Characterization of an Arabidopsis cDNA Encoding a Subunit of ...

Plant Cell Physiol. 42(11): 1274–1281 (2001) JSPP © 2001

Characterization of an Arabidopsis cDNA Encoding a Subunit of Serine Palmitoyltransferase, the Initial Enzyme in Sphingolipid Biosynthesis Kentaro Tamura 1, 3, Naoto Mitsuhashi 2, 4, Ikuko Hara-Nishimura 2 and Hiroyuki Imai 1, 5 1 2

Department of Biology, Graduate School of Natural Science, Konan University, Kobe, 658-8501 Japan Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-8502 Japan ;

Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine in the first step of de novo sphingolipid biosynthesis. In this study, we describe the cloning and functional characterization of a cDNA from Arabidopsis thaliana encoding the LCB2 subunit of SPT. The Arabidopsis LCB2 (AtLCB2) cDNA contains an open reading frame of 1,467 nucleotides, encoding 489 amino acids. The predicted polypeptide contains three transmembrane helices and a highly conserved motif involved in pyridoxal phosphate binding. Expression of this open reading frame in the Saccharomyces cerevisiae mutant strains defective in SPT activity resulted in the expression of a significant level of sphinganine, suggesting that AtLCB2 cDNA encodes SPT. Southern blot analysis and inspection of the complete Arabidopsis genome sequence database suggest that there is a second LCB2-like gene in Arabidopsis. Expression of a green fluorescent protein (GFP) fusion product in suspension-cultured tobacco BY-2 cells showed that AtLCB2 is localized to the endoplasmic reticulum. AtLCB2 cDNA may be used to study how sphingolipid synthesis is regulated in higher plants. Key words: Arabidopsis thaliana — 3-Ketosphinganine — LCB2 — Long-chain base — Serine Palmitoyltransferase (EC 2.3.1.50) — Sphingolipid synthesis. Abbreviations: DTT, dithiothreitol; ER, endoplasmic reticulum; EST, expressed sequence tag; GFP, green fluorescent protein; LCB, long-chain base; ORF, open reading frame; PMSF, phenylmethylsulfonyl fluoride; RT-PCR, reverse transcription-polymerase chain reaction; SPT, serine palmitoyltransferase; TLC, thin-layer chromatography. The nucleotide sequence reported in this paper has been submitted to the DDBJ, EMBL, and GenBank databases under the accession number AB046384.

Introduction Sphingolipids are membrane components in eukaryotic organisms and several bacteria. Sphingolipid contains a hydro3 4 5

phobic segment (ceramide) that consists of a sphingoid base (long-chain base, LCB) and a fatty acid. It has been proposed that sphingolipid catabolites, e.g. sphingosine (trans4-sphingenine), sphingosine-1-phosphate, and ceramide, are potential intra- and intercellular second messenger molecules in yeast and mammalian cells (Hannun 1996). In a plant pathogenesis study, fungal monoglucosylceramides, which contain a C-9-methylated LCB, have an elicitor activity that induces phytoalexins and the expression of pathogenesis-related proteins in rice leaves (Umemura et al. 2000). Furthermore, fungal metabolites with structural similarity to sphingosine, the fumonisins and Alternaria toxins (AAL toxins), act as toxins in plants (Gilchrist et al. 1992, Abbas et al. 1993, Nelson et al. 1993). Fumonisin B1 also elicits an apoptosis-like programmed cell death in Arabidopsis protoplasts (Wang et al. 1996, Asai et al. 2000). The mechanism of action of these phytotoxic compounds may involve the disruption of sphingolipid metabolism via the inhibition of ceramide synthesis (Abbas et al. 1994, Riley et al. 1993, Wang et al. 1991). However, little is known about the cellular mechanisms regulating the levels of sphingolipid molecules, and our understanding of de novo synthesis of sphingolipids in plant cells remains limited. Biosynthesis of sphingolipids is initiated by a reaction catalyzed by serine palmitoyltransferase (SPT; EC 2.3.1.50) that condenses L-serine with palmitoyl-CoA to form 3-ketosphinganine (Merrill and Jones 1990, Dickson and Lester 1999) (Fig. 1). 3-Ketosphinganine is immediately converted to D-erythrosphinganine by an NADPH-dependent 3-ketosphinganine reductase (D-erythro-sphinganine:NADP+ 3-oxidoreductase, EC 1.1.1.102). Several lines of evidence suggest that SPT is a ratelimiting enzyme in the sphingolipid synthetic pathway, and is therefore recognized as a key enzyme for regulating cellular sphingolipid content in mammalian tissues and cultured cell lines (Merrill and Jones 1990). Lynch and Fairfield (1993) first demonstrated in plant tissues that SPT activity in squash (Cucurbita pepo L.) pericarps exhibits a high degree of specificity for palmitoyl-CoA in a pyridoxal 5¢-phosphate-dependent reaction and that enzyme activity is found in the membrane fraction, specifically in the endoplasmic reticulum (ER). Therefore, the catalytic mechanism and subcellular localization of SPT may be conserved among various organisms including animals, fungi and plants.

