Báo cáo khoa học: Identification of a glycosphingolipid transfer protein GLTP1 in Arabidopsis thaliana ppt

Identification of a glycosphingolipid transfer
protein GLTP1 in Arabidopsis thaliana
Gun West
1,
*, Lenita Viitanen
1,
*, Christina Alm
2
, Peter Mattjus
1
, Tiina A. Salminen
1
and Johan Edqvist
3
1 Department of Biochemistry and Pharmacy, A
˚
bo Akademi University, Turku, Finland
2 Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden
3 IFM-Biology, Linko
¨
ping University, Sweden
Glycosphingolipids (GSLs) carry one or more sugar
units on a ceramide backbone [1]. These lipids are
major constituents of eukaryotic plasma membranes,
and function in several cellular processes such as cell
death [2,3], adhesion [4] and cell–cell recognition [5,6].
In plants, two types of GSLs are found: the neutral
cerebroside glucosylceramide (GlcCer), which has a
glucosyl residue at the primary hydroxyl group of
sphinganine, and the so-called phytoglycosphingolipids
or inositol phosphorylceramide, with a ceramide-

1-phosphate base, to which glycosylated inositol
residues are bound via a phosphodiester bond [7–10].
Typical mammalian sphingolipids, e.g. galactosylcera-
mide (GalCer), lactosylceramide (LacCer), neuraminic
Keywords
ceramide; GLTP; glycolipids; lipid transfer;
sphingolipids
Correspondence
J. Edqvist, IFM-Biology, Linko
¨
ping
University, SE-581 83 Linko
¨
ping, Sweden
Fax: +46 13 281399
Tel: +46 13 281288
E-mail:
*These authors contributed equally to this
study
(Received 24 January 2008, revised 12
March 2008, accepted 30 April 2008)
doi:10.1111/j.1742-4658.2008.06498.x
Arabidopsis thaliana At2g33470 encodes a glycolipid transfer protein
(GLTP) that enhances the intervesicular trafficking of glycosphingolipids
in vitro. GLTPs have previously been identified in animals and fungi but
not in plants. Thus, At2g33470 is the first identified plant GLTP and we
have designated it AtGTLP1. AtGLTP1 transferred BODIPY-glucosyl-
ceramide at a rate of 0.7 pmolÆs
)1
, but BODIPY-galactosylceramide and

BODIPY-lactosylceramide were transferred slowly, with rates below
0.1 pmolÆs
)1
. AtGLTP1 did not transfer BODIPY-sphingomyelin, monoga-
lactosyldiacylglycerol or digalactosyldiacylglycerol. The human GLTP
transfers BODIPY-glucosylceramide, BODIPY-galactosylceramide and BO-
DIPY-lactosylceramide with rates greater than 0.8 pmolÆs
)1
. Structural
models showed that the residues that are most critical for glycosphingolipid
binding in human GLTP are conserved in AtGLTP1, but some of the
sugar-binding residues are unique, and this provides an explanation for the
distinctly different transfer preferences of AtGLTP1 and human GLTP.
The AtGLTP1 variant Arg59Lys⁄ Asn95Leu showed low BODIPY-gluco-
sylceramide transfer activity, indicating that Arg59 and ⁄ or Asn95 are
important for the specific binding of glucosylceramide to AtGLTP1. We
also show that, in A. thaliana, AtGLTP1 together with At1g21360 and
At3g21260 constitute a small gene family orthologous to the mammalian
GLTPs. However, At1g21360 and At3g21260 did not transfer any of the
tested lipids in vitro.
Abbreviations
DGDG, digalactosyldiacylglycerol; GalCer, galactosylceramide; GlcCer, glucosylceramide; GLTP, glycolipid transfer protein; GSL,
glycosphingolipid; GST, glutathione S-transferase; LacCer, lactosylceramide; MGDG, monogalactosyldiacylglycerol; POPC, 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine; SM, sphingomyelin.
FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS 3421
(sialic) acid-containing ceramides (gangliosides) and
sphingomyelin (SM), have not been found in higher
plants, such as Arabidopsis thaliana [7–9].
A remarkable property of the GSLs is that they
have a high melting temperature due to the high sat-

uration of the hydrocarbon chains, and, furthermore,
the region between the polar head group and the
hydrophobic backbone contains chemical groups that
can function both as hydrogen bond donors and
hydrogen bond acceptors [11]. These properties allow
GSLs to self-associate and bring local order to
otherwise disordered and fluid membranes [12]. The
ordered membrane microdomains or lipid rafts,
which are enriched in GSLs and sterols, are believed
to play important roles in protein sorting, signal
transduction and infection by pathogens, as the rafts
appear to mediate a lipid-based sorting mechanism
that could facilitate protein–protein interactions by
selectively including or excluding proteins [13,14].
Membrane microdomains have mostly been studied
in animal and yeast cells; however, it was recently
suggested that similar lipid rafts enriched in GlcCer
and sterols also exist in plant plasma membranes
from tobacco leaves and BY2 cells as well as in
callus membranes [15,16].
Serine palmitoyltransferase catalyses the first step
in sphingolipid biosynthesis, which is the formation
of 3-ketosphinganine from the condensation of serine
and palmitoyl CoA [17,18]. The 3-ketosphinganine is
reduced to sphinganine, which is subsequently acyl-
ated to produce ceramide. In mammalian cells, cera-
mide is synthesized in the endoplasmic reticulum
(ER) and translocated to the Golgi compartment for
further conversions into more complex sphingolipids.
The ceramide transport protein mediates intracellular

trafficking of ceramide between ER and the Golgi in
a non-vesicular manner [19]. The biosynthesis of
GlcCer is catalyzed by a UDP-glucose:ceramide
glucosyltransferase (GlcCer synthase), which transfers
glucose to the ceramide backbone [20]. In mammalian
cells, GlcCer is synthesized at the cytosolic surface of
the Golgi membrane. In Drosophila melanogaster, the
GlcCer synthase GlcT-1 has also been identified in
pre-Golgi compartments including the ER, indicating
that ER to Golgi ceramide transport may not always
be necessary for GlcCer synthesis [2]. GlcCer synthase
has also been identified in plants, but the intracellular
location of the plant enzyme has not been determined
[21]. GlcCer is enriched in the plasma membrane and
endosomes, suggesting that there is a need for trans-
port of GlcCer from the Golgi to the plasma mem-
brane. Transport of GlcCer probably occurs via
transport vesicles and non-vesicular monomeric trans-
port through the cytosol [22]. Non-vesicular transport
may be mediated by glycolipid transfer proteins
(GLTPs), which accelerate the transfer of GSLs
between membranes in vitro. GLTPs are specific for
GSLs, such as GlcCer and GalCer for example,
which have sugar residues attached via b-linkages to
the lipid hydrocarbon backbone [23].
Glycolipid transfer protein was discovered initially
in membrane-free cytosolic extracts of bovine spleen
[24], and later in a wide variety of tissues [23,25]. It
is a ubiquitous, basic (pI 9), soluble protein of
24 kDa [26]. The crystal structures of apo-GLTP

