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Originally published In Press as doi:10.1074/jbc.M405915200 on August 26, 2004 Originally published In Press as doi:10.1074/jbc.M405915200 on August 24, 2004

J. Biol. Chem., Vol. 279, Issue 47, 49243-49250, November 19, 2004
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FVT-1 Is a Mammalian 3-Ketodihydrosphingosine Reductase with an Active Site That Faces the Cytosolic Side of the Endoplasmic Reticulum Membrane*

Akio Kihara and Yasuyuki Igarashi{ddagger}

From the Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812, Japan

Received for publication, May 27, 2004 , and in revised form, August 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sphingolipids are essential membrane components of eukaryotic cells. Their synthesis is initiated with the condensation of L-serine with palmitoyl-CoA, producing 3-ketodihydrosphingosine (KDS), followed by a reduction to dihydrosphingosine by KDS reductase. Until now, only yeast TSC10 has been identified as a KDS reductase gene. Here, we provide evidence that the human FVT-1 (hFVT-1) and mouse FVT-1 (mFVT-1) are functional mammalian KDS reductases. The forced expression of hFVT-1 or mFVT-1 in TSC10-null yeast cells suppressed growth defects, and hFVT-1 overproduced in cultured cells exhibited KDS reductase activity in vitro. Moreover, purified recombinant hFVT-1 protein exhibited NADPH-dependent KDS reductase activity. The identification of the FVT-1 genes enabled us to characterize the mammalian KDS reductase at the molecular level. Northern blot analyses demonstrated that both hFVT-1 and mFVT-1 mRNAs are ubiquitously expressed, suggesting that FVT-1 is a major KDS reductase. We also found the presence of hFVT-1 variants, which were differentially expressed among tissues. Immunofluorescence microscopic analysis revealed that hFVT-1 is localized at the endoplasmic reticulum. Moreover, a proteinase K digestion assay revealed that the large hydrophilic domain of hFVT-1, which contains putative active site residues, faces the cytosol. These results suggest that KDS is converted to dihydrosphingosine in the cytosolic side of the endoplasmic reticulum membrane. Moreover, the topology studies provide insight into the spatial organization of the sphingolipid biosynthetic pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sphingolipids are essential lipid components of the eukaryotic membrane. Recent studies have demonstrated that sphingolipids, together with cholesterol, form lipid microdomains or rafts that play important roles in membrane traffic and cell signaling (1). Moreover, some sphingolipid metabolites such as ceramide, sphingosine, and sphingosine 1-phosphate function as bioactive lipid molecules that regulate cell proliferation, differentiation, apoptosis, and cell migration (2).

Sphingolipids consist of a hydrophobic ceramide backbone and a hydrophilic polar head group. Mammalian sphingolipids include sphingomyelin, with phosphocholine as its head group, and hundreds of glycosphingolipids with carbohydrate head groups. Biosynthesis of these sphingolipids begins with the condensation of palmitoyl-CoA and L-serine by serine palmitoyltransferase, producing 3-ketodihydrosphingosine (KDS)1 (3, 4). The carbonyl group at the C-3 position of KDS is then reduced by KDS reductase to generate dihydrosphingosine (DHS) (3–5). Subsequently, DHS is N-acylated by ceramide (dihydroceramide) synthase to form dihydroceramide. A double bond is then formed between the C-4 and C-5 positions of dihydroceramide by dihydroceramide desaturase, creating ceramide (3, 4). This biosynthetic pathway is well conserved, even in lower eukaryotes like the yeast Saccharomyces cerevisiae, with the exception of the final step (4). In the yeast, dihydroceramide desaturase does not exist; hence sphingosine-type ceramide is not produced. Instead, dihydroceramide or phytoceramide serves as the hydrophobic backbone of the sphingolipids, with phytosphingosine (PHS) being generated by hydroxylation at the C-4 position of DHS by C-4 hydroxylase (6, 7).

Most of the enzymes involved in sphingolipid biosynthesis have been identified in recent years, and the contribution of yeast genetic approaches in these studies has been quite significant. LCB1 and LCB2 were identified as genes required for the serine palmitoyltransferase activity in yeast (8–11). Subsequent studies demonstrated that Lcb1p and Lcb2p are subunits of serine palmitoyltransferase (11). By their homology, mammalian LCB1 and LCB2 were identified and found to possess serine palmitoyltransferase activity (12–14). Similarly, Lag1p and Lac1p share homology and have overlapping functions in dihydroceramide/phytoceramide synthesis in yeast (15, 16). Their homologs, UOG1, TRH1, and TRH4, recently were demonstrated to be involved in dihydroceramide/ceramide synthesis in mammals (17–19). Interestingly, Sur2p, a C-4 hydroxylase found in yeast, shares the same desaturase/hydroxylase superfamily as DES1, which is a mammalian dihydroceramide desaturase (6, 7, 20).

The yeast gene TSC10 was identified in a screening for temperature-sensitive suppressors of the Ca2+-sensitive {Delta}csg2 mutant (5). This mutant exhibits a defect in the synthesis of mannosylinositol phosphorylceramide, one of three myo-inositol-containing sphingolipids in yeast (21). The temperature-sensitive tsc10 mutants accumulated KDS and lacked KDS reductase activity (5). Moreover, purified recombinant Tsc10p catalyzed the conversion of KDS to DHS in an NADPH-dependent manner, indicating that Tsc10p is a KDS reductase (5).

Although Tsc10p was identified in 1998 (5), experimental evidence identifying its mammalian homolog has remained lacking thus far. In the present study, we demonstrate that the KDS reductase in mammals is encoded by the follicular lymphoma variant translocation-1 (FVT-1) gene, a gene identified previously in follicular lymphoma as being juxtaposed to an immunoglobulin J{kappa} segment by chromosome translocation (22). With the identity of this mammalian gene and its product in hand, we are now at last able to establish the characteristics for this mammalian KDS reductase and examine its role in sphingolipid synthesis. Northern blotting demonstrated that FVT-1 is expressed throughout the body in all tissues examined, although its expression varied among tissues, suggesting that FVT-1 is a ubiquitous KDS reductase. Although FVT-1 had been predicted to be a secreted protein (22), our immunofluorescence microscopic analysis showed that FVT-1 is localized in the endoplasmic reticulum (ER). Moreover, we also reveal that the domain encompassing its active site is exposed to the cytosol, indicating that sphingolipid synthesis proceeds on the cytosolic leaflet of the ER membrane, at least up to the production of DHS.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—Human embryonic kidney (HEK293T) cells and HeLa cells were grown in Dulbecco's modified Eagle's medium D6429 or D6046, respectively (Sigma) containing 10% fetal calf serum and supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin. HEK293T cells were grown on collagen-coated dishes. Transfection was performed using LipofectAMINE PlusTM Reagent (Invitrogen).

