able to redistribute all changes in phospholipid composition due to
biosynthetic processes between the two leaflets of the endoplasmic
reticulum membranes within a time scale of seconds.
The endoplasmic reticulum of eukaryotic cells is the site of synthesis of several phospholipids including phosphatidylcholine, phosphatidylethanolamine, and in part phosphatidylserine. These synthetic activities are mostly located on the cytoplasmic leaflet of the membrane(1, 2, 3) . Therefore translocation of newly synthetized phospholipids is likely to be necessary for proper biogenesis of the membrane. This suggest that a rapid transbilayer motion of phospholipids occurs in the endoplasmic reticulum(4) .
Many studies have been devoted to the measurement of this flip-flop activity in the ER(1) . Although all agree for a relatively rapid translocation of phospholipid in isolated microsomes as compared with many other membranes, discrepancies exist with regard to both the rate and the lipid specificity of the transverse diffusion process. Half-times ranging from 2-3 (5) to 45 min (6) have been measured using various methods. A much more rapid translocation of glycerophospholipids compared with sphingomyelin was found in one study (6) and not in another(7) .
The mechanism of this
rapid flip-flop is also a subject of controversy. Two proposals have
been made in this regard. Bishop and Bell (8) found that the
translocation of short chain PC is protein-mediated and
suggested the occurrence of a PC transporter in the endoplasmic
reticulum membrane. On the other hand Van Duijn et al.(9) have suggested that the nonbilayer structures present
in microsomal membrane as detected by
P-NMR (10) are responsible for the transverse diffusion.
In the
present study, we have reinvestigated the rate, selectivity, and
mechanism of the translocation of phospholipids in rough and smooth
endoplasmic reticulum membranes. For this purpose we have used both
spin-labeled and radioactive long chain phospholipids with various head
groups in conjunction with a new lipid transport assay adapted to the
measurement of rapid flip-flop processes. We have also studied in
parallel the involvement of nonbilayer lipid structure in the
microsomal membrane by P-NMR.
Liver microsomes were
prepared from male Wistar rats of 300-350-g body weight, which
were starved for 16 h prior to slaughtering but had full access to
water. Rough and smooth microsomes were isolated from rat liver as
described(16) . Membranes were resuspended at 1 mM MgSO, 50 mM Tris-HCl, 250 mM sucrose, pH 7.3, and pretreated with 4 mM diisopropylfluorophosphate before use in order to prevent
phospholipase A
activity. Protein concentration was
determined by the Coomassie Blue protein reagent (Pierce). The purity
of rough and smooth microsome fraction was assessed by the specific
activity of glucose-6-phosphatase (16) with inorganic phosphate
determination according to Rouser et al.(17) and
found to be similar to those of other preparations. The integrity of
microsomal membranes was checked through the mannose-6-phosphatase
activity in the presence and the absence of
taurocholate(5, 18) . A latency of 91% was found in
agreement with previous results(5, 6, 18) .
Figure 1:
Outside-inside transbilayer motion of
SL phospholipid in rough ER. Membranes (0.8 mgml
of protein) were incubated at 20 °C with 2% (mol/mol relative
to total phospholipid) of SL-PC (a), SL-PS (b), SL-PE (c), SL-SM (d), and SL-LPC (e). At desired
times, 50-µl aliquots were taken and submitted to the BSA
extraction-rapid filtration-ESR procedure described under
``Experimental Procedures'' in order to assay separately the
spin-label in the outer (closed symbols) and the inner leaflet (open symbols).
Fig. 2shows the dependence of the
initial rate of phospholipid translocation upon concentration. All
three glycerophospholipids show a similar saturable behavior with an
apparent K corresponding to a spin label mole
fraction of 0.03 with regard to total phospholipids. The catalytic
constants that can be calculated range from 37.5
nmol
min
mg
for SL-PC
and SL-PS to 45 nmol
min
mg
for SL-PE. On the other hand, SM shows no evidence for such
saturation. However, saturation would be difficult to detect due to the
low rates of SM translocation.
Figure 2:
SL phospholipid dependence of transbilayer
motion. Rough microsomes were incubated as in Fig. 1for 20 s in
the presence of varying amount of SL-PC (), SL-PS (
),
SL-PE (
), and SL-SM (
) and assayed for spin-label
transmembrane distribution. The solid lines are for SL-PC and
SL-SM. Initial rates were calculated from the amount of phospholipids
translocated in 20 s averaged from BSA and SDS extraction values and
measures in triplicate. The mannose-6-phosphatase latency of microsome
was measured to be 88% after labeling with 10% SL-PC
(mol/mol).
