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J. Biol. Chem., Vol. 277, Issue 33, 30048-30054, August 16, 2002

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Identification and Characterization of a Saccharomyces cerevisiae Gene, RSB1, Involved in Sphingoid Long-chain Base Release^*

Akio Kihara and Yasuyuki Igarashi

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, April 9, 2002, and in revised form, May 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sphingoid long-chain bases (LCBs) and long-chain base phosphates (LCBPs) act as signaling molecules in eukaryotic cells. Accumulation of LCBPs results in cell growth inhibition in yeast, although the mechanism is unknown. Here, we identified a novel yeast gene, RSB1 (resistance to sphingoid long-chain base), by screening a multicopy suppressor of the LCB-sensitive phenotype of the LCBP lyase mutant. RSB1 encodes a polypeptide of 354 amino acids with a molecular mass of 40.4 kDa. Rsb1p is predicted to be an integral membrane protein with seven transmembrane-spanning domains. We demonstrated that cells overproducing Rsb1p showed a decrease in accumulation of exogenously added sphingosine and dihydrosphingosine because of their increased release. This release was ATP-dependent, and a mutant of the predicted ATP binding motif had no activity. Substrate specificity analysis of Rsb1p demonstrated that it is active on LCBs but not on LCBPs or other hydrophobic compounds. These results suggest that Rsb1p is a transporter or flippase that translocates LCBs from the cytoplasmic side toward the extracytoplasmic side of the membrane.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sphingolipids are components of eukaryotic cell membranes composed of a hydrophobic segment, ceramide, coupled to one of a variety of polar head groups. The ceramide moiety consists of a sphingoid long-chain base (LCB)¹ (sphingosine (SPH) in animals, phytosphingosine (PHS) in yeast and plants), which is N-fatty acylated. In addition to the structural functions of sphingolipids in maintaining cell membrane integrity, recent discoveries have further revealed important roles for their metabolites such as ceramide, SPH, and sphingosine 1-phosphate (SPH1P) in such diverse biological responses as cell growth, differentiation, motility, apoptosis, and stress responses in mammalian cells (1-3).

Biosynthesis of sphingolipids begins with the condensation of serine with palmitoyl-CoA to form 3-ketodihydrosphingosine, which is reduced to dihydrosphingosine (DHS) and then acylated to dihydroceramide. In mammalian cells, a 4,5-trans-double bond is then incorporated to form ceramide, which is further converted to sphingomyelin or glycosphingolipids. Not a component in this synthetic pathway, sphingosine is encountered only as a product of sphingolipid turnover, but is then available also for phosphorylation.

Synthesis of sphingolipids in the yeast Saccharomyces cerevisiae is similar to that of mammalian sphingolipids (Fig. 1). However, SPH is not produced in yeast, because dihydroceramide desaturase is not present. Instead, yeast contains PHS as the main sphingoid base. Phytoceramide is synthesized by attachment of a very-long-chain fatty acid to the amine of the LCB and converted to myo-inositol-containing sphingolipids, inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide (MIPC), and mannosyldiinositol phosphorylceramide (M(IP)₂C). Because sphingolipids are essential for growth, yeast strains harboring mutations in the serine palmitoyltransferase gene, LCB1 or LCB2, cannot grow (4, 5). However, addition of LCBs to the nutrient medium restores the growth defect of these mutant cells, indicating that yeast can incorporate exogenous LCBs into sphingolipids (4, 5). Because the endoplasmic reticulum (ER) is the site of ceramide synthesis, exogenously added LCBs must be delivered to the ER. Recently, it has been shown that a mutation of LCB3, which encodes a sphingoid long-chain base phosphate (LCBP) phosphatase associated with the ER membrane, or that of LCB4, a LCB kinase, reduces this incorporation drastically (6-8). These results suggest that conversion of LCBs to LCBPs is essential for delivery of LCBs from the plasma membrane to the ER membrane.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Pathway of sphingolipid metabolism in S. cerevisiae. Shown are the pathways for de novo sphingolipid biosynthesis as well as the metabolism of endogenous and exogenous DHS and PHS, including the genes involved at each step. The very-long-chain fatty acid moiety of ceramide is mostly hydroxylated by Scs7p (42).

