The BST1 Gene of Saccharomyces cerevisiaeIs the Sphingosine-1-phosphate Lyase

Sphingolipids elicit a wide variety of eukaryotic cellular responses, most involving regulation of cell growth, differentiation, and apoptosis. Sphingosine 1-phosphate, a sphingolipid catabolite, is mitogenic in fibroblasts and inhibits the chemotactic mobility and invasiveness of human tumor cells. Sphingosine 1-phosphate degradation requires cleavage at the C2–3 carbon bond by sphingosine phosphate lyase. A yeast genetic approach was used to clone the first sphingosine phosphate lyase gene, BST1. BST1 overexpression conferred resistance to sphingosine in yeast.BST1 deletion produced sensitivity to exogenousd-erythro-sphingosine and phytosphingosine and intracellular accumulation of sphingosine 1-phosphate upon exposure to exogenous sphingosine. This study confirms that sphingoid base metabolism is similar in all eukaryotes and suggests that yeast genetics may be useful in the isolation and identification of other genes involved in sphingolipid signaling and metabolism. Sphingolipids elicit a wide variety of eukaryotic cellular responses, most involving regulation of cell growth, differentiation, and apoptosis. Sphingosine 1-phosphate, a sphingolipid catabolite, is mitogenic in fibroblasts and inhibits the chemotactic mobility and invasiveness of human tumor cells. Sphingosine 1-phosphate degradation requires cleavage at the C2–3 carbon bond by sphingosine phosphate lyase. A yeast genetic approach was used to clone the first sphingosine phosphate lyase gene, BST1. BST1 overexpression conferred resistance to sphingosine in yeast.BST1 deletion produced sensitivity to exogenousd-erythro-sphingosine and phytosphingosine and intracellular accumulation of sphingosine 1-phosphate upon exposure to exogenous sphingosine. This study confirms that sphingoid base metabolism is similar in all eukaryotes and suggests that yeast genetics may be useful in the isolation and identification of other genes involved in sphingolipid signaling and metabolism. Sphingolipids and their metabolic derivatives elicit a wide variety of eukaryotic cellular responses (1Saba J. Obeid L. Hannun Y. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1996; 351: 233-244Crossref PubMed Scopus (46) Google Scholar, 2Speigel S. Merrill A. FASEB. 1996; 10: 1388-1397Crossref Scopus (650) Google Scholar). Although the stimuli and biological end points differ in each cell type, the role of sphingolipid by-products as second messengers in specific, growth regulatory signal transduction pathways appears to be a universal theme among eukaryotic cells (3Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar). Sphingosine and sphingosine 1-phosphate (S-1-P) 1The abbreviation used is: S-1-P, sphingosine 1-phosphate. are both catabolites of sphingolipid breakdown, which have been shown to modulate DNA synthesis and cellular proliferation in mammalian cells (4Olivera A. Spiegel S. Nature. 1993; 365: 557-559Crossref PubMed Scopus (818) Google Scholar). Evidence suggests that S-1-P is largely responsible for these effects. In addition, S-1-P has recently been shown to inhibit the growth, motility, and invasiveness of tumor cells (5Sadahira Y. Ruan F. Hakomori S. Igarashi Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9686-9690Crossref PubMed Scopus (237) Google Scholar, 6Spiegel S. Olivera A. Zhang H. Thompson E. Su Y. Berger A. Breast Cancer Res. Treat. 1994; 31: 337-348Crossref PubMed Scopus (41) Google Scholar). Free sphingosine and S-1-P are maintained at very low levels in mammalian cells (7Merrill A. Wang E. Mullins R. Jamison W. Nimkar S. Liotta D. Anal. Biochem. 1988; 171: 373-381Crossref PubMed Scopus (305) Google Scholar), consistent with the notion that potent second messengers would be tightly regulated in the absence of a particular stimulus. The mechanism(s) by which the intracellular levels of sphingosine and S-1-P are regulated have not been established. Such control could occur at the synthetic stage, via regulation of the activities of ceramidases and sphingosine kinase (8Buehrer B. Bell R. Adv. Lipid Res. 1993; 26: 59-67PubMed Google Scholar). Alternatively, control could occur at the catabolic stage, through regulation of the activity of sphingosine phosphate lyase (9Veldhoven P.V. Mannaerts G. Adv. Lipid Res. 1993; 26: 69-98Google Scholar). Sphingolipids exist in yeast where they provide vital yet unknown functions (10Wells G. Lester R. J. Biol. Chem. 1983; 258: 10200-10203Abstract Full Text PDF PubMed Google Scholar). In this study, we cloned the sphingosine phosphate lyase gene through its ability to suppress sphingosine-induced growth suppression when present on a high-copy vector in Saccharomyces cerevisiae. The following yeast strains were used: SGP3 (leu2–3,112 trp1 ura3–52 his3 ade8 ras1::HIS3), JS14 (SGP3 pRS202-BST1); JS16 (SGP3 bst1Δ::NEO); JS29 (SGP3bst1Δ::NEO pYES2-BST1); Jk93d (ura3–52 leu2–3,112 his4 trp1 rme1). The pRS202 library used in all transformations was constructed by Connelly and Heiter. 