To investigate whether the
transbilayer diffusion process is vectorial, we also measured
transbilayer diffusion of phospholipids from the inner leaflet to the
outer leaflet of the rough endoplasmic reticulum membrane. Microsomal
membranes were labeled with SL-PE and incubated in order to reach a
stationary transbilayer distribution. BSA, which first extracted
external phospholipids, was then added. The BSA incubation was
continued for the indicated times in order to extract phospholipid back
translocated to the outer leaflet. The results are shown in Fig. 3for SL-PE and indicate that transbilayer diffusion occurs
with similar characteristics in both directions. At 1% SL-PE the rates
of out-in and in-out translocation were 16.8 ± 0.9 and 17.0
±.08
nmolmin
mg
,
respectively.
Figure 3:
Inside-outside transbilayer motion of SL
phospholipids. 500 µl of rough microsomes were labeled with SL-PE,
incubated, and assayed for phospholipid in the outer () and
inner (
) leaflet as in Fig. 1. Additionally, after 5.25 min
at 20 °C, a 5000-µl fraction of the membranes was mixed with
500 µl of BSA 20% (w/v) and further incubated. At desired times,
100-µl aliquots were taken and assayed for spin-labeled
phospholipid transmembrane distribution in the outer (
) and inner
leaflet (
) in parallel.
As shown in Fig. 4, the transbilayer diffusion
of the radioactive analogs, RL-PC and RL-SM, is identical to that of
the corresponding SL-analogs. As an example at a 0.02 mole fraction,
the rate of translocation of RL-PC and SL-PC were 22.1 ± 0.9 and
19.4 ± 1.0
nmolmin
mg
,
respectively. Fig. 5shows the results of competition
experiments in which translocation of RL phospholipids was studied in
the presence of SL phospholipids. In the presence of increasing
concentrations of SL-PE, the initial rate of transbilayer diffusion of
RL-PC was progressively slowed down indicating competition for
transport (Fig. 5a). A smaller but definite effect was
found for RL-PC in the presence of SL-SM, suggesting that competition
also occurs between these two phospholipids (Fig. 5b).
In contrast, no effect of SL-LPC on the flip-flop of RL-PC was found,
demonstrating an absence of competition between these two phospholipids (Fig. 5c). This also indicates that the effects shown
in Fig. 5(a and b) are indeed due to
competition and not to a membrane fluidity variation associated with
the spin label added in excess.
Figure 4:
Outside-inside transbilayer motion of RL
phospholipids in rough ER. The experiment was done identically as
described in the legend to Fig. 1except that 2% (mol/mol)
radioactive RL-SM (a) and RL-PC (b) were used and
assayed in the outer () and inner (
) leaflets by
scintillation counting.
Figure 5:
Competition between RL and SL
phospholipids. Membranes were labeled with 0.25% radioactively labeled
phospholipid alone or with 4% spin-labeled phospholipid, incubated, and
assayed for radioactive label transmembrane distribution. a,
RL-PC in the presence () and the absence (
) of SL-PE; b, RL-PC in the presence (
) and the absence (
) of
SL-SM; c, effect of SL-PC (
), SL-PE (
), SL-SM
(
), and SL-LPC (
) concentration upon the initial rates of
RL-PC translocation. Initial rates were calculated as described in the
legend to Fig. 2.
Figure 6:
Relationship between P-NMR
spectra and transverse diffusion in ER. Membranes were assayed for
P-NMR at 30 mg/ml (a, b, c, and d) and for transmembrane distribution (e, f, g, and h) of SL-PC and SL-SM in the outer (
,
) and the inner (
,
) leaflets at 0.8 mg/ml. a and e, rough ER membranes; b and f,
rough ER membranes in the presence of dibucaine; c and g, rough ER membranes pretreated with 3% (v/v)
Me
SO; d and h, smooth ER
membranes.
Two
distinct treatments of the microsomes were found to lead to a decrease
of the nonbilayer peak. This included addition of dibucaine as already
reported by De Kruijff et al.(21) and washing of the
membrane with 3% (v/v) MeSO. As shown in Fig. 6(b and c), the isotropic 100 Hz wide
peak almost completely disappeared in both treated membranes.
Me
SO treatment also led to the disappearance of the narrow
metabolic peaks, presumably due to transient permeabilization of the
vesicles. Despite of these NMR changes, we found that neither dibucaine
nor Me
SO treatment affected to any extent the transbilayer
diffusion of SL phospholipids as shown in Fig. 6(f and g) for SM and PE. Control enzymatic measurements indicated
that the integrity and the polarity of the vesicles was preserved at
90% after these treatments.