Construction of {Delta}tsc10 Yeast Strains—The TSC10 gene was amplified by PCR using yeast genomic DNA and primers TSC10-F (5'-ACTAGTACTGGCAGCAGCTGCAGAC-3') and TSC10-R (5'-CCTGCGTTCCCTGAGCACGGTCCTG-3') and then cloned into pGEM-T Easy (Promega Corp., Madison, WI) to generate pAK568. The {Delta}tsc10::LEU2 construct was generated by replacement of the 0.7-kb BclI-EcoRV region in the TSC10 gene with the LEU2 marker and was used to transform the S. cerevisiae diploid cells KA31 (MATa/{alpha} ura3/ura3 leu2/leu2 his3/his3 trp1/trp1) (23). Transformants were selected on synthetic complete (SC; 0.67% yeast nitrogen base and 2% glucose) plates lacking leucine. One of the clones obtained, designated KHY623, exhibited a TSC10+/{Delta}tsc10::LEU2 genotype. The KHY623 cells were sporulated, and the resulting tetrads were dissected onto YPD (1% yeast extract, 2% peptone, and 2% glucose) plates containing 5 µM PHS and 0.0015% Nonidet P-40 as a dispersant. The haploid spores were then allowed to form colonies at 30 °C. One of the clones, KHY625 (MATa ura3 {Delta}tsc10::LEU2 his3 trp1), was used for further studies.

Plasmids—The pAK80 plasmid is a yeast cloning vector for TDH3 promoter-dependent expression (24). The pAK157 plasmid, a derivative of pAK80, was constructed to produce an N-terminally FLAG-tagged fusion protein. For construction of the pAK287 plasmid (FLAG-TSC10), the TSC10 gene was amplified using yeast genomic DNA and primers 5'-GGATCCATGAAGTTTACGTTAGAAGACC-3' and 5'-TGAGAGTGGATGTGCTCTCAGTTGG-3'. The amplified fragment was cloned into pGEM-T Easy to generate pAK270. The pAK287 plasmid was produced by cloning the 1.0-kb BamHI-NotI fragment of pAK270 into the BamHI-NotI site of pAK157.

The mouse FVT-1 (mFVT-1) cDNA was amplified by PCR using total cDNA prepared from the mouse teratocarcinoma cell line F9 and primers mFVT1-F1 (5'-TCGGCAGAGATGTTGCTGTTGGCCG-3') and mFVT1-R1 (5'-GGGGCAAGGTTTAGGCAGTTTTGTC-3'). The amplified fragment was cloned into pGEM-T Easy to create pAK565. The obtained nucleotide sequence has been deposited with GenBankTM with accession number AY634684 [GenBank] . For expression in yeast, pAK572 was constructed by cloning the 1.1-kb NotI-NotI fragment of pAK565 into the NotI site of pAK80.

The human FVT-1 (hFVT-1) cDNA was amplified from a cDNA mixture, prepared from adult male human liver (Stratagene, La Jolla, CA), using primers hFVT1-F1 (5'-GGAGCGATGCTGCTGCTGGCTGCCGCC-3') and hFVT1-R1 (5'-AAGAAGATTAGGCAGTTTTGTCTGC-3'). The amplified fragment was cloned into pGEM-T Easy to generate pAK588. To express the hFVT-1 gene in yeast, the 1.1-kb NotI-NotI fragment of pAK588 was cloned into pAK80 to produce pAK574.

The pAK600 (FLAG-hFVT-1) and pAK602 (FLAG-{Delta}N-hFVT-1) plasmids were constructed as follows. First, full-length hFVT-1 and N-terminally truncated hFVT-1 ({Delta}N-hFVT-1) cDNAs were amplified from the pAK588 using a common primer (5'-TTTCTAGAAGATTAGGCAGTTTTGTCTGC-3') and respective primers (for pAK600, 5'-GGATCCATGCTGCTGCTGGCTGCCGCC-3', and for pAK602, 5'-GGATCCAAGCCCCTCGCCCTGCCCG-3'). These were cloned into pGEM-T Easy to generate pAK591 and pAK592 plasmids, respectively. The pAK600 and pAK602 plasmids were then constructed by cloning the BamHI-XbaI fragment of pAK591 or pAK592 into the BamHI-XbaI site of pAK157.

The pCE-puro hFVT-1 plasmid, designed to express hFVT-1 in mammalian cells, was constructed by cloning the hFVT-1 cDNA from pAK588 into the pCE-puro plasmid (25). Finally, the pAK603 plasmid (MBP-{Delta}N-hFVT-1) was constructed by cloning the 0.9-kb BamHI-XbaI fragment of pAK592 into the BamHI-XbaI site of pMAL-c2E (New England Biolabs, Inc., Beverly, MA), an Escherichia coli expression vector constructed to produce a fusion protein with an N-terminal maltose-binding protein (MBP) domain.

Preparation of Soluble and Membrane Fractions—HEK293T cells transfected with the indicated plasmids were washed twice with phosphate-buffered saline, suspended in buffer A (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 1 mM EDTA, 1x protease inhibitor mixture (CompleteTM, Roche Diagnostics), and 1 mM phenylmethylsulfonyl fluoride (PMSF)), and sonicated. After centrifugation at 300 x g for 3 min at 4 °C, the resulting supernatant was used as a total fraction. For preparing soluble and membrane fractions, the total fraction was subjected to centrifugation at 100,000 x g for 1 h at 4 °C.

