Lipid regulation of cell membrane structure

and function
PHILIP L. YEAGLE
Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214, USA

and early 1970s. Only recently has a clear picture of cell

ABSTRACT membrane structure and function emerged.
The major features of cell membrane structure that
Recent studies of structure-function relationships in we know to be important to their function in living or-
biological membranes have revealed fundamental con- ganisms have been described in detail. For example, all
cepts concerning the regulation of cellular membrane cell membranes contain a lipid bilayer. The structure of
function by membrane lipids. Considerable progress the lipid bilayer is determined in large part by the
has been made in understanding the roles played by hydrophobic effect (which also controls protein struc-
two membrane lipids: cholesterol and phosphatidyl- ture). In particular, it is the repulsion of the lipid
ethanolamine. Cholesterol has been shown to regulate hydrocarbon chains by the water structure that drives
ion pumps, which in some cases show an absolute de- these chains into an environment sequestered from
pendence on cholesterol for activity. These studies sug- water. The amphipathic structure of the polar mem-
gest that an essential role that cholesterol plays in brane lipids then directly determines the bilayer struc-
mammalian cell biology is to enable crucial membrane ture by providing a hydrophobic environment in the
enzymes to provide function necessary for cell survival. middle of the bilayer for the hydrocarbon chains, with
Studies of phosphatidylethanolamine regulation of the lipid polar head groups encountering the aqueous
membrane protein activity and regulation of mem- phase.
brane morphology led to hypotheses concerning the Cell membranes usually work best when the lipid bi-
roles for this particular lipid in biological membranes. layer is in the liquid crystalline state. As indicated by
New information on lipid-protein interactions and on the term liquid crystal the interior of a lipid bilayer
the nature of the lipid head groups has permitted the is distinctly different from liquid hydrocarbon, although
development of mechanistic hypotheses for the regula- both are hydrophobic. Dynamically, the lipid bilayer is
tion of membrane protein activity
by phosphatidyl- highly anisotropic; much of the interior of a bilayer is
ethanolamine. In addition, intermediates
in the lamellar- well ordered, and only a small region in the middle is
nonlamellar phase transitions of membrane systems liquid-like. The conformation of the lipid hydrocarbon
containing phosphatidylethanolamine, or other lipids chains (as well as the conformation of many of the lipid
with similar properties, have recently been implicated head groups) in the bilayer are well described.
in facilitating membrane fusion. Finally, studies of All cell membranes contain protein. The membrane
transmembrane movement of lipids have provided new proteins may be integrated into the lipid bilayer or may
insight into the regulation of membrane lipid asym- simply be associated with the membrane. When the
metry and the biogenesis of cell membranes. These membrane proteins are integrated into the bilayer, the
kinds of studies are harbingers of a new generation of transmembrane portions of the protein consist pre-
progress in the field of cell membranes.-YEAGLE, dominantly of hydrophobic amino acids, making the
P. L. Lipid regulation of cell membrane structure and transmembrane segment compatible with the hydro-
function. FASEBJ. 3: 1833-1842; 1989. phobic interior of the lipid bilayer. These transmem-
brane segments may adopt the a-helical conformation.
Key Words: lipid bilayer . hydrophobic region . membrane At the end of these hydrophobic transmembrane regions,
cholesterol calcium pump hexagonal (II) phase head a relatively high incidence of charged amino acids is
group protein . cell membrane fusion 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 STUDY OF CELL MEMBRANES, their structure and the hydrophobic portions of the protein are incompati-
function, is a relatively new field. Although a number ble with water.
of important contributions date back to the early days Lipids and proteins can diffuse in the plane of the
of this century, intense activity and progress in this field membrane. This may involve relatively free diffusion
is marked primarily from the period of the late 1960s over long distances, or the membrane proteins may be