and lactosylceramide-bound GLTP show a topology
dominated by a-helices with a single binding site for
the GSL [27]. So far, no phenotypes have been asso-
ciated with a lack of functional GLTP in metazoans.
The HET-C2 protein from the filamentous fungi
Podospora anserina shows sequence similarity to the
mammalian GLTPs, GSL transfer activity [28] and a
functional GSL binding site similar to that of mam-
malian GLTPs [29]. Inactivation of the het-c2 gene
leads to abnormal ascospore formation, and it has
been suggested that HET-C2 is involved in regulating
cell-compatibility interactions during the hetero-
karyon formation that occurs during hyphal fusion
between different strains [30,31]. The mammalian
four-phosphate adaptor protein 2 (FAPP2) protein
contains a domain with similarity to GLTPs, con-
nected to a pleckstrin homology domain. Two recent
studies have indicated that GlcCer synthesis in early
Golgi compartments, as well as its transport by
FAPP2 to distal Golgi compartments, is required for
protein transport out of the distal compartments
[32,33].
The lethal recessive knockout of the A. thaliana gene
ACCELERATED CELL DEATH 11 (ACD11) shows
activation of programmed cell death. ACD11 shares
30% similarity to mammalian GLTPs, and has been
suggested to be orthologous to mammalian GLTPs.
However, ACD11 does not translocate GSLs in vitro;
instead, it facilitates the intermembrane transfer of
single-chain sphingosine [34]. Our aim was to deter-

mine whether plants encode and express GLTPs with
specificity for GSLs. We have identified three genes in
the A. thaliana genome, At1g21360, At2g33470 and
At3g21260, that, based on sequence analysis, encode
GLTP-like sequences. At2g33470 and At3g21260 were
also recently identified as putative GLTPs by Jouhet
et al. [35]. According to our structural models,
At1g21360 and At2g33470 have the necessary features
for binding GSL. In vitro lipid transfer assays con-
firmed that At2g33470 (designated as AtGLTP1) is in
fact a GLTP with specificity for GlcCer.
Arabidopsis glycolipid transfer protein G. West et al.
3422 FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS
Results
Identification of A. thaliana glycolipid transfer
proteins
We used the amino acid sequences of bovine and
human GLTPs to search databases for GLTP-like pro-
teins from other eukaryotic organisms. Putative
GLTPs were detected in vertebrates, insects and nema-
todes, but also in the cnidarians Hydra magnipapillata
and Nematostella vectensis, in the choanoflagelate Mo-
nosiga ovata, in fungi classified as zygomycota, basid-
omycota and ascomycota, in green algae and land
plants, in species of the phylum Apicomplexa, such as
Cryptosporidum and Plasmodium, and in the diplomo-
nad Giardia lamblia. We could not identify any GLTP-
like proteins in the yeasts Schizosaccharomyces pombe
and Saccharomyces cerevisae, in the slime mold Dictyo-
stelium discoideum, in ciliates, or in Trypanosoma and

Leishmania (Kinetoplastida).
There have been no reports of any plant GLTP with
specificity for GSLs, and therefore it was particularly
interesting to discover that A. thaliana proteins from
five genes (ACD11, At1g21360, At2g33470, At3g21260
and At4g39670; Fig. 1A), gave blast e-values below
5e-05 when the amino acid sequence of human GLTP
was used as bait. These five genes encode proteins with
amino acid sequences that show 18–25% identity and
A
B
Fig. 1. Analysis of the amino acid
sequences of putative Arabidopsis thaliana
GLTPs. (A) Percentage of amino acid
sequence similarity and identity from pair-
wise comparisons of the identified putative
A. thaliana GLTPs and human (Hs) GLTP. In
each case, the value before the solidus indi-
cates identity, and that after the solidus indi-
cates similarity. (B) Multiple amino acid
sequence alignment of AtGLTP1,
At1g21360, At3g21260, ACD11 and
At4g39670. The amino acid sequences of
human GLTP, the fungus Podospora anseri-
na HET-C2 and the red alga Galdieria sulphu-
raria GLTP are also included. Black boxes
indicate that identical amino acids are pres-
ent in at least four of the sequences, while
shaded boxes indicate that amino acids with
similar physicochemical properties are pres-

ent in at least four of the sequences.
G. West et al. Arabidopsis glycolipid transfer protein
FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS 3423
32–39% similarity to human GLTP (Fig. 1B). The
highest similarity scores between the A. thaliana puta-
tive sphingolipid transporters are found between
At1g21360 and At3g21260, which share 54% identity
and 70% similarity, and between ACD11 and
At4g39670, with 45% similarity and 61% identity. The
putative molecular masses of these A. thaliana proteins
range from 17 kDa for At3g21260 to 27 kDa for
At4g39670. ACD11 has previously been shown to
facilitate the intermembrane transfer of single-chain
sphingosine in vitro, but does not transfer GSLs
in vitro [34]. The lethal recessive knockout of ACD11
shows activation of programmed cell death. There are
no reports on the physiological function, biochemical
activity or regulation of the other proteins identified.
To investigate the relationship between these A. tha-
liana proteins and known and putative eukaryotic
GLTPs, a phylogenetic tree (Fig. 2) was constructed
from the amino acid sequences using the maximum-
likelihood method [36]. The phylogenetic analysis sug-
gests that At1g21360, At2g33470 and At3g21260 share
a common origin with metazoan and fungal GLTPs.
The tree indicates a close relationship between
At1g21360 and At3g21260, and suggests that this gene
pair evolved from a duplication event that occurred
after the split of monocotyledons and dicotyledons.
Fig. 2. Phylogenetic tree of glycolipid trans-

fer protein amino acid sequences recon-
structed by the maximum-likelihood
method. Numbers indicate the percentage
of 100 bootstrap re-samplings that support
the inferred topology. Only bootstrap values
over 50% are shown. Sequences are identi-
fied by gene names or by National Center
for Biotechnology Information GI numbers.
Arrows indicate amino acid sequences of
putative GLTPs from A. thaliana.
Arabidopsis glycolipid transfer protein G. West et al.
3424 FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS
The tree further indicates that a gene duplication that
occurred before the split of gymnosperms and angio-
sperms is responsible for the formation of the
At2g33470 and At1g21360 ⁄ At3g21260 gene families.
ACD11 and At4g39670 group close together in a sepa-
rate plant-specific branch, containing sequences from
other land plants and green algae. The evolutionary
relationship of this plant-specific branch to the GLTP
branch of the tree is unclear. On the basis of the
results of the sequence analyses, we concluded that
At1g21360, At2g33470 (here on referred to as
AtGLTP1) and At3g21260 are possible candidates for
A. thaliana GLTPs. We therefore focused our attention
on gaining insight into the biological role, activity and
ligand specificity of these proteins.
Structural modeling of putative A. thaliana
GLTPs
Structural models of AtGLTP1 (At2g33470),