Purification of MBP-{Delta}N-hFVT-1 Protein—The E. coli cells Rosetta (Novagen (EMD Biosciences, Inc.), La Jolla, CA) bearing the pAK603 plasmid were grown at 37 °C in LB (1% tryptone, 0.5% yeast extract, 1% NaCl (pH 7.2)) medium containing 50 µg/ml ampicillin and 20 µg/ml chloramphenicol to early log phase. Isopropyl-{beta}-D-thiogalactopyranoside (final concentration, 1 mM) was added to the culture to induce expression of MBP-{Delta}N-hFVT-1. After incubation at 37 °C for 2 h, cells were chilled on ice and collected by centrifugation. Cells were suspended in buffer B (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20% glycerol, 10 mM 2-mercaptoethanol, 1x protease inhibitor mixture, and 1mM PMSF) and incubated on ice for 30 min with 1/10 volume of 1 mg/ml lysozyme, suspended in 0.1 M EDTA (pH 8.0). Cells were lysed by sonication, and cell debris was removed by centrifugation at 20,000 x g for 5 min at 4 °C. The resulting supernatant was further centrifuged at 100,000 x g for 30 min at 4 °C, and the membrane fraction (pellet) was suspended in buffer B. After incubation with Triton X-100 (final concentration, 1%) at 4 °C for 30 min, solubilized proteins were collected by centrifugation at 100,000 x g for 30 min at 4 °C. The resulting supernatant was loaded onto an amylose resin (New England Biolabs, Inc.), washed with buffer C (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20% glycerol, 10 mM 2-mercaptoethanol, 1 mM EDTA, and 0.2% Triton X-100), and eluted with buffer C containing 10 mM maltose.

In Vitro KDS Reductase Assay—The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10% glycerol, 150 mM NaCl, 1 mM EDTA, 10 mM 2-mercaptoethanol, 1x protease inhibitor mixture, 1 mM PMSF, 0.2% Triton X-100, 100 µM NADPH, 50 µM KDS, and purified MBP-{Delta}N-hFVT-1 protein. When total membrane fractions were used instead of the purified protein, 1 mM NADPH and 100 µM KDS were included. All reactions were performed by incubating the samples at 37 °C. The reactions were terminated by adding 1 M NH4OH, and the oxidation of NADPH was monitored by measuring the decreased absorbance at 340 nm. Alternatively, for lipid extractions the reactions were terminated by 3.75 volumes of chloroform/methanol (1:2, v/v), followed by 1.25 volumes of chloroform and 1.25 volumes of 1% KCl. Phases were separated by centrifugation, and the organic phase was recovered, dried, and suspended in chloroform/methanol (2:1, v/v). The lipids were resolved by TLC on Silica Gel 60 high performance TLC plates (Merck) with chloroform, methanol, 2 N NH4OH (40:10:1, v/v). KDS and DHS were visualized by spraying the plates with 0.3% ninhydrin in 1-butanol/acetic acid (100:3, v/v) and heating at 180 °C for 1 min. KDS and DHS were purchased from Matreya, Inc. (Pleasant Gap, PA) and Sigma, respectively.

Northern Blotting—Poly(A)+ RNA blots containing 1 µg of RNA from human tissues (Clontech, BD Biosciences, Palo Alto, CA) and total RNA blots containing 20 µg of RNA from mouse tissues (Seegene Inc., Seoul, Korea) were used. The hFVT-1 fragments were amplified using the pAK567 plasmid and primers hFVT1-F1 and hFVT1-R1, whereas the mFVT-1 fragments were amplified using pAK565 and primers mFVT1-F1 and mFVT1-R1. These fragments were labeled with [32P]dCTP by random priming using the random primer DNA labeling kit, version 2 (TAKARA Bio Inc., Shiga, Japan), to generate their probes. Hybridization was carried out in ExpressHyb buffer (Clontech, BD Biosciences) for 2 h at 68 °C.

Immunoblotting and Immunofluorescence Microscopy—Rabbit polyclonal anti-hFVT-1 antiserum was raised against the middle region of the hFVT-1 protein (amino acid residues 26–292) following expression of the MBP-fused protein. Immunoblotting was performed as described previously (25). Anti-hFVT-1 antiserum (1:1000 dilution), anti-calnexin (H-10) antibody (0.2 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and a peroxidase-conjugated donkey anti-rabbit IgG F(ab')2 fragment (1:7500 dilution) were used. Immunofluorescence microscopic analysis was done as described previously (26). Anti-hFVT-1 antiserum (1:100 dilution), anti-KDEL antibody (1 µg/ml; Stressgen Biotechnologies, Inc., San Diego, CA), Alexa 488-conjugated anti-rabbit antibody (5 µg/ml; Molecular Probes, Inc., Eugene, OR), and Alexa 594-conjugated anti-mouse antibody (5 µg/ml; Molecular Probes, Inc.) were used.

Proteinase K Digestion Assay—HEK293T cells transfected with pCE-puro hFVT-1 were washed with phosphate-buffered saline twice, suspended in buffer D (20 mM HEPES/NaOH (pH 7.5), 0.25 M sucrose, 1 mM dithiothreitol, 1x protease inhibitor mixture, and 1 mM PMSF), and lysed using an electric Potter homogenizer for 10 strokes. After removal of cell debris by centrifugation at 300 x g for 3 min at 4 °C, the supernatant was subjected to ultracentrifugation at 100,000 x g for 1 h at 4 °C. The resulting pellet was suspended in buffer D (without the protease inhibitor mixture or PMSF) and treated with 0.5 mg/ml proteinase K at 4 °C for 2 h in the presence or absence of 1% Triton X-100. After termination of the digestion with 1 mM PMSF, total proteins were precipitated with 5% trichloroacetic acid on ice for 20 min. Protein precipitates were washed with 5% trichloroacetic acid and with acetone and then suspended in SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and a trace amount of bromphenol blue) containing 1 mM PMSF, separated by SDS-PAGE, and subjected to immunoblotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of FVT-1—To identify a mammalian KDS reductase, the GenBankTM data base was searched using the BLAST program for sequences similar to yeast Tsc10p. Although no apparent homologs could be found, various members of the short-chain dehydrogenase/reductase (SDR) family, to which Tsc10p belongs, exhibited moderate sequence similarities (~20% identity). These included FVT-1, 11-{beta}-hydroxysteroid dehydrogenase type 3, some short-chain dehydrogenase/reductase 10 isoforms, 17-{beta}-hydroxysteroid dehydrogenase type 2, and sepiapterin reductase. Of these, we chose human FVT-1 (23.5% identity and 40.7% similarity with Tsc10p) as a candidate for being a mammalian KDS reductase because its amino acid length (332 amino acids) is similar to that of Tsc10p (320 amino acids) and because its function was unknown. Additionally, we found a putative mouse homolog of hFVT-1. The mFVT-1 sequence indicated 92.8% identity and 97.3% similarity to hFVT-1 and 20.8% identity and 39.5% similarity to Tsc10p (Fig. 1).



