Lateral organization in
Acholeplasma laidlawii
lipid bilayer
models containing endogenous pyrene probes
Patrik Storm
1
,LuLi
2
, Paavo Kinnunen
3,4
and A
˚
ke Wieslander
1
1
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden;
2
Wallenberg Laboratory for
Cardiovascular Research, Go
¨
teborg University, Sweden;
3
Department of Medical Chemistry, Institute of Biomedicine,
Helsinki University, Finland;
4
Memphys – Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark
In membranes of the small prokaryote Acholeplasma laid-
lawii bilayer- and nonbilayer-prone glycolipids are major
species, similar to chloroplast membranes. Enzymes of the
glucolipid pathway keep certain important packing proper-
ties of the bilayer in vivo, visualized especially as a monolayer

properties and how these are maintained by the lipid
synthesizing enzymes. Membrane lipids are synthesized in
two competing pathways, both using phosphatidic acid (PA)
as a precursor, with one branch resulting in glucolipids and
the other in phosphatidylglycerol (PG) as shown in the
diagram below.
At least five enzymes constitute the glucolipid pathway.
Phosphatidic acid phosphatase (PAP) makes diacylglycerol
(DAG) from PA. 1,2-diacylglycerol-3-glucosyltransferase
Correspondence to A
˚
ke Wieslander, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
Fax: + 46 8 15 36 79, Tel.: + 46 8 16 24 63, E-mail:
Abbreviations: bis-PyrPC, 1,2-bis-[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphatidylcholine; bis-PyrPG, 1,2-bis-[10-(pyren-1-yl)]decanoyl-sn-
glycero-3-phospho-rac-glycerol; CL, cardiolipin; 1,2-DOG, 1,2-dioleoylglycerol; DGlcDAG, 1,2-diacyl-3-O-[a-
D
-glucopyranosyl-(1fi2)-O-a-
D
-glucopyranosyl]-sn-glycerol; MADGlcDAG, 1,2-diacyl-3-O-[a-
D
-glucopyranosyl-(1fi2)-O-(6-O-acyl-a-
D
-glucopyranosyl)]-sn-glycerol;
MAMGlcDAG, 1,2-diacyl-3-O-[6-O-acyl(a-
D
-glucopyranosyl)]-sn-glycerol; MGlcDAG, 1,2-diacyl-3-O-(a-
D
-glucopyranosyl)-sn-glycerol; PA,
phosphatidic acid; PC, phosphatidylcholine; PD, pyrenedecanoic acid; PG, phosphatidylglycerol; PyrDAG, 1-palmioyl-2-pyrenedecanoyl-
glycerol; PyrPA, 1-palmioyl-2-pyrenedecanoyl phosphatidic acid; PyrPG, 1-palmioyl-2-pyrenedecanoyl-phosphatidylglycerol.

MGlcDAG synthase (I) is activated by approximately
20 mol/100 mol PG or 10 mol/100 mol cardiolipin (CL),
but is not critically dependent on the nature of the
phosphate moiety and can be activated by other negatively
charged lipids, however, not as efficiently [4–7]. The
activation by CL indicates no specificity for the PG
headgroup, but that the negatively charged phosphate is
important for the enzyme.
DGlcDAG synthase (II) is activated by PG or CL in the
same way as MGlcDAG synthase, but also by other
phosphate-containing species such as certain metabolites
and dsDNA [8]. However, PG is the strongest activator
among the naturally occurring lipids (strain A-EF22 does
not make CL). As for the MGlcDAG synthase, this process
is cooperative with respect to PG amounts and has a fairly
high Hill coefficient (4–6 for MGlcDAG synthase and 3–7
for DGlcDAG synthase) [4]. Substrate fractions of MGlc-
DAG up to five mol/100 mol raise the activity, which then
levels out, most likely due to saturation of the active site [8].
More DGlcDAG is made from MGlcDAG in membranes
with more unsaturated or longer acyl chains, increased
temperature or increased amount of cholesterol. The shift in
this lipid ratio stems from the more pronounced nonlamellar
tendency of the membrane, compensated by making more
DGlcDAG (from the nonbilayer prone MGlcDAG). This
curvature sensitivity implies a sensing mechanism of mem-
brane perturbation of nonbilayer-prone lipids. Analogous
sensing features have been proposed for CTP:phosphocho-
line cytidylyltransferase [9–11] or protein kinase C [12].
Could lipids adopting a heterogeneous lateral distribution

