Lipid
regulation
of cell membrane
structure
and function
PHILIP
L. YEAGLE
Department
of Biochemistry,
State
University
of New
ABSTRACT
Recent
studies
of structure-function
relationships
in
biological
membranes
have revealed
fundamental
concepts concerning
the regulation
of cellular
membrane
function
by membrane
lipids. Considerable
progress
has been made in understanding
the roles played by
two membrane
lipids:
cholesterol
and phosphatidylethanolamine.
Cholesterol
has been shown to regulate
ion pumps,
which in some cases show an absolute
dependence
on cholesterol
for activity. These studies suggest that an essential
role that cholesterol
plays in
mammalian
cell biology is to enable
crucial membrane
enzymes
to provide function
necessary
for cell survival.
Studies
of phosphatidylethanolamine
regulation
of
membrane
protein
activity
and regulation
of membrane
morphology
led to hypotheses
concerning
the
roles for this particular
lipid in biological
membranes.
New information
on lipid-protein
interactions
and on
the nature
of the lipid head groups has permitted
the
development
of mechanistic
hypotheses
for the regulation
of membrane
by phosphatidylin the lamellarnonlamellar
phase
transitions
of membrane
systems
containing
phosphatidylethanolamine,
or other lipids
with similar
properties,
have recently
been implicated
in facilitating
membrane
fusion.
Finally,
studies
of
transmembrane
movement
of lipids have provided
new
insight
into the regulation
of membrane
lipid asymmetry
and the biogenesis
of cell membranes.
These
kinds of studies are harbingers
of a new generation
of
progress
in the field of cell membranes.-YEAGLE,
P. L. Lipid regulation
of cell membrane
structure
and
function.
FASEBJ.
3: 1833-1842;
1989.
ethanolamine.
Key
group
lipid
bilayer
calcium
pump
Words:
cholesterol
protein
activity
In addition,
protein
.
intermediates
.
hydrophobic
hexagonal
region
(II)
.
membrane
phase
head
cell membrane fusion
STUDY
OF CELL
MEMBRANES,
their structure
and
function,
is a relatively
new field. Although
a number
of important
contributions
date back to the early days
of this century,
intense activity and progress
in this field
is marked
primarily
from the period of the late 1960s
THE
0892-6638l89/0003-1833/$o1.50.
© FASEB
York at Buffalo,
Buffalo,
New
York
14214,
USA
and early 1970s. Only recently has a clear picture of cell
membrane
structure
and function
emerged.
The major features
of cell membrane
structure
that
we know to be important
to their function
in living organisms have been described
in detail. For example,
all
cell membranes
contain a lipid bilayer. The structure
of
the lipid bilayer
is determined
in large part by the
hydrophobic
effect (which also controls
protein
structure).
In particular,
it is the repulsion
of the lipid
hydrocarbon
chains by the water structure
that drives
these chains
into an environment
sequestered
from
water. The amphipathic
structure
of the polar membrane lipids then directly determines
the bilayer structure by providing
a hydrophobic
environment
in the
middle of the bilayer for the hydrocarbon
chains,
with
the lipid polar head groups encountering
the aqueous
phase.
Cell membranes
usually work best when the lipid bilayer is in the liquid crystalline
state. As indicated
by
the term “liquid crystal’
the interior
of a lipid bilayer
is distinctly
different from liquid hydrocarbon,
although
both are hydrophobic.
Dynamically,
the lipid bilayer is
highly anisotropic;
much of the interior
of a bilayer is
well ordered,
and only a small region in the middle is
liquid-like.
The conformation
of the lipid hydrocarbon
chains (as well as the conformation
of many of the lipid
head groups)
in the bilayer are well described.
All cell membranes
contain
protein.
The membrane
proteins
may be integrated
into the lipid bilayer or may
simply be associated
with the membrane.
When
the
membrane
proteins
are integrated
into the bilayer, the
transmembrane
portions
of the protein
consist
predominantly
of hydrophobic
amino
acids, making
the
transmembrane
segment
compatible
with the hydrophobic
interior
of the lipid bilayer.
These transmembrane segments
may adopt the a-helical
conformation.
At the end of these hydrophobic
transmembrane
regions,
a relatively
high incidence
of charged
amino
acids is
often found.
As a result, the transmembrane
proteins
are firmly locked in their position
in the lipid bilayer by
the hydrophobic
effect;
the charged
regions
cannot
enter the hydrophobic
interior
of the membrane
and
the hydrophobic
portions
of the protein
are incompatible with water.
Lipids and proteins
can diffuse in the plane of the
membrane.
This may involve relatively
free diffusion
over long distances,
or the membrane
proteins
may be
1833
limited
to a finite region of the membrane
in which
diffusion
can occur. Alternatively,
some plasma
membrane proteins
may be anchored
to the membrane
skeleton and show little capability
of lateral diffusion.
Early in the modern
period of membrane
studies, the
fluid-mosaic
model was presented
to describe
a structure that was at once dynamic
yet ordered
(1). This was
an important
hypothesis
that directed
much work in the
field. More recently
we have come to understand
in
some depth just what this dynamic
yet ordered
membrane structure
is.
Most cell membranes
are not well described
by the illustrations
commonly
used to represent
membrane
structure.
The protein
content
is sufficiently
high in
most cellular membranes
so that the membrane
surface
area occupied
by the membrane
proteins
is as extensive
or more so as the surface area occupied
by the lipids in
the bilayer. It is estimated
that in many cellular membranes
only about
three layers of lipid separate
the
proteins
at the point
of closest approach
of nonaggregated
membrane
proteins.