Yeast does not contain SPH or SPH1P, but two de novo synthesized LCBs, DHS and PHS, as well as their phosphorylated derivatives, LCBPs, have similarly been implicated as signaling molecules. LCBs are transiently increased upon heat shock and induce G₀/G₁ arrest and ubiquitin-dependent proteolysis (8-14). LCBPs are also induced by heat shock and function in heat stress resistance, diauxic shift, and Ca²⁺ mobilization (7, 15-18). Elimination of the Dpl1 lyase or the Lcb3 phosphatase pathway results in a 2-5-fold and a 10-12-fold increase in the LCBP level, respectively (19, 20). Although a single deletion of DPL1 or LCB3 elicits no growth defect, the concomitant deletion of DPL1 and LCB3 causes cell death or a severe growth defect, which is dependent on strain background. In contrast, dpl1 lcb3 lcb4 triple mutants exhibit normal growth, indicating that hyperaccumulation of LCBPs is toxic to cells (19, 20). However, the mechanism of this toxicity, as well as the target molecules of the LCBPs, has not been determined.

In an attempt to identify genes for which products may be involved in LCBs/LCBPs signaling or transport, we screened a multicopy suppressor gene of the LCB-sensitive phenotype of the dpl1 cells. We obtained a novel gene, RSB1, which encodes a highly hydrophobic protein. Overproduction of this protein, Rsb1p, conferred on cells a resistance to SPH, PHS, DHS, and L-threo-dihydrosphingosine, but not to other hydrophobic compounds tested. This indicated that the effect of Rsb1p was LCB-specific. Cells overproducing Rsb1p also demonstrated an increase in LCB release, an ATP-dependent reaction. Together, these results suggest that Rsb1p may be a transporter or flippase that translocates LCBs outward across the plasma membrane.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Yeast Strains and Media-- S. cerevisiae strains used were SEY6210 (MAT leu2-3, 112 ura3-52 his3-200 trp1-901 lys2-801 suc2-9; Ref. 21), KHY128 (MATa/a leu2-3, 112/leu2-3, 112 ura3-52/ura3-52 his3-200/his3-200 trp1-901/trp1-901 lys2-801 ade2-101 suc2-9/suc2-9), KHY13 (SEY6210, dpl1::TRP1), KHY21 (SEY6210, dpl1::TRP1 lcb3::LEU2), KHY24 (SEY6210, dpl1::TRP1 lcb3::LEU2 ysr3::URA3), and KHY91 (SEY6210, dpl1::TRP1 rsb1::HIS3). KHY128 cells were constructed by mating SEY6210 with SEY6211 (21) cells. The dpl1::TRP1, lcb3::LEU2, ysr3::URA3, and rsb1::HIS3 cells were constructed to replace the 0.47-kb SmaI-BglII region in the DPL1 gene, the 0.84-kb BamHI-HpaI region in the LCB3 gene, the 0.39-kb HindIII-StuI region in the YSR3 gene, and the 0.58-kb EcoRV-EcoRV region in the RSB1 gene with the TRP1, LEU2, URA3, or HIS3 marker, respectively. Cells were grown either in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or in synthetic complete (SC) medium containing nutritional supplements.

Cloning of RSB1 Gene-- A yeast genomic library was constructed by ligating 4-10-kb Sau3AI fragments of KHY24 genomic DNA to the BamHI site of pRS426 (22). A pool of 100,000 Ura⁺ clones, obtained after transformation of KHY13 cells with the library, was plated on YPD agar containing 5 µM SPH or 10 µM PHS. Nonidet P-40 (0.0015% w/v, final concentration) was also included in the plates as a dispersant.

Plasmid Construction-- pAK80, a pRS316-derived yeast cloning vector, was designed for protein expression under the control of the TDH3 promoter, encoding glyceraldehyde-3-phosphate dehydrogenase. The TDH3 promoter region was first amplified from the pKT10 plasmid (23) using the following primers: 5'-CAGCTGAAGCTTTTCTGCTGTAACCCGTACATGCC-3' and 5'-GTTAACAGATCTTTATGTGTGTTTATTCGAAAC-3'. The resulting fragments were cloned into pGEM-T Easy (Promega) to generate the pAK76 plasmid, and the 0.37-kb EcoRI fragment of pAK76 was then cloned into the EcoRI site of pRS316 (24) to generate pAK80.

The pAK90 plasmid, which encodes RSB1 under the control of the TDH3 promoter, was constructed in the following manner. Using the pAK69 plasmid, which contains a 3.1-kb SacI-PstI fragment of RSB1 region in pBluescript II SK⁺, as a template and the primers 5'-AGATCTTGGTATGGTACCGAACCTTCG-3' and 5'-CCAGGGTTTTCCCAGTCACGACG-3', the RSB1 region was amplified and then cloned into pGEM-T Easy to generate the pAK75 plasmid. Finally, pAK90 was constructed by cloning a 2.8-kb BglII-NotI fragment of pAK75 into the BamHI-NotI site of pAK80.