lipids in monolayer experiments [32,33]. Analogous features
are also recorded for plant galactolipids. The importance of
the glycolipids for these properties are highlighted by the
lower lateral diffusion for A. laidlawii in vivo glucolipids
compared to the E. coli phospholipids [34].
To investigate whether lateral heterogeneity exists in the
fluid glucolipid-rich membrane of A. laidlawii A-EF22 as a
function of headgroup composition, liposomes were made
where composition of five different lipids (major lipids in the
membrane of A. laidlawii A-EF22), all with di-18:1c acyl
chains, was varied according to a chemometrical experi-
mental design. Pyrene-derivatives of the same lipids, inclu-
ding endogenous major glucolipids synthesized by
A. laidlawii, were used as fluorescent probes. A potential
influence on the MGlcDAG synthase, the first regulating
enzyme in the glucolipid pathway, was also investigated.
Materials and methods
Lipids and probes
MGlcDAG and DGlcDAG were prepared from A. laidla-
wii cells grown in a lipid-depleted medium supplemented
with oleic acid [35]. 1,2-dioleoylglycerol (1,2-DOG) was pur-
chased from Larodan (Malmo
¨
, Sweden). Phosphatidylgly-
cerol (PG) was purchased from Avanti polar Lipids (USA).
Pyrenedecanoic (PD) acid, 1-palmitoyl-2-pyrenedecanoyl-
phosphatidylglycerol (PyrPG) and 1,2-bis-[10-(pyren-1-yl)]
decanoyl-sn-glycero-3-phosphatidylcholine (bis-PyrPC) was
purchased from Molecular Probes Inc. (Oregon, USA).
1-palmioyl-2-pyrenedecanoyl-glycerol (PyrDAG) and

[37]. Cell growth was monitored by absorbance and by
phase contrast light microscopy. Contamination by any
other bacteria was analyzed on standard bacteriological
agar plates.
Extraction and analysis of lipids
Cells were harvested by centrifugation, washed twice in
buffer, and frozen at )80 °C. Membrane lipids were
extracted from the cell pellets using chloroform/methanol
(2 : 1, v/v).
One-dimensional thin layer chromatography (TLC) was
used to separate and characterize the different lipids in the
membrane. The TLC plates coated with silica gel 60
(Merck, Darmstadt, Germany) were developed in chloro-
form/methanol/water (80 : 25 : 4, v/v/v). [
14
C]-labeled
lipids were visualized with electronic autoradiography
(Packard Instant Imager). Excised gel lipid spots were
digested in Soluene-350 (Packed) for 30 min at 37 °Cand
quantified by double-channel liquid scintillation counting.
To purify the pyrenyl lipids, the TLC plate was developed
first in chloroform/methanol/water (80 : 25 : 4, v/v/v) and
then in chloroform/methanol/ammonia (91 : 35 : 10, v/v/
v). Compared with a one-dimensionally developed TLC
plate of extracted lipids from medium 18:1c/PD
120 l
M
:30l
M
, the spots of pyrenyl lipids could be

yield of synthesized pyrenyl glucolipids was determined
from standard fluorescence intensity curves, obtained from
synthetic PyrDAG and bis-PyrPG.
Enzymes and assays
Mixed lipid micelles were made by swelling dry lipid to a
final concentration of 10 m
M
(1 m
M
substrate) in a buffer of
110 m
M
Tris pH 8, 22 m
M
Chaps, 22 m
M
Mg
2+
.Purified
MGlcDAG synthase (50 lL) or DGlcDAG synthase was
incubated with 40 lL lipid micelles at 4 °Cfor30min.The
enzyme reaction was started by adding 10 lLof10m
M
(0.5 CiÆmol
)1
)UDP-[
14
C]glucose. The reactions were ter-
minated by the addition of 375 lL methanol/chloroform
(2 : 1, v/v). Synthesized MGlcDAG or DGlcDAG was

