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J Biol Chem, Vol. 273, Issue 46, 30688-30694, November 13, 1998
Mutant*
,From the Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Saccharomyces cerevisiae csg2 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).
mutants accumulate the sphingolipid inositolphosphorylceramide, which
renders the cells Ca2+-sensitive. Temperature-sensitive
mutations that suppress the Ca2+ sensitivity of
csg2 mutants were isolated and characterized to identify
genes that encode sphingolipid synthesis enzymes. These
temperature-sensitive csg2
suppressors (tsc) fall into 15 complementation groups. The
TSC10/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 from
tsc10 mutants have low 3-ketosphinganine reductase
activity. His6-tagged Tsc10p was expressed in
Escherichia 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.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (17K):
[in a new window]
Fig. 1.
Synthesis of mannosylinositol
phosphorylceramide from acetyl CoA.
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).
Aur1p catalyzes the transfer of the phosphoinositol head of phosphatidylinositol to OH-phytoceramide to form inositol- phosphorylceramide (IPC-C) in the endoplasmic reticulum (6). In the Golgi, IPC is mannosylated to form mannosylinositol- phosphorylceramide (7, 8). This reaction requires two genes, CSG1 and CSG2 (1, 9).
The CSG1 and CSG2 genes were originally identified in a screen for mutants unable to grow in medium containing 100 mM Ca2+ (10). Deletion of either gene causes the accumulation of IPC-C and renders the cells Ca2+-sensitive (1, 9). Mutations that decrease the synthesis of IPC-C or alter its structure suppress the Ca2+ 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 Ca2+-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 Ca2+ 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
Ca2+-sensitive phenotype of csg2 mutants and the
use of tsc10 mutants to identify the gene that encodes
3-ketosphinganine reductase.
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EXPERIMENTAL PROCEDURES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Yeast Strains and Growth--
The yeast strains used in this
study were TDY2037 (Mat lys2 ura3-52 trp1 leu2),
TDY2038 (Mat lys2 ura3-52 trp1 leu2 csg2::LEU2), TDY2039 (Mata ade2-101
ura3-52 trp1 leu2), TDY2040 (Mata
ade2-101 ura3-52 trp1 leu2 csg2::LEU2), and DBY947 (Mat ade2-101 ura3-52). Media were prepared
and cells were grown using standard procedures (12). Phytosphingosine and dihydrosphingosine were purchased from Sigma and added from a 50 mg/ml stock solution (in ethanol) to autoclaved media containing 0.2% tergitol.
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 3150-base 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 A600 units of cells (A600 of 0.5-1.0) were pelleted, washed in water, and resuspended by vortexing in 0.2 ml of 0.5 M NH4OH. LCBs were extracted by a modification of the method of Williams et al. (16). Two ml of CHCl3: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 CHCl3, 2 ml of 0.5 M NH4OH, 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 N2. Extract corresponding to 10 A600 units of cells was spotted on a silica gel TLC plate, which was developed using CHCl3:MeOH:2 M NH4OH (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.
NaBH4 Reduction of Long Chain Bases-- The long chain base extract (from 10 A600 units of cells) or the 3-ketosphinganine standard (50 µg) was dried and resuspended in 200 µl of 95% ethanol. NaBH4 (0.2 g) was added, and the sample was incubated at room temperature for 20 min. Hydrochloric acid (100 µl of 0.1 N) was added followed after 5 min by NH4OH (100 µl of 2 M), and LCBs were re-extracted as described above.
Membrane Preparation--
Exponentially growing cells (750 A600 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 × g for 10 min. The supernatant was centrifuged at
40,000 × 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 + 33% glycerol at a protein concentration of about 12 mg/ml and stored
at 
80 °C.
Assay of Serine Palmitoyltransferase and 3-Ketosphinganine Reductase-- The reaction was initiated by the addition of 1 mg of protein and 60 µmol of palmitoyl CoA to a 300-µl reaction mixture containing 0.1 M Hepes (8.3), 5 mM dithiothreitol, 2.5 mM EDTA, 1 mM serine (10 µCi/ml [3H]serine), 50 µM pyridoxal 5'-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 NH4OH to 0.25 M. The LCB was extracted by successively adding 1.5 ml of CHCl3:MeOH (1:2), 30 µg of carrier dihydrosphingosine, 1 ml of CHCl3, 2 ml of 0.5 M NH4OH 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 CHCl3:MeOH:2 M NH4OH (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 5'-GGCCCCATGGAGTTTACGTTAGAAGACCAAGTTGTG-3' (the NH2-terminal primer for both fragments), 5'-GGCCTCTAGATTGTTGGCCTTCTTGCCGTCATTTTCAC-3' (the COOH-terminal primer for the full-length Tsc10p construct), and 5'-GGCCTCTAGATTCGGAACAAAGCGGCTTTTCTTTGCGG-3' (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-- The transformed cells were grown in LB + ampicillin to an A600 of 0.8. Cells were pelleted and resuspended (A600 of 4) in M10 medium with 0.5% arabinose for 2 h. Harvested cells were resuspended in 4 ml (100 A600/ml) of 50 mM Tris (pH 7), 1 mM phenylmethylsulfonyl fluoride, 10 µg/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 × 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 polyhistidine-tagged 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 µM NADPH, 0.2% tergitol, 80 µM
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.
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Isolation of Mutants That Display Both Suppression of
Ca2+ Sensitivity of csg2 Mutants and Temperature
Sensitivity--
Suppressors of the Ca2+-sensitive
phenotype of csg2 mutants were isolated by streaking
csg2 (null) mutant cells (TDY 2038 and TDY 2040) onto YPD
plates containing 20 mM CaCl2 with (the CJY mutant strains) or without (the LHY mutant strains) 80 µM
bathocuproine disulfonate at 26 °C. Bathocuproine disulfonate is a
copper chelator; it inhibits hydroxylation of IPC-C thereby increasing
the Ca2+ 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 Ca2+ 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 mutations 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.