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  		FIG. 1.
Sequence comparison of mammalian and yeast KDS reductases. An alignment of the amino acid sequences of hFVT-1, mFVT-1, and Tsc10p was generated using the ClustalV (50) and BOX-SHADE programs (Institute for Animal Health, Surrey, United Kingdom). The corresponding GenBankTM accession numbers are CAA45197 [GenBank] (hFVT-1), AY634684 [GenBank] (mFVT-1), and CAA85228 [GenBank] (Tsc10p). Black boxes indicate identical residues, and gray boxes show amino acid similarity. The putative transmembrane segments are indicated by lines. Dotted residues indicate the putative active site motif, Tyr-X-X-X-Lys, which is conserved in the SDR family. The predicted Gly-X-X-X-Gly-X-Gly segment functioning in NAD(H) or NADP(H) binding is marked with a dashed line.

 
Both FVT-1 proteins contain a 25-amino acid extension at the N terminus, which is absent in Tsc10p. This N-terminal stretch is considerably hydrophobic and is predicted to function as a transmembrane segment (Fig. 1). In addition, two putative transmembrane segments are present in hFVT-1 and mFVT-1 at their C termini (Fig. 1). The large hydrophilic domain located between the putative first and second transmembrane segments of FVT-1 contains a region (corresponding to amino acid residues 35–269 of hFVT-1) that exhibits similarity to SDR family members. In the SDR family only a single Tyr residue is strictly conserved, and site-directed mutagenesis and x-ray crystallography have demonstrated that this Tyr residue is crucial to enzyme activity (27–31). A Lys residue four positions downstream is also conserved in most SDR members (32), so a catalytic mechanism based on the Tyr-X-X-X-Lys motif has been proposed (33, 34). Tyr-186 and Lys-190 in hFVT-1 correspond to this motif. The same motif is found in mFVT-1 and Tsc10p (Fig. 1, dotted residues). SDR members require NAD(H) or NADP(H) as a coenzyme for their enzyme activity. A Gly-X-X-X-Gly-X-Gly segment, generally characteristic of the coenzyme-binding fold in dehydrogenases (32, 34, 35), is also present in FVT-1 and Tsc10p proteins (Fig. 1, dashed line). A single Lys residue is observed at the -3 position in the C termini of both hFVT-1 and mFVT-1. Lys is known to be important for ER membrane proteins to be retained at the ER, although typically ER retention signals are KKXX and KXKXX sequences (34). Tsc10p contains a KKXX type sequence at its C terminus (Fig. 1).

FVT-1 Is a KDS Reductase—First we examined whether ectopic expression of hFVT-1 or mFVT-1 in the TSC10-null yeast cells complemented the growth phenotype. Because sphingolipids are essential for cell viability, {Delta}tsc10 cells cannot grow normally, unless the addition of DHS or PHS to the medium bypasses the requirement of de novo sphingolipid synthesis (5). KHY625 ({Delta}tsc10) cells bearing a vector plasmid could not grow on SC plates lacking uracil (SC - URA plates) in the absence of PHS but grew once it was added to the medium (Fig. 2A). The introduction of pAK287, encoding N-terminally FLAG-tagged TSC10, allowed KHY625 cells to grow in the absence of PHS (Fig. 2A). Moreover, both pAK572 and pAK574 plasmids, encoding the mFVT-1 and hFVT-1 genes, respectively, enabled the growth of KHY625 cells (Fig. 2A). These results indicate that hFVT-1 and mFVT-1 are functional homologs of Tsc10p.



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  		FIG. 2.
hFVT-1 and mFVT-1 overcome the growth defect of {Delta}tsc10 cells. KHY625 ({Delta}tsc10) cells bearing plasmids were streaked on SC plates lacking uracil (SC - URA plates) in the absence (left panels) or presence (center panels) of 5 µM PHS and incubated for 3 days at 30 °C. A, KHY625 cells bearing pAK80 (vector), pAK287 (FLAG-TSC10), pAK572 (mFVT-1), or pAK574 (hFVT-1) plasmids. B, KHY625 cells harboring pAK600 (FLAG-hFVT-1) or pAK602 (FLAG-{Delta}N-hFVT-1) plasmids.

 
We next investigated the KDS reductase activity of hFVT-1 in vitro. HEK293T cells were transiently transfected with a pCE-puro hFVT-1 plasmid or a control vector plasmid. Cell lysates were prepared and subjected to immunoblotting using anti-hFVT-1 antibody. The hFVT-1 protein was detected in the lysates from pCE-puro hFVT-1 plasmid-transfected cells as a 36-kDa band, consistent with its predicted molecular mass of 36.2 kDa, whereas it was absent in mock-transfected cells (Fig. 3A).



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  		FIG. 3.
KDS reductase activity is increased in cells overexpressing hFVT-1. A, HEK293T cells were transfected with pCE-puro (lane 1) or pCE-puro hFVT-1 (lane 2) plasmid, and after 24 h, lysates were prepared. Fixed amounts of protein (2 µg) were separated by SDS-PAGE and subjected to immunoblotting using an anti-hFVT-1 antibody. B, total lysates prepared from HEK293T cells transfected with pCE-puro hFVT-1 were separated by ultracentrifugation at 100,000 x g for 1 h into membrane (lane 1) and soluble (lane 2) fractions. C, buffer only (lanes 3 and 4) or membrane fractions (2.5 µg) prepared from HEK293T cells transfected with pCE-puro (lanes 5 and 6) or with pCE-puro hFVT-1 (lanes 7 and 8) were incubated at 37 °C for 1 h in the presence (lanes 4, 6, and 8) or absence (lanes 3, 5, and 7)of100 µM KDS in buffer containing 1 mM NADPH. Lipids were then extracted, separated by TLC, and detected by ninhydrin staining. Lane 1, DHS standard (10 nmol); lane 2, KDS standard (10 nmol). D, indicated amounts of membrane fractions prepared from HEK293T cells transfected with pCE-puro hFVT-1 were incubated with 100 µM KDS and 1 mM NADPH at 37 °C for 1 h. Lipids were separated by TLC, followed by detection with ninhydrin. The dots indicate nonspecific background.

 
Cell fractionation by centrifugation at 100,000 x g for 1 h demonstrated that most of the hFVT-1 was localized in the membrane fraction (Fig. 3B). Therefore, we performed an in vitro KDS reductase assay using the membrane fractions. In assays using membrane fractions (2.5 µg of proteins) prepared from HEK293T cells transfected with the vector plasmid, no significant decrease in KDS levels was observed, and little DHS was produced (Fig. 3C, lane 6). In contrast, the membrane fractions (2.5 µg of proteins) from HEK293T cells overproducing hFVT-1 converted all of the KDS to DHS (Fig. 3C, lane 8). This conversion was highly efficient and dose-dependent so that as little as 0.125 µg of protein converted most of the KDS present (Fig. 3D). Thus, the expression of hFVT-1 generated cellular KDS reductase activity.