At1g21360 and At3g21260 in apo form were con-
structed (supplementary Fig. S1) in order to examine
whether they have similar structural features to human
GLTP, supporting the theory that the proteins are
GLTPs. Based on the sequence alignments used for
modeling, AtGLTP1 shares a sequence identity of
23% with human GLTP and 27% with Galdieria
sulphuraria GLTP (GsGLTP). The overall folding of
the human apo-GLTP and apo-GsGLTP X-ray struc-
tures is very similar, but they are clearly different at
the N- and C-termini. The longer N-terminal part of
GsGLTP forms an a-helix. The C-terminal part of
GsGLTP is a long unstructured loop stretching away
from the sugar-binding site, whereas the C-terminus in
human GLTP is much shorter and participates in
ligand binding in the complex structures [27,37].
The AtGLTP1 (supplementary Fig. S1A) and
At1g21360 models have a two-layer all-a-helical topol-
ogy, with a binding pocket for a sugar moiety lined by
polar amino acids and a hydrophobic tunnel suitable
for binding the hydrocarbon chains of lipids. The
hydrophobic nature of the tunnel is highly conserved,
although only a few of the amino acids are totally con-
served. At3g21260 is considerably shorter than
AtGLTP1 and At1g21360, missing residues 1–57 and
1–74, respectively (Fig. 1B). This means that the model
of At3g21260 lacks the first layer of a-helices and
consequently half of the hydrophobic tunnel.
In human GLTP, the residues Asp48, Asn52, Trp96
and His140 have been shown by point mutations to be

the most important residues for the recognition of
sugar-amide moieties [27,38] (Fig. 3B). In AtGLTP1,
these residues are conserved and correspond to Asp52,
Asn56, Trp99 and His138 (Figs 1B and 3C–E), and
are also totally conserved in At1g21360 (Fig. 1B and
supplementary Fig. S1B). At3g21260 lacks the aspar-
tate and the asparagine, as it is much shorter at the
N-terminus, but has the conserved tryptophan and his-
tidine (Fig. 1B and supplementary Fig. S1D). When
we compared the other residues (Lys55, Leu92, Tyr207
and Val209) that interact with GSLs in human GLTP–
GSL complex structures, some interesting differences
were identified between human GLTP and the putative
A. thaliana GLTPs. Firstly, Lys55 in human GLTP is
replaced by Arg59 in the sugar recognition center of
AtGLTP1, and there is also an arginine in this posi-
tion in At1g21360 and At3g21260. Secondly, the resi-
due equivalent to Leu92 in human GLTP is Asn95 in
AtGLTP1 (Figs 1B and 3C–E and supplementary
Fig. S1B). This residue is replaced by an arginine in
both At1g21360 and At3g21260 (Fig. 1B). Thirdly, the
residue corresponding to Tyr207 in the human GLTP
is a lysine in both AtGLTP1 (Lys200) and At3g21260,
but an arginine in At1g21360. This makes the sugar-
binding pocket of At1g21360 very rich in arginines.
Fourthly, the residue corresponding to Val209 in
human GLTP corresponds to Ser202 in AtGLTP1, a
methionine in At1g21360 and a proline in At3g21260
(Fig. 1B). In summary, the modeling shows that the
AtGLTP1 and At1g21360 proteins are probably

GLTPs, as they share extensive structural similarities
with human GLTP. Amino acid replacements in the
sugar recognition center suggest that AtGLTP1 and
At1g21360 may have different sugar-binding properties
compared to human GLTP. At3g21260 has an incom-
plete hydrophobic binding cavity, which indicates that
it is not able to bind GSLs.
Lipid transfer capability of AtGLTP1
In order to examine whether AtGLTP1, At1g21360
and At3g21260 show a glycolipid transfer activity simi-
lar to that found for mammalian GLTPs [39], we
expressed the Arabidopsis proteins in Escherichia coli.
To test the lipid transfer capacity of the proteins, we
used a previously established transfer assay, which
relies on resonance energy transfer between a transfer-
able (energy donor) fluorescent lipid and a non-trans-
ferable (energy acceptor) fluorescent lipid from a
donor vesicle population to an acceptor population
[40–43]. The intervesicular trafficking of three
BODIPY-labeled glycolipids, GlcCer, GalCer and
LacCer, and a BODIPY-labeled SM was monitored as
a function of time between donor 1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphocholine (POPC) vesicles and
POPC acceptor vesicles (in a tenfold excess) using 4 lg
G. West et al. Arabidopsis glycolipid transfer protein
FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS 3425
of protein. AtGLTP1 is able to efficiently transfer
BODIPY-GlcCer (Fig. 4A, black trace), whereas
BODIPY-GalCer and BODIPY-LacCer are transferred
only to a limited extent (Fig. 4A, red and green

traces). BODIPY-SM was not transferred at all (yellow
trace, Fig. 4A). On the basis of its capacity to enhance
the translocation of BODIPY-GlcCer, we decided to
designate At2g33470 as AtGLTP1. At1g21360 and
At3g21260 are not able to transfer any of the
BODIPY-labeled lipids under the conditions of the
resonance energy transfer assay (Fig. 4B). Human
GLTP is able to efficiently move all three labeled gly-
colipids, but not BODIPY-SM (Fig. 4D). Numerical
values for the transfer rates are given in Table 1.
Using a competition assay, we also analyzed the
substrate specificity of AtGLTP1 for monogalactosyl-
diacylglycerol (MGDG) and digalactosyldiacylglycerol
(DGDG; supplementary Fig. S2). There was no change
in the transfer of BODIPY-GlcCer after addition of
MGDG and DGDG, which indicates that neither
MGDG nor DGDG are substrates for AtGLTP1.
Addition of POPC vesicles was used as a reference.
Human GLTP appears to be able to transfer DGDG
and MGDG to some extent (supplementary Fig. S2),
A
DEF
BC
Fig. 3. The human GLTP crystal structure and the sugar-binding pocket of AtGLTP1 (At2g33470). (A) The fold of the human GLTP crystal
structure with bound LacCer in yellow. (B) Close-up of the sugar-binding pocket in the crystal structure of human GLTP with bound GalCer
in yellow. Binding residues are shown in grey. (C–E) Sugar-binding pocket in the structural models of AtGLTP1 (At2g33470) in complex with
GlcCer (C), LacCer (D) and GalCer (E). The difference between the glucosyl and galactosyl units [in (B), (C) and (E)] is the orientation of the
OH4 hydroxyl (marked with arrow). (F) Sugar-binding pocket in the structural model of the AtGLTP1 Arg59Lys ⁄ Asn95Leu mutant. Conserved
residues are shown in grey and non-conserved residues are shown in green (C–F). GSLs are in yellow.
Table 1. AtGLTP1 and human GLTP in vitro lipid transfer activity.

The GLTP-mediated (4 lg) BODIPY-labeled lipid transfer was exam-
ined using a fluorescence assay, and the values given are
means ± SD from at least four analyses. Rates are given as pmol
transferred per second.
Protein Lipid
Transfer rate
(pmolÆs
)1
)
AtGLTP1 BODIPY-GlcCer 0.65 ± 0.06
BODIPY-GalCer 0.08 ± 0.04
BODIPY-LacCer 0.02 ± 0.01
Human GLTP BODIPY-GlcCer 1.16 ± 0.09
BODIPY-GalCer 0.93 ± 0.06
BODIPY-LacCer 0.83 ± 0.04
Arg59Lys ⁄ Asn95Leu mutant BODIPY-GlcCer 0.006 ± 0.01
BODIPY-GalCer 0.012 ± 0.01
Arabidopsis glycolipid transfer protein G. West et al.
3426 FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS
which is in agreement with previous findings using
porcine GLTP purified from brain [44]. Control
experiments (supplementary Fig. S3) with addition of
unlabeled GlcCer to the AtGLTP1 transfer assay
indicate that GlcCer competes for the labeled
BODIPY-GlcCer substrate, as the transfer trace tapers
off significantly after addition of the GlcCer vesicles.
The BODIPY-GlcCer transfer continues at the same
rate after addition of POPC, MGDG and DGDG.
Analysis of the GSL transfer specificity of
AtGLTP1