Present address: Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-8502 Japan. Present address: Department of Biological Science, Nara Women’s University, Nara, 630-8506 Japan. Corresponding author: E-mail, [email protected]; Fax, +81-78-435-2539. 1274

Serine palmitoyltransferase in Arabidopsis

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Here we describe the isolation and characterization of an Arabidopsis cDNA homologous to S. cerevisiae LCB2. The gene corresponding to this cDNA clone was designated AtLCB2. To our knowledge, this is the first achievement of cDNA cloning and characterization of enzymes involved in the synthesis of sphingolipids in plants. We also examined the intracellular localization of a fusion protein between the synthetic green fluorescent protein (sGFP; S56T) and AtLCB2 protein in suspension-cultured cells of tobacco, and demonstrate that AtLCB2 protein is localized on the ER. Some of the present work has been reported previously in preliminary form (Tamura et al. 2000).

Results

Fig. 1 bases.

The pathway for the synthesis of sphingolipid long-chain

Although the structure and metabolism of LCB in plants are essentially similar to those in other eukaryotes, little is known about the regulation of de novo synthesis of sphingolipids in plants. In the yeast Saccharomyces cerevisiae, two genes, LCB1 and LCB2, that encode subunits of SPT have been isolated by the complementation of mutant strains defective in SPT activity (Buede et al. 1991, Nagiec et al. 1994). Zhao et al. (1994) independently isolated the SCS1/LCB2 gene, as one of suppressor genes of the Ca2+-sensitive growth phenotype of cgs2 mutant cells. This suggests that sphingolipid metabolism in S. cerevisiae is related to Ca2+ homeostasis. Recently, a TSC3 gene that suppresses a high-temperature-sensitive lethal phenotype of cgs2 mutants was identified (Gable et al. 2000). TSC3 protein is a membrane-associated protein that stimulates SPT activity through association with LCB1 and LCB2 proteins. Mammalian cDNA homologs of both LCB1 and LCB2 have also been identified and functionally characterized (Nagiec et al. 1996, Hanada et al. 1997, Weiss and Stoffel 1997). Hanada et al. (2000) expressed the hamster LCB1 protein in a Chinese hamster ovary cell mutant strain lacking endogenous LCB1, and purified SPT from the cells. Endogenous LCB2 protein was co-purified with tagged LCB1 protein, suggesting that the LCB1 and LCB2 proteins may function as an enzyme complex in vivo in mammalian cells. Ikushiro et al. (2001) reported that SPT from a Gram-negative obligatory aerobic bacterium Sphingomonus paucimobilis is a water-solubule enzyme, suggesting that cellular localization of Sphingomonus SPT is distinct from that of eukatyotic SPT.