The pAK162 yeast vector was constructed to produce N-terminally triple hemagglutinin (3×HA)-tagged fusion proteins. The 3×HA epitope was amplified from the pKHR45 plasmid (25), using the primers 5'-GCAGCCCGGGGGAACCATGAGTTACCC-3' and 5'-TTGTTAACGAGCTCTAAAGCGTAATCTGG-3'. This PCR fragment was first cloned into pGEM-T Easy to generate pAK147, and the 0.14-kb SmaI-NotI fragment of pAK147 was cloned into the same site of pAK80 to generate pAK162. The plasmid pAK170, which encodes 3×HA-RSB1, was further constructed by cloning a 2.8-kb BglII (blunt-ended by T4 DNA polymerase)-NotI fragment into the HpaI-NotI site of pAK162. Finally, pAK186 (3×HA-rsb1 (G282W)) was constructed by site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene) and primers (5'-GCGTTTAAATGGAGAAGCACCTCG-3' and 5'-CGAGGTGCTTCTCCATTTAAACGC-3').

Assay for LCB Uptake-- Yeast strains were grown to 1 A₆₀₀ unit/ml in SC medium lacking uracil at 30 °C. Cells were treated with [3-³H]SPH (23.5 Ci/mmol; PerkinElmer Life Sciences) or [4,5-³H]DHS (50 Ci/mmol; American Radiolabeled Chemical, Inc.), both of which had been complexed with 1 mg/ml fatty acid-free bovine serum albumin (BSA) (Sigma, A-6003) and incubated for indicated time periods. 0.1 µCi of [³H]SPH and 1 µCi of [³H]DHS were used for labeling 1 A₆₀₀ cells. At each time point, cells equivalent to 0.45 A₆₀₀ were chilled on ice, washed with cold SC medium containing 1 mg/ml BSA, and suspended in 50 µl of 20 mM HEPES, pH 7.5, containing 10 mM EDTA. Glass beads were added, and cells were lysed by vortexing vigorously. Lipids were extracted by successive addition and mixing of 187.5 µl of chloroform/methanol/HCl (100:200:1, v/v), 62.5 µl of chloroform, and 62.5 µl 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 labeled lipids were resolved by thin layer chromatography (TLC) on Silica Gel 60 high performance TLC plates (Merck) with 1-butanol/acetic acid/water (3:1:1, v/v).

DHS Release Assay-- Yeast strains were grown to 1 A₆₀₀ unit/ml in SC medium lacking uracil at 30 °C. Cells were treated with [³H]DHS (1 µCi/1 A₆₀₀ cells), which had been complexed with 1 mg/ml BSA, and incubated for 1 h at 30 °C. Cells were then washed with SC medium containing 1 mg/ml BSA two times, suspended in SC medium containing 1 mg/ml BSA, and incubated for the indicated time periods at 30 °C. Cells and medium were separated by centrifugation, and lipids were extracted from the medium and separated by TLC as described above.

Immunoblotting-- Cells equivalent to 2 A₆₀₀ were recovered by centrifugation and suspended in 100 µl of 0.2 N NaOH, 0.5% 2-mercaptoethanol. After incubation for 15 min on ice, samples were treated with 1 ml of cold acetone and incubated for 30 min at 20 °C. After centrifugation at 15,000 × g for 5 min at 4 °C, the resulting pellets were suspended in 100 µl of 2× SDS sample buffer (125 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 5% 2-mercaptoethanol, and a trace amount of bromphenol blue). Lysates equivalent to 0.2 A₆₀₀ were subjected to SDS-PAGE and transferred to Immobilon^TM polyvinylidene difluoride membrane (Millipore). The resulting membrane was incubated with a 1:1000 dilution of anti-HA antibodies, Y-11 (Santa Cruz) for 1 h and then with a 1:7500 dilution of horseradish peroxidase-conjugated donkey anti-rabbit IgG F(ab')₂ fragment (Amersham Biosciences) for 1 h. Labeling was detected by the ECL detection method (Amersham Biosciences).