We have also compared rough and smooth
microsomes with regard to both P-NMR and phospholipid
flip-flop. The
P-NMR spectrum of smooth microsomes is
characterized by a much decreased nonbilayer peak as compared with
rough microsomes. In contrast, it was found that transbilayer diffusion
of SL phospholipids was identical in both microsomal fractions (compare Fig. 6, e and h).
The passive transverse diffusion of phospholipids in lipid bilayers or in most biological membrane usually occurs within time scales of hours or days (for review see Zachowski(22) ). All studies devoted to the measurement of flip-flop in ER membranes have indicated a more rapid process. However, half-times ranging from 3 to 45 min have been reported for PC. In addition, the transverse diffusion of SM has been found to be similar to that of PC in one study (7) and much slower in another(6) . Such discrepancies may in principle arise from two origins. The first is related to the type of phospholipid probe used for the assay of transbilayer diffusion. Although a few studies have used natural phospholipids(5, 6) , others have used either spin-labeled phospholipids carrying one short chain (7) or soluble phospholipids with two short chains(8, 23) . Although such probes probably behave qualitatively as natural phospholipids, quantitative differences in their transverse diffusion rates cannot be excluded. A second origin for the differences may come from the slow time scales of the methods used to assay phospholipid transport. Most approaches have measured the disappearance of external leaflet phospholipid using either BSA extraction or phospholipid exchange protein extraction followed by a slow separation step using centrifugation. An exception is the study by Bishop and Bell(8) , which used a rapid filtration assay to recover external soluble short chain phospholipids. However, it is also possible that such short chain phospholipids have slower transport rates than long chain phospholipids because their diffusion from the aqueous phase to the membrane may be a limiting factor.
In the
present study, we have attempted to take these two potential
limitations into account. First, we have used two types of phospholipid
probe carrying a long chain fatty acid at the sn-1 position
and a spin-labeled (7) or radioactive fatty acid at the sn-2 position. The fact that identical results are obtained
with both probes gives us confidence that these are relatively faithful
reporters of natural phospholipids. Second, in order to assay the
translocation of these membrane bound phospholipids with a rapid time
scale, we have adapted the standard BSA extraction procedure into a
rapid filtration assay having a time resolution of 30 s.
With these improvements, it is shown that for glycerophospholipids, the transbilayer diffusion is even more rapid than previously concluded. Indeed, half-times for diffusion of the order of 25 s were found for PC, PE, and PS. Considering that this value corresponds to the time resolution of our method, it is possible that this flip-flop might be even faster. This rate is about 10 times more rapid than the fastest transbilayer diffusion rates previously reported in the ER(8) . Such rapid flip-flop can occur in both directions with a similar efficiency.
Using identical spin-labeled phospholipids, Herrmann et al.(7) found much lower transverse diffusion rates of glycerophospholipids in ER, with half-times of 20 min at 37 °C. These author used a slow BSA extraction assay (1-min incubation followed by 2.5-min centrifugation) and measured only the disappearance of external leaflet phospholipids. To understand this discrepancy, we have performed control experiments using our method but with the 3-min incubation time of the labeled microsomes with BSA (data not shown). Under such conditions, an apparent half-time of 23 min for SL-PC flip-flop was found at 37 °C in agreement with Herrmann et al.(7) . This suggests that the apparently slow transverse diffusion reported by these authors was due to the use of a translocation assay with too long a time scale compared with the 25-30-s half-time of glycerophospholipid transverse diffusion in ER.
There appears to be little specificity in the very fast transverse diffusion of glycerophospholipids, with PE transport being slightly more rapid than PC and PS. This result is in contrast with the very high selectivity of the transbilayer diffusion of SM and LPC. SM diffuses transversally an order of magnitude slower than glycerophospholipids. The relative differences in rates found for PC and SM are in agreement with those found by Zilversmit et al.(6) . The other phospholipid that displays a slow flip-flop in the ER is the spin-labeled analog of LPC. LPC is an important intermediate in the phospholipid biosynthetic routes of the ER. Our results contrast with those of Kawashima and Bell(23) , who found a very rapid transport of a short chain LPC in microsomes. Of course, it cannot be excluded that the presence of the nitroxide group perturbs LPC transport, although this does not appear to be the case for glycerophospholipids.
This rapid flip-flop of glycerophospholipids has consequences in relation to the topography of phospholipids synthesis in ER. Bishop and Bell (8) pointed out that the rapid PC transport that they observed (half-time, 5 min) was sufficient to ensure an even biogenesis of both membrane leaflets in the ER, because it was more efficient than the main biosynthetic routes for phospholipids that occur on the cytoplasmic surface(24) . Our finding of an even faster flip-flop of all glycerophospholipids further suggests that transbilayer diffusion can in fact redistribute within seconds any change in composition due to the activity of biosynthetic enzymes. In agreement with this, several authors reported that appearance of phospholipids on one leaflet occurred very rapidly after their synthesis on the other leaflet(4) .