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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 wild-type (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 GenBank
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 = 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).
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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 3 and 4 from the COOH terminus may
function to localize Tsc10p to the endoplasmic retention (21) (Fig.
2).
Since serine palmitoyltransferase deficiency suppresses the
Ca2+-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.
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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 dihydrosphingosine (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).
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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 ninhydrin-reactive lipid (data not shown). Based upon its mobility on thin layer chromatography, its color after reaction with ninhydrin, its reduction by NaBH4, 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 [3H]3-ketosphinganine from palmitoyl-CoA and [3H]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, [3H]3-ketosphinganine is reduced to [3H]dihydrosphingosine, which is subsequently hydroxylated to [3H]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.
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To confirm that Tsc10p catalyzes 3-ketosphinganine reduction, Tsc10p containing a Myc epitope and a His6 domain fused at its COOH terminus was expressed in E. coli under control of the arabinose-inducible pBAD promoter, isolated using Ni-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.
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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.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Mutations that decrease the rate of IPC-C synthesis or alter its
structure suppress the Ca2+-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 3 and 4 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.
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ACKNOWLEDGEMENT |
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We thank Michael Seidel (Matreya Lipids and Biochemicals, Pleasant Gap, PA) for the synthesis of the 3-ketosphinganine.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 51891 and Uniformed Services University of the Health Sciences Grants CO71Cw and CO71DC.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.
This manuscript is dedicated to the memory of Troy J. Beeler.
Deceased June 9, 1998.
The abbreviations used are:
LCB, long chain
base; IPC, inositol- phosphorylceramide; ORF, open reading frame; NTA<
nitrilotriacetic acid, PCR, polymerase chain reaction; IPC-C, inositolphosphoryl(-hydroxy)phytoceramide.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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S. Wullschleger, R. Loewith, W. Oppliger, and M. N. Hall Molecular Organization of Target of Rapamycin Complex 2 J. Biol. Chem., September 2, 2005; 280(35): 30697 - 30704. [Abstract] [Full Text] [PDF]
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 J. P. Miller, R. S. Lo, A. Ben-Hur, C. Desmarais, I. Stagljar, W. S. Noble, and S. Fields Large-scale identification of yeast integral membrane protein interactions PNAS, August 23, 2005; 102(34): 12123 - 12128. [Abstract] [Full Text] [PDF]
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T. Kobayashi, H. Takematsu, T. Yamaji, S. Hiramoto, and Y. Kozutsumi Disturbance of Sphingolipid Biosynthesis Abrogates the Signaling of Mss4, Phosphatidylinositol-4-phosphate 5-Kinase, in Yeast J. Biol. Chem., May 6, 2005; 280(18): 18087 - 18094. [Abstract] [Full Text] [PDF]
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A. Kihara and Y. Igarashi FVT-1 Is a Mammalian 3-Ketodihydrosphingosine Reductase with an Active Site That Faces the Cytosolic Side of the Endoplasmic Reticulum Membrane J. Biol. Chem., November 19, 2004; 279(47): 49243 - 49250. [Abstract] [Full Text] [PDF]
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Q. Lisman, D. Urli-Stam, and J. C. M. Holthuis HOR7, a Multicopy Suppressor of the Ca2+-induced Growth Defect in Sphingolipid Mannosyltransferase-deficient Yeast J. Biol. Chem., August 27, 2004; 279(35): 36390 - 36396. [Abstract] [Full Text] [PDF]
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K. Baetz, L. McHardy, K. Gable, T. Tarling, D. Reberioux, J. Bryan, R. J. Andersen, T. Dunn, P. Hieter, and M. Roberge Yeast genome-wide drug-induced haploinsufficiency screen to determine drug mode of action PNAS, March 30, 2004; 101(13): 4525 - 4530. [Abstract] [Full Text] [PDF]
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 K. Gable, S. Garton, J. A. Napier, and T. M. Dunn Functional characterization of the Arabidopsis thaliana orthologue of Tsc13p, the enoyl reductase of the yeast microsomal fatty acid elongating system J. Exp. Bot., February 1, 2004; 55(396): 543 - 545. [Abstract] [Full Text] [PDF]
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S. Uemura, A. Kihara, J.-i. Inokuchi, and Y. Igarashi Csg1p and Newly Identified Csh1p Function in Mannosylinositol Phosphorylceramide Synthesis by Interacting with Csg2p J. Biol. Chem., November 14, 2003; 278(46): 45049 - 45055. [Abstract] [Full Text] [PDF]
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 A. K.A. deHart, J. D. Schnell, D. A. Allen, J.-Y. Tsai, and L. Hicke Receptor Internalization in Yeast Requires the Tor2-Rho1 Signaling Pathway Mol. Biol. Cell, November 1, 2003; 14(11): 4676 - 4684. [Abstract] [Full Text] [PDF]
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)
and LHYa56 (tsc10-1)(
) cells were grown in YPD at
26 °C (A and B) or at 26 °C and then
shifted to 37 °C for 3 h (C and D) prior
to membrane preparation. Following a 10-min incubation, the LCBs were
extracted and separated by thin layer chromatography. Each lane of the
TLC was divided into 20 equal fractions, and the silica gel was removed
and counted. The position of migration of the 3-ketosphinganine
(3KS), dihydrosphinganine (DHS), or
phytosphingosine (PS) standards is indicated.