In order to obtain an understanding of the differences in
the ligand transfer activities between the plant and
mammalian GLTPs, we attempted to change the GSL
binding specificity of AtGLTP1 through mutagenesis.
Models of AtGLTP1 in complex with GSLs were con-
structed in order to identify amino acids suitable for
mutagenesis. In the human GLTP–GSL complex struc-
tures [27,37], the first sugar unit stacks with Trp96 and
forms a network of hydrogen bonds with Asp48, Asn52,
Lys55 and Tyr207 (Fig. 3B). In AtGLTP1, Trp99,
Asp52 and Asn56 are conserved, while the lysine and
tyrosine are replaced by Arg59 and Lys200 (Fig. 3C–E).
In the AtGLTP1–GlcCer and AtGLTP1–GalCer
models, Arg59 could hydrogen bond with the sugar unit
similarly to the corresponding residue Lys55 in human
GLTP (Fig. 3C,E). Interestingly, however, in the
AtGLTP1–LacCer model, Arg59 appears to sterically
hinder binding of the lactosyl group (Fig. 3D). In the
human GLTP–LacCer structure [27], Leu92 forms a
hydrophobic interaction with the Gal ring, while the
corresponding Asn95 residue in AtGLTP1 cannot form
similar hydrophobic contacts (Fig. 3D). On the other
hand, Asn95 appears to play an important role in the
specific binding of GlcCer, as its amine group binds the
OH4 hydroxyl of Glc in our AtGLTP1–GlcCer model
(Fig. 3C). In agreement with the low GalCer transfer
activity of AtGLTP1 (Table 1), Asn95 cannot bind the
OH4 hydroxyl of Gal in our AtGLTP1–GalCer model,
as it points away from the amine group of Asn95
(Fig. 3E).

A
B
C
D
Fig. 4. Representative time-course traces for BODIPY-labeled Glc-
Cer, GalCer, LacCer and SM transfer by (A) AtGLTP1 (At2g33470),
(B) At1g21360 and At3g21260 (no activity), (C) AtGLTP1 Arg59Lys ⁄
Asn95Leu (no activity), and (D) human GLTP, from donor to accep-
tor vesicles. The donors contained 0.5 mol% of BODIPY-GlcCer,
BODIPY-GalCer, BODIPY-LacCer or BODIPY-SM and 3 mol% DiI-
C18 in a POPC matrix, and the acceptor vesicles contained 100%
POPC. The assay was run at 37 °C in sodium phosphate buffer
containing 140 m
M NaCl.
G. West et al. Arabidopsis glycolipid transfer protein
FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS 3427
As the structural models indicated that Arg59 and
Asn95 could be responsible for the altered GSL trans-
fer specificity of AtGLTP1, these residues were chosen
for site-directed mutagenesis to the corresponding
human GLTP residues, Lys and Leu, respectively.
According to the ligand docking results, the AtGLTP1
Arg59Lys ⁄ Asn95Leu mutant generated has a similar
GlcCer binding mode as the wild-type AtGLTP1 (data
not shown). The activity of the AtGLTP1 mutant was
tested in the lipid transfer assay, which showed that it
had lost the specific GlcCer transfer capability of
AtGLTP1, without gaining any increased capacity for
GalCer transfer (Fig. 4C and Table 1). Seemingly,
Arg59 and ⁄ or Asn95 are responsible for the specific

binding of GlcCer to AtGLTP1. However, substituting
Arg59 and Asn95 with the corresponding residues of
human GLTP did not increase the overall GSL trans-
fer activity of AtGLTP1, and thus this difference can-
not explain why AtGLTP1 shows much lower GSL
transfer activity compared to human GLTP (Table 1).
Expression of AtGLTP1, At1g21360 and
At3g21260 during development
To assess the expression pattern of AtGLTP1,
At1g21360 and At3g21260, we retrieved relevant data
from microarray analyses of gene expression during
A. thaliana development, accessible in public databases
(, evestigator.
ethz.ch, ). Figure 5 shows
the expression of AtGLTP1, At1g21360 and
At3g21260 in 63 samples from various tissues or
Fig. 5. Expression of AtGLTP1, At1g21360 and At3g21260 in A. thaliana tissues. The data are from the microarray experiment
AtGenExpress expression atlas of A. thaliana [45] (). The investigated tissue samples are from roots (RO; sam-
ples 1–7), stems (ST; samples 8–10), leaves (LE; samples 11–25), whole plants (WP; samples 26–36), shoot apex (SA; samples 37–40), floral
organs (FL; samples 41–55) and seeds (samples 56–63) of A. thaliana Col-0. Plants were grown on soil, unless an alternative growth sub-
strate is indicated. (1) root, 7 days; (2) root, 17 days; (3) root, 1· MS agar, 1% sucrose, 15 days; (4) root, 8 days, 1· MS agar; (5) root,
8 days, 1· MS agar, 1% sucrose; (6) root, 1· MS agar, 21 days; (7) root, 1· MS agar, 1% sucrose, 21 days; (8) hypocotyl, 7 days; (9) 1st
node, ‡ 21 days; (10) 2nd internode, ‡ 21 days; (11) cotyledons, 7 days; (12) leaf numbers 1 + 2, 7 days; (13) rosette leaf number 4,
10 days; (14) rosette leaf number 2, 17 days; (15) rosette leaf number 4, 17 days; (16) rosette leaf number 6, 17 days; (17) rosette leaf num-
ber 8, 17 days; (18) rosette leaf number 10, 17 days; (19) rosette leaf number 12, 17 days; (20) petiole leaf number 7, 17 days; (21) proximal
half of leaf number 7, 17 days; (22) distal half of leaf number 7, 17 days; (23) leaf, 1· MS agar, 1% sucrose, 15 days; (24) senescing leaves,
35 days; (25) cauline leaves, ‡ 21 days; (26) seedling, green parts, 7 days; (27) seedling, green parts, 1· MS agar, 8 days; (28) seedling,
green parts, 1· MS agar, 1% sucrose, 8 days; (29) seedling, green parts, 1· MS agar, 21 days; (30) seedling, green parts, 1· MS agar, 1%
sucrose, 21 days,; (31) rosette after transition to flowering, but before bolting, 21 days; (32) rosette after transition to flowering, but before
bolting, 22 days; (33) rosette after transition to flowering, but before bolting 23 days; (34) vegetative rosette, 7 days; (35) vegetative rosette,