The fast
transverse diffusion of glycerophospholipids appears to promote a
nearly symmetric transmembrane distribution of all head group species
between the two leaflets. As pointed out by Herrmann et
al.(7) , the average size of microsomal vesicles
corresponds to a slight area difference between the two leaflets so
that a symmetric distribution corresponds to 45% of the
phospholipid on the internal leaflet. Our results indicate that PC, PS,
and PE are distributed respectively to 40, 40, and 45% in the inner
leaflet. These values may be slightly underestimated due to the
occurrence of a very limited back translocation during the
translocation assay (see ``Experimental Procedures'') as well
as to a possible leakiness of some of the membranes (in view of the 91%
mannose-6-phosphatase latency). Therefore our results indicate that the
glycerophospholipid transverse distribution is symmetric or nearly
symmetric in the ER. Studies that assayed the endogenous phospholipid
asymmetry in the ER found a more asymmetric distribution of
glycerophospholipids both in rough and smooth
ER(25, 26, 27) . The discrepancy may be
explained by the use of methods with a slow time scale in these reports
as compared with the very rapid flip-flop rates in the ER.
Our data
also allow us to have an insight into the molecular mechanism of the
rapid phospholipid flip-flop in the ER. The results described above
indicate that this mechanism displays a strong structural selectivity
for the glycerol backbone and for the -position but exhibits
little discrimination between different head groups. Furthermore, our
data show that this transbilayer motion is not a simple diffusion but a
transport process involving discrete sites. Indeed for all three
glycerophospholipids, a saturable behavior was observed as a function
of phospholipid concentration. Competition for transport could be
detected between different glycerophospholipids. Competition is also
observed between PC and SM, suggesting that the latter is also
transported by the same pathway, although with a much lower efficiency.
Indeed it must be emphasized that the diffusion of SM in ER remains
significantly faster than in other systems(12) . On the other
hand, no competition was found between glycerophospholipids and LPC.
These data confirm that a protein-mediated transport of phospholipids
occurs in the ER membrane. It appears to be an ATP-independent (data
not shown) bidirectional transport. Such a transport activity has
already been demonstrated by Bishop and Bell (8) with short
chain phospholipids and reconstituted in liposomes by Baker and
Dawidowicz(28) . Several of our results suggest that both
transport systems are related without definitely proving their
identity. Both appear to be sensitive to N-ethylmaleimide and
to trypsin although to a different extent. There exists a definite
inhibition of spin-labeled phospholipids transport in the presence of
diC
PC, suggesting competitive behavior. The limited extent
of this inhibition may be due to the fact that the membrane-bound state
of the SL phospholipids promotes a much higher affinity for the
transport sites than the soluble nature of diC
PC. Our data
do not constitute a definitive proof of the identity of these two
transporters. However, they suggest that the ``PC flippase''
transports all membrane glycerophospholipids at a time scale of the
order of seconds, as well as SM with a very low efficiency. On the
other hand we cannot confirm that the protein also transports LPC as
suggested by Kawashima and Bell(23) .
The enhancing effect
of trinitrobenzenesulfonic acid treatment of the ER on both the
intensity of the isotropic peak in the P-NMR spectrum and
the rate of PC flip-flop in the ER led Van Duijn et al.(9) to suggest that nonbilayer structures present in the
membrane were responsible for the rapid flip-flop. Such structures have
indeed been shown to enhance transbilayer diffusion in other
systems(29, 30) . In contrast, Bishop and Bell found
that trinitrobenzenesulfonic acid inhibits diC
PC transport.
Herrmann et al.(7) suggested that such nonbilayer
structures may occur in ER in conjunction with a protein. Here we have
found three situations where the
P-NMR peak, previously
attributed to nonbilayer structures, is severely reduced in intensity
but where both the rate and specificity of phospholipids transport are
unchanged as compared with intact ER. Reduction of the isotropic
component of
P-NMR spectra of ER by dibucaine had already
been reported (20) and attributed to the cone shape of this
molecule. The fact that Me
SO treatment removes the
isotropic structure peak in
P-NMR spectra of ER may be due
to the fact that a molecule responsible for the stabilization of such
structures is extracted from the membrane. This is currently under
investigation in our laboratory. The reduced isotropic
P-NMR peak in smooth ER membranes might indicate that it
is related to ribosome-membrane interactions. In any case, these data
indicate that the particular lipid organizations giving rise to this
P-NMR peak are not involved in the rapid flip-flop of
phospholipids in the ER membrane. Our data rather favor a purely
protein-mediated facilitated transport, selective for
glycerophospholipids.