0892-6638l89/0003-1833/$o1.50. FASEB 1833

limited to a finite region of the membrane in which These and other structural features of cell mem-
diffusion can occur. Alternatively, some plasma mem- branes have been elaborated over the past 2 decades of
brane proteins may be anchored to the membrane skel- research (further reading and references in these
eton and show little capability of lateral diffusion. general areas can be found in ref 2). What is now com-
Early in the modern period of membrane studies, the ing into its own is the study of the mechanism by which
fluid-mosaic model was presented to describe a struc- structural elements of a cell membrane are exploited to
ture that was at once dynamic yet ordered (1). This was regulate the function of that cell membrane.
an important hypothesis that directed much work in the The following discussion concentrates on several rep-
field. More recently we have come to understand in resentative areas of investigation in which recent
some depth just what this dynamic yet ordered mem- progress has been made in uncovering regulatory rela-
brane structure is. tionships between cell membrane structure and cell
Most cell membranes are not well described by the il- membrane function. In karticular, regulation of mem-
lustrations commonly used to represent membrane brane function by mefrtbrane lipids is an area of intense
structure. The protein content is sufficiently high in current investigation and will be emphasized in this
most cellular membranes so that the membrane surface review. For clarity, specific examples will be used to II-
area occupied by the membrane proteins is as extensive lustrate each point rather than a comprehensive com-
or more so as the surface area occupied by the lipids in pendium of all the papers published on each subject.
the bilayer. It is estimated that in many cellular mem-
branes only about three layers of lipid separate the LIPID REGULATION OF MEMBRANE
proteins at the point of closest approach of nonag-
PROTEIN FUNCTION
gregated membrane proteins. Therefore, most draw-
ings of biological membranes show too much lipid Lipids and proteins are known to coexist as closely
bilayer. Figure 1 is an attempt to schematically packed neighbors in cell membranes. The lipid compo-
describe a more accurate relationship between the in- sition of most cell membranes is complex and fairly
tegral membrane proteins and the lipids in a cell mem- tightly regulated metabolically. In this context, one
brane. Both a hypothetical side view and top view are might expect cell membrane lipids to play a role in cell
presented. 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 con-
tacting the hydrophobic transmembrane region of the
membrane proteins. Therefore, separation of the mem-
brane enzymes from their native environment to iden-
tify 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 /3-
hydroxybutyrate 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 phosphatidyl-
ethanolamine, will be discussed.

I Cholesterol modulation of ion pumps

Cholesterol modulation of two ion pumps has been
studied in detail by several groups of workers: Na,K-
ATPase and Ca2-ATPase. The Na,K-ATPase of
plasma membranes is the enzyme responsible for
Figure 1. Schematic representation of the relationship between the pumping sodium out of the cell and potassium into the
amount of lipid bilayer and the amount of protein in a typical cellu- cell against their respective concentration gradients. In
lar membrane. Top shows the side view and the bottom a surface the erythrocyte membrane, the ratio is 3:2 (Na4/K), so
view of the membrane. The membrane lipids are represented by the
that the pump is electrogenic. These properties place
balls with two chains attached to each and the large Structures are
the proteins. The inner mitochondrial membrane would have an
this enzyme in a central role in a number of cellular
even higher protein content and thus less lipid bilayer than this processes, including sodium cotransport systems and
figure represents. The myelin membrane would have a lower pro- the establishment of electrical potentials along the
tein content and more lipid bilayer than represented in this figure. plasma membrane.