14 days; (36) vegetative rosette, 21 days; (37) shoot apex, vegetative + young leaves, 7 days; (38) shoot apex, vegetative, 7 days; (39) shoot
apex, transition (before bolting), 14 days; (40) shoot apex, inflorescence (after bolting), 21 days; (41) flower, stage 9; (42) flower,
stage 10 ⁄ 11; (43) flower, stage 12; (44) flower, stage 15; (45) flower, 28 days; (46) pedicel, stage 15; (47) sepal, stage 12; (48) sepal,
stage 15; (49) petal, stage 12; (50) petal, stage 15; (51) stamen, stage 12; (52) stamen, stage 15; (53) pollen, 6 weeks; (54) carpel, stage 12;
(55) carpel, stage 15; (56) siliques, with seeds stage 3; mid-globular to early heart embryos (57) siliques, with seeds stage 4; early to late
heart embryos (58) siliques, with seeds stage 5; late heart to mid-torpedo embryos (59) seeds, stage 6, without siliques; mid to late-torpedo
embryos (60) seeds, stage 7, without siliques; late-torpedo to early walking-stick embryos (61) seeds, stage 8, without siliques; walking-stick
to early curled cotyledons embryos; (62) seeds, stage 9, without siliques; curled cotyledons to early green cotyledons embryos; (63) seeds,
stage 10, without siliques; green cotyledons embryos.
Arabidopsis glycolipid transfer protein G. West et al.
3428 FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS
stages of development. The data are from the
AtGenExpress expression atlas (gel-
world.org/resources/microarray/AtGenExpress) [45].
AtGLTP1 mRNA is ubiquitous in all tissues and at
all stages of life of the plant. The highest levels of
AtGLTP1 mRNA were found in floral tissues
(Fig. 5, samples 41–55), such as petals (sample 50),
stamens (sample 51) and sepals (sample 48), and in
stems (Fig. 5, samples 8–10). The At3g21260 tran-
script was most abundant in roots (sample 6), but
was also detectable in most other tissues and devel-
opmental stages. The levels of At3g21260 transcripts
were lower in all analyzed tissues compared to
AtGLTP1 mRNA. The transcription of At1g21360 is
more restricted, as the transcript was only detected
in roots (samples 3–7). The expression of AtGLTP1
and At1g21360 was also analyzed using RT-PCR
(supplementary Fig. S4), and AtGLTP1 mRNA was
found to be ubiquitous in all tissues and at all

developmental stages tested. At1g21360 mRNA was
also detectable in all tested tissue samples (supple-
mentary Fig. S4), suggesting that At1g21360 mRNA
is expressed at a low level in the whole plant.
We fused the AtGLTP1 and At1g21360 promoters
to the GUS reporter gene. The constructs were
transformed into A. thaliana, and the temporal and
spatial patterns of expression from these gene fusions
were assessed during plant growth and development
(Fig. 6). In young seedlings carrying AtGLTP1::GUS,
staining in roots was restricted to the tips
(Fig. 6A,B). In the roots of more mature plants,
staining was still found in the tips, but also in the
stelar tissue of the elongation zone (Fig. 6D). The
root cap did not show any GUS expression. In
young seedlings, GUS activity was also present in
the tips of the cotyledons and in the first leaf pri-
mordia (Fig. 6A,C). Additionally, staining was seen
in hydathodes and epidermis of cotyledons (Fig. 6E)
and rosette leaves (Fig. 6F). In leaf epidermis, GUS
staining appeared to be more intense in stomatal
cells (Fig. 6F). GUS staining was also seen in floral
tissues, such as the receptacle (Fig. 6G,I), petals
(Fig. 6G), floral buds (Fig. 6G,H), styles (Fig. 6H)
and anther filaments (Fig. 6I). Staining was most evi-
dent in distal regions of the floral tissues. Expression
from At1g21360::GUS was only detected in roots
(Fig. 6J–O). In young At1g21360::GUS seedlings,
GUS staining was restricted to cells in the region of
the root, from which root hairs develop, and to root

hairs (Fig. 6J–L). In older At1g21360::GUS plants,
GUS activity could also be detected in the basal
regions of lateral roots.
Discussion
In this report, we identified three A. thaliana paralogs,
At1g21360, At2g33470 and At3g21260, as orthologs to
mammalian GLTPs. At1g21360, At2g33470 and
At3g21260 form a small gene family that has its origin
in a gene duplication event before the split of gymno-
sperms and angiosperms, and another duplication that
occurred after the split of monocotyledons and dicoty-
ledons. We designated At2g33470 as AtGTLP1
because we had shown that it was a true GLTP with
capacity to stimulate the in vitro transfer of GSLs from
donor to acceptor vesicles. AtGLTP1 could efficiently
transfer GlcCer, but the transfer of GalCer and Lac-
Cer was negligible. Human GLTP efficiently moved all
three tested glycolipids. It appears that amino acid
replacements that narrow the GSL transfer repertoire
have been tolerated in AtGLTP1 due to the lack of
GalCer and LacCer in Arabidopsis tissues.
Based on modeling of the AtGLTP1 structure, we
concluded that the Lys55 ⁄ Arg59 and Leu92 ⁄ Asn95
replacements most likely mediate the differences in
GSL transfer specificity between human GLTP and
AtGLTP1. We therefore constructed an AtGLTP1
Arg59Lys ⁄ Asn95Leu mutant (Fig. 3F), which had a
very low transfer activity for both GlcCer and GalCer
(Table 1), confirming that Arg59 and ⁄ or Asn95 in
AtGLTP1 are extremely important for specific GlcCer

binding (Fig. 3C). Lys200 and Ser202 (Tyr207 and
Val209 in human GLTP, Fig. 3B) are the only differ-
ences with respect to human GLTP in the sugar-bind-
ing site of the AtGLTP1 Arg59Lys ⁄ Asn95Leu mutant
(Fig. 3F). In the human GLTP–LacCer crystal struc-
ture [27], the ceramide amide group is oriented by
hydrogen bonds, which are aligned by the hydrophobic
contacts between Val209 and the initial three-carbon
ceramide segment of LacCer, but Ser202 in AtGLTP1
cannot form similar hydrophobic contacts. Lys200 in
AtGLTP1 is equivalent to Tyr207 in human GLTP,
which forms a hydrogen bond with the glucose ring of
LacCer [27]. The role of Tyr207 in the GSL transport
of human GLTP has been studied by point mutation
to a leucine, which had a slight effect on transfer activ-
ity [27], but there is no documentation regarding the
importance of Val209 on GSL transfer activity. In con-
clusion, we have shown that Asn95 and ⁄ or Arg59 are
involved in GlcCer binding. Further mutational studies
will be conducted in order to pinpoint the residues
responsible for the specific binding of GlcCer to
AtGLTP1 and to determine the reason for the lower
overall GSL transfer activity of AtGLTP1 compared
to human GLTP.
G. West et al. Arabidopsis glycolipid transfer protein
FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS 3429
B
C
D
E

F
G
H
G
I
J
K
M
N
O
L
A
Arabidopsis glycolipid transfer protein G. West et al.
3430 FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS
At1g21360 might also encode a genuine GLTP. The
residues (Asp48, Asn52, Trp96 and His140) that are
definitely required for sugar binding are totally
conserved between human GLTP, AtGLTP1 and
At1g21360 (Fig. 1B). The sugar-binding sites of
AtGLTP1 and At1g21360, however, exhibit structural
differences suggesting different ligand preferences (sup-
plementary Fig. S1B,C). At1g21360 could not transfer
any of the tested GSLs and we cannot therefore
confirm that this protein is a GLTP. At3g21260 could
not transfer any of the tested ligands either, and
because At3g21260 completely lacks two of the key
sugar-binding residues (supplementary Fig. S1D) and
half of the hydrophobic ligand-binding cavity, it is
quite unlikely that At3g21260 is a GLTP.
Glycolipid transfer protein orthologs are encoded in

