Tài liệu Báo cáo khoa học: Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes ppt

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

curvature stress (Ôspontaneous curvatureÕ). Two key enzymes
depend in a cooperative fashion on substantial amounts of
the endogenous anionic lipid phosphatidylglycerol (PG)
for activity. The lateral organization of five unsaturated
A. laidlawii lipids was analyzed in liposome model bilayers
with the use of endogenously produced pyrene-lipid probes,
and extensive experimental designs. Of all lipids analyzed,
PG especially promoted interactions with the precursor
diacylglycerol (DAG), as revealed from pyrene excimer ratio
(Ie/Im) responses. Significant interactions were also recorded
within the major nonbilayer-prone monoglucosylDAG
(MGlcDAG) lipids. The anionic precursor phosphatidic
acid (PA) was without effects. Hence, a heterogeneous lateral
lipid organization was present in these liquid-crystalline
bilayers. The MGlcDAG synthase when binding at the PG
bilayer interface, decreased acyl chain ordering (increase of
membrane free volume) according to a bis-pyrene-lipid
probe, but the enzyme did not influence the bulk lateral lipid
organization as recorded from DAG or PG probes. It is
concluded that the concentration of the substrate DAG by
PG is beneficial for the MGlcDAG synthase, but that
binding in a proper orientation/conformation seems most
important for activity.
Keywords: Acholeplasma; chemometrics; lipid heterogeneity;
pyrene.
Acholeplasma laidlawii A-EF22 is a simple cell-wall-less
prokaryotic parasite. Its membrane lipid composition is
metabolically adjusted in response to environmental and
lipid-supply conditions. Due to this, A. laidlawii has been
used as a model system to study plasma membrane

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.

Enzymes: 1,2-diacylglycerol-3-glucosyltransferase (MGlcDAG synthase; EC 2.4.1.157); 1,2-diacylglycerol-3-a-glucose (1fi2)-a-glucosyl
transferase (DGlcDAG synthase; EC 2.4.1.208).
(Received 30 November 2002, revised 22 January 2003, accepted 19 February 2003)
Eur. J. Biochem. 270, 1699–1709 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03527.x
(MGlcDAG synthase) (EC 2.4.1.157; I above), makes
monoglucosyl diacylglycerol (MGlcDAG) from DAG plus
UDP-Glc. 1,2-diacylglycerol-3-a-glucose (1fi2)-a-glucosyl
transferase (DGlcDAG synthase) (EC 2.4.1.208), II above,
makes diglucosyl diacylglycerol (DGlcDAG) from MGlc-
DAG and UDP-Glc. Under certain circumstances, when
MGlcDAG turns bilayer-prone by saturated chains, the
more acylated and more nonbilayer-prone minor glu-
colipids, i.e. MAMGlcDAG and MADGlcDAG, are
synthesized [1]. Likewise, substantial amounts (20–30 mol/
100 mol) of the normally minor precursor 1,2-DAG may
accumulate in membranes with many saturated acyl chains [2].
It has been shown that the lipid composition is regulated
to maintain certain properties: (a) a balance between bilayer
and nonbilayer lipids (e.g. MGlcDAG/DGlcDAG) yielding
phase equilibria close to a bilayer/nonbilayer transition; (b)
a certain surface charge density through the ratio between
the glucolipids (MGlcDAG, DGlcDAG and more acylated
variants) and the charged lipids (PG and the phosphoryl-
ated glucolipids). In vivo it has been shown that the ratio
between DGlcDAG and PG is nearly constant [3]. The
regulation of these properties is sensed and performed by
the lipid synthesizing enzymes. Each enzyme acts on a lipid
substrate with a specific headgroup, but are also sensitive to
the type of acyl chains and lipid composition in the
membrane [2].