1834 Vol. 3 May 1989 The FASEB Journal YEAGLE

 Several groups have studied cholesterol modulation tivity of enzymes (such as the Na,K-ATPase men-
of the enzyme from the human erythrocyte. Early re- tioned above, or protein kinase) (13) crucial to the
constitution experiments hoted an inhibition of the growth and development of the cell. The way in which
enzyme by cholesterol (4). Subsequently, studies of the cholesterol requirement is manifest might be
NaF,K+ATPase in human erythrocyte membranes through cholesterol-protein interactions, which are
showed inhibition of activity by high levels of mem- mediated by cholesterol-specific sites on the cholesterol-
brane cholesterol (5, 6). sensitive proteins.
These results were echoed in other membranes. For The calcium pump provides another interesting ex-
example, in rabbit erythrocyte membranes (7), guinea ample of cholesterol modulation of membrane-bound
pig erythrocyte membranes (8), rat liver membranes enzyme activity. The calcium pump in question is the
(9), and kidney basolateral membranes (10), high cho- Ca2-ATPase of the rabbit fast twitch muscle sarco-
lesterol levels (above those found in the native mem- plasmic reticulum. This enzyme has been observed to
branes) inhibit the ouabain-sensitive ATP hydrolyzing optimally pump two calcium ions per ATP hydrolyzed
activity. and can maintain transmembrane gradients of three or
This inhibition probably resUlts from the general four orders of magnitude in ion concentration. This en-
physical effects of cholesterol on a membrane. Choles- zyme has been extensively studied not only for its own
terol leads to an increase in the anisotropic motional or- intrinsic role in muscle contraction, but also as a more
dering of the lipid bilayer of the membrane due to the generic example of an ion pump because of the success
effects of its rigid sterol structure on the lipid compo- of investigators in a number of laboratories in recon-
nents of the membranes (11). This general increase in stituting the activity of this enzyme in bilayers of
ordering may also lead to an increase in the ordering defined lipid composition.
of the conformation of the Na,K-ATPase. A reduction Early data suggested that the level of cholesterol in
in the capability of the Na,K-ATPase to undergo con- the membrane did not affect the activity of the calcium
formational transitions would thereby inhibit its func- pump. Although this conclusion was questioned, subse-
tion, perhaps by inhibiting the E, to E2 conformational quent work supported the conclusion that the calcium
change suggested to be integral to its catalytic cycle. pump protein is not sensitive to cholesterol. The mech-
Cholesterol, however, has another remarkable effect anism by which the calcium pump was rendered im-
on the Na,K-ATPase (10). When low levels of cho- mune to the presence of cholesterol was suggested to be
lesterol are present in the membrane, cholesterol stimu- the exclusion of cholesterol from the immediate vicinity
lates the enzyme. Stimulation is not readily explained (lipid annulus) of the protein (14). This suggestion is a
by a bulk cholesterol effect on the membrane lipid direct manifestation of the hypothesis that lipid effects
properties, because it is difficult to postulate that inhibi- on membrane proteins are mediated through direct
tion of conformational transitions of the protein might binding to the protein of the lipid in question.
both inhibit and stimulate the enzyme. Furthermore, In reconstitution experiments in which phosphatidyl-
the stimulation was shown to be structurally specific: ethanolamine (PE) was used as a dominant lipid com-
lanosterol was less capable than cholesterol of stimulat- ponent in the membrane, cholesterol appeared to
ing the enzyme and ergosterol had virtually no capabil- stimulate the calcium pump (15). Thus, under special
ity to stimulate the enzyme. This stimulatory effect is circumstances, cholesterol appears capable of stimulat-
best explained by a direct sterol-protein interaction, ing function of the calcium pump protein. It is not
with a site (or sites) on the enzyme that would provide known at this time whether cholesterol interacts with or
the structural specificity and the mechanism for stimu- binds to the calcium pump protein under these condi-
lation of the enzyme activity. Further experimentation tions of reconstitution. Therefore, the hypothesis out-
is needed to test this hypothesis. lined above requires further testing.
The acetylcholine receptor is another example of a
membrane protein that appears to require cholesterol PE regulation of membrane protein activity
to function properly (12).
These results may provide a clue to the essential role Phospholipids as well as cholesterol are capable of
of cholesterol in mammalian cells, which is that cho- regulating the activity of membrane proteins. One sys-
lesterol is required for certain important membrane tem, which has been studied by several groups with
functions. This essential role of cholesterol has been considerable agreement in results, is the regulation of
found at the cellular level (13). For example, cells with the calcium pump protein by PE.
a requiremhnt for exogenous cholesterol need at least The first report of an effect of PE on calcium pump
small amounts of,the necessary sterol to exhibit growth. activity was published in 197 about a reconstituted
Although other sterols may operate synergistically at system containing soy PE and egg phosphatidylcholine
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. Abbreviations: DGDG, digalactosyldiglyceride; ESR, electron
spin resonance; MGDG, monogalactosyldiglyceride; NMR,
Therefore, it appears that recent evidence supports
nuclear magenetic resonance; PC, phosphatidylcholine; PE, phos-
the hypothesis that cholesterol-requiring cells (such as phatidylethanolamine; PS, phosphatidylserine; SPM, sphingomye-
mammalian cells) need cholesterol to maintain the ac- un, ROS, rod outer segment.

REGULATION OF CELL MEMBRANES 1835

(PC) (16). At low to moderate PE levels, PE appeared 50
to stimulate the calcium pump. However, important in-
formation about the Structure of the reconstituted sys-
40
tem and its lipid composition was not available then.
Subsequent studies (17) have shown that important
nonbilayer structures could form from membranes con- CO 30