eukaryotes ranging from animals to fungi, plants,
apicomplexans and diplomonads (Fig. 2). However,
there are some eukaryotes that appear to lack GLTPs,
such as the yeasts S. cerevisae and Sc. pombe. These
yeasts do not encode GlcCer synthase, and hence do
not synthesize GlcCer [46]. GlcCer-producing yeasts
[46], such as Eremothecium gossypii, do encode and
express GLTP. This correlation between GlcCer syn-
thesis and GLTPs indicates that GLTPs play an
important role in GlcCer function and metabolism.
GLTPs were also not identified in the kinetoplastid
protozoans Trypanosoma and Leishmania. These proto-
zoans are generally considered to synthesize inositol
phosphorylceramides rather than GlcCer [47], but
Trypanosoma cruzi, at least, does contain GlcCer [48],
which suggests that either the GLTP was not identified
due to sequence divergence, or that some GlcCer-
producing organisms may function without GLTP.
The functions of sphingolipids in plants are still
rather poorly characterized even though the sphingo-
lipids are major components of the tonoplast and
plasma membrane of plant cells. The A. thaliana LCB1
was recently shown to encode one of the subunits of
serine palmitoyltransferase. T-DNA disruption of
LCB1 results in embryo lethality, which indicates that
sphingolipids are essential for the viability of A. thali-
ana [18]. Furthermore, partial suppression of LCB1
resulted in dwarfing and altered leaf morphology,
probably due to reduced cell expansion [18]. AtGLTP1
is expressed in a wide range of tissues, from roots to

floral organs. In these tissues, AtGLTP1 expression is
not evenly distributed, but instead is located to the
distal regions of organs (Fig. 6). The activity of the
promoter–GUS fusion indicates that At1g21360 is
specifically expressed in the hair-forming region of the
young root, suggesting that its main function is during
root hair formation. Root hairs are tubular out-
growths of root epidermal cells. The initiation site for
a developing root hair, identified by a bulge in the cell
wall, normally occurs in the apical end of the root
hair-forming cells (trichoblasts). Before bulge forma-
tion starts, the trichoblasts must break their ordinary
symmetric growth pattern and initiate cell growth at a
restricted area [49]. At present, we think that Arabid-
opsis GLTP is probably involved in transferring GSLs
from the ER to the plasma membrane or the tono-
plast. It should be considered that GLTPs are stochio-
metric with respect to the transported lipid molecules,
whereas vesicles can transfer hundreds, or perhaps
thousands, of lipid molecules per transport step. Thus,
it is unlikely that GLTP-mediated transfer accounts
for the bulk flow of GSLs to the plasma membrane.
Rather, we suggest that GLTPs may play a role in
directing and regulating vesicle transport to specific
domains of the plasma membrane and the tonoplast,
or, alternatively, that they play a role in maintenance
of GSL-enriched membrane domains.
Experimental procedures
Chemicals
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)

was obtained from Avanti Polar Lipids (Alabaster, AL,
USA). Whole wheatflour monogalactosyldiglyceride
(MGDG) and digalactosyldiglyceride (DGDG) were
obtained from Sigma (St Louis, MO, USA). The fluores-
cent lipids N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-
s-indacene-3-dodecanoyl)sphingosyl-1-b-d-glucopyranoside
(BODIPY-C12-glucosylceramide, BODIPY-GlcCer) and
Fig. 6. Localization of GUS protein in transgenic A. thaliana plants expressing GUS from (A) to (I) the AtGLTP1 promoter, or (J–O) the
At1g21360 promoter. In plants expressing GUS from the A. thaliana GLTP1 promoter, staining was found in (A, B) root tips and (A) leaf pri-
mordia of 2-day-old seedlings, (C) leaf primordia of a 1-week old plant, (D) tips and stelar tissue of the elongation zone of roots from 2-week
old plants, (E) hydathode and epidermis of the cotyledon from a 1-week old plant, (F) the epidermis of a rosette leaf from a 2-week-old plant,
(G) the receptacle, petals and floral buds of 3-week-old plants, (H) the style of 3-week-old plants, and (I) the receptacle and anther filaments
of 6-week-old plants. In (F), GUS staining appeared to be more intense in stomatal cells, and staining was most evident in distal regions of
floral organs (G–I). In plants expressing GUS from the At1g21360 promoter, staining was detected in (J, K) roots of 2-day-old seedlings, (L)
root hairs of 6-day-old seedlings, (M) root hairs of 2-week-old seedlings. (N) lateral roots of 1-week-old plants and (O) lateral roots of 4-week-
old plants. At least five independent lines for each construct were analyzed, and representative images are shown.
G. West et al. Arabidopsis glycolipid transfer protein
FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS 3431
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-d iaza-s-indacene-
3-dodecanoyl)sphingosyl-1-b-d-galactopyranoside (BODIPY-
C12-galactosylceramide, BODIPY-GalCer), and the quen-
cher 1,1¢-dioctadecyl-3,3,3¢,3¢-tetramethylindocarbocyanine
perchlorate (DiI-C18) were purchased from Invitrogen
(Carlsbad, CA, USA). Triton X-100 was purchased from
ICN Biomedicals (Aurora, OH, USA). BODIPY-
C16-lactosylceramide was synthesized by dissolving lyso-
lactosylceramide (d-lactosyl-b1-1’-d-erythro-sphingosine),
BODIPY-FL C16 [N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,
4a-diaza-s-indacene-3-hexadecanoic acid)] and N-N-dicyclo-
hexylcarbodiimide in dichloromethane ⁄ methanol. Triethyl-

amine and hydroxytoluene were added, and the vial was
saturated with argon gas. The reaction was carried out for
6 h in the dark at 37 °C. The lipid probe was purified by
reverse-phase HPLC on a preparative Supelco (Bellefonte,
PA, USA) RP-18 column with 100% methanol as an eluent
and UV detection at 502 nm. A Micromass Quatro II mass
spectrometer (Waters, Milford, MA, USA) was used for
the verification of the product mass.
Plant materials and bacterial strains
We used A. thaliana ecotype Columbia (Col-0). Seeds were
surface-sterilized (washed in 70% ethanol for 2 min and in
15% chlorine and 0.5% SDS for 10 min, followed by at
least four washes in sterile distilled water) and sown on 1·
Murashige and Skoog (MS) medium [50]. Before cultiva-
tion, seed dormancy was broken by 3–4 days of cold treat-
ment (4 °C). Plants grown under non-sterile conditions
were planted on soil mixed with vermiculite (2 : 1). The
plants were cultivated in controlled environmental cham-
bers at 20–22 °C under long-day conditions (16 h light ⁄ 8h
darkness). Escherichia coli strains DH5a and TOP10 were
used for general cloning and blue ⁄ white screening. E. coli
BL21 was used for heterologous expression of At1g21360,
At2g33470 and At3g21260.
Sequence analyses
Putative GLTP sequences were identified and analyzed as
described previously [51]. Multiple sequence alignments
were created using clustal x [52]. Protein sequence align-
ments were performed using the following parameters:
gap opening penalty 10.0, gap extension penalty 0.20, and
a Gonnet protein weight matrix. To reconstruct phyloge-