To exclude the possibility that FVT-1 is not a KDS reductase but an activator, we tried to purify recombinant hFVT-1 protein from E. coli, which does not contain sphingolipids or KDS reductase. The full-length hFVT-1 fused N-terminally to MBP (MBP-hFVT-1) failed to express in E. coli (data not shown). However, removal of the N-terminal hydrophobic stretch, which is not conserved in Tsc10p, allowed the expression of the truncated form of MBP-hFVT1 (MBP-{Delta}N-hFVT-1). This N-terminal truncation did not abrogate the function of hFVT-1 because FLAG-tagged {Delta}N-hFVT-1 (FLAG-{Delta}N-hFVT-1) could enable the growth of {Delta}tsc10 cells in the absence of PHS, similarly to its full-length control (FLAG-hFVT-1) (Fig. 2B). Once expressed in E. coli, MBP-{Delta}N-hFVT-1 was recovered in the membrane fraction. The crude membrane fraction was solubilized with Triton X-100, and MBP-{Delta}N-hFVT-1 was purified using an amylose resin. Based on Coomassie staining MBP-{Delta}N-hFVT-1 was purified to near homogeneity (Fig. 4A). Using the purified protein, we performed an in vitro KDS reductase assay. KDS was unchanged upon incubation with buffer (Fig. 4B, lane 4), whereas inclusion of the MBP-{Delta}N-hFVT-1 protein efficiently converted the KDS to DHS (Fig. 4B, lane 3). This reaction was completely dependent on NADPH (Fig. 4B, lane 1), as NADH could not substitute for NADPH (Fig. 4B, lane 2). Using different concentrations of KDS or NADPH (Fig. 5) we estimated Km values for the reductase activity of the purified protein to be 3 and 28 µM for KDS and NADPH, respectively. These results indicated that hFVT-1 itself possesses an NADPH-dependent KDS reductase activity.



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  		FIG. 4.
Recombinant hFVT-1 protein exhibits KDS reductase activity. A, purified MBP-{Delta}N-hFVT-1 (0.2 µg) was separated by SDS-PAGE and stained with Coomassie Brilliant Blue. B, KDS (50 µM) was mixed with 0.5 µg of purified MBP-{Delta}N-hFVT-1 (lanes 1–3) or buffer (lane 4) in the presence or absence of NADPH or NADH as indicated and incubated at 37 °C for 1 h. Lipids were extracted, separated by TLC, and visualized by ninhydrin staining. Lane 5, DHS standard (10 nmol); lane 6, KDS standard (10 nmol).

 



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  		FIG. 5.
Substrate kinetics of the recombinant hFVT-1 protein with NADPH and KDS. Purified MBP-{Delta}N-hFVT-1 (0.1 µg) was incubated with 200 µM NADPH and varying amounts of KDS (A) or with 100 µM KDS and varying amounts of NADPH (B) at 37 °C for 10 min. The oxidation of NADPH was monitored by measuring the decreased absorbance at 340 nm.

 
Tissue Distribution of FVT-1—To investigate tissue-specific expression patterns of mFVT-1 and hFVT-1, we performed high stringency Northern blot analysis using mRNA extracted from different tissues. A predominant 2.2-kb mFVT-1 mRNA was detected in every tissue examined, although its level varied (Fig. 6A). The highest expression was observed in placenta. Lung, kidney, stomach, and small intestine expressed high levels of mFVT-1 mRNA, whereas only low levels of expression were detected in heart, spleen, and skeletal muscle (Fig. 6A). Similarly, hFVT-1 mRNA was expressed ubiquitously in the tissues examined (Fig. 6B). In contrast to mFVT-1, hFVT-1 mRNA was detected as two bands (2.7 and 2.5 kb) (Fig. 6B). In most tissues expression of the 2.5-kb mRNA was higher than that of the 2.7-kb mRNA (Fig. 6B). However, the 2.7-kb mRNA was the major species in placenta, and nearly equal levels of the two mRNAs were observed in liver (Fig. 6B). Moreover, the expression profile of hFVT-1 was different from that of mFVT-1. The levels of the hFVT-1 mRNAs were the highest in skeletal muscle and heart and lowest in the colon, thymus, and peripheral blood leukocytes (Fig. 6B). A 1.3-kb hFVT-1 transcript, possibly another splice variant, was detected in the heart and skeletal muscle tissues (Fig. 6B).



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  		FIG. 6.
Northern blot analyses of mFVT-1 and hFVT-1 expression. 32P-Labeled mFVT-1 (A) and hFVT-1 (B) probes were hybridized to total RNA blots containing 20 µg of RNA from mouse tissues (A) or poly(A)+ RNA blots containing 1 µg of RNA from human tissues (B). PBL, peripheral blood leukocyte.

 
FVT-1 Is Localized in the ER—To investigate the subcellular localization of hFVT-1, indirect immunofluorescence microscopic analysis was performed using HeLa cells transfected with a pCE-puro hFVT-1 plasmid for 24 h. hFVT-1 was detected as a reticular structure with a pattern similar to that of ER (Fig. 7A). Therefore, we applied double staining of the cells with the anti-FVT-1 antibody and an antibody directed to the ER retention signal of the ER luminal protein KDEL. The staining pattern of hFVT-1 showed a complete overlap with the ER marker KDEL (Fig. 7B), indicating that hFVT-1 is an ER-resident protein.



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  		FIG. 7.
Localization of FVT-1 in the ER. HeLa cells transfected with pCE-puro hFVT-1 were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and immunostained with an anti-hFVT-1 antibody (A) or double-stained with anti-hFVT-1 and anti-KDEL antibodies (B). Calibration bar, 10 µm.

