The Saccharomyces cerevisiae TSC10/YBR265w Gene Encoding 3-Ketosphinganine Reductase Is Identified in a Screen for Temperature-sensitive Suppressors of the Ca2+-sensitive csg2Δ Mutant*

Saccharomyces cerevisiae csg2Δmutants accumulate the sphingolipid inositolphosphorylceramide, which renders the cells Ca2+-sensitive. Temperature-sensitive mutations that suppress the Ca2+ sensitivity ofcsg2Δ mutants were isolated and characterized to identify genes that encode sphingolipid synthesis enzymes. Thesetemperature-sensitive csg2Δ suppressors (tsc) fall into 15 complementation groups. TheTSC10/YBR265w gene was found to encode 3-ketosphinganine reductase, the enzyme that catalyzes the second step in the synthesis of phytosphingosine, the long chain base found in yeast sphingolipids. 3-Ketosphinganine reductase (Tsc10p) is essential for growth in the absence of exogenous dihydrosphingosine or phytosphingosine. Tsc10p is a member of the short chain dehydrogenase/reductase protein family. The tsc10 mutants accumulate 3-ketosphinganine and microsomal membranes isolated fromtsc10 mutants have low 3-ketosphinganine reductase activity. His6-tagged Tsc10p was expressed inEscherichia coli and isolated by nickel-nitrilotriacetic acid column chromatography. The recombinant protein catalyzes the NADPH-dependent reduction of 3-ketosphinganine. These data indicate that Tsc10p is necessary and sufficient for catalyzing the NADPH-dependent reduction of 3-ketosphinganine to dihydrosphingosine.

Sphingolipids are essential components of eukaryotic membranes. They are composed of a polar head attached to a hydrophobic ceramide base. Ceramide contains a fatty acid attached in amide linkage to a long chain base (LCB). 1 Sphingolipid synthesis begins with the formation of the long chain base dihydrosphingosine (Fig. 1), which requires two enzymes. The first enzyme, serine palmitoyltransferase, catalyzes the condensation of serine and palmitoyl-CoA to form 3-ketosphinganine. This enzyme contains at least two subunits (Lcb1p and Lcb2p) (1,2,3). The second enzyme reduces 3-ketosphinganine to dihydrosphingosine (sphinganine) which is subsequently hydroxylated on C4 by Sur2p forming phytosphingosine (4).
Phytoceramide is synthesized by the attachment of a very long chain fatty acid to the amine of the LCB. Scs7p hydroxylates the fatty acid moiety to form ␣-OH-phytoceramide (4,5). Scs7p hydroxylates the ceramide less efficiently in a sur2 null mutant (which accumulates dihydroceramide rather than phytoceramide), indicating that hydroxylation of the very long chain fatty acid occurs after formation of ceramide (4).
The CSG1 and CSG2 genes were originally identified in a screen for mutants unable to grow in medium containing 100 mM Ca 2ϩ (10). Deletion of either gene causes the accumulation of IPC-C and renders the cells Ca 2ϩ -sensitive (1,9). Mutations that decrease the synthesis of IPC-C or alter its structure suppress the Ca 2ϩ sensitivity of csg1 and csg2 mutants (1,4,5,9,11). Therefore, the csg1 and csg2 mutants provide a positive selection for sphingolipid synthesis mutants since they are suppressors of the Ca 2ϩ -sensitive phenotype.
Many of the genes that can mutate to cause suppression are expected to be essential since sphingolipids are required for viability. Thus, a screen for temperature-sensitive suppressors of Ca 2ϩ sensitivity was conducted. Suppressing mutations that confer temperature sensitivity are experimentally powerful for several reasons. 1) They provide a restrictive condition that allows the wild-type gene to be cloned by complementation; 2) analysis of sphingolipid synthesis in the mutants after shifting to the restrictive condition may uncover defects that are not apparent at the permissive temperature; 3) suppressors of the temperature sensitivity are expected to identify new genes involved in sphingolipid synthesis; and 4) finally, the suppressor mutant collection is more likely to identify genes in sphingolipid synthesis that are essential.