Immunofluorescence Microscopy-- Cells were fixed with 5% formaldehyde in potassium phosphate (pH 6.5), collected by centrifugation, and suspended in a mixture of 100 mM HEPES, pH 7.5, 1 M sorbitol, and 5 mM sodium azide. Cells were then converted to spheroplasts by adding 0.002 volume of 2-mercaptoethanol and 0.1 mg/ml Zymolyase 100T (Seikagaku Co.) and incubating for 30 min at 30 °C. Spheroplasts were laid on multiwell glass slides (Cel-Line/Erie Scientific Co.) coated with polylysine, then permeabilized by treatment with 0.1% Triton X-100 in PBS for 10 min, and blocked with 1% BSA in PBS for 10 min. The cells were incubated with primary antibodies diluted in PBS containing 0.1% BSA for 1 h. Anti-HA monoclonal antibody HA7 (Sigma) and anti-Kar2 antiserum (a gift from T. Yoshihisa, Nagoya University, Nagoya, Japan) were used at 1:100 and 1:500 dilutions, respectively. After washing, the cells were incubated with the secondary antibodies diluted in PBS containing 0.1% BSA for 1 h. For secondary staining the antibodies Alexa Fluor 488 goat anti-mouse IgG (H+L) conjugate (10 µg/ml; Molecular Probes) and Alexa Fluor 594 goat anti-rabbit IgG (H+L) conjugate (10 µg/ml; Molecular Probes) were used. Cells were washed, mounted with a Slow Fade Light Antifade kit (Molecular Probes), and observed under a fluorescence laser scanning microscope (LSM510; Zeiss).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of the RSB1 Gene-- In searching for a new gene involved in LCBs/LCBPs signaling or in their transport, we screened for a multicopy suppressor gene that would rescue a LCB-sensitive phenotype. Because overaccumulation of LCBPs is toxic to yeast cells, the LCBP lyase mutant (dpl1) KHY13 cells are sensitive to exogenous LCBs.

First, to prevent the isolation of previously established genes for LCBP lyase (DPL1) and LCBP phosphatase (LCB3, YSR3) genes, we constructed a yeast genomic library using genomic DNA prepared from KHY24 (dpl1 lcb3 ysr3) cells in a high copy number vector pRS426. A pool of KHY13 cells was then transformed with this genomic library and plated on YPD plates containing 5 µM SPH or 10 µM PHS. Several clones were obtained, but restriction-enzyme mapping, nucleotide sequencing, and subcloning revealed that all clones carried the same gene. This gene, termed RSB1, was found to be identical to an open reading frame indicated as YOR049c (GenBank^TM accession no. CAA99241) in the S. cerevisiae data base.

The RSB1 gene encodes a putative polypeptide of 354 amino acids (Fig. 2A) with a deduced molecular mass of 40.4 kDa. This protein, Rsb1p, is highly hydrophobic, and both the TopPredII 1.1 (26) and the SOSUI (27) programs predict it to be an integral membrane protein with seven membrane-spanning segments (Fig. 2B). Rsb1p has four homologs (Rtm1p, Yer185w, Rta1p, and Ylr046c) in the yeast data base. Rtm1p and Rta1p have been isolated as conferring resistance in yeast to the toxicity of molasses (28) and 7-aminocholesterol (29), respectively, whereas functions for YER185w and YLR046c are unknown. Rsb1p shows 23-28% identity and 40-48% similarity to these proteins. Of these homologs, Rsb1p is the largest and contains an extended loop between predicted transmembrane domains 5 and 6 and a long C-terminal tail. A Walker A motif, GX₄G(K/R/H)(T/S), generally known to mediate ATP binding and hydrolysis (30), is present within the junction between the predicted transmembrane domain 7 and the C-terminal tail of Rsb1p. However, this motif is not found in other homologs.

View larger version (80K): [in this window] [in a new window]   Fig. 2.   Sequence and hydropathic profile of Rsb1p. A, alignment of amino acid sequences of Rsb1p, Rtm1p, Yer185w, Rta1p, and Ylr046c. GenBankTM accession nos. are CAA99241 (RSB1), AAA81533 (RTM1), AAB64712 (YER185w), CAA97240 (RTA1), and CAA97576 (YLR046c). Amino acid identities and similarities of these homologs to Rsb1p are 28%/48% (Rtm1p), 24%/44% (Yer185w), 23%/46% (Rta1p), and 23%/40% (Ylr046c). The alignment was generated using the Clustal V (43) and BOXSHADE (Institute for Animal Health, Surrey, United Kingdom) programs. Black boxes indicate identical residues, whereas gray boxes show amino acid similarity. The dashed line indicates the putative Walker A motif in Rsb1p. B, hydropathy plot (window size of 15; Ref. 44) of Rsb1p. TM1-TM7 indicate transmembrane segments predicted by the TopPred II 1.1 program (26).