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

have a bearing on the activity of the A. laidlawii glucosyl-
transferases, in that substrates or surface charges become
locally concentrated? Mammalian plasma membranes show
transverse and lateral asymmetry. In the outer leaflet, ÔraftsÕ
can form by the tight packing of saturated glycosphingo-
lipids and cholesterol in a L
o
phase, possible to isolate
[13–16]. The biological function seems to be enrichment of
certain proteins, e.g. doubly acylated or GPI anchored in
the ÔraftsÕ [17–21] involved in signaling and transport over
the membrane. In the inner leaflet, lateral heterogeneity can
form with phosphatidylserine and diacylglycerol, activating
protein kinase C [22]. In this respect DAG is special in the
interspacing, dehydration and altering conformation of lipid
headgroups, as well as conferring a nonbilayer propensity
for the membrane [23].
The reason for lateral heterogeneity is preferential
interaction between headgroups (Coulombic forces, hydro-
gen bonding, divalent cations, hydration level) or acyl
chains (London forces) of certain lipids [24,25]. It is known
that acyl hydrocarbon chain mismatch can cause lateral
segregation, either by the length or the degree of saturation
[5,26–30]. Indeed, stability of ÔraftsÕ demands a critical
mismatch, as POPE (1-palmitoyl-2-oleoyl-sn-glycerophos-
phatidylethanolamine) but not PDPE (1-palmitoyl-
2-docosahexanoyl-sn-glycerophosphatidylethanolamine)
mix with raft lipids [31]. In vivo lipid mixtures from
Micrococcus luteus and A. laidlawii, both containing gly-
colipids and PG, reveal interactions between the individual

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

1,2-bis-[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phospho-rac-
glycerol (bis-PyrPG) and 1-palmioyl-2-pyrenedecanoyl
phosphatidic acid (PyrPA) were from KKV Bioware
(Espos, Finland).
Organism and growth conditions
A. laidlawii strain A-EF22 was grown at 30 °C in a lipid-
depleted tryptose/bovine serum albumin medium [36]. The
1700 P. Storm et al.(Eur. J. Biochem. 270) Ó FEBS 2003
fatty acids, oleic (18:1c) and palmitic (16:0) were supple-
mented from sterile ethanol stock solutions and pyrene-
decanoyl (PD) acid was supplemented from sterile
dimethyl sulfoxide stock solution. Total concentration of
fatty acids was 150 l
M
in the growth medium. Fatty acids
were radiolabeled with 10 lCiÆL
)1
[
14
C]palmitic and
100 lCiÆL
)1
[
3
H]oleic acid (Amersham Pharmacia Biotech,
Uppsala, Sweden), respectively, after four consecutive
inoculations. 2-hydroxy-propyl-b-cyclodextrin (10 m
M
)
was used in the medium as a carrier for the PD acid

[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

separated better and become more concentrated in two
dimensions. Excised gel spots of pure pyrenyl glucolipids
(MGlcDAG and DGlcDAG) were extracted by chloro-
form/methanol (2 : 1, v/v), and typical fluorescence spec-
tra of mono-pyrenyl and bis-pyrenyl glucolipids visualized
(Fig. 2B,C).
Incorporation of PD and synthesis of pyrenyl
glucolipids
in vivo
No growth of A. laidlawii could be observed with only 16:0
or PD, separately or together (Table 1). The presence of
18:1c fatty acid was very important for both the growth of
A. laidlawii and the incorporation of PD into pyrenyl lipids.
However, the yield of pyrenyl lipids was quite low (less than
10% on the basis of added fatty acids) compared to the
nonpyrenyl lipids (30%)40%), but incorporation into
MGlcDAG and DGlcDAG was fairly similar.
The yield of nonpyrenyl MGlcDAG from A. laidlawii
strain A-EF22 was much lower than that of nonpyrenyl
DGlcDAG when PD in growth media; revealed by
quantitation of nonpyrenyl lipids from excised gel spots
(data not shown).
In the same membrane 18:1c fatty acid preferred to
incorporate PD acid to produce mono-pyrenyl glucolipid
rather than nonpyrenyl glucolipid, whereas 16:0 dominated
in the latter (data not shown). The Ie/Im ratio from the
fluorescence spectra increased for the extracted lipid mixture
from cells when increasing the PD ratio in the medium,
showing that more bis-pyrenyl lipids were synthesized at
higher PD acid to fatty acid ratios (data not shown). The