In this paper we report the isolation and characterization of temperature-sensitive mutants that suppress the Ca 2ϩ -sensitive phenotype of csg2 mutants and the use of tsc10 mutants to identify the gene that encodes 3-ketosphinganine reductase.
Cloning the TSC10 Gene-Strain LHYa56 (Mata ade2-101 ura3-52 trp1⌬ leu2⌬ csg2::LEU2 tsc10 -1) was transformed with a YCp50-based yeast genomic plasmid library (14) selecting for uracil prototrophs. The transformants were replica-plated to YPD and placed at 37°C, and one transformant had acquired a plasmid that complemented temperature sensitivity. Hybridization studies showed that the complementing genomic fragment resides on ATTC contiguous clone 70147. Subcloning experiments demonstrated that the YBR265W open reading frame was responsible for complementation. Linkage studies confirmed that YBR265W is the ORF that is mutated in the tsc10 mutants. A 3150base pair SalI fragment extending from a site within YBR263W to a site within YBR266C was cloned into pRS306 (13). The resulting plasmid was linearized at the MscI site in YBR265W and integratively transformed into LHYa56 (tsc10 -1). The temperature-sensitive phenotype was complemented. Diploids constructed by mating this haploid transformant to a wild-type haploid (DBY 947) were sporulated and dissected. All products of meiosis were temperature-resistant, demonstrating that the YBR265W-carrying plasmid integrated at a site linked to the tsc10 -1 locus.
Construction of the tsc10::TRP1 Null Mutant Allele-The 3150-base pair SalI fragment carrying the YBR265W ORF was cloned into pUC19 and a blunt-ended PCR fragment carrying the TRP1 gene was ligated into the MscI site in codon 252 of YBR265w/TSC10. The disrupting allele, liberated with SalI, was transformed into a diploid (formed by crossing TDY2038 with TDY2040), and tryptophan prototrophs were selected (15). Sporulation and dissection of the diploid demonstrated that the TSC10 gene is essential for viability.
Analysis of Long Chain Base-Fifty A 600 units of cells (A 600 of 0.5-1.0) were pelleted, washed in water, and resuspended by vortexing in 0.2 ml of 0.5 M NH 4 OH. LCBs were extracted by a modification of the method of Williams et al. (16). Two ml of CHCl 3 :MeOH (1:2) and glass beads (0.5 volume) were added, and the cells were vortexed for 1 min and sonicated for 15 min in a Bransonic 42 bath sonicator. The supernatant was removed to a fresh tube, and 1 ml of CHCl 3 , 2 ml of 0.5 M NH 4 OH, and 0.02 ml of 3 M KCl were added with vortexing after each addition. The bottom layer was washed twice with 2 ml of 30 mM KCl and dried under N 2 . Extract corresponding to 10 A 600 units of cells was spotted on a silica gel TLC plate, which was developed using CHCl 3 : MeOH:2 M NH 4 OH (40:10:1) as solvent (17). Phytosphingosine, dihydrosphingosine and sphingosine standards were purchased from Sigma. The 3-ketosphingosine standard was custom-synthesized by Matreya, Inc. (Pleasant Gap, PA). LCBs were visualized by spraying the plates with 0.2% ninhydrin in ethanol and heating at 180°C for 10 min.
NaBH 4 Reduction of Long Chain Bases-The long chain base extract (from 10 A 600 units of cells) or the 3-ketosphinganine standard (50 g) was dried and resuspended in 200 l of 95% ethanol. NaBH 4 (0.2 g) was added, and the sample was incubated at room temperature for 20 min. Hydrochloric acid (100 l of 0.1 N) was added followed after 5 min by NH 4 OH (100 l of 2 M), and LCBs were re-extracted as described above.