Determination of the Role of Rsb1p in Resistance to LCBs-- Rsb1p was previously predicted to be a transporter, based on its sequence (31). Therefore, we examined the effect of Rsb1p on the intracellular accumulation of LCBs (SPH or DHS) added exogenously. We also investigated the conversion of the imported LCB to LCBP, the step necessary for incorporation of exogenous LCB into sphingolipid. To measure the uptake and conversion of LCBs, we used the dpl1 lcb3 double deletion-containing KHY21 cells transformed with an empty vector pAK80 (control) or the same vector carrying the RSB1 gene, pAK90. ³H-Labeled SPH or DHS was added to the culture medium and incubated at 30 °C. At several time points, cells were separated from the medium by centrifugation, and lipids were extracted and separated by TLC. As shown in Fig. 3, yeast cells harboring the pAK80 vector efficiently transported both exogenous SPH (Fig. 3, A (lanes 1-4) and C (closed circles)) and DHS (Fig. 3, B (lanes 1-4) and C (open circles)) into the cells. By 20 min most of the exogenous SPH and DHS had been imported (Fig. 3C, closed and open circles). At 60 min, SPH1P and DHS1P were apparent (Fig. 3, A (lane 4) and B (lane 4)). On the other hand, cells overproducing Rsb1p (via pAK90) had much lower accumulations of exogenous SPH (Fig. 3, A (lanes 5-8) and C (closed squares)) and DHS (Fig. 3, B (lanes 5-8) and C (open squares)). These cells imported approximately 40% of the total SPH and 50% of the total DHS at 60 min, and initial transport rates of SPH and DHS were reduced by 6- and 5-fold, respectively, compared with control cells harboring the pAK80 vector (Fig. 3C). Again, SPH1P and DHS1P were detected at 60 min, although their amounts were also reduced compared with the control cells (Fig. 3, A (lane 8) and B (lane 8)).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Decrease in LCB accumulation by Rsb1p. KHY21 cells (dpl1 lcb3) harboring the pAK80 (vector only) (A and B, lanes 1-4; C, circles) or pAK90 (vector with RSB1) plasmids (A and B, lanes 5-8; C, squares) were grown in SC medium lacking uracil at 30 °C. Cells were treated with [3H]SPH (A and C, closed symbols) or [3H]DHS (B and C, open symbols), both of which had been complexed with 1 mg/ml BSA, and incubated for 0 (lanes 1 and 5), 5 (lanes 2 and 6), 20 (lanes 3 and 7), and 60 min (lanes 4 and 8). At each time point, cells were chilled on ice, washed with cold SC medium containing 1 mg/ml BSA. Lipids were extracted and separated by TLC. Radioactivity associated with imported SPH and DHS were quantified and expressed as percentages of total radioactivity in C.

To determine whether the reduced accumulation of exogenous DHS is caused by decrease in uptake or by an increase in release, we next performed an in vivo LCB release assay. The yeast cells carrying pAK80 or pAK90 were first incubated with [³H]DHS for 1 h to allow cells to accumulate DHS. Cells were then washed, resuspended in medium containing 1 mg/ml BSA, and incubated for 0, 10, and 30 min at 30 °C. At each time point, medium was separated from cells by centrifugation. Lipids were extracted from the medium and separated by TLC. Control cells harboring pAK80 released only 6% of the accumulated DHS by 10 min (Fig. 4, A (lane 2) and B (circles)). The amount of released DHS slightly decreased to 3% at 30 min (Fig. 4A, lane 3) probably because of competition by the import reaction. In contrast, cells overproducing Rsb1p released 25% of the accumulated DHS at 10 min, which was an ~4-fold increase compared with control cells (Fig. 4, A (lane 5) and B (squares)). The released DHS was slightly increased to 30% at 30 min (Fig. 4A, lane 6).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Involvement of Rsb1p in DHS release. KHY13 (dpl1)/pAK80 (vector only) (A, lanes 1-3; B, circles), KHY13/pAK90 (vector with RSB1) (A, lanes 4-6; B, squares), and KHY91 (dpl1 rsb1)/pAK80 (A, lanes 7-9; B, triangles) yeast cells were grown in SC medium lacking uracil at 30 °C. Cells were then treated with [3H]DHS complexed with 1 mg/ml BSA. After a 1-h incubation at 30 °C, cells were washed with SC medium containing 1 mg/ml BSA two times and suspended in SC medium containing 1 mg/ml BSA. Cells were then incubated for 0 (A, lanes 1, 4, and 7), 10 (lanes 2, 5, and 8), and 30 min (lanes 3, 6, and 9) at 30 °C. At each time point, medium was separated from cells by centrifugation. Lipids were extracted from the medium and separated by TLC. Radioactivities associated with the released DHS were quantified and expressed as percentages of total radioactivity in B.