Therefore,
most drawings of biological
membranes
show too much
lipid
bilayer.
Figure
1 is an attempt
to schematically
describe
a more accurate
relationship
between
the integral membrane
proteins
and the lipids in a cell membrane.
Both a hypothetical
side view and top view are
presented.
I
Figure
Vol. 3
May 1989
LIPID
REGULATION
PROTEIN
FUNCTION
OF
MEMBRANE
Lipids
and proteins
are known
to coexist
as closely
packed neighbors
in cell membranes.
The lipid composition of most cell membranes
is complex
and fairly
tightly
regulated
metabolically.
In this context,
one
might expect cell membrane
lipids to play a role in cell
membrane
protein
activity.
It has not proved
easy to
explore
this hypothesis,
however.
Membrane-bound
enzymes
have
extensive
hydrophobic
regions
and
usually
require
a lipid bilayer to maintain
activity.
In
some cases, detergent
micelles
can substitute
for the
lipid bilayer, protecting
the aqueous
media from contacting the hydrophobic
transmembrane
region of the
membrane
proteins.
Therefore,
separation
of the membrane enzymes
from their native environment
to identify the details of the lipid requirements
for an enzyme
is difficult and has hindered
progress
in this field. As a
result, an absolute
requirement
for a particular
lipid to
support
the activity of a membrane-bound
enzyme
has
been difficult to document.
A notable
exception
is /3hydroxybutyrate
dehydrogenase
(EC 1.1.1.30) for which
an absolute
requirement
for the choline head group has
been described
(3). In this section,
recent progress
in
understanding
the modulation
of membrane
proteins
by two membrane
lipids, cholesterol
and phosphatidylethanolamine,
will be discussed.
Cholesterol
1. Schematic
representation
of the relationship
between
the
amount
of lipid bilayer and the amount
of protein
in a typical cellular membrane.
Top shows the side view and the bottom
a surface
view of the membrane.
The membrane
lipids are represented
by the
balls with two chains attached
to each and the large Structures
are
the proteins.
The inner mitochondrial
membrane
would have an
even higher
protein
content
and thus less lipid bilayer
than this
figure represents.
The myelin membrane
would have a lower protein content
and more lipid bilayer than represented
in this figure.
1834
These
and other
structural
features
of cell membranes have been elaborated
over the past 2 decades of
research
(further
reading
and
references
in these
general areas can be found in ref 2). What is now coming into its own is the study of the mechanism
by which
structural
elements
of a cell membrane
are exploited
to
regulate
the function
of that cell membrane.
The following discussion
concentrates
on several representative
areas
of investigation
in which
recent
progress
has been made in uncovering
regulatory
relationships
between
cell membrane
structure
and cell
membrane
function.
In karticular,
regulation
of membrane function
by mefrtbrane
lipids is an area of intense
current
investigation
and will be emphasized
in this
review. For clarity, specific examples
will be used to IIlustrate
each point rather than a comprehensive
compendium
of all the papers published
on each subject.
modulation
of ion pumps
Cholesterol
modulation
of two ion pumps
has been
studied in detail by several groups of workers:
Na,KATPase
and
Ca2-ATPase.
The
Na,K-ATPase
of
plasma
membranes
is the enzyme
responsible
for
pumping
sodium out of the cell and potassium
into the
cell against their respective
concentration
gradients.
In
the erythrocyte
membrane,
the ratio is 3:2 (Na4/K),
so
that the pump is electrogenic.
These properties
place
this enzyme
in a central
role in a number
of cellular
processes,
including
sodium
cotransport
systems
and
the establishment
of electrical
potentials
along
the
plasma
membrane.
The FASEB Journal
YEAGLE
Several groups have studied
cholesterol
modulation
of the enzyme
from the human
erythrocyte.
Early reconstitution
experiments
hoted
an inhibition
of the
enzyme
by cholesterol
(4). Subsequently,
studies
of
NaF,K+ATPase
in human
erythrocyte
membranes
showed
inhibition
of activity
by high
levels
of membrane cholesterol
(5, 6).
These results were echoed in other membranes.
For
example,
in rabbit erythrocyte
membranes
(7), guinea
pig erythrocyte
membranes
(8), rat liver membranes
(9), and kidney basolateral
membranes
(10), high cholesterol
levels (above those found in the native membranes)
inhibit the ouabain-sensitive
ATP hydrolyzing
activity.
This inhibition
probably
resUlts from the general
physical
effects of cholesterol
on a membrane.
Cholesterol leads to an increase in the anisotropic
motional
ordering of the lipid bilayer of the membrane
due to the
effects
of its rigid sterol structure
on the lipid components of the membranes
(11). This general
increase
in
ordering
may also lead to an increase
in the ordering
of the conformation
of the Na,K-ATPase.
A reduction
in the capability
of the Na,K-ATPase
to undergo
conformational
transitions
would thereby
inhibit
its function, perhaps
by inhibiting
the E, to E2 conformational
change
suggested
to be integral
to its catalytic
cycle.
Cholesterol,
however,
has another
remarkable
effect
on the Na,K-ATPase
(10). When
low levels of cholesterol are present in the membrane,
cholesterol
stimulates the enzyme.
Stimulation
is not readily
explained
by a bulk cholesterol
effect on the membrane
lipid
properties,
because it is difficult to postulate
that inhibition of conformational
transitions
of the protein
might
both inhibit
and stimulate
the enzyme.
Furthermore,
the stimulation
was shown to be structurally
specific:
lanosterol
was less capable than cholesterol
of stimulating the enzyme and ergosterol
had virtually
no capability to stimulate
the enzyme.