mathematical model, e.g. Y ¼ m + Xb + e;wereX is the
model terms/variables, b is the coefficient of effect and e is
the residuals.
We have used the
MODDE
3.0 package (Umetri AB,
Umea
˚
, Sweden). Here, variables were changed from low to
high and the response was plotted and analyzed in the
computer to give a measure of effects. Variables in this case
are the amounts of different lipid headgroups (as all acyl
chains are 18:1c) and the amount of MGlcDAG synthase.
Responses are excimer formation of pyrene-labeled phos-
pholipid or anisotropy of diphenylhexatriene (DPH).
DGlcDAG, considered the matrix lipid, was set as a filler
and a full factorial design was chosen. In the simple case
of three variables (dimensions, factors), a full factorial
design is a cube in the experimental space, where data points
are in the corners and center of the cube (Fig. 1), resulting
in a linear interaction model. In a couple of cases the
investigation was expanded to a response surface model,
i.e. composite face-centered (CCF) design, where the design-
cube also has data points on the face of the sides, making
quadratic models possible to obtain. For the investigation
of chain ordering for pure lipids a mixture (
D
-optimal)
design was chosen, where matrix lipid DGlcDAG is not set
as a filler.

data, range of the factors, etc., need to be investigated. The
fitted model can then be presented as a response surface
(Fig. 1B), a curve or a table.
Furthermore, an important aspect of experimental design
is that interaction effects can be detected; this would not be
possible if only one variable at a time was changed. Inter-
action means that the response of a variable is dependent on
the level of another variable in a nonadditive fashion.
Preparation of large unilamellar vesicles
For each sample 0.25 lmol of total lipid was mixed to the
desired composition according to the experimental design,
where DOPG was varied 0–40 mol/100 mol, DOPA
0–10 mol/100 mol, MGlcDAG 0–30 mol/100 mol, DOG
0–10 mol/100 mol, and DGlcDAG was used as the (bal-
ance) matrix. The content of fluorescence probe was
constant at 1 mol/100 mol for mono-pyrenyl lipids, and
0.5 mol/100 mol for bis-pyrenyl lipids or DPH. The mixture
was then dried under a nitrogen flow and then under
reduced pressure (vacuum) overnight. The resulting lipid
film was hydrated with intermittent vortexing during 45 min
in filtered and deoxygenated 10 m
M
Hepes pH 8.0 with
5m
M
MgCl
2
, and then extruded with a LiposoFast Basic
extruder (Avestin Inc., Canada) 19 times through two
stacked polycarbonate filters (Millipore; pore diameter

398 nm (Im). Enzyme (MGlcDAG synthase) was incubated
with 50 lL liposomes (protein : lipid 1 : 700–1 : 70) on ice
for 30 min prior to measurement at room temperature in a
1 · 0.2 cm quartz fluorescence cuvette using a Perkin-
Elmer LB50 spectrofluorimeter.
DPH anisotropy [42,43] was analyzed using a Spex
Fluorolog 12 fluorometer (Department of Biophysical
Chemistry, Umea
˚
University), where bandwidths were
3.6 nm for excitation and 7.2 nm for emission. Sample
Fig. 1. Experimental design. (A) Full factorial design cube with cen-
terpoint. Variables, e.g. lipids, are changed from low to high amounts.
(B) Example of a response surface plot showing response variation
when varying two variables. The purpose of the design is to extract
maximum information from a minimum number of experiments.
1702 P. Storm et al.(Eur. J. Biochem. 270) Ó FEBS 2003
solution was equilibrated for five minutes in the cuvette
holder (no magnetic stirrer) to reach a temperature of 28 °C
prior to measurement. Absorption at the excitation wave-
length was less than 0.09, thus a minimal reabsorption.
Anisotropy r ¼ (I
I
) I
^
)/(I
I
+2I
^
) for each datapoint was