To determine the phenotypic consequences of a complete loss of RSB1 function, we constructed a null mutant allele of rsb1 by replacing approximately half of RSB1 with the HIS3 gene. The rsb1 cells were viable and showed normal growth in YPD medium, indicating RSB1 function is not required for vegetative growth of the cells (data not shown). We next performed the in vivo DHS release assay using the rsb1 cells. Released DHS was reduced by 4-fold at 10 min and 10-fold at 30 min compared with those released from wild-type cells (Fig. 4, A (lanes 8 and 9) and B (triangles)). These results indicate that Rsb1p is involved in DHS release.

Characterization of Rsb1p-Dependent LCB Release-- We next examined the energy dependence of the DHS release by Rsb1p. Carbonylcyanide m-chlorophenylhydrazone (CCCP), which disrupts the membrane potential, and 2-deoxy-D-glucose and sodium azide, which deplete intracellular ATP, were used. As shown in Fig. 5, treatment of the yeast cells with CCCP had little effect (Fig. 5A, lane 4), whereas treatment with 2-deoxy-D-glucose and sodium azide inhibited the Rsb1p-dependent DHS release (Fig. 5A, lane 6). These results suggest that Rsb1p may be an ATP-dependent transporter protein.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Energy dependence of Rsb1p-dependent DHS release. KHY13 cells bearing pAK80 (vector only) plasmid (A, lanes 1, 3, and 5; B, open columns) or pAK90 (vector with RSB1) (A, lanes 2, 4, and 6; B, closed columns) were grown in SC medium lacking uracil at 30 °C. Cells were incubated with [3H]DHS complexed with 1 mg/ml BSA for 1 h at 30 °C and then treated with dimethyl sulfoxide, 50 µM CCCP, or 50 mM 2-deoxy-D-glucose and 3 mM sodium azide for 10 min at 30 °C. Cells were washed with SC medium containing 1 mg/ml BSA two times, suspended in SC medium containing 1 mg/ml BSA and respective reagents, and incubated for 10 min at 30 °C. Lipids were extracted from medium and separated by TLC. Radioactivities associated with the released DHS were quantified and expressed as percentages of total radioactivity in B.

As shown in Fig. 2, Rsb1p contains a possible Walker A ATP binding motif located at the beginning of the C-terminal tail. To investigate whether this predicted ATP-binding motif is required for Rsb1p activity, Gly-282 was mutated by site-directed mutation to a large but similarly hydrophilic residue, Trp. For detection of Rsb1p by immunoblotting, we constructed pAK170 (3×HA-RSB1⁺) and pAK186 (3×HA-rsb1(G282W)) plasmids, which express N-terminally 3×HA-tagged Rsb1p or its mutant, respectively, under the control of a TDH3 promoter. Using anti-HA antibodies, a 43-kDa protein was detected in cell lysates prepared from cells bearing pAK170 (Fig. 6A, lane 2) and pAK186 (Fig. 6A, lane 3), but not those bearing the null plasmid pAK162 (Fig. 6A, lane 1). HA-Rsb1p was functional in vivo, because cells carrying pAK170 could suppress the growth defect of KHY13 on YPD plates containing 10 µM PHS (Fig. 6B). However, KHY13 cells overproducing the mutated Rsb1(G282W) could not form a single colony on the same plate (Fig. 6B). These results suggest that the predicted ATP-binding motif is required for the Rsb1p activity.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   ATP-binding motif mutant of Rsb1p had no activity. A, KHY13 cells bearing pAK162 (vector only; lane 1), pAK170 (vector with 3×HA-RSB1+; lane 2), or pAK186 (vector with 3×HA-rsb1(G282W); lane 3) were grown in SC medium lacking uracil at 30 °C. Total proteins were separated by SDS-PAGE and detected by immunoblotting with anti-HA antibodies. The asterisk indicates nonspecific background. B, KHY13 cells bearing pAK162, pAK170, and pAK186 were grown on YPD plate containing 10 µM PHS and 0.0015% Nonidet P-40 at 30 °C.