This stimulatory
effect is
best explained
by a direct
sterol-protein
interaction,
with a site (or sites) on the enzyme
that would provide
the structural
specificity
and the mechanism
for stimulation of the enzyme
activity.
Further
experimentation
is needed
to test this hypothesis.
The acetylcholine
receptor
is another
example
of a
membrane
protein
that appears
to require
cholesterol
to function
properly
(12).
These results may provide a clue to the essential
role
of cholesterol
in mammalian
cells, which is that cholesterol
is required
for certain
important
membrane
functions.
This essential
role of cholesterol
has been
found at the cellular level (13). For example,
cells with
a requiremhnt
for exogenous
cholesterol
need at least
small amounts
of,the necessary
sterol to exhibit growth.
Although
other sterols may operate
synergistically
at
higher
sterol contents,
removal
of the specific
sterol
from the media inhibits
growth.
Other
sterols cannot
substitute
for this obligatory
requirement
at low sterol
content.
Therefore,
it appears
that recent evidence
supports
the hypothesis
that cholesterol-requiring
cells (such as
mammalian
cells) need cholesterol
to maintain
the acREGULATION OF CELL MEMBRANES
of enzymes
(such as the Na,K-ATPase
mentioned
above,
or protein
kinase)
(13) crucial
to the
growth and development
of the cell. The way in which
the cholesterol
requirement
is manifest
might
be
through
cholesterol-protein
interactions,
which
are
mediated
by cholesterol-specific
sites on the cholesterolsensitive
proteins.
The calcium
pump provides
another
interesting
example of cholesterol
modulation
of membrane-bound
enzyme
activity. The calcium
pump in question
is the
Ca2-ATPase
of the rabbit
fast twitch
muscle
sarcoplasmic
reticulum.
This enzyme
has been observed
to
optimally
pump two calcium
ions per ATP hydrolyzed
and can maintain
transmembrane
gradients
of three or
four orders of magnitude
in ion concentration.
This enzyme has been extensively
studied not only for its own
intrinsic
role in muscle contraction,
but also as a more
generic example
of an ion pump because of the success
of investigators
in a number
of laboratories
in reconstituting
the activity
of this enzyme
in bilayers
of
defined
lipid composition.
Early data suggested
that the level of cholesterol
in
the membrane
did not affect the activity of the calcium
pump. Although
this conclusion
was questioned,
subsequent work supported
the conclusion
that the calcium
pump protein is not sensitive to cholesterol.
The mechanism by which
the calcium
pump
was rendered
immune to the presence
of cholesterol
was suggested
to be
the exclusion
of cholesterol
from the immediate
vicinity
(lipid annulus)
of the protein
(14). This suggestion
is a
direct manifestation
of the hypothesis
that lipid effects
on membrane
proteins
are mediated
through
direct
binding
to the protein
of the lipid in question.
In reconstitution
experiments
in which phosphatidylethanolamine
(PE)’ was used as a dominant
lipid component
in the membrane,
cholesterol
appeared
to
stimulate
the calcium
pump (15). Thus,
under special
circumstances,
cholesterol
appears
capable of stimulating function
of the calcium
pump
protein.
It is not
known at this time whether
cholesterol
interacts
with or
binds to the calcium
pump protein
under these conditions of reconstitution.
Therefore,
the hypothesis
outlined above requires
further
testing.
tivity
PE regulation
of membrane
protein
activity
Phospholipids
as well as cholesterol
are capable
of
regulating
the activity of membrane
proteins.
One system, which has been studied
by several
groups
with
considerable
agreement
in results,
is the regulation
of
the
calcium
The first
activity
system
pump
report
protein
by PE.
of an effect of PE
on calcium
pump
was published
in 197 about a reconstituted
containing
soy PE and egg phosphatidylcholine
‘Abbreviations:
DGDG,
digalactosyldiglyceride;
ESR,
spin resonance;
MGDG,
monogalactosyldiglyceride;
nuclear magenetic resonance; PC, phosphatidylcholine;
phatidylethanolamine;
un, ROS, rod outer
PS, phosphatidylserine;
segment.
SPM,
electron
NMR,
PE, phossphingomye-
1835
(PC) (16). At low to moderate
PE levels, PE appeared
to stimulate
the calcium pump. However,
important
information
about the Structure
of the reconstituted
system and its lipid composition
was not available
then.
Subsequent
studies
(17) have shown
that important
nonbilayer
structures
could
form
from
membranes
con-
taining the lipid components
used in the 1975 reconstitution studies. In particular,
a substantial
region of the
phase diagram
of this lipid system represents
hexagonal
II phase, and other regions contain nonbilayer
isotropic
structures.
Therefore,
it was suggested
that
these
changes in structure
should be considered
in relation to
the noted effects of PE on the calcium
pump protein.
More recently,
two studies again pointed
to the role
of PE in stimulating
the calcium pump protein.
In one
study, the PE content
of sarcoplasmic
reticulum
membranes was altered by chemically
labeling
the PE head
groups (18). This chemical
alteration
led to a decrease
in calcium
pump activity. To the extent that this loss of
function
resulted
from the reduction
in PE (and not
from the introduction
of the chemical
label), this study
implied
a role of PE in the native membrane
on calcium pump function.
In the second study, reconstituted
membranes
were
used, and the increase
in pump activity with increase
in PE content
was once again demonstrated,
although
actual PE content
of the reconstituted
membranes
was
not reported
(19).
What mechanism
might be operating
to promote
calcium pump function
in the presence
of PE in the membrane?
The suggestion
that it was a preferential
and
specific interaction
between
PE and the calcium
pump
protein
was challenged
by the finding that monogalactosyldiglyceride
(MGDG)
also stimulated
the pump activity, analogous
to PE (19).