netic trees by maximum likelihood, the multiple sequence
alignments were analyzed using phyml [36] by submitting
the alignments to the phyml server (http://atgc.
lirmm.fr/phyml) [53]. The JTT substitution matrix was
used for calculation of the amino acid substitutions [54].
A discrete gamma distribution with four categories was
used to account for variable substitution rates among
sites. The gamma distribution parameter was estimated
by phyml. A BIONJ distance-based tree was used as the
starting tree for refinement by the maximum-likelihood
algorithm. The number of generated bootstrapped pseudo
data sets was 100.
Expression and purification of A. thaliana GLTP
in E. coli
The cDNA clones U50148 and U66003 carrying cDNA
encoding At2g33470 and At1g21360, respectively, inserted
into vector pUni51 were obtained from the Arabidopsis
Biological Resource Center (Columbus, OH, USA). The
cDNA was released from U50148, inserted into pcDNA3.1,
and subsequently ligated into pGEX-5X-2 (GE Healthcare,
Little Chalfont, UK) to obtain a gene fusion between
glutathione S-transferase (GST) and AtGLTP1 in the
plasmid pGEX-GLTP1. The cDNA was amplified from
U66003 using primers U660035Eco2 (5¢-AATAGA
GAATTCAGAGAAAGAGATACGAGATGGA-3¢) and
U660033Not (5¢-TCATAAGGCGGCCGCCTACATCG
ATCTTAATCTGCTCAA-3¢). A fragment carrying
At3g21260 cDNA was amplifed from A. thaliana RNA by
RT-PCR. RNA was isolated from A. thaliana tissues using
an RNeasy plant mini kit (Qiagen, Hilden, Germany). The

cDNA was prepared as described elsewhere [55]. PCR
amplification of the obtained cDNA was performed using
primers GLTP3NE (5¢-ACTGGAATTCTGTGGGAATCT
GATCCTCTTGT-3¢) and GLTP3CN (5¢-TCATGGCGG
CCGCTTAGACTTTGTTACAATAACCAA-3¢). A syn-
thetic gene encoding the AtGLTP1 Arg59Lys ⁄ Asn95Leu
mutant inserted into vector pUC57 was obtained from
GenScript Corp (Piscataway, NJ, USA). The DNA was
amplified by PCR using the primers 5¢-CGATGGATCC
GAAGGCACCGTG-3¢ and 5¢-CGTAGAATTCTTACTC
GCTTTC-3¢. The PCR fragment encoding the AtGLTP1
Arg59Lys ⁄ Asn95Leu mutant was digested using the restric-
tion enzymes BamHI and EcoRI, and then subcloned into
the BamHI ⁄ EcoRI sites of vector pGEX-6p1 to yield the
plasmid pGEX-GLTP1Lys59Leu95.
The obtained PCR fragments carrying cDNA encoding
At1g21360 or At3g21260 were digested with restriction
enzymes EcoRI and NotI, and subcloned into the EcoRI ⁄
NotI site of vector pET-32b (Novagen ⁄ Merck, Darmstadt,
Germany). The inserts in the obtained plasmids pGEX-
GLTP1, pET-At1g21360 and pET-At3g21260 were con-
firmed by DNA sequencing. Plasmids pGEX-GLTP1 and
pGEX-GLTP1Lys59Leu95 were transformed into E. coli
BL21, while pET-At1g21360 and pET-At3g21260 were
transformed to E. coli BL21(DE3)-CodonPlus. The bacte-
ria were cultured at 29 °C. When the attenuance at 600 nm
(D
600
) reached 1.1, expression of the recombinant proteins
was induced by the addition of 0.3 mm isopropyl thio-b-d-

galactoside. Purification of the GST–GLTP1 fusion protein
and cleavage with Factor Xa was performed according to
the method described in the handbook for the GST Gene
Fusion System (GE Healthcare) as described previously
Arabidopsis glycolipid transfer protein G. West et al.
3432 FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS
[55]. The GST–AtGLTP1Lys59Leu95 protein was also puri-
fied according to the method described in the GST Gene
Fusion System handbook and cleaved with PreScission
protease (GE Healthcare). Purification of the fusion pro-
teins encoded by pET-At1g21360 and pET-At3g21260 was
performed by His affinity chromatography (Qiagen).
Lipid transfer assays
The concentration of the various phospholipids was deter-
mined by the Bartlett method [55] and gravimetrically for
probes (MT5; Mettler-Toledo, Columbus, OH, USA),
which we found to agree well with determinations based on
molar extinction coefficients. Small unilamellar vesicles
were prepared from a lipid suspension with a total concen-
tration of 0.4 mm that was sonicated for 10 min on ice
using a Branson (Danbury, CT, USA) 250 sonifier, and
then centrifuged for 15 min at 15 000 g to remove titanium
probe particles, multilamellar vesicles and undispersed lipid
(negligible amount). Light-scattering measurements (Mal-
vern 4700; Malvern Instruments, Malvern, UK) on the
probe-sonicated vesicles showed an average diameter of
36 nm (data not shown).
Calculations of the transfer rate were achieved by fitting
a first-order exponential behaviour as previously described
[40], and are given as pmol per second for the first minute

after GLTP addition. Briefly, by adding Triton X-100 (final
concentration 1%) to the transfer reaction mixture after the
initial BODIPY-glycolipid transfer, the total fluorescence
intensity can be obtained. Addition of Triton X-100 causes
the two fluorescently labeled lipids to become dispersed into
lipid–Triton X-100 micelles considerably beyond their Fo
¨
r-
ster distances. The intensity after Triton addition (with the
value for a Triton blank subtracted) corresponds to the
total concentration of BODIPY-glycolipid used in each
transfer reaction. By comparing the levels of the fluores-
cence intensity without GLTP, that seen 1 min after GLTP
was injected, and that seen after the addition of detergent,
we can calculate the amount BODIPY-glycolipid trans-
ferred by GLTP (pmol transferred per second).
To analyze the ability of plant AtGLTP1 to transfer
unlabeled MGDG and DGDG, a competition assay was
used [55–57]. Although indirect, this assay enables determi-
nation of whether unlabeled lipids interfere with the trans-
fer of BODIPY-GlcCer in an experimental set-up that does
not require the large amount of material that is often
required in conventional binding assays. A resonance
energy transfer assay with BODIPY-GlcCer, which has
been shown to be a substrate, was started by addition of
AtGLTP1. One minute after injection of the protein, when
the BODIPY-GlcCer transfer was still ongoing, we intro-
duced small unilamellar vesicles containing 10 mol%
MGDG or DGDG in POPC (400 nmol glycolipid). If the
added lipid is a substrate for AtGLTP1, this results in a

decrease in the rate of transfer of BODIPY-GlcCer because
of competition from the presence of a new distinct transfer-
able lipid pool, whereas if the added lipid is not a substrate
for AtGLTP1, no deviation in the slope of the transfer rate
will occur. No additional increase in fluorescence intensity
or light scattering was detected upon addition of unlabeled
vesicles, which confirms that there is no fusion or aggrega-
tion of the assay components during the time frame of the
measurements.
Structural modeling of putative A. thaliana
GLTPs
Structural models for the apo form of At2g33470,
At1g21360 and At3g21260 were constructed based on the
crystal structure of human GLTP (Protein Data Bank code
1SWX) [27]. Models of AtGLTP1 in complex with LacCer
and GlcCer were constructed based on the crystal structure
of human GLTP with LacCer (Protein Data Bank code
1SX6) [27]. Prior to modeling of the AtGLTP1–GlcCer
complex, the LacCer molecule in the human GLTP–LacCer
complex structure was modified to GlcCer. The model of
AtGLTP1 in complex with GalCer was constructed based
on the crystal structure of human GLTP bound to GalCer
(Protein Data Bank code 2EUK) [37]. The structures of
human apo-GLTP and the apo form of the glycolipid
transfer-like protein from the alga Galdieria sulphuraria
(GsGLTP; Protein Data Bank code 2I3F) were super-
imposed to create a structure-based sequence alignment.
The sequence alignment used for modeling was refined
slightly based on structural features derived from the struc-
ture-based sequence alignment. The programs vertaa and