PCR cloning and characterization of AtLCB2 A TBLASTN (Altschul et al. 1990) search in the EST database of Arabidopsis cDNA at the National Center for Biotechnology Information (NCBI) for sequences homologous to the LCB2 subunit of S. cerevisiae SPT (Nagiec et al. 1994) retrieved an Arabidopsis EST clone (accession no. C99820) that was significantly similar to the C-terminal domain of the S. cerevisiae LCB2. Accordingly, the gene corresponding to this clone was designated AtLCB2. However, this EST clone missed 1,100-bp of the 5¢ half of the ORF sequence. Therefore, we amplified the 5¢-missing sequence by PCR as described in Materials and Methods. Eventually, the longest PCR product (750 bp) was cloned, and its nucleotide sequence determined. The longest AtLCB2 cDNA isolated was 1,973 bp long (accession no. AB046384), with an ORF of 1,467 bp that encodes a protein of 489 amino acid residues. Alignment of the deduced amino acid sequences of different LCB2 proteins (Thompson et al. 1994) shows that the sequence from Arabidopsis shares 51% and 44% identity with those from human and S. cerevisiae, respectively (Fig. 2). The predicted AtLCB2 protein has a molecular mass of 54,288 Da, and an isoelectric point (pI) of 8.84. Homology studies also suggest that the AtLCB2 gene can be assigned to Arabidopsis chromosome 5 (accession no. AB025633) and has 11 (putative) introns, the sequences of which contain consensus GT and AG dinucleotides at the 5¢ and 3¢ splice sites, respectively (Hanley and Schuler 1988). We also found that the AtLCB2 protein contains a highly conserved motif, 307GTFTKSFG314 (Fig. 2), that is involved in pyridoxal phosphate binding. To evaluate the complexity of AtLCB2-related genes in the Arabidopsis genome, we performed Southern blot hybridization of Arabidopsis genomic DNA using AtLCB2 cDNA as a probe. DNA digestion with BamHI, EcoRV, and KpnI resulted in the identification of unique DNA fragments between 6.6 and 23.1 kb in size that hybridized with AtLCB2 cDNA (Fig. 3). Digestion with EcoRV and KpnI revealed two fragments. There are no BamHI, EcoRV, or KpnI sites in either the introns or exons of the AtLCB2 sequence. This indicates that at least two

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Fig. 2 Comparison of the deduced amino acid sequences of LCB2. LCB2 protein sequences from A. thaliana (AtLCB2), Homo sapiens (HsLCB2), and S. cerevisiae (ScLCB2) were compared using the CLUSTAL W program. Identical amino acid residues in the three proteins are highlighted, and similar residues are shaded. The putative motif for a pyridoxal 5¢-phosphate-binding site (GTFTKSFG) is underlined, and an asterisk indicates the lysine residue that forms a Schiff base with pyridoxal 5¢-phosphate.

Fig. 3 Genomic DNA gel-blot analysis of the AtLCB2 gene. Genomic DNA (10 mg per lane) from Arabidopsis leaves was digested with BamHI, EcoRV, or KpnI, separated electrophoretically on agarose gel, blotted, and probed with alkaline phosphatase-labeled AtLCB2 cDNA according to the instructions of the AlkPhos Direct Labelling and Detection System with CDP-Star (Amersham Pharmacia Biotech). DNA marker sizes are indicated on the left (in kbp).

DNA regions with high homology to the AtLCB2 gene exist in the Arabidopsis genome. Consistent with this, there is a sequence on chromosome 3 of the Arabidopsis genome with the potential to encode a protein with 89% identity and 93% similarity to AtLCB2 (accession no. CAB87906). This may be particularly noteworthy because the other LCB2 genes cloned so far are reported to be single-copy genes. To examine the organ-specific expression of the AtLCB2 gene, RT-PCR/Southern hybridization analysis was undertaken using total RNA prepared from leaves, stems, roots, flowers, and mature seeds of Arabidopsis. Transcripts were detected in equal amounts in leaves, stems, roots, and flowers, but at lower levels in mature seeds (Fig. 4). As a control, the level of expression of the ROC1 gene (Lippuner et al. 1994) was also examined using the same RNA samples. A 628-bp fragment, corresponding to ROC1 cDNA, was amplified at similar levels in all organs tested. Expression of AtLCB2 in an lcb2, mutant strain of S. cerevisiae During the initial stages of this study, we tried in vain to complement the LCB requirement of an lcb2 mutant strain


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Fig. 4 Organ-specific accumulation of the AtLCB2 transcripts. The level of AtLCB2 mRNA was analyzed by RT-PCR/Southern hybridization. Total RNA samples were prepared from leaves (lane 1), stems (lane 2), roots (lane 3), flowers (lane 4), and mature seeds (lane 5). Transcript levels of the cytosolic form of cyclophilin (ROC1) were also analyzed as a loading control.