One property
that PE and MGDG
have in common
is the ability to form the hexagonal
(II) phase. Therefore, it was necessary
to investigate
whether
the formation of hexagonal
(II) phase
was important
to the
stimulation
of the calcium
pump.
Figure
2 shows an
example
of such a study in which the calcium
pump
was reconstituted
into membranes
containing
various
levels of PE. Two different
PEs were used. One would
undergo
the transition
to the hexagonal
(II) phase under
the conditions
of the experiments
and the other would
not. Both PEs stimulated
the calcium
pump. However,
the loss of transport
activity
when one of the systems
lost its bilayer structure
was apparent.
Therefore,
the
stimulation
appears
unrelated
to the formation
of the
hexagonal
(II) phase.
It is not clear why PE is capable of significant
stimulation of the calcium
pump
protein.
One possibility
that remains
to be investigated
is the role of the bilayer
surface in controlling
membrane
protein function.
The
surface of PE bilayers is distinctly
different
than that of
many other phospholipids.
The PE surface
is poorly
hydrated
and tends
to interact
with other
surfaces,
whether
they are on other bilayers or proteins,
rather
than
interact
directly
with the aqueous
phase
(see
below) (20). The possibility
of interactions
between
the
bilayer surface
and the extramembranous
portions
of
1836
Vol. 3
May 1989
50
40
CO
30
0.
to
0
20
10
0
0
1
0.5
PC/(PC+PE)
Figure 2. Calcium
taining Ca-ATPase
function of the PE
recovery of calcium
molar
ratio
uptake in reconstituted
membrane
vesicles confrom rabbit muscle sarcoplasmic
reticulum as a
content of the vesicles. Plot of the percentage of
uptake at 37#{176}C
as a function of the PC/(PC +PE)
for vesicles
reconstituted
with
transphosphatidylated
(from egg PC) PE/egg PC (El) or soybean PE/egg pc (U) lipid mixtures. Because of the greater level of unsaturation
in the soy PE,
lipid
mixtures
hexagonal
(II)
and higher,
with
phase
that
lipid
will undergo
the
transition
more readily.
At 75%
the predominant
form is the isotropic
lamellar-toPE content
structures
or the
hexagonal
(II) phase, and the vesicles can no longer trap calcium.
In the other reconstituted
membranes,
the system remains
lamellar
throughout
and the monotonic
increase
in the stimulation
by PE is
apparent
for all levels of PE content.
(From
K. -H. Cheng,
S. W.
Hui, and P. L. Yeagle, unpublished
results.)
the calcium pump protein should be examined
as a possible mechanism
for lipid regulation
of this enzyme.
LIPID-PROTEIN
INTERACTIONS
It is likely that the interaction
between
lipids and proteins in membranes
is one of the mechanisms
for the
regulation
of membrane
protein
function
by membrane
phospholipids.
This interaction
might
involve:
1) a specific binding
of the lipid to sites on the protein;
2) a more general,
nonspecific
interaction
such as has
been embodied
in the term “lipid annulus”;
or 3) a
surface-surface
interaction
involving
the surface of the
bilayer
and
the extramembranous
portion
of the
protein.
The first two concepts have been extensively
studied.
The following will review the current
state of this field.
Do membrane
lipids
bind
to membrane
proteins?
Observations
have been
reported
that suggest
that
membrane
lipids bind to membrane
proteins.
For example, glycophorin
from the human
erythrocyte
membrane is isolated with tightly bound lipids that cannot
be removed
without
extreme
conditions.
The lipids
bound
to glycophorin
appear
to be enriched
in the
phosphatidylinositols
(21). Cytochrome
oxidase
(EC
1.9.3.1) from mitochondria
is another
example
in which
tightly bound lipids that cannot be easily removed
are
found with the protein
after isolation.
In this case the
The FASEB Journal
VEAGLE
bound lipids are enriched
in diphosphatidylglycerol
or
cardiolipin
(22). Titration
of the calcium
pump protein
with phospholipids
indicates
that about 30 lipid molecules are required
for full activation
of the enzyme (23).
Magnetic
resonance
has been heavily
exploited
to
study the problem
of lipid binding
to membrane
proteins. The story has grown complex
as the number
of
studies has multiplied.
Many of the early studies used
electron
spin resonance
(ESR) and spin-labeled
lipids
(24). The ESR spectra
of these spin labels show two
spectral
components
in membranes
containing
some
membrane
proteins.
Such data were interpreted
in
terms of two lipid environments
in the membrane
in
the presence
of membrane
proteins.
One spectral component was similar, though not identical,
to the spectra
obtained
from spin labels in lipid bilayers without
protein. The other spectral
component
was representative
of a motion-restricted
lipid environment.
Model studies
suggested
that the motion-restricted
lipid environment
resulted
from the lipid encountering
the surface of proteins in the membrane.
The population
of the motionrestricted
lipid environment
was proportional
to the
protein
content
of the membrane.
Model calculations
suggested
that the population
of the motion-restricted
environment
was suitable
to form one ring of lipid
around
the protein.
(However,
because
recent studies
have indicated
that many membrane
proteins
exist as
dimers in the membrane
(25), the details of these calculations should be reexamined.
For example,
under conditions where the calcium
pump protein
is a dimer, the
population
of the motion-restricted
environment
is adequate to surround
the dimer with a single layer of lipid,
but not enough
to surround
a monomer
of the protein)
(26). From these studies came the concept of a lipid annulus: lipid in a special environment
produced
by the
membrane
protein
that is sufficient
to cover the hydrophobic
surface
of the protein.