 
The Active Site of FVT-1 Faces the Cytosolic Side of the ER—Both FVT-1 proteins contain a large hydrophilic domain between the putative first and second transmembrane-spanning segments. This domain exhibits similarity to domains of other members in the SDR family and contains the putative NADPH binding site and the active site motif Tyr-X-X-X-Lys. Determination of the membrane topology of FVT-1 is important for identifying on which side of the ER membrane DHS is produced. For this purpose, intact organelles were prepared from HEK293T cells transfected with pCE-puro hFVT-1 and were treated with proteinase K. Calnexin is a type I ER membrane protein with an N-terminal large domain that is exposed to the lumen of the ER (36). We chose calnexin as a control to assess the proper orientation of the isolated ER. Protease treatment reportedly results in the production of C-terminally truncated forms of calnexin (calnexin-{Delta}C) (36). Accordingly, upon treatment with proteinase K, calnexin was converted to calnexin-{Delta}C, which reacted with an antibody raised against the N-terminal region of calnexin (amino acid residues 1–70) (Fig. 8A, lane 5). This band disappeared after disrupting the ER membrane with Triton X-100 (Fig. 8A, lane 6), indicating that calnexin-{Delta}C is protected by the ER membrane. On the other hand, the FVT-1 protein was not protected against cleavage by proteinase K in the absence of Triton X-100 (Fig. 8A, lane 2). Because the anti-hFVT-1 antibody was raised against an epitope between residues 26 and 292 in the middle region of the hFVT-1 protein, this result suggests that the large hydrophilic domain of FVT-1 is exposed to the cytosolic side of the ER membrane.



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  		FIG. 8.
Determination of the membrane topology of the FVT-1 protein. A, crude membrane fractions were prepared from HEK293T cells transfected with pCE-puro hFVT-1 and treated with 0 (lanes 1 and 4) or 0.5 mg/ml (lanes 2, 3, 5, and 6) proteinase K (Prot. K)at4 °Cfor2h in the absence (lanes 1, 2, 4, and 5) or presence (lanes 3 and 6) of 1% Triton X-100. Proteins were separated by SDS-PAGE, followed by immunoblotting with anti-hFVT-1 (lanes 1–3) or anti-calnexin (lanes 4–6) antibodies. B, a model of the proposed topology of FVT-1 relative to the established topology of calnexin (36). FVT-1 is proposed to be an integral membrane protein with three transmembrane domains. The N terminus is located in the lumen of the ER, whereas the large hydrophilic domain containing the active site residues and the C terminus is located in the cytosol.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
During the last decade, most of the mammalian genes responsible for sphingolipid biosynthesis have been identified (12–14, 17–20, 37, 38). However, the identification of the gene for KDS reductase, which is involved in the second step of de novo sphingolipid synthesis, has remained a missing link. We searched for a mammalian gene encoding a product similar to Tsc10p and found FVT-1 as a functional mammalian KDS reductase. First, in {Delta}tsc10 yeast cells forced expression of either human or mouse isoforms of FVT-1 restored the growth defect in the absence of PHS (Fig. 2A). Second, expression of hFVT-1 conferred KDS reductase activity to HEK293T cells (Fig. 3C). Third, purified recombinant MBP-{Delta}N-hFVT-1 exhibited unequivocal KDS reductase activity (Fig. 4B). Together, these results provide strong support for the hypothesis that FVT-1 is a mammalian KDS reductase.

We found that FVT-1 is localized at the ER (Fig. 7), which is consistent with previous results that sphingolipid biosynthesis, up to the point of ceramide formation, takes place in the ER (39).

FVT-1 appears to be ubiquitous and somewhat conserved because the mouse and human genes share sequence homology and, to some extent, expression patterns. Although it is possible that multiple KDS reductases exist in mammalian cells, the ubiquitous expression of both hFVT-1 and mFVT-1 (Fig. 6) suggests that FVT-1 is a major KDS reductase. Interestingly, two predominant FVT-1 mRNA isoforms (2.5 and 2.7 kb) were detected in human tissues. Expression of the 2.5-kb mRNA was higher than that of the 2.7-kb form in most tissues. However, in the liver their levels were nearly equal, whereas in the placenta the 2.7-kb form was the major species. We found that the cDNA in the GenBankTM with accession number AK025120 [GenBank] comprises a full-length hFVT-1 cDNA. This cDNA is composed of 2044 bp in addition to the poly(A) sequence. Using the BLAST program, we compared the sequence of AK025120 [GenBank] with other human expressed sequence tag clones containing hFVT-1 cDNA and found that most of the expressed sequence tag clones, including AI754522 [GenBank] , BQ010432 [GenBank] , and BQ020986 [GenBank] , have a 3'-untranslated region identical to that of AK025120 [GenBank] . However, some expressed sequence tag clones, such as BM995782 [GenBank] , BQ182543 [GenBank] , and CA438131 [GenBank] , contain an extra 100 bp in the 3'-untranslated region. Therefore, the two mRNA species detected by Northern blotting (Fig. 6) may reflect these differences in the length of the 3'-untranslated region.

FVT-1 was originally identified in a study searching for genes involved in tumor progression (22). Variant t(2;18)(p11; q21) chromosome translocation, noted in follicular lymphomas, induced the expression of the BCL-2 proto-oncogene, although rearrangement of BCL-2 was not observed by Southern blot analysis (40). Subsequent study revealed that in this translocation, a J{kappa} segment is juxtaposed to the FVT-1 gene, which is localized 10 kb upstream of the BCL-2 locus (22). FVT-1 was weakly expressed in all of the normal hematopoietic tissues tested, whereas high levels of expression were observed in some T-cell malignancies (22). At the present time, any involvement of FVT-1 in tumorigenesis remains unclear.

In the report in which hFVT-1 was first identified, hFVT-1 was predicted to be a secreted protein based on the presence of the N-terminal hydrophobic stretch (22), although its localization was not examined. However, this N-terminal region lacks the features characteristic of signal sequences. Signal sequences are typically composed of a short positively charged N-terminal segment (n-region), a central hydrophobic segment (h-region), and a more polar C-terminal segment (c-region) (41). Neither human nor mouse FVT-1 has basic amino acids at its N terminus; thus both are lacking the n-region. The distribution of charged residues on either side of the hydrophobic core of signal sequences or transmembrane segments is important for determining the membrane orientation. The "positive-inside rule" indicates that flanking sequences with positive charges are predominantly localized to the cytosolic side of the membrane (42). Thus, basic amino acids in the n-region of signal sequences are important in orientating the signal sequence to Ncyt/Cexo (cytoplasmic N and exoplasmic C termini). Near the N-terminal hydrophobic stretch, hFVT-1 and mFVT-1 both contain a Lys residue, which is downstream to the hydrophobic core, but lack acidic amino acids. Thus, this hydrophobic stretch is predicted to be oriented Nexo/Ccyt, an inverse alignment to that of a signal sequence. Therefore, the N terminus does not appear to be a signal sequence but rather a transmembrane domain. Indeed, we obtained corroborating evidence that FVT-1 is an ER membrane protein (Fig. 7).