in combination. A. laidlawii cells became much bigger and
less aggregated, and the density of the culture became
lower,whentheratioofPDinfattyacidswasincreased.
One-dimensional thin layer chromatography developed in
chloroform/methanol/water (80 : 25 : 4, v/v/v) was used to
characterize the lipids extracted from the cells (Fig. 2). The
R
f
values of different lipids on a TLC plate were compared
according to the standard samples characterized by NMR
[1,51]. Fluorescent spots (under UV light) are marked by
rings in Fig. 2A. Combined with the data from radiolabel
analysis, it is obvious that without a pyrene group in the
hydrocarbon chain of the lipid, there was no fluorescence.
With one PD acyl chain and the other chain 16:0 or 18:1c,
as in mono-pyrenyl lipids, both fluorescence and isotope
signals were detected (data not shown). With two pyrenyl
chains, only fluorescence but no isotope signal could be
detected from the spot of the bis-pyrene lipid on the TLC
plate (Fig. 2A). Note that mono-pyrenyl lipid migrated a
little further than the nonpyrenyl lipid, and bis-pyrenyl
lipid migrated even further, as expected from the larger
hydrocarbon regions of the pyrenyl-containing lipids
(Fig. 2A).
Purified MGlcDAG synthase and partially purified
DGlcDAG synthase were used to study the potential
disturbance of the polar headgroup organization by the
purified pyrenyl-labeled glucolipids in vitro (Fig. 3). The
enzymatic products, pyrenyl-MGlcDAG or pyrenyl-DGlc-
DAG, from the in vitro enzyme reactions, were extracted

14
C]glucose by purified
DGlcDAG synthase. The contents of pyrenyl glucolipids were less
than 1% (mol/mol). (s)[
14
C]DGlcDAG produced from di-18:1c-
MGlcDAG; (m) mono-pyrenyl DGlcDAG produced from mono-
pyrenyl MGlcDAG; and (d) bis-pyrenyl DGlcDAG produced from
bis-pyrenyl MGlcDAG, respectively, by the DGlcDAG synthase.
Ó FEBS 2003 Lateral organization of A. laidlawii lipids (Eur. J. Biochem. 270) 1703
acting on phosphatidylinositol lipids [52]. This seems logical
in that the MGlcDAG synthase is attached to the
membrane interface [7] and does not recognize the acyl
chain region close to the bilayer center, where the pyrene
moiety is.
Lipid organization as seen with pyrene derivatives
Potential interactions between the A. laidlawii membrane
lipids were analyzed in liposome bilayer models containing
various pyrene-labeled probes of synthetic and in vivo
origin. Excimer formation (Materials and Methods) for
lipids with one pyrene-acyl chain is an intermolecular event,
depending on the collision rate, and Ie/Im hence monitor
lateral mobility and concentration of these molecules. A
series of full factorial designs were made, where the
compositions (87 conditions in total) were varied to cover
the limits occurring in vivo for the five important lipids of the
glucolipid pathway in the membrane of A. laidlawii (PG,
PA, DAG, MGlcDAG and DGlcDAG). In in vitro bilayer
(liposome) models, the lipids adopted a heterogeneous
organization, as was seen with excimer formation of the