After a period of these spin label studies,
2H-NMR
studies of 2H-labeled
lipids in membranes
containing
proteins
were used to examine
the same questions.
The
first such study suggested
that results similar to those of
the ESR studies would be obtained
(27). However,
this
was later retracted
in favor of results that revealed
only
one spectral component
(28). The presence
of one spectral component
was interpreted
variously
in terms of
1) no interactions
between
the lipids and the proteins
or 2) rapid exchange
of lipids between
sites next to the
protein
and sites in the lipid bilayer of the membrane
giving rise to a single, averaged
spectral
component.
Considering
the results
and conclusions
of the ESR
studies and the observations
of lipids tightly bound to
membrane
proteins,
the first alternative
is not a useful
interpretation.
The second interpretation
rested on the
absence of a second spectral component
in the 2H-NMR
spectra.
Subsequent
publications
demonstrated
that a
component
resulting
from lipids interacting
strongly
with membrane
proteins
would not normally
be visible
in the 2H-NMR
spectra
due to artifactual
loss of such
a component
in the collection
of the data (29-31).
Therefore,
the interpretation
of single-component
2HNMR spectra in terms of lipid-protein
interactions
was
RECULATION
OF CELL MEMBRANES
hazardous.
Another
approach
or approaches
to the
question
was required.
Phospholipid
head groups contain
the chemically
interesting
charged
structures
that might be expected
to
interact
with proteins
(for example,
choline
of acetylcholine that binds to the receptor
of the same name, is
also found in the head group of one of the common
phospholipids),
and so they are an important
region of
the lipid molecule
to explore for a better understanding
of lipid-protein
interactions.
31P-NMR
provides
an excellent, nonperturbing
way to study the behavior
of the
lipid head groups.
31P-NMR
has been used in the most recent studies
of lipid-protein
interactions
in biological
membranes
and reconstituted
systems (32-34).
In some cases, two
spectral
components
are observed.
One spectral
component is similar, though not identical,
to the spectrum
arising
from pure
phospholipid
bilayers.
The other
spectral
component
is characteristic
of a motionrestricted
phospholipid
head group environment.
These spectra can be deconvoluted
into two spectral
components
by subtraction
of a normal bilayer component from the spectrum
of the whole membrane.
The
resulting
difference
spectrum
is a broad (about twice as
broad
as a normal
phospholipid
bilayer
powder
pattern) but axially
symmetrical
powder
pattern
that is
characteristic
of axial rotation,
but indicative
of a substantially
more ordered
environment
than is found in
pure lipid bilayers.
This latter spectral
shape was observed
most
clearly
from
the phosphatidylinositol
tightly bound to glycophorin
reconstituted
as a proteinphospholipid
complex
in a glycolipid
bilayer
(35). In
this experiment,
only the phospholipid
that was tightly
bound to the protein contributed
to the 3tP-NMR
spectrum. So it was not necessary
to use any of the assumptions appropriate
when spectra
must be deconvoluted
to obtain the individual
components.
However,
deconvolution
(that is, subtraction
of the normal
phospholipid bilayer powder
pattern
from the total spectrum)
produced
the same (as in the case of reconstituted
glycophorin)
broad
axially
symmetrical
powder
pattern
of
protein-bound
phospholipid
from the 31P-NMR
spectrum of the native and functional
light sarcoplasmic
reticulum
membrane
(B. S. Selinsky
and P. L. Yeagle,
unpublished
results).
Therefore,
this broad (relative
to
pure phospholipid
bilayers) but axially symmetrical
3tP
powder pattern
may be representative
of the phospholipid head group environment
when phospholipids
are
bound to membrane
proteins
in biological
membranes.
Using magnetization
transfer
experiments,
the exchange
rate of phospholipids
between
the motionrestricted
environment
and the lipid bilayer
environment was explicitly
measured
in sarcoplasmic
reticulum in the only example
to date of a direct measurement of such lipid exchange
rates. An exchange
rate of
1 s1 was observed
(36).
These more recent experiments
appear
to complement the picture
of lipid-protein
interactions
derived
from the ESR data. Both approaches
identify a motionrestricted
lipid environment
induced
by some membrane proteins.
ESR data show that the environment
is
1837
enriched
in particular
phospholipids
in the case of
Na,K-ATPase
(37). 31P-NMR
studies have revealed a
modest
exchange
rate between
the two lipid environments
for the sarcoplasmic
reticulum,
whereas
for
human
erythrocyte
glycophorin,
the exchange
rate is
very slow.
On the basis of these studies, the possibility
of phospholipid
regulation
of membrane
protein
function
through
binding
to the membrane
protein
is still a viable hypothesis.
However,
the field has not progressed
sufficiently
to provide
many complete
examples
of this
hypothesis.
PROTEIN
REGULATION
OF
TRANSMEMBRANE
LIPID
DISTRIBUTION
One of the fascinating
mysteries
of membrane
biology
is the creation
and maintenance
of transmembrane
lipid asymmetry.
The erythrocyte
membrane
is the
best-documented
example
available
of the asymmetrical distribution
of phospholipid
classes (38). The consensus of published
data on the erythrocyte
membrane
is that the PE and phosphatidylserine
(PS) are located
on the inside (cytoplasmic
face) of the membrane
and
the sphingomyelin
(SPM) is located almost entirely
on
the exterior,
whereas
the PC is disproportionately
distributed
toward
the exterior
face of the membrane.
How these inhomogeneities
of phospholipid
distributions are created
or maintained
had not previously
been explained.