(LCJ201) of S. cerevisiae with the plasmid pYE-AtLCB2, which carries the ORF of AtLCB2 cDNA under the control of the GAL1 promoter. Under similar conditions, the pYES2ScLCB2 plasmid, a control plasmid that expresses the S. cerevisiae LCB2 gene, complemented the Lcb– phenotype. Therefore, to prove that cloned AtLCB2 cDNA encodes a component of SPT, in vitro SPT activity was measured with membrane fractions from S. cerevisiae LCJ201 cells transformed either with pYE-AtLCB2 or with vector alone, using L[14C]serine with palmitoyl-CoA as substrates. As shown in Table 1, LCJ201 cells transformed with pYE-AtLCB2 exhibited about 20-fold more SPT activity than did LCJ201 cells or vector-transformed LCJ201 cells. These low levels of activity constitute the limits of detection. These data clearly demonstrate that recombinant AtLCB2 protein itself has SPT activity. By contrast, BY4743, a parental strain of LCJ201, exhibited an enzyme activity of 149 pmol min–1 (mg protein)–1, suggesting that the level of SPT activity of AtLCB2-transformed LCJ201 cells was too low to rescue the lcb2 defect of the LCJ201 mutant strain (discussed below). When a protein blot was probed with mouse antiserum against the 13.3-kDa hydrophilic domain (Ala45 to Gly160) of the AtLCB2 protein, a 54-kDa polypeptide was detected in the microsomal fraction of the LCJ201 cells carrying pYEAtLCB2 (Fig. 5). This result indicates that the expressed

Table 1

Fig. 5 Expression of the AtLCB2 protein in the lcb2-null mutant LCJ201 strain. Cell-free extracts prepared from LCJ201 cells containing the vectors pYES2 or pYE-AtLCB2 and BY4743 cells were fractionated by differential centrifugation. Both the cytosolic (Su) and microsomal (Pe) proteins (50 mg) were applied to SDS-PAGE, blotted onto a PVDF membrane, and analyzed by immunoblotting using antiserum against the 13.3-kDa hydrophilic domain (Ala45 to Gly160) of the AtLCB2 protein.

AtLCB2 protein is localized in the membrane fraction, which is similar to the site of expression of native LCB2 protein in S. cerevisiae (Gable et al. 2000). Localization of sGFP-AtLCB2 fusion protein in the ER Analysis of the AtLCB2 with a Kyte and Doolite hydrophobicity plot and a transmembrane (TM) prediction algorithm (http://www.ch.embnet.org/software/TMPRAD_form.html) suggested that the AtLCB2 protein contains three transmembrane helices. The distribution of SPT activity among subcellular fractions has been characterized in mouse liver (Mandon et al. 1992) and squash pericarps (Lynch and Fairfield 1993) using differential centrifugation and marker enzymes, indicating that SPT activity is localized to the ER. Therefore, to examine whether AtLCB2 protein is localized to the ER, tobacco BY-2 cells were transformed with a chimeric gene encoding a fusion

SPT activity in different cell types

Source of enzyme activity N. tabacum BY-2 (suspension-cultured cells) S. cerevisiae LCJ201 S. cerevisiae LCJ201 (pYES2) S. cerevisiae LCJ201 (pYE-AtLCB2) S. cerevisiae BY4743 S. cerevisiae INVSC1 A. thaliana (etiolated seedlings)

Sphinganine produced (pmol (mg protein)–1 min–1) 19.71±2.46 0.33±0.02 0.54±0.06 8.94±0.62 149.27±3.44 144.53±1.87 6.31±0.79

Each value represents the mean ± SD from three separate experiments.

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Fig. 6 The GFP-AtLCB2 fusion protein was localized to the ER in tobacco BY-2 cells. (A) The sGFP-AtLCB2 protein construct that was expressed in tobacco BY-2 cells. (B) Total proteins from the transformants or wild-type cells were applied to SDS-PAGE, blotted onto a PVDF membrane, and analyzed with an anti-GFP antibody (upper panel) or an anti-AtLCB2 antibody (lower panel). The 82-kDa protein was accumulated in the transformed cells. (C) GFP fluorescence was observed in the ER networks and nuclear membranes of the transformants.

between the synthetic green fluorescent protein (sGFP; S56T) and AtLCB2 protein. As shown in Fig. 6C, the resultant transformant designated sGFP-AtLCB2/BY2 showed fluorescence in the ER network and nuclear envelope, which is similar to our previous study with a fusion protein including an ER-retention signal, HDEL (Mitsuhashi et al. 2000). These results suggest that AtLCB2 may be localized to the ER in BY-2 cells, and probably in Arabidopsis cells. Immunoblots of the extracts of these cells with GFP-specific or AtLCB2-specific antibodies showed that an 82-kDa protein accumulated in the transformed cells (Fig. 6B). The molecular mass of 82 kDa is consistent with the calculated mass of 81,286 Da for sGFP-AtLCB2.