Membrane Preparation-Exponentially growing cells (750 A 600 units) were harvested and washed in water. The cells were resuspended at 2 g/ml by vortexing in 50 mM Tris (7.5), 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM ␤-mercaptoethanol (TEPM buffer). Glass beads were added to the meniscus, and the cell walls were disrupted by vortexing eight times for 30 s with cooling on ice between vortexing. The homogenate was transferred to a centrifuge tube, and the glass beads were washed with TEPM buffer until the wash was clear. The washes were pooled and centrifuged at 4000 ϫ g for 10 min. The supernatant was centrifuged at 40,000 ϫ g for 40 min. The pellet was resuspended in the TEPM buffer using a Dounce homogenizer and centrifuged at 40,000 g X for 40 min. This final pellet was resuspended in TEPM ϩ

TABLE I
The TSC mutant collection Mutants were placed in complementation groups by mating the Mat␣ tsc mutant isolates with the Mata tsc isolates. In the cases where only one representative was found, the mutant was back-crossed to wild-type and tsc haploids in the other mating type were obtained for complementation testing. The suppressors were isolated on YPD ϩ 20 mM Ca 2ϩ with (CJY isolates) or without (LHY isolates) 80 M bathocuproine disulfonate. The gene that is mutated in the isolates of each complementation is listed according to its nomenclature in the Saccharomyces Genome Database.
33% glycerol at a protein concentration of about 12 mg/ml and stored at Ϫ80°C.

Assay of Serine Palmitoyltransferase and 3-Ketosphinganine
Reductase-The reaction was initiated by the addition of 1 mg of protein and 60 mol of palmitoyl CoA to a 300-l reaction mixture containing 0.1 M Hepes (8.3), 5 mM dithiothreitol, 2.5 mM EDTA, 1 mM serine (10 Ci/ml [ 3 H]serine), 50 M pyridoxal 5Ј-phosphate (16). In some samples, an NADPH-regenerating system was added (2.4 mM glucose 6-phosphate, 2-8 units of glucose-6-phosphate dehydrogenase, 1 mM NADPH) to allow reduction of the 3-ketosphinganine intermediate. After 10 min at 37°C, the reaction was terminated by addition of NH 4 OH to 0.25 M. The LCB was extracted by successively adding 1.5 ml of CHCl 3 :MeOH (1:2), 30 g of carrier dihydrosphingosine, 1 ml of CHCl 3 , 2 ml of 0.5 M NH 4 OH with vortexing after each addition. The sample was centrifuged, and the upper aqueous layer was removed. The lower phase was washed two times with 2 ml of 30 mM KCl. The extracted LCBs were dried and run on a silica gel TLC plate developed with CHCl 3 :MeOH:2 M NH 4 OH (40:10:1).

Expression of Tsc10p in Escherichia coli-The TSC10
ORF was subcloned into the pBAD/Myc-His B plasmid (Invitrogen) resulting in arabinose-inducible expression of Tsc10p with a Myc and polyhistidine tag at the COOH terminus. A construct having the full-length Tsc10p, as well as one that lacks the COOH-terminal 38 amino acids, was made. The PCR primers used to amplify the Tsc10-containing fragments were 5Ј-GGCCCCATGGAGTTTACGTTAGAAGACCAAGTTGTG-3Ј (the NH 2 -terminal primer for both fragments), 5Ј-GGCCTCTAGATTGTT-GGCCTTCTTGCCGTCATTTTCAC-3Ј (the COOH-terminal primer for the full-length Tsc10p construct), and 5Ј-GGCCTCTAGATTCGGAAC-AAAGCGGCTTTTCTTTGCGG-3Ј (the COOH-terminal primer for the construct lacking the COOH-terminal 38 amino acids). The PCR-generated fragments were digested with NcoI and XbaI (sites shown in bold on the PCR primers) and ligated into pBAD-Myc-His-B. The underlined ATG in the NcoI site is the start codon, and codon 2 is changed from lysine to glutamate in the recombinant protein. The plasmids were transformed into TOP10 cells (Invitrogen).