before that decreasing amounts of DGlcDAG (the balance
here) upon increasing PG, increased the collision rate
between pyrenes [5]. All other lipids had an insignificant
effect on excimer formation. As a comparison, increasing
PG amounts in a matrix with DGlcDAG did not change the
Fig. 4. Glucosyltransferases and lipid organization. (A) Response of
pyrene-derivatives of activator PG and substrate DOG lipids, and
order-sensing bis-PyrPC, upon increase in the DOPG content. (B)
Normalized enzyme activities (adapted from Dahlqvist et al.[3])ofthe
MGlcDAG and DGlcDAG synthases.
Table 2. Lateral interactions between A. laidlawii lipids in liposome bilayers. Changes in Ie/Im of pyrene probes upon variation in lipid amounts
according to a factorial design or, in the case of PyrDAG and PyrPA probes, to a composite face-centered design (Materials and methods). The
balance (matrix) in the various lipid mixtures was always DGlcDAG. Only statistically significant changes are shown. NT, not tested. R
2
and Q
2
are
measures of model fit and vary between 0 and 1 (Materials and methods).
Probe
MGlcDAG
0–30 mol/100 mol
DAG
0–10 mol/100 mol
PG
0–40 mol/100 mol
PA
0–10 mol/100 mol
Replicate
error (Ie/Im) R
2

probe (Table 2), probably reflecting exclusion from the
domains formed.
Bilayer chain ordering
A starting point is the response at a homogenous distribu-
tion of probe in the membrane, as is the case for
1 mol/100 mol PyrPC in a matrix of DOPC [46]. This gave
an Ie/Im of 0.07 in PC-matrix and 0.06 in DGlcDAG-
matrix (data not shown). The difference may reflect ease
of diffusion. Diffusion is also indirectly related to chain
ordering in the membrane. This property decreased with
increasing content of DGlcDAG and increased with
increasing content of DOPG or DOG when measured
with a bis-PyrPC probe. For bis-pyrenyl lipid probes the
Ie/Im reflects an intramolecular event, where an increase
corresponds to increased chain-chain contacts (collision
rates). The Ie/Im was 1.4 at 40 mol/100 mol DOPG,
and 1.05 at 0 mol/100 mol DOPG (Table 3), indicating
a decreased V
f
by increased PG (or increased V
f
by
DGlcDAG). A complete inverse of this property was
indicatedwhenmeasuredwithDPH(Table3).Steadystate
anisotropy r for DPH was between 0.10 at high DOPG (low
DGlcDAG) content and 0.14 at low DOPG content (high
DGlcDAG). DOG had the same effect as DOPG, although
the smaller fraction in the membrane made its effect less
pronounced. This has been observed before [26] and was
addressed to chain splaying motions of the bis-PyrPC. The

¼ 0.41) with LUVs
composed of DOPG (0–40%), CL (0–20%), DAG (0–
10%), bis-PyrPC (0.1%) and DGlcDOG as balance, and
1 : 700–1 : 70 (mol/mol) enzyme : lipid (Fig. 5). PG and
DAG increase the order, as seen in Table 3, as do CL. There
was also synergism between PG and the enzyme, and
antagonism between DAG and enzyme, with respect to
chain-ordering effects (Fig. 5). Hence, interfacial binding of
fairly large amounts of the MGlcDAG enzyme reduced
chain order (increased V
f
) but did not detectably change the
lateral distribution of the A. laidlawii A-EF22 polar lipid
species.
Table 3. Lipid composition and chain ordering. Excimer formation (Ie/Im) and anisotropy (r) were monitored by bis-PyrPC and DPH, respectively.
A mixture design was made (Materials and methods) where DOPG 0–40 mol/100 mol, DODAG 0–10 mol/100 mol, DODGlcDAG 40–90 mol/
100 mol (40–99 mol/100 mol with DPH as a probe), DOMGlcDAG 0–30 mol/100 mol, DOPA 0–10 mol/100 mol. Only lipids with a significant
effect are shown, with variables from the modeled data. The model is linear with R
2
¼ 0.95 and Q
2
¼ 0.91 for bis-PyrPC as the probe, and
R
2
¼ 0.89 and Q
2
¼ 0.79 for DPH as the probe.
Lipid Content level Ie/Im r
DOPG low (0 mol%) 1.09 0.122
high (40 mol%) 1.37 0.101