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

extracted according to a modified Bligh and Dyer method
[38] and separated from other lipids by TLC. The
14
C-
labeled glucolipid products were quantified using electronic
autoradiography (Packard Instant Imager). Homogeneous
MGlcDAG synthase for liposome binding was purified
from detergent-solubilized A. laidlawii cells by three column
chromatography methods, including ion exchange, gel
filtration and hydroxyapatite chromatography [39].
Experimental design
Chemometrics is how to design an experimental series in
order to extract the maximum information from the
minimum number of experiments [40]. Chosen factors are
varied simultaneously in a randomized run order to reduce
or eliminate unknown or uncontrolled influence on data.
Table 1. Incorporation of PD and yield of A. laidlawii pyrenyl lipids.
Fatty acids (l
M
) PD/FA
% Incorporation
of PD into lipids16:0 18:1c PD Initial Harvest
1 120 30 0 – – –
2 90 30 30 0.25 0.024 3.83
3 30 30 90 1.5 0.21 8.13
4 0 60 90 1.5 0.31 6.17
5 0 30 120 4 0.68 7.03
6 0 10 140 14 1.62 4.22
Ó FEBS 2003 Lateral organization of A. laidlawii lipids (Eur. J. Biochem. 270) 1701
The response(s) Y is then fitted to the variables by a

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.

Partial least squares (PLS) was used to fit the model.
PLS finds the relationship between a matrix Y (response
variables) and a matrix X (model terms). Measure of model
fit is R
2
¼ 1 ) (RSS/YSS), where RSS is residual sum of
squares and YSS is the response sum of squares. Internal
validation (crossvalidation or prediction ability) is measured
by the Q
2
value, i.e. Q
2
¼ 1 ) (PRESS/YSS), where PRESS
is the predicted residual sum of squares. Rules of thumb are
that R
2
should be at least 0.8 and Q
2
above 0.3 for linear
models and even closer to one for quadratic models. R
2
and
Q
2
are the overall parameters for model accuracy, which
encompass analysis of variance (
ANOVA
), lack of fit, normal
distribution of residuals. If these parameters are not
satisfactory, then outliers, wrong metric, inhomogeneous

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

100 nm). This yields large unilamellar vesicles (LUV) with
an average diameter of nearly 100 nm [41]. The quality of
vesicles for all data points was verified with dithionite
quenching of an NBD-probe, showing that all vesicles were
LUVs, as only the outer leaflet is quenched and roughly
50% of the signal remained after quenching (data not
shown).
Fluorescence and absorbance methods
Absorbance measurements were performed with a Beckman
DU 70 spectrophotometer.
Fluorescence measurements with labeled vesicles were
carried out so that 50 lL of prepared liposomes were added
to 1950 lL buffer in an optical 1 · 1 cm fluorescence
cuvette, and fluorescence measured with a Spex Fluoro-
Max-2 fluorometer with magnetic stirrer and temperature
control (28 °C). Samples with pyrene probes, were excited at
344 nm and emission spectra collected between 360 and
500 nm. Slits had a bandwidth of 1 nm for excitation and
4 nm for emission (step width 1 nm, integration time 0.5 s).
Four scans were sampled, averaged, and subtracted by a
blank consisting of the buffer, in order to obtain the
fluorescence curve. Vesicles without a probe do not
particularly affect the spectra, as verified in a control design
showing only noise that was virtually the same as the blank;
therefore no such reference was used in any of the runs.
Ie/Im (excimer ratio) was calculated as the ratio between
excimer emission at 480 nm (Ie), when two pyrenes are
in close proximity ( 3.5 A
˚
), and monomer emission at

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

calculated and averaged in connection to the measurement
by a computer program.
Results
Enzymes recognize pyrene derivatives
For analysis of potential interactions between the various
A. laidlawii membrane lipids we chose fluorescence spectro-
scopy, with pyrene-labeled lipids containing one normal and
one pyrene-labeled chain as proximity probes, as the studied
phenomena may be transient and not possible to isolate,
and too small (<300 nm) to be detected with microscopy.
Pyrene-decanoyl chains locate in the membrane hydro-
phobic core and are virtually nonperturbing at a fraction of
1 mol/100 mol or less, partitioning preferentially in the fluid
phase [44–49]. To investigate chain ordering, i.e. membrane
free volume (V
f
), a bis-pyrenyl lipid, with a pyrene on both
acyl chains, was used [47,50].
Native A. laidlawii pyrene-labeled glucolipids (not com-
mercially available) were produced in vivo,andtestedas
lipid enzyme substrates in vitro to monitor the impact of the
pyrenyl chain moiety on headgroup organization. Pyrenyl-
decanoic acid (PD) was used for the incorporation of the
pyrene group into the lipids. 16:0 and 18:1c fatty acid were
chosen for their approximately similar chain length to PD.
Different compositions of growth medium fatty acids were
used to optimize the incorporation of PD acid, with or
without 16:0 and 18:1c, into the glucolipids (Table 1). Cell
growth and size of cells were checked by routine light
microscopy. PD or 16:0 could not support growth, alone or