However,
it has been known for some
time that transmembrane
movement
of lipids must occur. The active sites of the enzymes
involved
in lipid
biosynthesis
are found on the cytoplasmic
face of the
endoplasmic
reticulum.
Therefore,
the newly synthesized lipid must be located initially on that cytoplasmic
face of the membrane,
and some select portion
of the
newly synthesized
pool must subsequently
be translocated to the lumenal
face of the endoplasmic
reticulum.
Several investigators
have explored
these important
issues and shed new light on the problem
of membrane
lipid asymmetry.
Recent
experiments
have revealed
that
proteins
probably
participate
in the transmembrane
movement
of lipids and in the maintenance
of the asymmetry
of
erythrocyte
and endoplasmic
reticulum
membranes.
Devaux
(39) observed
rapid movement
in the erythrocyte, outside to inside, of labeled phospholipid
analogs
of PE and PS. The movement
is dependent
on ATP.
The PE and PS analogs compete with each other for the
transport
system,
with a greater
affinity for the translocater shown by the PS analog. Sulfydryl
group modification inhibits the ability of the membrane
to promote
translocation
of the PE and PS analogs. Results such as
these suggest that PE and PS translocation
across the
erythrocyte
membrane
is enhanced
by a protein
in the
membrane.
PC and sphingomyelin
analogs translocate
much more slowly by a pathway
independent
of the
pathway for PE and PS translocation.
Thus, the affinity
of the transport
system for these phospholipid
analogs
appears to be in the order: PS > PE >> PC, SPM. Such
1838
Vol. 3
May 1989
a differential
affinity is helpful in explaining
the exclusive location
of PS and the nearly exclusive
location
of
PE on the inside of the erythrocyte
membrane.
The
proteins
involved
in this translocation
in the erythrocyte have not been isolated,
but preliminary
efforts in
this area are under way.
A novel assay was developed
to explore
the same
question
regarding
the
endoplasmic
reticulum
(40).
Previous
work showed
evidence
for relatively
rapid
transmembrane
movement
of phospholipids
in microsomal preparations
(41). More recently,
the activity
of
phospholipid
translocation
was reconstituted
into lipid
vesicles from rat liver microsomes.
Although
the protein likely to be involved has not yet been identified,
it
appears
that a new class of membrane
proteins
may be
involved
in facilitating
the translocation
of phospholipids across membranes.
The question
of which ones
are energy dependent
and which are not will have to be
addressed.
From a thermodynamic
view, translocation
processes
that are involved in the establishment
and/or
maintenance
of transmembrane
lipid asymmetry
presumably
require
cellular energy.
LIPID
REGULATION
MORPHOLOGY
OF
MEMBRANE
As indicated
at the beginning
of this review, it is presumed that biological
membranes
have a lipid bilayer
as their fundamental
structural
component.
The lipid
bilayer imparts
the essential permeability
control to the
cell. Passive permeability
through
a lipid bilayer is slow
for most solutes
(except
water),
so that membrane
transport
functions
control
the entry
and exit of
nutrients
from the cell. The loss of integrity
of the lipid
bilayer
would make the cell freely permeable
to all
solutes and lead to cell death.
The stability of the lipid bilayer is an important
issue
to explore
in this context.
It is well known that some
lipids can readily adopt nonlamellar
phase structures.
Lipids that promote
this behavior
are detrimental
to
cell viability,
although
they probably
have other important roles to play in cell membrane
function
(see the
discussion
of stimulation
of the Ca-ATPase
by PE, a
hexagonal
(II) phase-forming
lipid,
above).
In fact,
aberrant
lipid metabolism
could lead to an imbalance
between lamellar
and nonlamellar
phase-forming
lipids
in a membrane
and lead directly to a pathological
state
involving
membrane
degeneration.
It is important
then
to understand
how lipids regulate
the stability
of a cell
membrane
lipid bilayer.
The formation
of hexagonal
II phase by PE is a wellstudied phenomenon.
From its structure,
one can readily conclude
that the hexagonal
II phase would be disastrous to a cell membrane
function.
Three
issues have
been addressed
recently
concerning
this phenomenon
of nonlamellar
phase formation:
1) the driving
force for
forming
hexagonal
II phase; 2) how a membrane
might
adjust to maintain
bilayer
stability
in the face of the
potential
of nonlameilar
phase formation;
and 3) how
metabolic
events within the cell might lead to at least
The FASEB Journal
YEAGLE
transient
favorable
Forces
bilayer instabilities
biological
functions.
governing
hexagonal
that
(II)
may
phase
have
important
formation
The formation
of the hexagonal
(II) phase structure
has
been carefully
studied
by Gruner
and colleagues
(42)
and by Siegel (43), who introduced
the concept
of intrinsic
radius
of curvature,
R0, to describe
quantitatively the formation
of the cylinders
of the hexagonal
(II) phase. R0 reflects the radius of the channel
formed
by the close packing of the lipid head groups in the hexagonal (II) phase. Thus,
each phospholipid
has an intrinsic
radius
of curvature
that can be measured
by
X-ray diffraction.
Factors that affect the tendency
of a
phospholipid
dispersion
to enter
the hexagonal
(II)
phase have predictable
effects on R0.
A small value of R0 will favor the formation
of the
hexagonal
(II) phase. For example,
unsaturation
in the
lipid hydrocarbon
chains
favors the formation
of the
hexagonal
(II) phase, and the hexagonal
(II) phase is
favored
at higher
temperatures.
Conversely,
methylation of the amino function
will lead to an increase
in
R0 to the extent that R0 is nearly infinite for PC (i.e.,
three methyl groups),
which reflects the stability
of the
lamellar
phase formed
by PC.