0.
taining the lipid components used in the 1975 reconsti-
tution studies. In particular, a substantial region of the to
0 20
phase diagram of this lipid system represents hexagonal
II phase, and other regions contain nonbilayer isotropic
structures. Therefore, it was suggested that these 10
changes in structure should be considered in relation to
the noted effects of PE on the calcium pump protein.
0
More recently, two studies again pointed to the role 0 0.5 1
of PE in stimulating the calcium pump protein. In one
study, the PE content of sarcoplasmic reticulum mem- PC/(PC+PE)
branes was altered by chemically labeling the PE head Figure 2. Calcium uptake in reconstituted membrane vesicles con-
groups (18). This chemical alteration led to a decrease taining Ca-ATPase from rabbit muscle sarcoplasmic reticulum as a
in calcium pump activity. To the extent that this loss of function of the PE content of the vesicles. Plot of the percentage of
recovery of calcium uptake at 37#{176}C
as a function of the PC/(PC +PE)
function resulted from the reduction in PE (and not
molar ratio for vesicles reconstituted with transphosphatidylated
from the introduction of the chemical label), this study (from egg PC) PE/egg PC (El) or soybean PE/egg pc (U) lipid mix-
implied a role of PE in the native membrane on cal- tures. Because of the greater level of unsaturation in the soy PE,
cium pump function. lipid mixtures with that lipid will undergo the lamellar-to-
In the second study, reconstituted membranes were hexagonal (II) phase transition more readily. At 75% PE content
used, and the increase in pump activity with increase and higher, the predominant form is the isotropic structures or the
in PE content was once again demonstrated, although hexagonal (II) phase, and the vesicles can no longer trap calcium.
In the other reconstituted membranes, the system remains lamellar
actual PE content of the reconstituted membranes was
throughout and the monotonic increase in the stimulation by PE is
not reported (19). apparent for all levels of PE content. (From K. -H. Cheng, S. W.
What mechanism might be operating to promote cal- Hui, and P. L. Yeagle, unpublished results.)
cium pump function in the presence of PE in the mem-
brane? The suggestion that it was a preferential and
specific interaction between PE and the calcium pump the calcium pump protein should be examined as a pos-
protein was challenged by the finding that monogalac- sible mechanism for lipid regulation of this enzyme.
tosyldiglyceride (MGDG) also stimulated the pump ac-
tivity, analogous to PE (19). LIPID-PROTEIN INTERACTIONS
One property that PE and MGDG have in common
is the ability to form the hexagonal (II) phase. There- It is likely that the interaction between lipids and pro-
fore, it was necessary to investigate whether the forma- teins in membranes is one of the mechanisms for the
tion of hexagonal (II) phase was important to the regulation of membrane protein function by mem-
stimulation of the calcium pump. Figure 2 shows an brane phospholipids. This interaction might involve:
example of such a study in which the calcium pump 1) a specific binding of the lipid to sites on the protein;
was reconstituted into membranes containing various 2) a more general, nonspecific interaction such as has
levels of PE. Two different PEs were used. One would been embodied in the term lipid annulus; or 3) a
undergo the transition to the hexagonal (II) phase under surface-surface interaction involving the surface of the
the conditions of the experiments and the other would bilayer and the extramembranous portion of the
not. Both PEs stimulated the calcium pump. However, protein.
the loss of transport activity when one of the systems The first two concepts have been extensively studied.
lost its bilayer structure was apparent. Therefore, the The following will review the current state of this field.
stimulation appears unrelated to the formation of the
hexagonal (II) phase. Do membrane lipids bind to membrane proteins?
It is not clear why PE is capable of significant stimu-
lation of the calcium pump protein. One possibility Observations have been reported that suggest that
that remains to be investigated is the role of the bilayer membrane lipids bind to membrane proteins. For ex-
surface in controlling membrane protein function. The ample, glycophorin from the human erythrocyte mem-
surface of PE bilayers is distinctly different than that of brane is isolated with tightly bound lipids that cannot
many other phospholipids. The PE surface is poorly be removed without extreme conditions. The lipids
hydrated and tends to interact with other surfaces, bound to glycophorin appear to be enriched in the
whether they are on other bilayers or proteins, rather phosphatidylinositols (21). Cytochrome oxidase (EC
than interact directly with the aqueous phase (see 1.9.3.1) from mitochondria is another example in which
below) (20). The possibility of interactions between the tightly bound lipids that cannot be easily removed are
bilayer surface and the extramembranous portions of found with the protein after isolation. In this case the