Discussion The Arabidopsis LCB2 protein contains a unique sequence, 307GTFTKSFG314, which is related to a pyridoxal phosphate-binding motif that is commonly found in members of the a-oxoamine synthase subfamily, such as 2-amino-3ketobutyrate CoA ligase (Mukherjee and Dekker 1990) and 8amino-7-oxononanoate synthase (Alexeev et al. 1998). The lysine residue underlined has been shown to form a Schiff base with pyridoxal phosphate (Mukherjee and Dekker 1990). The

GTFTKSFG motif is completely conserved among the predicted LCB2 proteins from Arabidopsis, human, and S. cerevisiae, suggesting that these LCB2 polypeptides constitute catalytic subunits of SPT. The Arabidopsis protein is estimated to be about 9 kDa smaller than those of human and S. cerevisiae (Fig. 2). While the reason why AtLCB2 does not have a similar N-terminal sequence is unclear, the extra N-terminal domain in the human and S. cerevisiae LCB2 proteins is not involved in protein targeting. In the present study, the full-length AtLCB2 cDNA did not complement the Lcb– phenotype of LCJ201. This is consistent with the results of other workers who found that human LCB2 cDNA did not complement the Lcb– phenotype of a S. cerevisiae lcb2D strain (Nagiec et al. 1996). However, as shown in Table 1, membrane fractions of S. cerevisiae LCJ201 cells expressing the AtLCB2 had SPT activity. Therefore, it is not known exactly why AtLCB2 was unable to complement the Lcb– phenotype of LCJ201 cells. Because the level of expression is not high, it is possible that the amount of enzyme is not sufficient to synthesize enough amounts of sphinganine to rescue the Lcb– phenotype. Nagiec et al. (1996) suggested that lack of complementation does not rule out functional homology, and in this context lack of complementation may be attributable to a lack of interaction between AtLCB2 and the S. cerevisiae LCB1 subunit of SPT, a failure of the protein to localize to the ER, or the metabolic instability of the chimeric protein. In higher plants, monoglucosylceramides have been idenetified as a major lipid component of the plasma membrane and tonoplast (Yoshida and Uemura 1986, Cahoon and Lynch 1991, Norberg et al. 1991). We recently reported that major components of LCBs in Arabidopsis monoglucosylceramides are 8-unsaturated-4-hydroxy-sphingenines (Imai et al. 2000). Although it has been speculated that these glycosphingolipids might play important physiological roles in plants in addition to structural roles in those membranes (Ohnishi et al. 1988, Kasamo et al. 2000), even the mechanisms of monoglucosylceramide synthesis are not well understood. Recently, genes were identified in plants for a sphingolipid hydroxylase that catalyzes the formation of 4-hydroxylated LCBs (Sperling et al. 2001), which should be valuable in establishing the role of these lipids in plants. In summary, we have cloned and characterized the first plant SPT. Sequence analysis reveals that Arabidopsis SPT is a member of the a-oxoamine synthase subfamily. The cloning of a plant SPT cDNA and the identification of its gene will contribute significantly to clarifying how the regulation of SPT influences sphingolipid synthesis in plant cells.

Materials and Methods Plant materials Seeds of Arabidopsis thaliana (ecotype Columbia) were surfacesterilized in 2% (w/v) NaClO and 0.02% (w/v) Tween 20, then rinsed five times with sterile, distilled water. Sterile seeds were placed in