We propose a model for the topology of FVT-1 (Fig. 8B) in which FVT-1 has three transmembrane segments, with the N terminus facing the lumen of the ER and the large catalytic domain and C terminus located in the cytosol. This model is supported by experimental data and predictions. First, proteinase K digestion demonstrated that the large hydrophilic domain, which contains the active site residues, faces the cytosol (Fig. 8A). This result is consistent with the predicted Nexo/Ccyt orientation of the putative first transmembrane domain described above. Second, our results on the topology of Tsc10p are consistent with the topology model for FVT-1. FVT-1 and Tsc10p share a similar hydropathy profile, except that Tsc10p lacks the N-terminal putative transmembrane segment. Proteinase K digestion using N-terminally FLAG-tagged Tsc10p in conjunction with the results of a C-terminal reporter demonstrated that both the N terminus and C terminus of Tsc10p are exposed to the cytoplasm.2 Finally, both Tsc10p and FVT-1 contain putative ER retention signals at their C termini. This signal is known to function in the cytosolic side of the ER membrane (43).

Previous biochemical analyses using mouse liver tissues suggested that sphingolipid synthesis is initiated in the cytoplasmic leaflet of the lipid bilayer of the ER membrane and proceeds to the point of dihydroceramide synthesis (39). This conclusion is based on the sensitivity of the sphingolipid biosynthetic activities to exogenous proteases and the membrane-impermeable compound 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (39). However, the possibility cannot be excluded that some membrane proteins present residues essential for activity on both sides of the membrane, and the precise topology can only be determined by detecting specific protein domains rather than enzymatic activity. Such topological study, which was performed recently, indicated that the active site of the serine palmitoyltransferase catalyzing the first step of sphingolipid biosynthesis is indeed exposed to the cytosolic side of the membrane (44). In this study, we have demonstrated that the domain containing the active site of the KDS reductase also faces the cytosol. Therefore, these results are consistent with the hypothesis that production of KDS and DHS takes place on the cytosolic side of the ER membrane. However, the side of the ER on which ceramide is synthesized remains unclear. DHS and PHS are generated in the luminal side of the ER membrane by the yeast long-chain base 1-phosphate phosphatase Lcb3p and are used as precursors of ceramide (45). Moreover, galactosylceramide and glucosylceramide are synthesized on the luminal side of the ER and on the cytosolic side of the Golgi apparatus, respectively, suggesting that their precursor, ceramide, may exist in both sides (46–49). Thus, future topological studies are required to determine the precise orientation of ceramide synthase to reveal the complete trans-bilayer organization of the sphingolipid biosynthetic pathway.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY634684 [GenBank] .

* This work was supported by Grant-in-aid for Scientific Research on Priority Areas B 12140201 and Grant-in-aid for Young Scientists B 15770078 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 81-11-706-3970; Fax: 81-11-706-4986; E-mail: yigarash{at}pharm.hokudai.ac.jp.

1 The abbreviations used are: KDS, 3-ketodihydrosphingosine; DHS, dihydrosphingosine; PHS, phytosphingosine; FVT, follicular lymphoma variant translocation; ER, endoplasmic reticulum; HEK, human embryonic kidney; mFVT-1, mouse FVT-1; hFVT-1, human FVT-1; MBP, maltose-binding protein; PMSF, phenylmethylsulfonyl fluoride; TLC, thin layer chromatography; SDR, short-chain dehydrogenase/reductase. Back