This is part of the geometric
argument
that was advanced previously,
that PE is a wedge-shaped
molecule
(because
of its small head group),
and that wedgeshaped
molecules
pack well into the hexagonal
(II)
phase. However,
new theories
were called for when it
was reported
that some lipids with large head groups
also favored
the hexagonal
(II) phase (44). The latter
result suggested
that the relative hydration
of the head
group, or its ability to interact
with the aqueous
phase,
was an important
physical
property
to explore.
A complementary
thermodynamic
viewpoint
was developed
on the basis of related behavior
of hexagonal
(II) phase-forming
lipids (20). It was observed
that, in
the lamellar
phase, membranes
rich in PE tended to aggregate,
membrane
surface-to-membrane
surface,
to
partially
exclude water. This led to the conclusion
that
the surfaces
of PE bilayers
interacted
poorly with the
aqueous
phase
a good example
of the hydrophobic
effect.
It could
then be predicted
that altering
the
nature
of the aqueous
phase should alter the extent of
surface-surface
interactions
that were manifest
as vesicle aggregation.
Addition
of chaotropic
agents to the
aqueous
phase (which alter its nature)
also interrupted
the aggregation
of the PE vesicles.
Furthermore,
the
chaotropic
agents stabilized
the lamellar
phase of the
PE against
extremes
in temperature.
The tendency
to
form the hexagonal
(II) phase can then be understood
as a thermodynamic
consequence
of the poor interaction of PE with the aqueous
phase. Formation
of the
hexagonal
(II) phase severely limits the exposure
of the
lipid head groups to water (they are exposed only to the
small amount
of water within the tubes and the head
groups
are probably
more tightly packed than on the
surface of the lamellar
phase, again reducing
their ex-
REGULATION OF CELL MEMBRANES
posure to water).
Higher
temperature
or unsaturation
increases
the surface area each head group is forced to
occupy in the lamellar
phase, thereby
exacerbating
the
unfavorable
interaction
of the PE bilayer surface with
the water. This fundamental
physical
property
of PE,
manifest
as a modulation
of bilayer surface properties,
may be important
to its role in biological
membranes
in the larnellar
phase.
Biological
response
to hexagonal
(II)
phase
potential
laidlawii
have MGDG
and digalactosyldiglyceride
(DGDG)
as major lipid components.
These
lipids exhibit behavior
analogous
in some ways to PE
and PC. MGDG
is capable
of forming
the hexagonal
(II) phase,
as is PE. The head group
of MGDG
is
poorly hydrated,
as is PE. In contrast,
DGDG
is analogous in some ways to PC: It is more
extensively
hydrated
and is more stable in the lamellar
phase than
is MGDG
(45).
How do the Acholeplasma
respond
to stress induced
by
an increase
in growth temperature?
Increases
in temperature
will favor the hexagonal
(II) phase of MGDG.
Formation
of hexagonal
(II) phase would be disastrous
to the organism.
The response
of the organism
is to
decrease
the MGDG
and increase
the DGDG,
or to decrease the lipid favoring
the hexagonal
(II) phase and
increase the lipid favoring
the lamellar
phase. Structurally this alteration
in composition
will stabilize
the
lamellar
phase of the lipids in the membrane
against
the temperature-induced
tendency
to form the hexagonal (II) phase (46).
One of the big questions
in this field concerns
what
the hexagonal
(II) phase-forming
lipid is doing in biological membranes
if one of its effects is to destabilize
the structure
of those membranes.
This is currently
an
area of considerable
interest
and investigation.
Acholeplasma
Metabolic
regulation
of nonlamellar
phase
formation
A recent observation
in the area of bilayer stability
in
biological
membranes
is the ability of diacylglycerol
to
reduce
dramatically
the temperature
of the lamellarhexagonal
(II) phase transition
(47, 48). In some systems, diacylglycerol
levels as low as 1-3% (of the total
lipid content)
will reduce
the lamellar-hexagonal
(II)
phase transition
temperature
by tens of degrees centrigrade. This reduction
in the temperature
of the lamellarhexagonal
(II) phase boundary
can be understood
in
terms of a reduction
in R0. Therefore,
diacylglycerol
is
capable not only of activating
protein kinase C, but also
of disrupting
the lipid bilayer
of the membranes
in
which it is produced.
Diacylglycerol
is an intermediate
in lipid metabolism
and can be produced
by the action of phospholipase
C.
Phospholipase
C can be stimulated
by receptor
activation. The amount
of diacylglycerol
in the membrane
will regulate
the stability
of the lipid bilayer
of the
membrane.
Therefore,
a mechanism
for metabolic
regulation
of membrane
stability
through
lipid metab-
1839
olism is potentially
available
to the cell. As the discussion below suggests,
such a pathway
of regulation
may
be important
to biological
membrane
function.
Fusion of membranes
phase formation
and
regulation
by nonlamellar
Cell membrane
fusion is involved in a number
of cellular processes.
For example,
fusion is involved in receptormediated
endocytosis
and in intracellular
vesicular
transport;
it is involved
in secretion
and in viral infection of some enveloped
viruses.
At least three steps in the fusion process can be identified: 1) the close approach
of two membranes,
or
aggregation;
2) removal
of at least part of the water between the two membranes,
or partial dehydration;
and
3) a transient
destabilization
of the lipid bilayer structure to permit a partial,
short-lived
mixing of the structures of the individual
membranes
that can lead to
fusion of those membranes.
Lipid combinations
that produce
partial dehydration
of the membrane
surface and are capable
of introducing transient
nonlamellar
structures
into the lipid
bilayer might be expected
to enhance
the incidence
of
fusion. PE would be expected
to be a good candidate
in
this regard.