Serine palmitoyltransferase in Arabidopsis round 9-cm Petri dishes containing 4.6 mg ml–1 Murashige-Skoog salt (Wako Pure Chemicals, Osaka, Japan), 2% (w/v) sucrose, 3 mg ml–1 thiamine-HCl, 0.5 mg ml–1 pyridoxine, 5 mg ml–1 nicotinic acid, and 0.2% (w/v) Gellan Gum (Wako Pure Chemicals) under 16 h light/8 h dark fluorescent illumination at 23°C. Suspension-cultured cells of tobacco BY-2 (Nicotiana tabacum L. cv. bright yellow 2) were subcultured in Linsmaier-Skoog medium once a week at 26.5°C in the dark with a rotary shaker (Multi Shaker MMS-300, EYELA, Tokyo, Japan) (Mitsuhashi et al. 2000). Isolation of an Arabidopsis LCB2 cDNA A BLAST search (Altschul et al. 1990) of Arabidopsis sequence databases, using the sequence of the S. cerevisiae LCB2 protein, identified an expressed sequence tag (EST) clone (accession no. C99820) that contained a partial cDNA sequence for a putative Arabidopsis LCB2 proteins. To isolate cDNA containing a full-length open reading frame (ORF), the 5¢-end was isolated by PCR, using Taq DNA polymerase (Ex Taq, TaKaRa, Shiga, Japan) and a lgt11 cDNA library prepared from the roots and leaves of Arabidopsis, as template. An initial PCR was performed with the first gene-specific primer A1 (5¢TTTGATTCCGCTAAGGTCAC-3¢), and the lgt11-specific primer LG1 (5¢-GGTGGCGACGACTCCTGGAGCCCG-3¢). The PCR products were used as templates for nested PCR, using the second genespecific primer A1N (5¢-CTTGAATGAGATGCGGAGAT-3¢), and LG1. The third gene-specific primer A2 (5¢-TTATCAACCCTCCCTTTCCAATCAA-3¢) was designed from the sequence of the amplified PCR fragment, then the 5¢-coding region was amplified using the primers A2 and LG1. Amplified PCR fragments were subcloned into a pBluescript II (KS+) vector (Stratagene, La Jolla, CA), and sequenced using an ALF DNA sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden) and the BcaBEST Dideoxy Sequencing Kit (TaKaRa). Genome blot analysis Genomic DNA was extracted from leaves with a DNeasy Plant Mini Kit (Qiagen). The genomic DNA was digested with BamHI, EcoRV, or KpnI, separated by electrophoresis on 0.8% (w/v) agarose gel, and transferred to a positively charged nylon membrane (GeneScreen Plus, NEN Life Science Products, MA) by capillary blotting. A 1.5-kb fragment containing AtLCB2 cDNA was amplified by PCR using the primers A3 (5¢-CGGGATCCCGGATGATAACGATTCCTTATTTAACCGCTG-3¢) and A4 (5¢-CCCAAGCTTGGGGCGTTTTCCTGGATGCATCTCTATTT-3¢) and used as a hybridization probe. The probe was labeled with alkaline phosphatase according to the instructions of the AlkPhos Direct Labelling and Detection System with CDP-Star (Amersham Pharmacia Biotech). The membrane was hybridized and washed according to the manufacturer’s instructions. The hybridization signals were detected with a Typhoon 8600R imaging analyzer (Amersham Pharmacia Biotech). RT-PCR/Southern hybridization analysis RNAs were isolated from leaves, stems, roots, and flowers of 4week-old Arabidopsis plants and from mature seeds, using an RNeasy Plant Kit (Qiagen). RT-PCR analysis of AtLCB2 transcripts was carried out with SuperScript One-Step System (Invitrogen Life Technologies, Carlsbad, CA). A 256-bp fragment of the 3¢-untranslated region of AtLCB2 was amplified with the primer 5¢-GCAGAAGATGGGATTTGAGG-3¢ (forward) and the reverse primer A1 under 20 cycles of primer-annealing at 60°C. PCR products were separated on 1.5% (w/v) agarose gel and blotted onto nylon membrane. A 1.5-kb fragment containing AtLCB2 cDNA was amplified by PCR using the primers A3 and A4 and used as a hybridization probe. The probe was labeled with horseradish peroxidase according to the instructions of the ECL Direct Labelling and Detection System (Amersham Pharma-