Purification of the Recombinant Myc-His-tagged Tsc10 Proteins-  (25). The assignment of the ␣-helices and ␤-sheets in Tsc10p agrees with those assigned to Streptomyces 20-␤-hydroxysteroid dehydrogenase and human sepia-pterin reductase by Jornvall et al. (18). The amino acids that are conserved in all four proteins are indicated below the alignment. The lysine and the tyrosine present in the catalytic center of all the short chain dehydrogenase/reductase enzymes as well as the highly conserved Ser that is usually located 13 residues before the Tyr are indicated by asterisks. The hydrophobic tail of Tsc10p is underlined.
The transformed cells were grown in LB ϩ ampicillin to an A 600 of 0.8. Cells were pelleted and resuspended (A 600 of 4) in M10 medium with 0.5% arabinose for 2 h. Harvested cells were resuspended in 4 ml (100 A 600 /ml) of 50 mM Tris (pH 7), 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin and pepstatin A, 1 mg/ml lysozyme, and 0.1 mg/ml DNase I. After several cycles of freeze/thaw, the lysed cells were centrifuged at 10,000 ϫ g for 10 min. The supernatant was adjusted to 300 mM NaCl and 1 mM imidazole, and 2 ml of a 50% slurry of Ni-NTA agarose (Qiagen) was added for 1 h at 4°C to allow the polyhistidinetagged protein to bind. The resin was washed with 20 mM imidazole, 300 mM NaCl, 50 mM sodium phosphate (pH 7), and eluted with 250 mM imidazole, 300 mM NaCl, 50 mM sodium phosphate. The purification was followed by SDS-PAGE. The gels were either Coomassie-stained or transferred to nitrocellulose for Western blot analysis. Anti-Myc horseradish peroxidase-conjugated antibodies (Invitrogen), which recognize the Myc epitope on the recombinant proteins, were detected using the ECL system (Amersham Pharmacia Biotech).
Assay of the Recombinant Tsc10 Proteins-The activity of the recombinant Tsc10 proteins was measured in 50 mM Hepes (6.9), 133 M NADPH, 0.2% tergitol, 80 M 3-ketosphinganine at 25°C either by thin layer chromatography analysis to follow the conversion of 3-ketosphinganine to dihydrosphingosine, or by monitoring the decrease in NADPH absorbance at 340 nm.

Isolation of Mutants That Display Both Suppression of Ca 2ϩ
Sensitivity of csg2⌬ Mutants and Temperature Sensitivity-Suppressors of the Ca 2ϩ -sensitive phenotype of csg2 mutants were isolated by streaking csg2⌬ (null) mutant cells (TDY 2038 and TDY 2040) onto YPD plates containing 20 mM CaCl 2 with (the CJY mutant strains) or without (the LHY mutant strains) 80 M bathocuproine disulfonate at 26°C. Bathocuproine disulfonate is a copper chelator; it inhibits hydroxylation of IPC-C thereby increasing the Ca 2ϩ sensitivity of csg2⌬ mutant cells (9). Suppressors were tested for ability to grow on YPD plates at 37°C, a permissive condition for the starting csg2⌬ strains. The goal was to obtain mutants in which the same mutation that causes suppression of Ca 2ϩ sensitivity simultaneously confers temperature sensitivity.