2 A. Kihara and Y. Igarashi, unpublished observations. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Simons, K., and Ikonen, E. (1997) Nature 387, 569-572[CrossRef][Medline] [Order article via Infotrieve]
  2. Hannun, Y. A., Luberto, C., and Argraves, K. M. (2001) Biochemistry 40, 4893-4903[CrossRef][Medline] [Order article via Infotrieve]
  3. Merrill, A. H., Jr. (2002) J. Biol. Chem. 277, 25843-25846[Free Full Text]
  4. Sims, K. J., Spassieva, S. D., Voit, E. O., and Obeid, L. M. (2004) Biochem. Cell Biol. 82, 45-61[CrossRef][Medline] [Order article via Infotrieve]
  5. Beeler, T., Bacikova, D., Gable, K., Hopkins, L., Johnson, C., Slife, H., and Dunn, T. (1998) J. Biol. Chem. 273, 30688-30694[Abstract/Free Full Text]
  6. Haak, D., Gable, K., Beeler, T., and Dunn, T. (1997) J. Biol. Chem. 272, 29704-29710[Abstract/Free Full Text]
  7. Grilley, M. M., Stock, S. D., Dickson, R. C., Lester, R. L., and Takemoto, J. Y. (1998) J. Biol. Chem. 273, 11062-11068[Abstract/Free Full Text]
  8. Buede, R., Rinker-Schaffer, C., Pinto, W. J., Lester, R. L., and Dickson, R. C. (1991) J. Bacteriol. 173, 4325-4332[Abstract/Free Full Text]
  9. Pinto, W. J., Srinivasan, B., Shepherd, S., Schmidt, A., Dickson, R. C., and Lester, R. L. (1992) J. Bacteriol. 174, 2565-2574[Abstract/Free Full Text]
  10. Pinto, W. J., Wells, G. W., and Lester, R. L. (1992) J. Bacteriol. 174, 2575-2581[Abstract/Free Full Text]
  11. Nagiec, M. M., Baltisberger, J. A., Wells, G. B., Lester, R. L., and Dickson, R. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7899-7902[Abstract/Free Full Text]
  12. Nagiec, M. M., Lester, R. L., and Dickson, R. C. (1996) Gene (Amst.) 177, 237-241[CrossRef][Medline] [Order article via Infotrieve]
  13. Weiss, B., and Stoffel, W. (1997) Eur. J. Biochem. 249, 239-247[Medline] [Order article via Infotrieve]
  14. Hanada, K., Hara, T., Nishijima, M., Kuge, O., Dickson, R. C., and Nagiec, M. M. (1997) J. Biol. Chem. 272, 32108-32114[Abstract/Free Full Text]
  15. Guillas, I., Kirchman, P. A., Chuard, R., Pfefferli, M., Jiang, J. C., Jazwinski, S. M., and Conzelmann, A. (2001) EMBO J. 20, 2655-2665[CrossRef][Medline] [Order article via Infotrieve]
  16. Schorling, S., Vallee, B., Barz, W. P., Riezman, H., and Oesterhelt, D. (2001) Mol. Biol. Cell 12, 3417-3427[Abstract/Free Full Text]
  17. Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Sullards, M. C., Merrill, A. H., Jr., and Futerman, A. H. (2002) J. Biol. Chem. 277, 35642-35649[Abstract/Free Full Text]
  18. Riebeling, C., Allegood, J. C., Wang, E., Merrill, A. H., Jr., and Futerman, A. H. (2003) J. Biol. Chem. 278, 43452-43459[Abstract/Free Full Text]
  19. Guillas, I., Jiang, J. C., Vionnet, C., Roubaty, C., Uldry, D., Chuard, R., Wang, J., Jazwinski, S. M., and Conzelmann, A. (2003) J. Biol. Chem. 278, 37083-37091[Abstract/Free Full Text]
  20. Ternes, P., Franke, S., Zahringer, U., Sperling, P., and Heinz, E. (2002) J. Biol. Chem. 277, 25512-25518[Abstract/Free Full Text]
  21. Zhao, C., Beeler, T., and Dunn, T. (1994) J. Biol. Chem. 269, 21480-21488[Abstract/Free Full Text]
  22. Rimokh, R., Gadoux, M., Bertheas, M. F., Berger, F., Garoscio, M., Deleage, G., Germain, D., and Magaud, J. P. (1993) Blood 81, 136-142[Abstract/Free Full Text]
  23. Irie, K., Takase, M., Lee, K. S., Levin, D. E., Araki, H., Matsumoto, K., and Oshima, Y. (1993) Mol. Cell. Biol. 13, 3076-3083[Abstract/Free Full Text]
  24. Kihara, A., and Igarashi, Y. (2002) J. Biol. Chem. 277, 30048-30054[Abstract/Free Full Text]
  25. Kihara, A., Ikeda, M., Kariya, Y., Lee, E. Y., Lee, Y. M., and Igarashi, Y. (2003) J. Biol. Chem. 278, 14578-14585[Abstract/Free Full Text]
  26. Ogawa, C., Kihara, A., Gokoh, M., and Igarashi, Y. (2003) J. Biol. Chem. 278, 1268-1272[Abstract/Free Full Text]
  27. Ghosh, D., Weeks, C. M., Grochulski, P., Duax, W. L., Erman, M., Rimsay, R. L., and Orr, J. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10064-10068[Abstract/Free Full Text]
  28. Ensor, C. M., and Tai, H. H. (1991) Biochem. Biophys. Res. Commun. 176, 840-845[CrossRef][Medline] [Order article via Infotrieve]
  29. Varughese, K. I., Skinner, M. M., Whiteley, J. M., Matthews, D. A., and Xuong, N. H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6080-6084[Abstract/Free Full Text]
  30. Obeid, J., and White, P. C. (1992) Biochem. Biophys. Res. Commun. 188, 222-227[CrossRef][Medline] [Order article via Infotrieve]
  31. Albalat, R., Gonzàlez-Duarte, R., and Atrian, S. (1992) FEBS Lett. 308, 235-239[CrossRef][Medline] [Order article via Infotrieve]
  32. Jörnvall, H., Persson, B., Krook, M., Atrian, S., Gonzàlez-Duarte, R., Jeffery, J., and Ghosh, D. (1995) Biochemistry 34, 6003-6013[CrossRef][Medline] [Order article via Infotrieve]
  33. McKinley-McKee, J. S., Winberg, J. O., and Pettersson, G. (1991) Biochem. Int. 25, 879-885[Medline] [Order article via Infotrieve]
  34. Ghosh, D., Wawrzak, Z., Weeks, C. M., Duax, W. L., and Erman, M. (1994) Structure 2, 629-640[Medline] [Order article via Infotrieve]
  35. Rossmann, M. G., Liljas, A., Brändén, C. I., and Banaszak, L. J. (1975) in The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. 11, pp. 61-102, Academic Press, New York
  36. Ou, W. J., Bergeron, J. J., Li, Y., Kang, C. Y., and Thomas, D. Y. (1995) J. Biol. Chem. 270, 18051-18059[Abstract/Free Full Text]
  37. Huitema, K., Van Den Dikkenberg, J., Brouwers, J. F., and Holthuis, J. C. (2004) EMBO J. 23, 33-44[CrossRef][Medline] [Order article via Infotrieve]
  38. Yamaoka, S., Miyaji, M., Kitano, T., Umehara, H., and Okazaki, T. (2004) J. Biol. Chem. 279, 18688-18693[Abstract/Free Full Text]
  39. Mandon, E. C., Ehses, I., Rother, J., van Echten, G., and Sandhoff, K. (1992) J. Biol. Chem. 267, 11144-11148[Abstract/Free Full Text]
  40. Bertheas, M. F., Rimokh, R., Berger, F., Gaucherand, M., Machado, P., Vasselon, C., Calmard-Oriol, P., Jaubert, J., Guyotat, D., and Magaud, J. P. (1991) Br. J. Haematol. 78, 132-134[Medline] [Order article via Infotrieve]
  41. Nothwehr, S. F., and Gordon, J. I. (1990) BioEssays 12, 479-484[CrossRef][Medline] [Order article via Infotrieve]
  42. von Heijne, G., and Gavel, Y. (1988) Eur. J. Biochem. 174, 671-678[Medline] [Order article via Infotrieve]
  43. Nilsson, T., Jackson, M., and Peterson, P. A. (1989) Cell 58, 707-718[CrossRef][Medline] [Order article via Infotrieve]
  44. Yasuda, S., Nishijima, M., and Hanada, K. (2003) J. Biol. Chem. 278, 4176-4183[Abstract/Free Full Text]
  45. Kihara, A., Sano, T., Iwaki, S., and Igarashi, Y. (2003) Genes Cells 8, 525-535[Abstract]
  46. Coste, H., Martel, M. B., and Got, R. (1986) Biochim. Biophys. Acta 858, 6-12[Medline] [Order article via Infotrieve]
  47. Futerman, A. H., and Pagano, R. E. (1991) Biochem. J. 280, 295-302[Medline] [Order article via Infotrieve]
  48. Jeckel, D., Karrenbauer, A., Burger, K. N., van Meer, G., and Wieland, F. (1992) J. Cell Biol. 117, 259-267[Abstract/Free Full Text]
  49. Sprong, H., Kruithof, B., Leijendekker, R., Slot, J. W., van Meer, G., and van der Sluijs, P. (1998) J. Biol. Chem. 273, 25880-25888[Abstract/Free Full Text]
  50. Higgins, D. G., Bleasby, A. J., and Fuchs, R. (1992) Comput. Appl. Biosci. 8, 189-191[Abstract/Free Full Text]

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