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cia Biotech). The hybridization signals were detected by exposing the membrane to film (X-U, Fuji Photo Film, Tokyo, Japan). As a positive control, a ROC1 gene for the cytosolic form of cyclophilin (Lippuner et al. 1994) was amplified, using primers 5¢-ACTTCGACATGACCATCGAC-3¢ and 5¢-TTCCCATGAGAACACACACC-3¢. Construction of a lcb2-defective strain of S. cerevisiae An S. cerevisiae strain carrying an lcb2D::LEU2 deletion was constructed according to the method of Baudin et al. (1993). DNA fragments of LCB2 were amplified from the genomic DNA of S. cerevisiae by PCR, using the following combinations of primers: a 5¢LCB2 fragment using primers 5¢-CCCAAGCTTGGGGCCGAAGAGACACCAAAATG-3¢ and 5¢-AACTGCAGAACCAATGCATTGGAATGAAACTTACGGGCACTG-3¢ and a 3¢-LCB2 fragment using primers 5¢-CGGGATCCCGTGAAATCAAATTCCGGCAAATC-3¢ and 5¢-GACTAGTCTGCGAATATGATACGATAAAGTTG-3¢. The 5¢- and 3¢-LCB2 fragments were subcloned into the HindIII–PstI sites, and into the BamHI–SpeI sites, of pBluescript II (KS+), respectively, and the resulting plasmid was designated pBS-lcb2D. A PstI–BamHI fragment of the LEU2 gene from pJJ283 (Jones and Prakash 1990) was ligated into the PstI–BamHI sites of pBS-lcb2D. HindIII–SpeI digestion of the resultant plasmid produced the lcb2D::LEU2 cassette. An S. cerevisiae lcb2D deletion strain (MAT a/a, lcb2D::KanMax4, leu2D0/ leu2D0, his3D1/his3D1, ura3D0/ura3D0) (Research Genetics/Invitrogen, MA, U.S.A.), which was derived from the wild-type strain BY4743 (MAT a/a, leu2D0/leu2D0, his3D1/his3D1, ura3D0/ura3D0) (Wach et al. 1994), was transformed with the LCB2D::LEU2 cassette to produce a diploid version of an lcb2-null mutant strain with selection for LEU+ transformants, designated LCJ201 (MAT a/a, lcb2,::KanMax4, lcb2,::LEU2, his3,1/his3,1, ura3,0/ura3,0). Strain LCJ201 was grown at 30°C in YPD medium (1% yeast extract, 2% peptone, 2% glucose; Difco, U.S.A.) containing 200 mg ml–1 geneticin (Sigma-Aldrich) and 25 mM phytosphingosine (4-hydroxysphinganine) (Sigma-Aldrich). Expression of AtLCB2 protein in an lcb2-defective strain of S. cerevisiae The ORF of AtLCB2 cDNA was amplified by PCR using primers A5 (5¢-CGGGATCCCGAAAATGATAACGATTCCTTATTTAACCGCTG-3¢) and A6 (5¢-GGAATTCCGCGTTTTCCTGGATGCATCTCTATTTC-3¢). The amplified fragment was subcloned into the BamHI–EcoRI sites of the S. cerevisiae–E. coli shuttle vector pYES2 (Invitrogen, CA, U.S.A.). The resultant plasmid designated pYEAtLCB2 expresses AtLCB2 under the control of the galactosedependent GAL1 promoter. pYE-AtLCB2, and pYES2 as a control, were introduced into the LCJ201 strain using a lithium acetate method (Gietz et al. 1995). Transformed cells were grown overnight at 28°C in minimal medium (1´ Difco yeast nitrogen base without amino acids, 20 mg ml–1 L-histidine, 200 mg ml–1 geneticin, and 25 mM phytosphingosine) with 2% (w/v) glucose. Overnight cultures were centrifuged and the cell pellets resuspended in minimal medium containing 2% (w/v) galactose. Samples were incubated overnight at 28°C. The galactose-induced S. cerevisiae cells were collected by centrifugation, suspended in 100 mM HEPES-KOH (pH 8.0), 2.5 mM EDTA, 5 mM dithiothreitol (DTT), 10% (w/v) glycerol, and 1 mM phenylmethylsulfonyl fluoride (PMSF), and vortexed with glass beads. The homogenate was fractionated by centrifugation at 5,000´g for 10 min and then at 105,000´g for 1 h. Assay for SPT activity SPT assays were based on the method of Williams et al. (1984). Reactions were carried out in a 0.1 ml volume containing 100 mM HEPES-KOH (pH 8.0), 2.5 mM EDTA, 5 mM DTT, 50 mM pyridoxal