malign within the Bodil modeling environment [58] were
used for superimposing structures and for sequence align-
ments, respectively.
Based on the final sequence alignment, a set of ten
models for At2g33470, At1g21360, At3g21260 and each
AtGLTP1–GSL complex was produced based on the tem-
plate structure using the program modeller [59]. From the
set of ten models, the one with the lowest value of the
modeller objective function was chosen for further analy-
sis. For the models of AtGLTP1 in complex with LacCer,
GlcCer and GalCer, the rotamer of the residues Arg52,
Asn95 and Lys200 was altered within the Bodil modeling
environment [58] to a conformation that could (based on
visual inspection) form hydrogen bonds with the ligand.
For the complex models, hydrogen bonds between these
residues and the sugar unit were evaluated using the pro-
gram whatif [60] with a 3.5 A
˚
upper limit for the donor–
acceptor distance. In order to obtain a model of the
AtGLTP1 Arg59Lys ⁄ Asn95Leu mutant, Arg59 and
Asn95 in the AtGLTP1–GlcCer complex model were
mutated within the Bodil modeling environment [58] to
lysine and leucine (the corresponding residues in human
GLTP) and altered to the same conformation as in human
GLTP [27].
G. West et al. Arabidopsis glycolipid transfer protein
FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS 3433
Docking of GlcCer into the model of AtGLTP1
and the model of AtGLTP1 Arg59Lys ⁄ Asn95Leu

Ligand docking was used as a method to evaluate the puta-
tive differences in the GlcCer binding mode of wild-type
and Arg59Lys ⁄ Asn95Leu mutant AtGLTP1. Docking of
GlcCer into the AtGLTP1 and AtGLTP1 Arg59Lys ⁄
Asn95Leu models was performed using the program gold
version 3.2 [61,62]. Hydrogens were added to the models
using the program reduce [63]. The GlcCer structure was
derived from the AtGLTP1–GlcCer complex model. Hydro-
gens were added to the structure using the program sybyl
version 7.3 (Tripos International, St Louis, MO, USA).
Ten independent genetic algorithm runs were made in gold
for the ligand, using the default docking parameters. The
binding site in the models was restricted within a 20.0 A
˚
radius from the CD1 atom of Leu107. Hydrogen bond con-
straints were applied from the glucose ring to Asp52,
Asn56 and His138 in the same way as in the human
GLTP–LacCer complex structure [27]. Docking was discon-
tinued if the root mean square deviation between the three
best scoring solutions was within 1.5 A
˚
. The docking results
were visualized and examined within the Bodil modeling
environment.
Histochemical GUS activity assays
A 1.8 kb DNA fragment carrying the At2g33470 promoter
was amplified from the A. thaliana Col-0 genome using
primers GLTP1PROUXBA (5¢-AGACTGCTCTAGAATG
GGTTTCTAAACCAACACGT-3¢) and GLTP1PRON-
BAM (5¢-CTCCTTGGATCCGCCTGAGAATTGAAAAA

GGTGGG-3¢). A 1.3 kb fragment carrying the At1g21360
promoter was amplified using primers 21360PRUXBA
(5¢-AACGATCTAGATTAAGAATGTAATCACATTAGG
GT-3¢) and 21360PRNNBAM (5¢-GGAAGGATCCACTT
TATTACAAGACCAGCGTTAT-3¢). The promoter frag-
ments obtained were incubated with restriction enzymes
XbaI and BamHI and subsequently fused to the GUS
reporter gene by ligation into the XbaI ⁄ BamHI sites of vec-
tor pBI101 (provided by E. So
¨
derberg, Uppsala University,
Sweden). This resulted in plasmid pCA2, carrying the
At1g21360 promoter, and plasmid pCA8 carrying the
At2g33470 promoter. The floral dip method [64] was used
to transform A. thaliana Col-0 with Agrobacterium tumefac-
iens C58 carrying pCA2 or pCA8. Transformations and
selection of transformants were performed at the Uppsala
Transgenic Arabidopsis Facility, Sweden.
Histochemical GUS assays were performed as described
by Jefferson et al. [65]. Plant tissues were incubated in a
substrate solution containing 50 mm sodium phosphate buf-
fer (pH 7.0), 1 mm 5-bromo-4-chloro-3-indolyl-b-d-glucu-
ronic acid cyklohexyl ammonium salt (X-GlcA CHA;
Duchefa Biochemie, Haarlem, The Netherlands), 0.5 mm
K
4
Fe(CN)
6
, 0.5 mm K
3

Fe(CN)
6
and 0.01% w ⁄ v
Triton X-100 at 37 °C overnight. Stained samples were
incubated in 70% ethanol at room temperature to extract
the chlorophyll.
Isolation of RNA and RT-PCR
RNA was isolated from A. thaliana tissues using an
RNeasy plant mini kit (Qiagen). Plant tissues were dis-
rupted in RLC buffer, which contains guanidine isothiocya-
nate. Tissues were taken from mature plants (28 days old),
and from 3- and 7-day-old seedlings. One microgram of
total RNA was used for first-strand cDNA synthesis. Ran-
dom hexamers (Invitrogen) were used as primers for reverse
transcription, catalyzed with Superscript III (Invitrogen).
Incubation times and incubation temperatures were chosen
according to the manufacturer’s instructions (Invitrogen).
PCR amplification of the cDNA was performed in the
presence of gene-specific primers. The oligonucleotides
ATGLTPRT1 (5¢-ATGGAAGGGACTGTGTTCACGC
CT-3¢) and ATGLTPRT2 (5¢-AGAAGCTTTCATGTCA
TCCATACC-3¢) were used for amplification of AtGLTP1,
U660035Eco2 and U660033Not were used for amplification
of At1g21360, and UBL1 and UBL2 [55] were used for
amplification of the ubiquitin-conjugating enzyme E2
(At5g41340). Approximately 100 ng of plant total RNA
was used for each RT-PCR reaction.
Acknowledgements
Professor Mark Johnson is acknowledged for provision
of the excellent facilities at the Structural Bioinfor-

matics Laboratory, Department of Biochemistry and
Pharmacy, A
¨
bo Akademi University, Turku, Finland,
and Professor J. Peter Slotte for allowing us to use the
fluorescence facility at the Department of Biochemistry
and Pharmacy, A
˚
bo Akademi University. This
research was supported by grants from the Swedish
Research Council, the Carl Trygger Foundation, the
Magnus Bergvall Foundation, the Academy of
Finland, the Sigrid Juse
´
lius Stiftelse, the Magnus
Ehrnrooths Stiftelse, the Svenska Kulturfonden, the
Medicinska Understo
¨
dsfo
¨
reningen Liv och Ha
¨
lsa r.f.
and A
˚
bo Akademi University.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Structural models of AtGLTP1, At1g21360
and At3g21260 in apo form.

Fig. S2. AtGLTP1 lacks DGDG and MGDG transfer
activity.
Fig. S3. GlcCer, but not DGDG or MGDG, competes
with BODIPY-GlcCer for AtGLTP1 transfer.
Fig. S4. RT-PCR analysis of the accumulation of
AtGLTP1 and At1g21360 mRNA in A. thaliana tissues.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
G. West et al. Arabidopsis glycolipid transfer protein
FEBS Journal 275 (2008) 3421–3437 ª 2008 The Authors Journal compilation ª 2008 FEBS 3437