PE appears
to enhance
the rate of fusion in some
model membrane
systems. This may result in part from
the facilitation
by PE of the close apposition
of the two
membranes
to be fused.
However,
an important
new
role
that
PE may
play
in
the fusion event has been identified
(49). On comparison of the phase behavior
of a particular
lipid system,
it was suggested
that fusion was greatly
enhanced
by
the so-called
isotropic
structures
identified
in the 31PNMR
spectra
of that lipid system.
These
isotropic
structures
occurred
under the same conditions
as the
lipid particles in the freeze-fracture
electron microscopy
of the same preparations.
The data suggested
further
that if the system
became
hexagonal
(II), fusion no
longer occurred
and only extensive
leakiness
characterized the membranes
because
of the breakdown
of the
lipid bilayer. So the isotropic
lipidic particle appears
to
be an important
nonlamellar
structure
and may be a
candidate
for an intermediate
in the fusion event.
In this context,
the observations
reviewed
abovethat diacylglycerol
enhances
the formation
of the hexagonal (II) phase and concurrently
lowers the threshold
for formation
of the isotropic
structures
- suggest that
diacylglycerol
might
enhance
membrane
Once again there is the possibility
the regulation
of membrane
fusion
metabolic
events in the catabolism
lipids.
PROTEIN
REGULATION
MORPHOLOGY
OF
fusion
MEMBRANE
It was noted above that lipids and proteins
interact
each other in biological
membranes.
One possible
sequence
of such interactions
is that membrane
1840
Vol. 3
May 1989
(47).
of a mechanism
for
that involves known
of cell membrane
with
conpro-
teins might
influence
the morphological
behavior
of
membrane
lipids.
The retinal
rod outer
segment
(ROS)
disk membrane
provides
an interesting
example
of such
a
phenomenon.
The membranes
of the ROS disks are
rich in PE and in highly unsaturated
fatty acids. In
fact, the single largest population
of lipids in the disk
membrane
is PE and the most abundant
fatty acid is
22:6. It was no surprise then to observe that the isolated
disk lipids were able to enter the hexagonal
(II) phase
in the presence
of calcium
(50). What was surprising
was that the native membrane
containing
the photopigment rhodopsin
was stable to more extreme
stress (for
example,
higher
calcium
concentration)
than
that
necessary
to produce
hexagonal
(II) phase in the isolated lipids. In this case the membrane
proteins
appear
to stabilize
the lipid bilayer of that membrane.
SUMMARY
In this review, we have examined
some aspects of the
lipid regulation
of membrane
function.
The study of
cell membrane
structure
and function
has progressed
sufficiently
to begin examining
hypotheses
for the regulation of membrane
function
through
membrane
structural features.
Through
an examination
of regulation,
key issues of cellular
biology
begin
to become
unraveled.
Studies of cholesterol
regulation
of Na,K-ATPase,
as well as other membrane
proteins,
and of the regulation of cell growth have produced
clues to the role of
cholesterol
in mammalian
cell biology.
The available
cell biology and biochemistry
studies suggest that cholesterol is required
for the normal
function
of essential
membrane
enzymes.
This is a structurally
specific requirement.
For example,
in a cholesterol-requiring
cell,
ergosterol
cannot substitute
for cholesterol
as the essential sterol. The biochemical
basis of this is the dependence of crucial membrane
enzymes
on the presence
of
the essential
sterol in the membrane.
Without
the essential sterol, these crucial membrane
enzymes
cannot
function,
and without
their function,
the cell that requires those membranes
cannot grow, divide, or differentiate. What remains to be described
by future research
is the physical
basis for the biochemical
requirement
certain
membrane
enzymes
have for cholesterol.
The
structural
specificity
of the sterol requirement
suggests
that the essential
sterol binds to the enzyme
that requires it at a sterol-specific
binding
site on the protein.
The essential
sterol then activates
the enzyme
as a positive effector. However,
this physical mechanism
has yet
to be established.
Understanding
the role and mechanisms
by which
membrane
phospholipids
regulate
membrane
protein
activity presents
considerable
challenges.
Although
the
individual
species of lipids that are found in cellular
membranes
number
in the thousands,
the biological
reason for such variety has not been discovered.
And
the mechanism
by which regulation
of membrane
protein function
(which
has been observed
for several
lipids) occurs is still speculation.
Much more work is re-
The FASEB Journal
YEAGLE
quired.
Factors to consider
in such studies include
not
only specific lipid-protein
interactions
but also what individual
lipids or combinations
of lipids may do to the
surfaces of membranes
and to membrane
stability
and
internal
dynamics.
Studies
of the regulation
of lipid morphology
and
mechanisms
of membrane
fusion have led to new ideas
about the metabolic
control
of cellular
membrane
fusion. Intermediates
discovered
in the studies of phase
changes
between
lamellar
and nonlamellar
phases for
lipids
appear
to facilitate
membrane
fusion.
Other
studies have shown that products
(that can be generated
biologically
by receptor
excitation)
from lipid metabolism increase
the incidence
of the fusion-stimulating
structural
intermediates
in the lipid membrane.
What
remains
to be discovered
is whether
such a mechanism
exists in vivo for biological
control of membrane
fusion.
Finally,
research
on the influence
of phospholipid
metabolites
on the stability of lipid bilayers suggests the
existence
of regulatory
mechanisms
not only for normal states of a cell or tissue, but also for disease states.
If the balance between the lamellar phase and hexagonal
(II) phase is upset, then membrane
degeneration
is the
inevitable
result.
Cellular
destruction
would
be the
direct consequence
of such a disaster.
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