As shown above, we directly demonstrated that Rsb1p is involved in DHS release. Because RSB1 suppressed the SPH- and PHS-sensitive phenotypes of the LCBP lyase mutant (Table I), Rsb1p may also have releasing activities effective on SPH and PHS. To investigate whether the activity of Rsb1p is restricted to D-erythro isomers of LCBs, sensitivity to L-threo-dihydrosphingosine was tested. Cells overproducing Rsb1p showed a resistance to L-threo-dihydrosphingosine (Table I), indicating that Rsb1p is not stereospecific. Next, we examined whether Rsb1p can release intracellular LCBPs. KHY21 (dpl1 lcb3) cells were used for this purpose. In some yeast backgrounds, a double deletion of DPL1 and LCB3 genes causes a lethal phenotype resulting from hyperaccumulation of intracellular LCBPs (19, 20). KHY21 (dpl1 lcb3) cells did show a severe growth defect, but were able to grow. The plasmid pAK80 (vector only) or pAK90 (vector with RSB1) was introduced into KHY21 cells, and their growth was investigated. KHY21/pAK90 cells, which overproduced Rsb1p, showed a growth rate indistinguishable from the control cells, indicating that Rsb1p cannot release intracellular LCBPs. We also examined the effects of Rsb1p on the release of hydrophobic compounds unrelated to LCBs such as aureobasidin A (an inhibitor of IPC synthase) and deoxycholic acid. As shown in Table I, overproduction of Rsb1p had no effects on sensitivities to these compounds. Thus, Rsb1p is highly specific to LCBs.

Determination of Rsb1p Localization-- Rsb1p plays a role in releasing LCBs, suggesting that it is localized to the plasma membrane. To examine whether the localization of Rsb1p is limited to the plasma membrane, we performed an immunofluorescent microscopic analysis. Cells expressing 3×HA-Rsb1p were stained by anti-HA antibodies. By confocal imaging, HA-Rsb1p was detected at several membrane structures including the plasma membrane (Fig. 7A). Double staining with antibodies against Kar2p, an ER marker, revealed that Rsb1p was partially co-localized with Kar2p (Fig. 7B). These results indicated that Rsb1p is localized to the plasma membrane, the ER, and other unknown organelles, probably the endosomes.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Immunofluorescence staining of Rsb1p. KHY128 yeast cells harboring the pAK169 plasmid (3×HA-RSB1) were grown in SC medium lacking uracil at 30 °C. The cells were fixed by formaldehyde, converted to spheroplasts, and permeabilized with 0.1% Triton X-100. They were then stained with anti-HA antibodies (A). In B, cells were double-stained with anti-HA (left panel) and anti-Kar2p (middle panel) antibodies. In the merged image (right panel), yellow indicates co-localization.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we isolated a novel gene, RSB1, as a multicopy suppressor of the toxic effects of exogenous PHS and SPH. Rsb1p, its product, is highly hydrophobic and contains seven putative transmembrane segments. Rsb1p is predicted to be a transporter, although its substrates have been unclear. Here, we demonstrated that overproduction of Rsb1p caused an apparent decrease in the uptake of exogenous SPH and DHS (Fig. 3). Further analysis showed that this decrease was, in fact, the result of an increase in DHS release (Fig. 4). Two mechanisms for the acceleration of DHS release by Rsb1p can be considered. First is the possibility that Rsb1p is a transporter protein and directly releases the imported DHS to the medium. Alternatively, Rsb1p enhances the metabolism of the exogenous DHS, which in turn increases the DHS release. Recent studies have demonstrated that exogenous DHS, once imported, is metabolized to sphingolipid through conversion to DHS1P (6-8). DHS1P is dephosphorylated by a LCBP phosphatase, Lcb3p, at the ER, and the generated DHS is converted to ceramide. Ceramide is then converted to inositol-containing sphingolipids at the Golgi apparatus and transported to the plasma membrane. Therefore, it is possible that intracellular DHS accumulated by enhanced metabolism flows to the plasma membrane and is released to the medium. However, we reject the second possibility for following reasons. First, the kinetics of the DHS release is much faster compared with its conversion to DHS1P. The accelerated DHS release caused by Rsb1p competed with the import reaction, suggesting that the DHS release was occurring even at an earlier time point. However, the conversion to DHS1P was not detected until 60 min (Fig. 3). Moreover, because we used the dpl1 lcb3 cells for the import assay, the metabolic pathway for exogenous LCB would be blocked in these cells. Therefore, it is strongly suggested that Rsb1p directly releases the imported DHS.

The yeast S. cerevisiae contains four Rsb1p homologs, Rtm1p, Yer185w, Rta1p, and Ylr046c. RTM1 and RTA1 genes were also isolated as multicopy suppressors of toxic compounds, molasses (28) and 7-aminocholesterol (29), respectively. Thus, these homologs may constitute a transporter family. Among them, Rsb1p is the largest, and the only one possessing a predicted ATP-binding motif. We demonstrated that Rsb1p-dependent DHS release was ATP-dependent, and mutation of this motif abolished its activity. We also established that Rsb1p was highly specific to LCBs, and it could not act even on the phosphorylated LCBs, LCBPs. Rsb1p released all LCBs tested (phytosphingosine, D-erythro-sphingosine, D-erythro-dihydrosphingosine, L-threo-dihydrosphingosine). Therefore, the common amino and hydroxyl groups of LCBs may be important for recognition by Rsb1p, but some variations of the long-chain moieties may be permitted. From these results, we suggest that Rsb1p releases LCBs specifically and in an ATP-dependent manner.