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

from TLC plates. Similar yields were obtained for pyrenyl-
glucolipid and nonpyrenyl-glucolipid products, from both
the MGlcDAG synthase and DGlcDAG synthase reactions
(Fig. 3). Furthermore, the shape of the fluorescence spectra
of the product depends on which type of pyrenyl lipid was
used as substrate; mono- and bis-pyrenyl lipid substrate
produced mono- or bis-pyrenyl glucolipid products,
respectively (data not shown). Thus, these enzymes do not
discriminate between substrates with a pyrene moiety in the
acyl chain. Similar features have been observed for enzymes
Fig. 2. In vivo synthesis of pyrenyl lipids. (A) A. laidlawii
14
C/
3
H-
labeled glucolipids and pyrenyl-glucolipids after TLC separation.
Extracted lipids applied on TLC plates were developed in chloroform/
methanol/ammonia (91 : 35 : 10, v/v/v). The growth medium fatty
acid composition (ratio 16:0/18:1c/PD) was from left to right:
120 : 30 : 0; 90 : 30 : 30; 30 : 30 : 90; 0 : 60 : 90; 0 : 30 : 120 and
0 : 10 : 140. Encircled spots represent the fluorescent mono-
pyrenylMGlcDAG (lower) and bis-pyrenylMGlcDAG (upper),
respectively. The fluorescence spectra of purified mono-pyrenyl (B)
and bis-pyrenyl glucolipids (C) produced in vivo.Thesamples(0.1m
M
lipid) were excited at 344 nm in chloroform/methanol (2 : 1, v/v).
Synthetic mono-pyrenylDAG (B), and bis-pyrenylPG (C), were used
as references.
Fig. 3. Pyrenyl lipids as enzyme substrates in vitro. Synthesis of
DGlcDAG from MGlcDAG and UDP-[

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

pyrene-labeled probes for each lipid type. Table 2 lists lipid
composition and the statistically significant changes in Ie/Im
(the excimer ratio) for different pyrene derivatives as a
function of different headgroups, going from a low mole
content to a high mole content for each lipid, and where all
acyl chains are 18:1c (dioleoyl). In two cases this investiga-
tion was expanded to a composite face-centered (CCF)
design.
The distribution of DAG, as monitored by Ie/Imofthe
PyrDAG probe (Table 2) was affected significantly by PG
and DAG (increased Ie/Im), but none of the other lipids,
according to a CCF design. Interestingly, the model also
revealed a dependence (interaction) between the variables
PG and DAG (data not shown), i.e. increasing both PG and
DAG does not increase the Ie/Im additively. In accordance
with this model a one-variable titration, where DOPG was
varied 0–40 mol/100 mol (at constant 5 mol/100 mol
DOPA, 5 mol/100 mol DOG and DGlcDAG as balance),
showed a 1.5-fold increase in Ie/Im (0.080–0.124) for the
PyrDAG probe between 0 and 25 mol/100 mol PG, and
then a decrease to 0.105 between 25 and 40 mol/100 mol
PG (Fig. 4). We therefore conclude that DAG has a
heterogeneous distribution, strongly promoted by increas-
ing amounts of PG. It is interesting to note that increasing
the amount of CL (0–20 mol/100 mol) in a DGlcDAG
matrix with 5 mol/100 mol DOG and 1 mol/100 mol
PyrDAG, did not produce any change in the Ie/Im(data
not shown).
For the PyrPG probe Ie/Im increased 1.35-fold (0.051–
0.069) between 0 and 40% PG (Table 2). It has been noted

