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: Johed@ifm.liu.se *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 (http://www.arabidopsis.org, http://www.genevestigator. ethz.ch, http://www.weigelworld.org). 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] (http://www.weigelworld.org). 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 (http://www.weigel- 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 [...]... thaliana Col-0 genome using primers GLTP1PROUXBA (5¢-AGACTGCTCTAGAATG GGTTTCTAAACCAACACGT-3¢) and GLTP1PRONBAM (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 fragments obtained were incubated with restriction... 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¢)... 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¢)... al Arabidopsis glycolipid transfer protein 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 primordia of 2-day-old seedlings, (C) leaf primordia of a 1-week old plant, (D) tips and stelar... palmitoyltransferase Plant Cell 18, 3576– 3593 19 Hanada K, Kumagai K, Yasuda S, Miura Y, Kawano M, Fukasawa M & Nishijima M (2003) Molecular machinery for non-vesicular trafficking of ceramide Nature 426, 803–809 20 Ichikawa S, Sakiyama H, Suzuki G, Hidari KI & Hirabayashi Y (1996) Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid. .. assay enables determination of whether unlabeled lipids interfere with the transfer 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, ... Arabidopsis glycolipid transfer protein 17 Merrill AH Jr (1983) Characterization of serine palmitoyltransferase activity in Chinese hamster ovary cells Biochim Biophys Acta 754, 284–291 18 Chen M, Han G, Dietrich CR, Dunn TM & Cahoon EB (2006) The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase... 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 http://www.blackwell-synergy.com 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... Molecular features of phospholipids that affect glycolipid transfer proteinmediated galactosylceramide transfer between vesicles Biochim Biophys Acta 1758, 807–812 West G, Nylund M, Slotte JP & Mattjus P (2006) Membrane interaction and activity of the glycolipid transfer protein Biochim Biophys Acta 1758, 1732–1742 3436 44 Yamada K, Abe A & Sasaki T (1986) Glycolipid transfer protein from pig brain transfers... 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-weekold plants At least five independent lines for each . (5¢-TCATAAGGCGGCCGCCTACATCG ATCTTAATCTGCTCAA-3¢). A fragment carrying At3g21260 cDNA was amplifed from A. thaliana RNA by RT-PCR. RNA was isolated from A. thaliana. thaliana Col-0 genome using primers GLTP1PROUXBA (5¢-AGACTGCTCTAGAATG GGTTTCTAAACCAACACGT-3¢) and GLTP1PRON- BAM (5¢-CTCCTTGGATCCGCCTGAGAATTGAAAAA GGTGGG-3¢).