and biophysical properties of membranes. This is also
the first time mono- and bis-pyrenyl glucolipids from
A. laidlawii have been synthesized and purified.
Lateral organization of
A. laidlawii
lipids
The metabolism of glucolipids in A. laidlawii depends on
several factors such as growth temperature, presence of
foreign molecules, and unsaturation and length of the
fatty acids [2,3,56]. Not all lipids adopt a homogeneous
distribution in liquid-crystalline liposome model membranes
with A. laidlawii lipids having 18:1c acyl chains, as indicated
from the present investigation. The most prominent effect
was for DAG when increasing the molar fraction of PG.
There was a good agreement between results from the
factorial design models (Table 2) and a one-variable titra-
tion showing (at most) a 1.5-fold increase in the excimer
ratio (Ie/Im) (Fig. 4). Similar increase in Ie/Im was observed
when ceramide (DAG analogue) was enzymatically split
from sphingomyelin and forming microdomains [57,58], or
the patching of DOPG in liquid-crystalline PC due to a
hydrophobic (chain length) mismatch [5]. This is most
probably not due to an increased diffusion in the mem-
brane. Given an excited state lifetime of 100 ns for pyrene
and diffusion coefficient of lipids approximately 5 · 10
)8
cm
2
Æs
)1

modulation, membrane fusion and membrane physical
properties. For membrane physical properties, unsaturated
DAG imposes no phase separation at low molar fractions
but does increase the chain order in an unsaturated
phosphatidylcholine membrane. This ordering was also
Fig. 5. Enzyme binding and chain ordering. Plot showing the effects of varying amount of MGlcDAG synthase (MGS) and three different lipids in
LUVs with DGlcDAG as balance, going from low to high amount, and 0.1 mol/100 mol bis-PyrPC as probe (computed (R
2
¼ 0.93, Q
2
¼ 0.41) in
MODDE
3.0). Range: CL 0–20 mol/100 mol, DOG 0–15 mol/100 mol, DOPG 0–40 mol/100 mol and MGS/Lipid 1 : 700–1 : 70 (mol/mol).
Interaction effects between pairs of variables are also plotted, where positive value means synergism and negative value antagonism. The effect in
Ie/Im is the sum of the response difference between high and low variable levels divided by two. Effects for which error bars encompass zero are
insignificant.
1706 P. Storm et al.(Eur. J. Biochem. 270) Ó FEBS 2003
observed in the system investigated here (Table 3 and
Fig. 5). DAG also increases the spacing between phospho-
lipid (or glucolipid) headgroups, with its hydroxyl proton
participating in hydrogen bonding. This is part of the reason
why ceramide assemble laterally when enzymatically
released from sphingomyelin [58,61]. Hydrogen bonding
in the lipid interface plays a role in the interaction between
sugar headgroups, where a subtle difference as between
galactose and glucose may be important [62]. For other lipid
species, modulation by divalent cations (in our case Mg
2+
)
coordinating negatively charged lipids and decreasing the

have been varied simultaneously in vitro and that a
fluorescent glucolipid probe has been synthesized in vivo.
We find that DAG does not mix ideally but forms
microdomains, possibly in weak interaction with PG. As
PG is the strongest activator in vivo for the two glucolipid-
synthesizing enzymes, this phenomenon has a biological
relevance in concentrating DAG, the substrate for MGlc-
DAG synthase, which is rate-limiting in glucolipid synthesis.
Purified MGlcDAG synthase, free of lipids and detergent
[55], did not affect the organization (Ie/Im) for the tested
PyrDAG and PyrPG probes, but affects order in the
membrane. Activity therefore seems to depend more on the
ability to bind to the membrane in a proper orientation/
conformation as Fig. 5 suggests, and due to the fact that
CL, a strong activator, did not affect the Ie/Imfor
PyrDAG.
Acknowledgements
This work was supported by the Swedish Natural Science Research
Council, and the K & A Wallenberg foundation.
References
1. Andersson, A.S., Rilfors, L., Lewis, R.N., McElhaney, R.N. &
Lindblom, G. (1998) Occurrence of monoacyl-diglucosyl-diacyl-
glycerol and monoacyl-bis-glycerophosphoryl-diglucosyl-diacyl-
glycerol in membranes of Acholeplasma laidlawii strain B-PG9.
Biochim. Biophys. Acta 1389, 43–49.
2. Wieslander, A
˚
., Nordstro
¨
m,S.,Dahlqvist,A.,Rilfors,L.&