1836 Vol. 3 May 1989 The FASEB Journal VEAGLE

bound lipids are enriched in diphosphatidylglycerol or hazardous. Another approach or approaches to the
cardiolipin (22). Titration of the calcium pump protein question was required.
with phospholipids indicates that about 30 lipid mole- Phospholipid head groups contain the chemically in-
cules are required for full activation of the enzyme (23). teresting charged structures that might be expected to
Magnetic resonance has been heavily exploited to interact with proteins (for example, choline of acetyl-
study the problem of lipid binding to membrane pro- choline that binds to the receptor of the same name, is
teins. The story has grown complex as the number of also found in the head group of one of the common
studies has multiplied. Many of the early studies used phospholipids), and so they are an important region of
electron spin resonance (ESR) and spin-labeled lipids the lipid molecule to explore for a better understanding
(24). The ESR spectra of these spin labels show two of lipid-protein interactions. 31P-NMR provides an ex-
spectral components in membranes containing some cellent, nonperturbing way to study the behavior of the
membrane proteins. Such data were interpreted in lipid head groups.
terms of two lipid environments in the membrane in 31P-NMR has been used in the most recent studies
the presence of membrane proteins. One spectral com- of lipid-protein interactions in biological membranes
ponent was similar, though not identical, to the spectra and reconstituted systems (32-34). In some cases, two
obtained from spin labels in lipid bilayers without pro- spectral components are observed. One spectral com-
tein. The other spectral component was representative ponent is similar, though not identical, to the spectrum
of a motion-restricted lipid environment. Model studies arising from pure phospholipid bilayers. The other
suggested that the motion-restricted lipid environment spectral component is characteristic of a motion-
resulted from the lipid encountering the surface of pro- restricted phospholipid head group environment.
teins in the membrane. The population of the motion- These spectra can be deconvoluted into two spectral
restricted lipid environment was proportional to the components by subtraction of a normal bilayer compo-
protein content of the membrane. Model calculations nent from the spectrum of the whole membrane. The
suggested that the population of the motion-restricted resulting difference spectrum is a broad (about twice as
environment was suitable to form one ring of lipid broad as a normal phospholipid bilayer powder pat-
around the protein. (However, because recent studies tern) but axially symmetrical powder pattern that is
have indicated that many membrane proteins exist as characteristic of axial rotation, but indicative of a sub-
dimers in the membrane (25), the details of these calcu- stantially more ordered environment than is found in
lations should be reexamined. For example, under con- pure lipid bilayers. This latter spectral shape was ob-
ditions where the calcium pump protein is a dimer, the served most clearly from the phosphatidylinositol
population of the motion-restricted environment is ade- tightly bound to glycophorin reconstituted as a protein-
quate to surround the dimer with a single layer of lipid, phospholipid complex in a glycolipid bilayer (35). In
but not enough to surround a monomer of the protein) this experiment, only the phospholipid that was tightly
(26). From these studies came the concept of a lipid an- bound to the protein contributed to the 3tP-NMR spec-
nulus: lipid in a special environment produced by the trum. So it was not necessary to use any of the assump-
membrane protein that is sufficient to cover the hydro- tions appropriate when spectra must be deconvoluted
phobic surface of the protein. to obtain the individual components. However, decon-
After a period of these spin label studies, 2H-NMR volution (that is, subtraction of the normal phospho-
studies of 2H-labeled lipids in membranes containing lipid bilayer powder pattern from the total spectrum)
proteins were used to examine the same questions. The produced the same (as in the case of reconstituted glyco-
first such study suggested that results similar to those of phorin) broad axially symmetrical powder pattern of
the ESR studies would be obtained (27). However, this protein-bound phospholipid from the 31P-NMR spec-
was later retracted in favor of results that revealed only trum of the native and functional light sarcoplasmic
one spectral component (28). The presence of one spec- reticulum membrane (B. S. Selinsky and P. L. Yeagle,
tral component was interpreted variously in terms of unpublished results). Therefore, this broad (relative to
1) no interactions between the lipids and the proteins pure phospholipid bilayers) but axially symmetrical 3tP
or 2) rapid exchange of lipids between sites next to the powder pattern may be representative of the phospho-
protein and sites in the lipid bilayer of the membrane lipid head group environment when phospholipids are
giving rise to a single, averaged spectral component. bound to membrane proteins in biological membranes.
Considering the results and conclusions of the ESR Using magnetization transfer experiments, the ex-
studies and the observations of lipids tightly bound to change rate of phospholipids between the motion-
membrane proteins, the first alternative is not a useful restricted environment and the lipid bilayer environ-
interpretation. The second interpretation rested on the ment was explicitly measured in sarcoplasmic reticu-
absence of a second spectral component in the 2H-NMR lum in the only example to date of a direct measure-
spectra. Subsequent publications demonstrated that a ment of such lipid exchange rates. An exchange rate of
component resulting from lipids interacting strongly 1 s1 was observed (36).
with membrane proteins would not normally be visible These more recent experiments appear to comple-
in the 2H-NMR spectra due to artifactual loss of such ment the picture of lipid-protein interactions derived
a component in the collection of the data (29-31). from the ESR data. Both approaches identify a motion-
Therefore, the interpretation of single-component 2H- restricted lipid environment induced by some mem-
NMR spectra in terms of lipid-protein interactions was brane proteins. ESR data show that the environment is