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5¢-phosphate, 0.2 mM palmitoyl-CoA, 2 mM L-[U-14C]serine (5 mCi mmol–1), and 50–200 mg of protein. After incubation at 30°C for 20 min with gentle shaking, the reaction was terminated with 200 ml 0.5 M NH4OH, then 30 ml 10 mg ml–1 NaBH4 was added (Lynch and Fairfield 1993). The radiolabeled products were extracted by adding 1.5 ml chloroform-methanol (1 : 2, v/v), 1 ml chloroform, 0.5 ml 0.5 M NH4OH, and 20 mg sphinganine. After brief centrifugation, the lower organic phase was analyzed by thin-layer chromatography (TLC) (Silica Gel 60, Merck, Germany) with a solvent of chloroform/ methanol/2 M NH4OH (80 : 30 : 4, by volume). The radioactive spots corresponding to authentic sphinganine (Sigma-Aldrich) were identified and quantified using a Bio-Imaging Analyzer (BAS2000, Fuji Photo Film). The radioactivity of TLC scrapings was also counted on a liquid scintillation counter (LSC-5100, Aloka, Tokyo) using a scintisol (Scintiblender I, Nacalai Tesque, Kyoto). Antibody preparation The AtLCB2 cDNA fragment encoding a 13.3-kDa putative hydrophilic region (Ala45 to Gly160) was amplified by PCR using primers A7 (5¢-GGGGTACCCCCGCGCCGATCTGTTTGGGGCATGA-3¢) and A8 (5¢-CCCAAGCTTGGGTCCCACAAACCTAGTAACACATTCCTCA-3¢). The amplified fragment was subcloned into the KpnI– HindIII sites of the E. coli expression vector, pQE-40 (Qiagen). This plasmid, designated pQE-ALCB202, was introduced into E. coli strain M15 cells harboring pREP4. A 41-kDa polypeptide expressed in E. coli was electroeluted and subjected to antiserum preparation in mice. Construction of synthetic green fluorescent protein expression plasmids, transformation of tobacco BY-2 cells, and fluorescence microscopy The plasmid pUC-sGFP(S65T), which contains the cauliflower mosaic virus (CaMV) 35S promoter, the sequence coding synthetic green fluorescent protein [sGFP(S65T)], and the nopaline synthase terminator (nos3¢) in the multiple cloning site of pUC18 was generous gift from Dr. Y. Niwa of the University of Shizuoka (Chiu et al. 1996). The ORF of AtLCB2 cDNA was amplified by PCR using pYEAtLCB2 as template with the primers 5¢-TGTACAAAATGATAACGATTCCTTATTTAACCGCTG-3¢ and 5¢-GGGGAGGGCGTGAATGTA-3¢. The PCR product was subcloned into the BsrGI–NotI sites of pUC-sGFP(S65T). This plasmid, designated pUC-sGLCB2, was digested with BamHI and EcoRI, and ligated into the BamHI–EcoRI sites of pBI121. This plasmid, designated pBI-sGLCB2, was introduced into Agrobacterium tumefaciens (strain GV3101) by electroporation using a Gene-Pulse apparatus (BioRad Laboratories, CA, U.S.A.). BY-2 cells were transformed with the chimeric gene encoding sGFP-AtLCB2 via A. tumefaciens according to the method of Matsuoka and Nakamura (1991). The transformed BY-2 cells were inspected with a fluorescence microscope (Axiophot 2, Carl Zeiss, Jena, Germany) according to the method of Mitsuhashi et al. (2000). Immunoblotting Sample solutions from S. cerevisiae BY4743 cells or galactoseinduced S. cerevisiae LCJ201 cells transformed with pYE-AtLCB2 or pYES2 were separated by SDS-PAGE (Laemmli 1970) and blotted onto Immun-Blot PVDF Membrane (Bio-Rad Laboratories). The membrane was incubated with specific antiserum (diluted 1 : 500) against the 41-kDa polypeptide of AtLCB2 protein. Horseradish peroxidase-conjugated antibodies (1 : 10,000) raised in goat against mouse IgG (Chemicon International) were used as secondary antibodies. BY-2 cells transformed with the chimeric gene encoding sGFPAtLCB2 were immunoblotted with antibodies against GFP (1 : 100; Living Colors™ Peptide Antibody, Clontech), or with specific antiserum (1 : 1,000) against the 41-kDa polypeptide of AtLCB2 protein.

Horseradish peroxidase-conjugated antibodies (1 : 5,000) raised in goat against rabbit IgG (BioRad Laboratories) were used as secondary antibodies for GFP detection. ECL Plus Western Detection Reagents (Amersham Pharmacia Biotech) were used for immunodetection.

Acknowledgements We thank Dr. Niwa of the University of Shizuoka for his kind donation of the modified GFP gene. We also thank Dr. Nishikata of Konan University for his kind help in the preparation of antibodies. This work was supported in part by a Grant for Basic Science Research Projects (970659) to H.I. from The Sumitomo Foundation.

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(Received May 30, 2001; Accepted September 8, 2001)