7.Berg,S.,Edman,M.,Li,L.,Wikstro
¨
m, M. & Wieslander, A
˚
.
(2001) Sequence properties of the 1,2-diacylglycerol 3-glucosyl-
transferase from Acholeplasma laidlawii membranes. Recognition
of a large group of lipid glycosyltransferases in eubacteria and
archaea. J. Biol. Chem. 276, 22056–22063.
8. Vikstro
¨
m, S., Li, L., Karlsson, O.P. & Wieslander, A
˚
. (1999) Key
role of the diglucosyldiacylglycerol synthase for the nonbilayer-
bilayer lipid balance of Acholeplasma laidlawii membranes. Bio-
chemistry 38, 5511–5520.
9. Arnold, R.S., DePaoli-Roach, A.A. & Cornell, R.B. (1997)
Binding of CTP:phosphocholine cytidylyltransferase to lipid
vesicles: Diacylglycerol and enzyme dephosphorylation increase
the affinity for negatively charged membranes. Biochemistry 36,
6149–6156.
10. Cornell, R.B. & Northwood, I.C. (2000) Regulation of
CTP:phosphocholine cytidylyltransferase by amphitropism and
relocalization. Trends Biochem. Sci. 25, 441–447.
11. Davies, S.M., Epand,R.M., Kraayenhof, R. & Cornell, R.B. (2001)
Regulation of CTP:phosphocholine cytidylyltransferase activity
by the physical properties of lipid membranes: an important role
for stored curvature strain energy. Biochemistry 40, 10522–10531.
12. Newton, A.C. (2001) Protein kinase C: structural and spatial

13974.
23. Goni, F.M. & Alonso, A. (1999) Structure and functional
properties of diacylglycerols in membranes. Progr. Lipid Res. 38,
1–48.
24. Kinnunen, P.K. (1991) On the principles of functional ordering in
biological membranes. Chem. Phys, Lipids 57, 375–399.
25. Edidin, M. (1997) Lipid microdomains in cell surface membranes.
Curr. Opin. Struct. Biol. 7, 528–532.
26. Lehtonen, J.Y., Holopainen, J.M. & Kinnunen, P.K. (1996) Evi-
dence for the formation of microdomains in liquid crystalline large
unilamellar vesicles caused by hydrophobic mismatch of the
constituent phospholipids. Biophys. J. 70, 1753–1760.
27. Killian, J.A. (1998) Hydrophobic mismatch between proteins and
lipids in membranes. Biochim. Biophys. Acta 1376, 401–415.
28. Dumas, F., Lebrun, M.C. & Tocanne, J.F. (1999) Is the protein/
lipid hydrophobic matching principle relevant to membrane
organization and functions? FEBS Lett. 458, 271–277.
29. Piknova
´
,B.,Pe
´
rochon, E. & Tocanne, J F. (1993) Hydrophobic
mismatch and long-range protein/lipid interactions in bacterio-
rhodopsin/phosphatidylcholine vesicles. Eur. J. Biochem. 218,
385–396.
30. Tocanne, J.F., Cezanne, L., Lopez, A., Piknova, B., Schram, V.,
Tournier, J.F. & Welby, M. (1994) Lipid domains and lipid/pro-
tein interactions in biological membranes. Chem. Phys. Lipids 73,
139–158.
31. Shaikh, S.R., Brzustowicz, M.R., Gustafson, N., Stillwell, W. &