RECULATION OF CELL MEMBRANES 1837

enriched in particular phospholipids in the case of a differential affinity is helpful in explaining the exclu-
Na,K-ATPase (37). 31P-NMR studies have revealed a sive location of PS and the nearly exclusive location of
modest exchange rate between the two lipid environ- PE on the inside of the erythrocyte membrane. The
ments for the sarcoplasmic reticulum, whereas for proteins involved in this translocation in the erythro-
human erythrocyte glycophorin, the exchange rate is cyte have not been isolated, but preliminary efforts in
very slow. this area are under way.
On the basis of these studies, the possibility of phos- A novel assay was developed to explore the same
pholipid regulation of membrane protein function question regarding the endoplasmic reticulum (40).
through binding to the membrane protein is still a via- Previous work showed evidence for relatively rapid
ble hypothesis. However, the field has not progressed transmembrane movement of phospholipids in micro-
sufficiently to provide many complete examples of this somal preparations (41). More recently, the activity of
hypothesis. phospholipid translocation was reconstituted into lipid
vesicles from rat liver microsomes. Although the pro-
tein likely to be involved has not yet been identified, it
PROTEIN REGULATION OF appears that a new class of membrane proteins may be
TRANSMEMBRANE LIPID DISTRIBUTION involved in facilitating the translocation of phospho-
lipids across membranes. The question of which ones
One of the fascinating mysteries of membrane biology
are energy dependent and which are not will have to be
is the creation and maintenance of transmembrane
addressed. From a thermodynamic view, translocation
lipid asymmetry. The erythrocyte membrane is the
processes that are involved in the establishment and/or
best-documented example available of the asymmetri-
maintenance of transmembrane lipid asymmetry pre-
cal distribution of phospholipid classes (38). The con-
sumably require cellular energy.
sensus 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 LIPID REGULATION OF MEMBRANE
the sphingomyelin (SPM) is located almost entirely on MORPHOLOGY
the exterior, whereas the PC is disproportionately dis-
tributed toward the exterior face of the membrane. As indicated at the beginning of this review, it is pre-
How these inhomogeneities of phospholipid distribu- sumed that biological membranes have a lipid bilayer
tions are created or maintained had not previously as their fundamental structural component. The lipid
been explained. However, it has been known for some bilayer imparts the essential permeability control to the
time that transmembrane movement of lipids must oc- cell. Passive permeability through a lipid bilayer is slow
cur. The active sites of the enzymes involved in lipid for most solutes (except water), so that membrane
biosynthesis are found on the cytoplasmic face of the transport functions control the entry and exit of
endoplasmic reticulum. Therefore, the newly synthe- nutrients from the cell. The loss of integrity of the lipid
sized lipid must be located initially on that cytoplasmic bilayer would make the cell freely permeable to all
face of the membrane, and some select portion of the solutes and lead to cell death.
newly synthesized pool must subsequently be translo- The stability of the lipid bilayer is an important issue
cated to the lumenal face of the endoplasmic reticulum. to explore in this context. It is well known that some
Several investigators have explored these important is- lipids can readily adopt nonlamellar phase structures.
sues and shed new light on the problem of membrane Lipids that promote this behavior are detrimental to
lipid asymmetry. cell viability, although they probably have other impor-
Recent experiments have revealed that proteins tant roles to play in cell membrane function (see the
probably participate in the transmembrane movement discussion of stimulation of the Ca-ATPase by PE, a
of lipids and in the maintenance of the asymmetry of hexagonal (II) phase-forming lipid, above). In fact,
erythrocyte and endoplasmic reticulum membranes. aberrant lipid metabolism could lead to an imbalance
Devaux (39) observed rapid movement in the erythro- between lamellar and nonlamellar phase-forming lipids
cyte, outside to inside, of labeled phospholipid analogs in a membrane and lead directly to a pathological state
of PE and PS. The movement is dependent on ATP. involving membrane degeneration. It is important then
The PE and PS analogs compete with each other for the to understand how lipids regulate the stability of a cell
transport system, with a greater affinity for the trans- membrane lipid bilayer.
locater shown by the PS analog. Sulfydryl group modi- The formation of hexagonal II phase by PE is a well-
fication inhibits the ability of the membrane to promote studied phenomenon. From its structure, one can read-
translocation of the PE and PS analogs. Results such as ily conclude that the hexagonal II phase would be disas-
these suggest that PE and PS translocation across the trous to a cell membrane function. Three issues have
erythrocyte membrane is enhanced by a protein in the been addressed recently concerning this phenomenon
membrane. PC and sphingomyelin analogs translocate of nonlamellar phase formation: 1) the driving force for
much more slowly by a pathway independent of the forming hexagonal II phase; 2) how a membrane might
pathway for PE and PS translocation. Thus, the affinity adjust to maintain bilayer stability in the face of the
of the transport system for these phospholipid analogs potential of nonlameilar phase formation; and 3) how
appears to be in the order: PS > PE >> PC, SPM. Such metabolic events within the cell might lead to at least