In a screen of 946 suppressors, 59 were found to have a recessive temperature-sensitive phenotype. Complementation of the mutations was determined by testing the temperature sensitivity of diploids constructed by pair-wise mating of the tsc haploid mutant strains of opposite mating types. As shown in Table I, the 59 mutants fall into 15 complementation groups. These groups are named TSC for temperature-sensitive suppressors of csg2 mutants. With one exception (TSC6), tetrad analysis of members from each complementation group confirmed that the temperature-sensitive and suppressing muta- FIG. 4. The tsc10 mutants accumulate 3-ketosphinganine. LCBs were extracted from TDY2040 (csg2⌬), LHYa56 (tsc10 -1), and LHYa108 (tsc10 -2) cells grown in YPD medium at 26°C and analyzed by thin layer chromatography as described under "Experimental Procedures." A, the tsc10 mutants (lanes 7 and 8) accumulate a ninhydrinreactive lipid with the same mobility as 3-ketosphinganine (3KS). The 3-ketosphinganine standard (lane 4) is reduced by treatment with NaBH 4 to dihydrosphingosine (DHS) (lane 5). B, LCBs extracted from TDY2040 (csg2⌬), LHYa56 (tsc10 -1), and LHYa108 (tsc10 -2) were subjected to thin layer chromatography analysis before (Ϫ) or after (ϩ) treatment with NaBH 4 . The lipid that accumulates in the tsc10 mutants is reduced to a species that migrates with dihydrosphingosine (DHS) by NaBH 4 reduction. The identity of the species that migrates between phytosphingosine (PS) and dihydrosphingosine (DHS) in A, and with sphingosine (S) in B is unknown.
tions are genetically linked, indicating that suppression and temperature sensitivity arose from a single mutation (data not shown). In the case of the TSC6 complementation group (which consists of a single representative), the suppressing mutation is in the SCS7 gene (1,4,5) and the temperature-sensitive mutation is unlinked. The identities of several of the TSC genes have been determined and are listed in Table I. TSC10 Encodes 3-Ketosphinganine Reductase-The TSC10 gene was cloned by complementation of the temperature-sensitive phenotype of the LHYa56 (tsc10 -1) mutant as described under "Experimental Procedures." The complementing gene was found to reside on chromosome II between ORF YBR261c and YBR270c. Subcloning identified the complementing ORF as YBR265w. An integrating plasmid carrying the YBR265w ORF was integratively transformed into the tsc10 -1 mutant, which resulted in complementation of the temperature-sensitive phenotype. When this transformant was crossed to a wildtype (TSC10) haploid and the diploids were sporulated and dissected, all products of meiosis were temperature-resistant, indicating that YBR265w is the wild-type TSC10 gene.
Comparison of the sequence of Tsc10p with proteins in Gen-Bank identifies Tsc10p as a member of the short chain dehydrogenase/reductase protein family (Fig. 2). This family includes over 60 enzymes found in both prokaryotic and eukaryotic cells. Although these enzymes display only 15-30% sequence identity, the family is characterized by a YXXXK sequence (X ϭ any amino acid) found in the catalytic site (18). In many cases, a serine lies 13 residues amino-terminal to the conserved tyrosine. These three amino acids are believed to participate in catalysis by facilitating transfer of a proton from tyrosine to the substrate (19). In Tsc10p, the serine is at position 167, and the tyrosine and lysine residues are amino acids 180 and 184. The short chain dehydrogenase/reductase enzymes also contain a conserved sequence, GXXXGXG (amino acids 14 -20 of Tsc10p), which forms a turn between a ␤-strand and an ␣-helix that borders the NADPH-binding domain known as the Rossman fold (19,20).
There is a stretch of 28 non-charged, mostly hydrophobic amino acid residues (280 -307) close to the COOH terminus (320), which is not predicted to be part of the catalytic domain (1 to about 250) based on homology to other members of the short chain dehydrogenase/reductase family (Fig. 2). Perhaps the hydrophobic domain functions to anchor 3-ketosphingosine reductase to the endoplasmic reticulum membrane. The dilysine motif at position Ϫ3 and Ϫ4 from the COOH terminus may function to localize Tsc10p to the endoplasmic retention (21) (Fig. 2).