551.
38. Kates, M. (1972) Techniques of Lipidology: Isolation, Analysis and
Identification of Lipids. North-Holland Publishing Co., Amsterdam.
39. Karlsson, O.P., Dahlqvist, A., Vikstro
¨
m, S. & Wieslander, A
˚
.
(1997) Lipid dependence and basic kinetics of the purified 1,2-
diacylglycerol 3-glucosyltransferase from membranes of Achole-
plasma laidlawii. J. Biol. Chem. 272, 929–936.
40. Eriksson, L., Johansson, E., Kettaneh-Wold, N., Wikstro
¨
m, C. &
Wold, S. (2000) Design of Experiments: Principles and Applica-
tions. Learnways, Stockholm.
41. MacDonald, R.C., MacDonald, Ruby, I., Menco, Bert, PhM.,
Takeshita, Keizo, Subbarao, Nanda, K. & Hu, Lan-rong. (1991)
Small-volume extrusion apparatus for preparation of large uni-
lamellar vesicles. Biochim. Biophys. Acta 1061, 297–303.
42. Boldyrev, A.A., Lopina, O.D. & Prokopjeva, V.D. (1984)
Diphenylhexatriene and pyrene as tools for characterization of
biological membranes. Biochem. Int. 8, 851–859.
43. Gidwani, A., Holowka, D. & Baird, B. (2001) Fluorescence ani-
sotropy measurements of lipid order in plasma membranes and
lipid rafts from rbl-2h3 mast cells. Biochemistry 40, 12422–12429.
44. Galla, H.J. & Sackmann, E. (1974) Lateral diffusion in the
hydrophobic region of membranes: use of pyrene excimers as
optical probes. Biochim. Biophys. Acta 339, 103–115.
45. Jones, M.E. & Lentz, B.R. (1986) Phospholipid lateral organiza-

escent phosphoinositide derivatives reveal specific binding of gel-
solin and other actin regulatory proteins to mixed lipid bilayers.
Eur. J. Biochem. 263, 85–92.
53. Asgharian, B., Cadenhead, D.A., Mannock, D. & McElhaney,
R.N. (2000) A comparative monolayer film behaviour study of
monoglucosyl diacylglycerols containing linear, methyl iso-, and
x-cyclohexyl fatty acids. Langmuir 16, 7315–7317.
54. Eriksson, P.O., Rilfors, L., Wieslander, A
˚
., Lundberg, A. &
Lindblom, G. (1991) Order and dynamics in mixtures of mem-
brane glucolipids from Acholeplasma laidlawii studied by 2H
NMR. Biochemistry 30, 4916–4924.
55. Li, L. (2001) Functional Regulation of Glucolipid Synthases in
Membranes.Umea
˚
University, Sweden.
56. Vikstro
¨
m, S., Li, L. & Wieslander, A
˚
. (2000) The nonbilayer/
bilayer lipid balance in membranes. Regulatory enzyme in
1708 P. Storm et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Acholeplasma laidlawii is stimulated by metabolic phosphates,
activator phospholipids, and double-stranded DNA. J. Biol.
Chem. 275, 9296–9302.
57. Holopainen, J.M., Lehtonen, J.Y.A. & Kinnunen, P.K.J. (1997)
Lipid microdomains in dimyristoylphosphatidylcholine-ceramide
liposomes. Chem. Phys. Lipids 88, 1–13.

Rousseau, B., Beaucourt, J.P. & Tocanne, J.F. (1989) Transverse
and lateral distribution of phospholipids and glycolipids in the
membrane of the bacterium Micrococcus luteus. Biochemistry 28,
3728–3737.
66. Berg,S.&Wieslander,A
˚
. (1997) Purification of a phosphatase
which hydrolyzes phosphatidic acid, a key intermediate in gluco-
lipid synthesis in Acholeplasma laidlawii Amembranes.Biochim.
Biophys. Acta Bio. Membr. 4, 225–232.
Ó FEBS 2003 Lateral organization of A. laidlawii lipids (Eur. J. Biochem. 270) 1709