1838 Vol. 3 May 1989 The FASEB Journal YEAGLE

transient bilayer instabilities that may have important posure to water). Higher temperature or unsaturation
favorable biological functions. increases the surface area each head group is forced to
occupy in the lamellar phase, thereby exacerbating the
Forces governing hexagonal (II) phase formation unfavorable interaction of the PE bilayer surface with
the water. This fundamental physical property of PE,
The formation of the hexagonal (II) phase structure has manifest as a modulation of bilayer surface properties,
been carefully studied by Gruner and colleagues (42) may be important to its role in biological membranes
and by Siegel (43), who introduced the concept of in- in the larnellar phase.
trinsic radius of curvature, R0, to describe quantita-
tively the formation of the cylinders of the hexagonal Biological response to hexagonal (II) phase potential
(II) phase. R0 reflects the radius of the channel formed
by the close packing of the lipid head groups in the hex- Acholeplasma laidlawii have MGDG and digalactosyldi-
agonal (II) phase. Thus, each phospholipid has an in- glyceride (DGDG) as major lipid components. These
trinsic radius of curvature that can be measured by lipids exhibit behavior analogous in some ways to PE
X-ray diffraction. Factors that affect the tendency of a and PC. MGDG is capable of forming the hexagonal
phospholipid dispersion to enter the hexagonal (II) (II) phase, as is PE. The head group of MGDG is
phase have predictable effects on R0. poorly hydrated, as is PE. In contrast, DGDG is analo-
A small value of R0 will favor the formation of the gous in some ways to PC: It is more extensively
hexagonal (II) phase. For example, unsaturation in the hydrated and is more stable in the lamellar phase than
lipid hydrocarbon chains favors the formation of the is MGDG (45).
hexagonal (II) phase, and the hexagonal (II) phase is How do the Acholeplasma respond to stress induced by
favored at higher temperatures. Conversely, methyla- an increase in growth temperature? Increases in tem-
tion of the amino function will lead to an increase in perature will favor the hexagonal (II) phase of MGDG.
R0 to the extent that R0 is nearly infinite for PC (i.e., Formation of hexagonal (II) phase would be disastrous
three methyl groups), which reflects the stability of the to the organism. The response of the organism is to
lamellar phase formed by PC. decrease the MGDG and increase the DGDG, or to de-
This is part of the geometric argument that was ad- crease the lipid favoring the hexagonal (II) phase and
vanced previously, that PE is a wedge-shaped molecule increase the lipid favoring the lamellar phase. Structur-
(because of its small head group), and that wedge- ally this alteration in composition will stabilize the
shaped molecules pack well into the hexagonal (II) lamellar phase of the lipids in the membrane against
phase. However, new theories were called for when it the temperature-induced tendency to form the hex-
was reported that some lipids with large head groups agonal (II) phase (46).
also favored the hexagonal (II) phase (44). The latter One of the big questions in this field concerns what
result suggested that the relative hydration of the head the hexagonal (II) phase-forming lipid is doing in bio-
group, or its ability to interact with the aqueous phase, logical membranes if one of its effects is to destabilize
was an important physical property to explore. the structure of those membranes. This is currently an
A complementary thermodynamic viewpoint was de- area of considerable interest and investigation.
veloped on the basis of related behavior of hexagonal
(II) phase-forming lipids (20). It was observed that, in Metabolic regulation of nonlamellar phase formation
the lamellar phase, membranes rich in PE tended to ag-
gregate, membrane surface-to-membrane surface, to A recent observation in the area of bilayer stability in
partially exclude water. This led to the conclusion that biological membranes is the ability of diacylglycerol to
the surfaces of PE bilayers interacted poorly with the reduce dramatically the temperature of the lamellar-
aqueous phase - a good example of the hydrophobic hexagonal (II) phase transition (47, 48). In some sys-
effect. It could then be predicted that altering the tems, diacylglycerol levels as low as 1-3% (of the total
nature of the aqueous phase should alter the extent of lipid content) will reduce the lamellar-hexagonal (II)
surface-surface interactions that were manifest as vesi- phase transition temperature by tens of degrees centri-
cle aggregation. Addition of chaotropic agents to the grade. This reduction in the temperature of the lamellar-
aqueous phase (which alter its nature) also interrupted hexagonal (II) phase boundary can be understood in
the aggregation of the PE vesicles. Furthermore, the terms of a reduction in R0. Therefore, diacylglycerol is
chaotropic agents stabilized the lamellar phase of the capable not only of activating protein kinase C, but also
PE against extremes in temperature. The tendency to of disrupting the lipid bilayer of the membranes in
form the hexagonal (II) phase can then be understood which it is produced.
as a thermodynamic consequence of the poor interac- Diacylglycerol is an intermediate in lipid metabolism
tion of PE with the aqueous phase. Formation of the and can be produced by the action of phospholipase C.
hexagonal (II) phase severely limits the exposure of the Phospholipase C can be stimulated by receptor activa-
lipid head groups to water (they are exposed only to the tion. The amount of diacylglycerol in the membrane
small amount of water within the tubes and the head will regulate the stability of the lipid bilayer of the
groups are probably more tightly packed than on the membrane. Therefore, a mechanism for metabolic
surface of the lamellar phase, again reducing their ex- regulation of membrane stability through lipid metab-