2C. Connelly and P. Hieter, unpublished data. The vector is a modified version of the pRS306 vector, into which a 2-micron plasmid piece was inserted. Inserts from this library are approximately 6–8 kilobases in length. Wild type yeast were transformed with the high-copy library, selected for uracil prototrophy, and transformants were pooled and replated at a concentration of 106 cells/plate onto 1 mmd-erythro-sphingosine plates. Six transformants which grew large colonies on 1 mmd-erythro-sphingosine plates were grown in selective medium, and control SGP3 colonies were grown in minimal medium, at 30 °C until saturated. Absorbance at 660 nm was used to correct for small variations in cell concentration between cultures. Serial dilutions were performed, and cells were template-inoculated onto 1 mmd-erythro-sphingosine plates, incubated at 30 °C for 48 h. The creation of genomicbst1Δ alleles was performed by replacing BST1with kanMX, which confers resistance to G418. The kanMX marker replaced all sequence between nucleotide 396 and 1770 of the 1770-base pair open reading frame, creating a 1373-base pair deletion. Disruption was confirmed using polymerase chain reaction amplification of genomic DNA from G418-resistant clones, using primers to genomic sequence just 5′ and 3′ to the region replaced by the disruption. Uptake and metabolism studies were performed using C3-3Hsphingosine (NEN Life Science Products). Specific activity of sphingosine was 22 Ci/mmol, 0.1 mCi/ml. Cells were incubated in a volume of 5 ml for varying times with sphingosine at a final concentration of 1 μm, final specific activity of 0.4 mCi/μmol and cell density of 5 × 107 cells/ml. Cells were harvested, washed in sterile water, and subjected to Bligh-Dyer extraction (14Bligh E. Dyer W. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar). Radioactivity present in the aqueous (upper) phase of the Bligh-Dyer extraction of whole cells was determined using a standard scintillation counter. TLC conditions for initial evaluation of sphingosine recovery were butanol:acetic acid:water (3:1:1). Identification of the unknown lipid as sphingosine 1-phosphate was performed under the following conditions, with the resulting R F values: butanol:water:acetic acid (3:1:1), 0.47; chloroform:methanol:water (60:35:8), 0.22; chloroform:methanol:water:acetic acid (30:30:2:5), 0.33. Sphingosine phosphate lyase activity was determined essentially as described (11Veldhoven P.V. Mannaerts G. J. Biol. Chem. 1991; 266: 12502-12507Abstract Full Text PDF Google Scholar), using unlabeledd-erythro-dihydrosphingosine-1-phosphate (Biomol, Plymouth Meeting, PA) andd-erythro-dihydrosphingosine 4,5-3H1-phosphate (American Radiolabeled Chemicals, Inc., St. Louis, MO). Specific activity was 100 mCi/mmol. We had previously observed that long chain sphingoid bases inhibit proliferation of S. cerevisiae cells in liquid culture. To determine whether this inhibition could be employed in a selection scheme, 1 × 106 exponentially growing yeast cells were inoculated onto 1 mmd-erythro-sphingosine plates and incubated for several days. Under these conditions growth was completely inhibited, whereas no growth inhibition occurred with eitherl-erythro-sphingosine or stearylamine. The stereospecificity of sphingosine-induced growth inhibition indicated interaction with a biological target or receptor. Our strategy was to transform wild type yeast cells with a yeast genomic library carried on the pRS202 high-copy shuttle vector (12Sikorski R. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) containing a selectable nutritional marker (URA3) and then to subject pooled transformants tod-erythro-sphingosine selection. Utilizing this approach, we expected to identify S. cerevisiae genes involved in sphingolipid signal transduction and/or metabolism. Numerous clones which demonstrated between 100- and 1000-fold greater resistance to sphingosine than the parent strain were identified (Fig.1 A). The most highly represented insert, 13-1, was subcloned and sequenced identifying a novel gene on S. cerevisiae chromosome IV, which we namedBST1 (bestower of sphingosinetolerance) (GenBank™ accession number U51031; S. cerevisiae genome data base accession number YDR294C). TheBST1 nucleotide sequence encodes a predicted protein of 65,523 kilodaltons and 589 amino acids in length. This sequence is 23% identical to gadA and gadB, two nearly identicalE. coli genes encoding glutamate decarboxylase, a pyridoxal-5′-phosphate-dependent enzyme that catalyzes synthesis of the neurotransmitter γ-amino butyric acid. To explore the function of BST1, a deletion strain was created through homologous recombination using aNEO-selectable marker (13Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2241) Google Scholar). Whereas our original selection was performed in the SGP3 yeast strain due to its high transformation efficiency, we were concerned that the deletion of the RAS1gene in this strain might complicate phenotypic analysis ofBST1 deletion. Therefore, deletion of BST1 and all subsequent biological studies were performed in both SGP3 and another strain, JK93d. Heterozygous diploids were sporulated and spores segregated 2:2 for G418 resistance. Both G418-resistant and-sensitive progeny were viable, indicating that BST1 is not an essential gene. Despite weak nucleotide sequence homology between BST1 andgadA/gadB, analysis of glutamate decarboxylase activity in cytosolic extracts from wild type, BST1 overexpression andbst1Δ strains refuted the notion that BST1encodes the S. cerevisiae homologue of glutamate decarboxylase (data not shown). However, deletion of BST1was associated with another interesting phenotype, severe sensitivity to d-erythro-sphingosine (Fig. 1 B). Concentrations as low as 10 μm sphingosine completely inhibited growth of bst1Δ strains but had no effect on the viability of wild type cells. In comparison with the control strain, the bst1Δ strain also demonstrated greater sensitivity to 100 μm phytosphingosine, the long chain base endogenous to S. cerevisiae (Fig. 1 C). We did not appreciate a noticeable difference between the growth of wild type andBST1 overexpression strains on phytosphingosine, which is only minimally toxic to wild type cells at this concentration. To determine whether differences in sphingosine uptake or metabolism were responsible for these sensitivity differences, BST1wild type, overexpression and bst1Δ strains were exposed to 3H-labeled sphingosine and subjected to Bligh-Dyer extractions (14Bligh E. Dyer W. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar). There were no major differences in sphingosine recovery among the three strains. However, the aqueous phase from thebst1Δ strain contained a 10-fold increase in radioactivity over that of control and BST1 overexpression strains (Fig.2 A). Thin layer chromatography (TLC) analysis of the lipid fractions revealed a sphingosine band, which appeared equivalent in each strain. Interestingly, we observed the presence of another radioactive compound, distinct from sphingosine, in the extracts from the bst1Δ strain, but not in the wild type or BST1 overexpression strains (Fig.2 B). This compound accumulated rapidly, reaching a plateau by 60 min (Fig. 2 C). The presence of significant radioactivity in the aqueous phase and an unknown compound in the organic phase of bst1Δ strain extracts led us to consider whether the unknown lipid might be S-1-P, well known for its unusual solubility properties. We first confirmed that at neutral pH and ambient temperature, S-1-P partitions 65% in aqueous and 35% in organic phases of a (chloroform:methanol:water 1:1:0.9) mixture. We then evaluated the unknown lipid and an S-1-P standard under three separate TLC conditions and found the two compounds to comigrate in each case, confirming the lipid to be S-1-P. Hyperaccumulation of S-1-P and hypersensitivity tod-erythro-sphingosine could be explained by a failure to metabolize S-1-P. Degradation of S-1-P requires cleavage at the C2–3 carbon bond by a pyridoxal-5′-phosphate-dependent enzyme, sphingosine phosphate lyase. The enzyme is stereospecific for thed-erythro sphingoid base, but demonstrates less specificity toward the type of sphingoid base. It was, therefore, possible that we had isolated sphingosine phosphate lyase through a screen employing a nonendogenous but stereospecifically acceptable sphingolipid. Lyase activity in BST1 wild type, overexpression, and deletion strains correlated with BST1expression (Fig. 3, A andB), confirming BST1 to be the yeast homologue of sphingosine phosphate lyase. Cloning of the first sphingosine phosphate lyase gene from S. cerevisiae indicates that phosphorylated sphingoid base signaling is conserved throughout evolution, and thus isolation of other genes responsible for sphingolipid metabolism, transport and signaling should be possible in yeast. Our findings indicate that sphingosine phosphate lyase catalyzes a rate-limiting step in sphingolipid catabolism, and regulation of intracellular S-1-P levels should be possible by regulating lyase activity. Further analysis of the bst1Δstrain may provide insights into the role(s) of sphingosine 1-phosphate in eukaryotic cell signaling, and knowledge of the yeast sphingosine phosphate lyase sequence should provide a means for the isolation of the sphingosine phosphate lyase genes from higher eukaryotes. We extend thanks to Joseph Heitman and Babak Oskouian for many helpful discussions, Alexander Lucas for careful review of the manuscript, and Shirley Phelps for expert secretarial assistance.