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

/Q
2
PyrDAG – 0.078–0.087 (11.5%) 0.078–0.110 (41%) – ± 0.0017 0.97/0.72
PyrPG – – 0.051–0.069 (35%) – ± 0.0031 0.81/0.59
PyrPA 0.058–0.062 (7%) 0.058–0.064 (10%) – – ± 0.0027 0.90/0.51
PyrMGDG 0.081–0.107 (32%) – – – ± 0.0027 0.78/0.64
PyrPC 0.064–0.068 (6%) 0.064–0.065 (1.5%) 0.064–0.073 (14%) NT ± 0.0029 0.94/0.42
1704 P. Storm et al.(Eur. J. Biochem. 270) Ó FEBS 2003
excimer ratio for pyrene-labeled DGlcDAG, Ie/Imwas
always approximately 0.06. As the PyrPG signal increased
during these conditions (Table 2, Fig. 4), this may indicate
affinity between like molecules for these two lipids.
The Ie/Im for pyrene probes of MGlcDAG and PA were
not changed when increasing the amount of the enzyme
activator lipid PG (Table 2), indicating no interactions.
However, increasing the amounts of normal MGlcDAG
gave an increase in Ie/Im for PyrMGlcDAG probe
(Table 2), supporting an interaction between the MGlc-
DAG molecules (note that decreasing DGlcDAG amounts
was the balance). Also, headgroup interaction for MGlc-
DAG lipids is indicated in a monolayer study, showing a
much more compact fluid state than the equivalent
phosphatidylcholine (PC) lipid [53]. For PA, precursor to
both the glucolipid and phospholipid pathways, an increase
in Ie/Im for PyrPA probe was also observed when
increasing the amount of DAG or MGlcDAG (Table 2)
in a CCF design, supporting a heterogeneous distribution,
however, the effect was small. Note that there are only a few
mol percent of this lipid naturally occurring in the
membrane. A small effect was also seen with PyrPC as a

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

location of pyrene has nevertheless been determined to be
located close to the bilayer center. MGlcDAG, shown to
increase order [54], is organized laterally in this five-lipid
model system in a way that gives no significant contribution
to the ordering of the membrane bilayer, according to the
experimental design model.
Enzyme binding, lateral organization and chain order
The MGlcDAG synthase (purified without detergent [55]),
when binding to the lipid bilayers at initial protein : lipid-
ratios of 1 : 700, 1 : 350 or 1 : 70 (mol/mol), did not affect
the Ie/Im of PyrDAG or PyrPG (data not shown). Thus, the
substrate lipid DAG and the activator lipid PG were not
concentrated on a large scale by the enzyme. Yet, binding
and activity with liposomes, as here, was strongly correlated
with increasing amounts of anionic lipid ([55] and Li et al.
submitted), with a Hill coefficient of 4–6, which is the
potential number of PG molecules associated to the enzyme.
However, the number of lipid molecules bound under an
enzyme makes it very unlikely that these be two probe
molecules (1 mol/100 mol concentration), even at the
highest enzyme-lipid ratio. Binding was practically irrever-
sible and more enzymes bound to liposomes as a function of
mol/100 mol anionic lipid (promoted by nonbilayer lipid),
revealed by surface plasmon resonance experiments using
Biacore (Li et al. submitted). Furthermore, the Ie/Imfroma
bis-PyrPC probe was reduced by the addition of enzyme,
as seen in a design (R
2
¼ 0.93, Q
2