REGULATION OF CELL MEMBRANES 1839

olism is potentially available to the cell. As the discus- teins might influence the morphological behavior of
sion below suggests, such a pathway of regulation may membrane lipids.
be important to biological membrane function. The retinal rod outer segment (ROS) disk mem-
brane provides an interesting example of such a
Fusion of membranes and regulation by nonlamellar phenomenon. The membranes of the ROS disks are
rich in PE and in highly unsaturated fatty acids. In
phase formation
fact, the single largest population of lipids in the disk
Cell membrane fusion is involved in a number of cellu- membrane is PE and the most abundant fatty acid is
lar processes. For example, fusion is involved in receptor- 22:6. It was no surprise then to observe that the isolated
mediated endocytosis and in intracellular vesicular disk lipids were able to enter the hexagonal (II) phase
transport; it is involved in secretion and in viral infec- in the presence of calcium (50). What was surprising
tion of some enveloped viruses. was that the native membrane containing the photopig-
At least three steps in the fusion process can be iden- ment rhodopsin was stable to more extreme stress (for
tified: 1) the close approach of two membranes, or example, higher calcium concentration) than that
aggregation; 2) removal of at least part of the water be- necessary to produce hexagonal (II) phase in the iso-
tween the two membranes, or partial dehydration; and lated lipids. In this case the membrane proteins appear
3) a transient destabilization of the lipid bilayer struc- to stabilize the lipid bilayer of that membrane.
ture to permit a partial, short-lived mixing of the struc-
tures of the individual membranes that can lead to
SUMMARY
fusion of those membranes.
Lipid combinations that produce partial dehydration In this review, we have examined some aspects of the
of the membrane surface and are capable of introduc- lipid regulation of membrane function. The study of
ing transient nonlamellar structures into the lipid cell membrane structure and function has progressed
bilayer might be expected to enhance the incidence of sufficiently to begin examining hypotheses for the regu-
fusion. PE would be expected to be a good candidate in lation of membrane function through membrane struc-
this regard. tural features. Through an examination of regulation,
PE appears to enhance the rate of fusion in some key issues of cellular biology begin to become un-
model membrane systems. This may result in part from raveled.
the facilitation by PE of the close apposition of the two Studies of cholesterol regulation of Na,K-ATPase,
membranes to be fused. as well as other membrane proteins, and of the regula-
However, an important new role that PE may play in tion of cell growth have produced clues to the role of
the fusion event has been identified (49). On compari- cholesterol in mammalian cell biology. The available
son of the phase behavior of a particular lipid system, cell biology and biochemistry studies suggest that cho-
it was suggested that fusion was greatly enhanced by lesterol is required for the normal function of essential
the so-called isotropic structures identified in the 31P- membrane enzymes. This is a structurally specific re-
NMR spectra of that lipid system. These isotropic quirement. For example, in a cholesterol-requiring cell,
structures occurred under the same conditions as the ergosterol cannot substitute for cholesterol as the essen-
lipid particles in the freeze-fracture electron microscopy tial sterol. The biochemical basis of this is the depen-
of the same preparations. The data suggested further dence of crucial membrane enzymes on the presence of
that if the system became hexagonal (II), fusion no the essential sterol in the membrane. Without the es-
longer occurred and only extensive leakiness character- sential sterol, these crucial membrane enzymes cannot
ized the membranes because of the breakdown of the function, and without their function, the cell that re-
lipid bilayer. So the isotropic lipidic particle appears to quires those membranes cannot grow, divide, or differ-
be an important nonlamellar structure and may be a entiate. What remains to be described by future research
candidate for an intermediate in the fusion event. is the physical basis for the biochemical requirement
In this context, the observations reviewed above- certain membrane enzymes have for cholesterol. The
that diacylglycerol enhances the formation of the hex- structural specificity of the sterol requirement suggests
agonal (II) phase and concurrently lowers the threshold that the essential sterol binds to the enzyme that re-
for formation of the isotropic structures - suggest that quires it at a sterol-specific binding site on the protein.
diacylglycerol might enhance membrane fusion (47). The essential sterol then activates the enzyme as a posi-
Once again there is the possibility of a mechanism for tive effector. However, this physical mechanism has yet
the regulation of membrane fusion that involves known to be established.
metabolic events in the catabolism of cell membrane Understanding the role and mechanisms by which
lipids. membrane phospholipids regulate membrane protein
activity presents considerable challenges. Although the
PROTEIN REGULATION OF MEMBRANE individual species of lipids that are found in cellular
MORPHOLOGY membranes number in the thousands, the biological
reason for such variety has not been discovered. And
It was noted above that lipids and proteins interact with the mechanism by which regulation of membrane pro-
each other in biological membranes. One possible con- tein function (which has been observed for several
sequence of such interactions is that membrane pro- lipids) occurs is still speculation. Much more work is re-

1840 Vol. 3 May 1989 The FASEB Journal YEAGLE

quired. Factors to consider in such studies include not Biology of Cholesterol (Yeagle, P. L., ed) pp. 147-172, CRC Press,
only specific lipid-protein interactions but also what in- Boca Raton, FL
14. Warren, G. B., Houslay, M. D., Metcalfe, J. C., and Birdsall,
dividual lipids or combinations of lipids may do to the N. J. M. (1975) Cholesterol is excluded from the phospholipid
surfaces of membranes and to membrane stability and annulus surrounding an active calcium transport protein. Nature
internal dynamics. (London) 255, 684-687
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(1986) The role of cholesterol in the activity of reconstituted Ca-
mechanisms of membrane fusion have led to new ideas
ATPase vesicles containing unsaturated phosphatidylethanol-
about the metabolic control of cellular membrane fu- amine. j Biol. Chem. 261, 5081-5087
sion. Intermediates discovered in the studies of phase 16. Knowles, A. F., and Racker, E. (1975) Properties of a recon-
changes between lamellar and nonlamellar phases for stituted calcium pump. J. Biol. Chem. 250, 3508-3544
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1842 Vol. 3 May 1989 The FASEB Journal VEAGLE