Since serine palmitoyltransferase deficiency suppresses the Ca 2ϩ -sensitive phenotype of csg2⌬ mutants, it was believed that a deficiency in the next enzyme in the pathway, 3-ketosphinganine reductase, might also suppress. Tsc10p, a member of the short chain dehydrogenase/reductase family of enzymes that reduce ketones to hydroxyl groups, was considered a good candidate for 3-ketosphinganine reductase. Mutants in 3-ketosphinganine reductase are predicted to be rescued by the addition of either dihydrosphingosine or phytosphingosine to the growth medium. Indeed, the temperature-sensitive phenotype of the tsc10 mutants is reversed by exogenous phytosphingosine (Fig. 3) and dihydrosphingosine (data not shown). In contrast, 3-ketosphinganine, which bypasses the requirement for serine palmitoyltransferase (22), does not rescue the tsc10 mutants. This is consistent with tsc10 mutants having a 3-ketosphinganine reductase deficiency.
The genomic TSC10 gene was disrupted and the locus marked with a TRP1 gene as described under "Experimental Procedures." The tsc10⌬ mutants are inviable in the absence of the plasmid-borne TSC10 gene unless the growth medium contains phytosphingosine or dihydrosphingosine (data not shown and Fig. 3).
Normally 3-ketosphinganine does not accumulate to levels that are detectable by ninhydrin staining of lipid extracts analyzed by thin layer chromatography. However, the tsc10 mutant strains accumulate a ninhydrin-reactive lipid that has the same mobility as 3-ketosphinganine (Fig. 4A). The sphingosine, dihydrosphingosine, and phytosphingosine standards react with ninhydrin to form a purple-colored product typical of the adduct formed with primary amines. In contrast, the 3-ketosphinganine standard and the lipid that accumulates in the tsc10 mutants forms a brown-colored product. 3-Ketosphinganine reacts with sodium borohydride to produce dihydrosphin-gosine (Fig. 4A). The ninhydrin-reactive lipid that accumulates in the tsc10 mutants is also reduced to a lipid with the same mobility as dihydrosphingosine (Fig. 4B).
3-Ketosphinganine synthesis is catalyzed by serine palmitoyltransferase. Introduction of a temperature-sensitive allele of the TSC1/LCB2 gene into a tsc10 mutant to generate a tsc1tsc10 double mutant blocks accumulation of the ninhydrinreactive lipid (data not shown). Based upon its mobility on thin layer chromatography, its color after reaction with ninhydrin, its reduction by NaBH 4 , and its dependence on serine palmitoyltransferase for accumulation, it is concluded that this lipid is 3-ketosphinganine, consistent with Tsc10p encoding 3-ketosphinganine reductase. The activity of 3-ketosphinganine reductase in microsomal membranes isolated from wild-type and tsc10 mutants was compared. Wild-type microsomes contain both serine palmitoyltransferase and 3-ketosphinganine reductase activities (16,23,24). Serine palmitoyltransferase catalyzes the formation of [ 3 H]3-ketosphinganine from palmitoyl-CoA and [ 3 H]serine (Fig. 5A). Microsomes from wild-type and tsc10 -1 mutant cells have similar serine palmitoyltransferase activity. In the presence of NADPH and wild-type microsomes, [ 3 H]3-ketosphinganine is reduced to [ 3 H]dihydrosphingosine, which is subsequently hydroxylated to [ 3 H]phytosphingosine (Fig. 5, B and D). In contrast, microsomes prepared from the tsc10 -1 mutant cells after a 3-h shift to 37°C display no 3-ketosphinganine reductase activity (Fig. 5D). Even microsomes prepared from the tsc10 -1 mutant cells maintained at 26°C display reduced 3-ketosphinganine reductase activity (Fig. 5B). This experiment demonstrates that Tsc10p is required for 3-ketosphinganine reductase activity.
To confirm that Tsc10p catalyzes 3-ketosphinganine reduction, Tsc10p containing a Myc epitope and a His 6 domain fused at its COOH terminus was expressed in E. coli under control of the arabinose-inducible pBAD promoter, isolated using Ni- FIG. 7. Recombinant Tsc10p catalyzes the 3-ketosphinganinedependent oxidation of NADPH. A, partially purified recombinant truncated Tsc10-Myc-His 6 protein (10 g) was added to a cuvette containing varying amounts of 3-ketosphinganine (as indicated) in 750 l of the standard reaction buffer (see "Experimental Procedures") and the oxidation of NADPH was monitored by measuring the decreased absorbance at 340 nm. B, varying amounts of the partially purified recombinant truncated Tsc10-Myc-His 6 protein were added to the standard reaction containing 80 M 3-ketosphinganine and the reaction was measured as in A.