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

DODAG low (0 mol%) 1.2 0.115
high (10 mol%) 1.262 0.108
DODGlcDAG low (40 mol%) 1.38 0.103
high (90 or 99 mol%) 1.02 0.14
Replicate error ± 0.024 ± 0.0014
Ó FEBS 2003 Lateral organization of A. laidlawii lipids (Eur. J. Biochem. 270) 1705
Discussion
Synthesis of pyrenyl glucolipid
The existence of 18:1c fatty acid in the medium is very
important for both cell growth and the incorporation of PD
into glucolipids (Materials and Methods). A higher ratio of
PD in the growth medium produces more bis-pyrenyl
glucolipids, as A. laidlawii must choose PD as the side
chain, as there are not enough of the other fatty acids in the
medium. However, the cell cannot grow well with a high PD
ratio. In vivo it might be difficult to incorporate PD acid into
the precursor PA as the occupied volume and hydropho-
bicity are larger than for 18:1 or 16:0 fatty acids, resulting in
low yields of pyrenyl lipids. In our experiment, less than
10% of PD could be incorporated into lipids. Media with a
30 : 120 ratio of 18:1c to PD is appropriate to obtain a
reasonable yield of mono-pyrenyl and bis-pyrenyl gluco-
lipids. In vitro, using PyrDAG, purified mono-pyrenyl
MGlcDAG or bis-pyrenyl together with extra large
amounts of nonpyrenyl lipids as substrates, the product
yields were approximately the same for these two glucolipid
synthases (Fig. 2A). This suggests that the enzymes do not
discriminate between pyrenyl lipid and nonpyrenyl lipid in a
micelle system, which makes pyrenyl-glucolipid probes
possible to use in the study of metabolic stages of glucolipids

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

, the pyrene derivative move at most two lipid
diameters and should not form excited dimers unless
already being close [59]. This is supported by PyrPC
showing a small change in Ie/Im (Table 2) and by the
ordering of lipids seen with bis-PyrPC (Table 3). Indeed,
total lipid extracts from A. laidlawii grownattwodifferent
growth temperatures, thereby containing different lipid
compositions, show a fairly similar lateral diffusion coeffi-
cient [34]. An interesting observation is that anisotropy of
DPH is decreasing when Ie/Im of bis-PyrPC is increasing
(Table 3). However, Ie/Im increases when cholesterol is
included in the alloys with sugar lipids (data not shown),
giving reason to believe that bis-PyrPC gives a good
indication of the fluidity (free volume) in the membrane. No
further investigation was made into this phenomenon, but
hypothesizing that it may have to do with the matrix of
sugar lipids with 18:1c acyl chains, as a report by Kaiser and
London [60] states that DPH is located close to bilayer
center in DOPC bilayers.
Thus, DAG is segregated in what seems like micro-
domains by increasing the amount of PG in the membrane,
possibly due to favorable hydrogen bonding between DAG
and PG. This is not due to charge entirely, as PA had
no appreciable effect on the PyrDAG-probe response. The
decrease in Ie/Im after 25 mol/100 mol PG (Fig. 4) can be
that a microdomain formed preferably by the DAG lipid is
diluted. DAG is a special lipid, as pointed out in a review by
Goni and Alonso [23]. Constituting only a small fraction of
membranes, it can act as an intracellular second messenger
or metabolic intermediate and is involved in enzyme

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

headgroup repulsion, is also important. PG, which has a
flexible glycerol moiety in the headgroup, is shown to take
part in hydrogen bonding [63,64] and it has been noticed
that PG and dimannosyl-DAG is heterogeneously distri-
buted, due to interactions in the headgroup region [65].
Furthermore, lipids in total lipid mixtures from A. laidlawii
have a smaller interfacial area than any of the individual
lipids [33,65], due to interactions in the headgroup region
causing a lateral condensation. An interaction between
MGlcDAG or DGlcDAG species was revealed here from
the responses of the corresponding pyrene probes (Table 2
and Results above).
The patching of substrate lipid DAG with activator PG
(Fig. 4), in combination with an increased charge density
creating an electrostatic interaction between the membrane
and the enzyme (L. Li, unpublished observation), contribute
to the explanation of the anionic lipid preference for the
MGlcDAG synthase. An increase in acyl chain order also
precedes enzyme activity (bis-PyrPC signal in Fig. 4). For
the DGlcDAG synthase the patching of its substrate
MGlcDAG when the amount increases (Table 2) seems
not biologically relevant as PG is the only naturally
occurring activator, and that 3 mol/100 mol MGlcDAG
saturates the DGlcDAG synthase [8]. However, the order
increase probably contributes to the activity (Fig. 4). With
respect to the precursor PA, the PyrPA show an addi-
tively increased Ie/Im by DAG and MGlcDAG (Table 2),
but the biological function is less clear as the PA phospha-
tase is not regulated by changes in lipid composition [66].
In conclusion, this is the first time that up to five lipids

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.
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