Yeast cells that carry a rsb1 mutation exhibited a decrease in LCB release but no other apparent unique phenotype. Therefore, the physiological function of Rsb1p remains unclear. One possibility is that Rsb1p is a drug transporter responsible for releasing LCB-like compounds. Previous microarray analysis showed that RSB1 is one of the genes regulated by Pdr1p and Pdr3p (31). Pdr1p and Pdr3p are transcription factors that control expressions of various transporter proteins such as ABC transporters. The other possibility is that Rsb1p is a flippase rather than a transporter. Because LCBs are natural components of yeast membranes and are embedded in the lipid bilayer, we favor this possibility. Lipids can move within the lipid bilayer, a process termed flip-flop, where flip means translocation toward the cytosolic membrane leaflet. Because this process is generally slow, it may be driven by proteins termed flippases in cells. In our assay for LCB release, BSA was included in the medium to prevent nonspecific binding of LCB to the cell surface. Thus, LCB "flopped" toward the outer leaflet of the plasma membrane by Rsb1p could be extracted by exogenous BSA. LCBs are regarded as signaling molecules, and their transient increase induces G₀/G₁ arrest and ubiquitin-dependent proteolysis (12, 14). Therefore, their orientation changes in lipid bilayers may be important for regulation of LCB signaling. Alternatively, it is possible that "flop" of LCBs is an important step in the de novo sphingolipid synthesis. Previous biochemical results using mouse liver suggested that sphingolipid synthesis is initiated and proceeds on the cytoplasmic side of the ER membrane until dihydroceramide synthesis begins (32). On the other hand, galactosylceramide and sphingomyelin were shown to be synthesized in the luminal side of the ER and the Golgi apparatus, respectively (33, 34). Therefore, a flop reaction is required in the sphingolipid synthesis pathway, and it is assumed that ceramide is a candidate for having a role in this step. Recently, most of the enzymes involved in the sphingolipid synthesis have been cloned (35-40). Therefore, the topology of their active sites can be re-examined by more direct methods such as antibody binding assays, fusion to a topological reporter protein, and protease site introduction/digestion experiments. Such topological analysis has been performed only for the yeast IPC synthase, Aur1p, illustrating that its active site is located on the luminal side of the Golgi apparatus (41). Recently, we also demonstrated that the active site of the LCBP phosphatase, Lcb3p, is located in the lumen of the ER,² suggesting that LCBPs are dephosphorylated at the luminal side and the resulting LCBs are released into the luminal leaflet. Because LCBs generated in this manner are then metabolized to ceramide and sphingolipids, LCBs in the luminal leaflet may be substrates for ceramide synthase. Therefore, we hypothesize that translocation of DHS (or the earlier intermediate, 3-ketodihydrosphingosine) toward the luminal side of the ER membrane is an essential process in sphingolipid synthesis. Indirect immunofluorescence microscopy demonstrated that Rsb1p is localized not only to the plasma membrane but also to the ER and other unknown organelles. Although it is possible that staining at the ER is artifact resulting from overexpression, it is possible that Rsb1p also functions in de novo sphingolipid synthesis or its regulation as one of the flippases that translocates LCBs toward the luminal side of the ER membrane. However, further analysis is required for elucidation of the precise physiological function of Rsb1p.

	ACKNOWLEDGEMENTS

We thank T. Yoshihisa (Nagoya University, Nagoya, Japan) for the anti-Kar2p antiserum and J. Inokuchi (this laboratory) for discussion.

	FOOTNOTES

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

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

Published, JBC Papers in Press, May 28, 2002, DOI 10.1074/jbc.M203385200

² A. Kihara, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: LCB, long-chain base; SPH, sphingosine; PHS, phytosphingosine; SPH1P, sphingosine 1-phosphate; PHS1P, phytosphingosine 1-phosphate; DHS, dihydrosphingosine; IPC, inositol phosphorylceramide; ER, endoplasmic reticulum; LCBP, long-chain base phosphate; SC, synthetic complete; HA, hemagglutinin; BSA, bovine serum albumin; CCCP, carbonylcyanide m-chlorophenylhydrazone; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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