FIG. 8. Recombinant
Tsc10p catalyzes the NADPH-dependent reduction of 3-ketosphinganine to dihydrosphingosine. The reaction conditions are similar to those described for Fig. 7 except tergitol was omitted. The reaction was monitored by following the decrease in absorbance of NADPH at 340 nm (A). At the indicated times (0, 15 min, etc.), 0.5-ml aliquots were removed and long chain base was extracted and analyzed by thin layer chromatography (see "Experimental Procedures") along with 3-ketosphinganine (3KS) and dihydrosphingosine (DHS) standards (B). NTA chromatography, and assayed for reductase activity. Two constructs were made; in one, the Myc and polyhistidine tags followed the last amino acid of the full-length protein. In the other, the carboxyl-terminal 38 amino acids (containing the potential membrane anchoring domain) of Tsc10p were omitted. As shown in Fig. 6, both tagged proteins were partially purified by Ni-NTA chromatography.
The full-length and the carboxy-truncated purified proteins catalyze a 3-ketosphinganine-dependent oxidation of NADPH to NADP (Fig. 7). NADH does not substitute for NADPH in the reduction. The long chain bases extracted from aliquots of the reaction were analyzed by thin layer chromatography. 3-Ketosphinganine is converted to dihydrosphingosine (Fig. 8B) in a reaction that depends on the addition of NADPH confirming that the recombinant protein is 3-ketosphinganine reductase. The reaction is stimulated 10-fold by 0.2% tergitol, but in this reaction (Fig. 8) detergents were omitted since they interfere with the long chain base extraction and the separation on thin layer chromatography. These experiments also demonstrate that the carboxyl-terminal 38 amino acids are not required for enzymatic activity.

DISCUSSION
Mutations that decrease the rate of IPC-C synthesis or alter its structure suppress the Ca 2ϩ -sensitive phenotype of csg2⌬ mutants (1,4,5,9,11). Since IPC is essential for growth, it was expected that a temperature-sensitive suppressor collection would identify essential genes required for IPC synthesis. This expectation is confirmed by the finding that temperature-sensitive alleles of the serine palmitoyltransferase genes (TSC1 and TSC2) are among the most represented members of the collection.
The TSC10 gene was cloned, and its sequence identified it as a member of the short chain reductase/dehydrogenase enzyme family. The results presented in this report demonstrate that Tsc10p is the enzyme that converts 3-ketosphinganine to dihydrosphinganine. Although the enzymes in this family show low sequence identity and varied substrates, their well conserved secondary structures show they possess a single domain comprising the cofactor-binding and active sites. Tsc10p has a stretch of 28 hydrophobic amino acids (residues 280 -307) at its carboxyl terminus, which could anchor the protein to the membrane. There are 13 additional amino acids including a KK motif located at position Ϫ3 and Ϫ4 from the end of the protein.
In some cases, dilysine motifs at the carboxyl terminus specify retention of the protein within the endoplasmic reticulum (21). Tsc10p, which fractionates with the microsomes, is expected to be an endoplasmic reticulum protein since that is the location of IPC synthesis. These amino acids are not required for enzymatic activity.
Based on our analysis of the TSC genes, it is expected that several other genes required for sphingolipid synthesis will be identified from the continued study of the temperature-sensitive suppressors. Many of the complementation groups are represented by a single mutant isolate, making it clear that the collection is far from saturated. This work promises to provide new information about the enzymes required for sphingolipid synthesis, regulation of the biosynthetic pathway, and functions of sphingolipids.