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J Biol Chem, Vol. 275, Issue 11, 7597-7603, March 17, 2000
From the Department of Biochemistry and Molecular Biology,
Uniformed Services University of the Health Sciences,
Bethesda, Maryland 20814
Serine palmitoyltransferase catalyzes the first
step of sphingolipid synthesis, condensation of serine and palmitoyl
CoA to form the long chain base 3-ketosphinganine. The
LCB1/TSC2 and LCB2/TSC1 genes encode homologous
proteins of the Serine palmitoyltransferase (EC 2.3.1.50)
(SPT)1 catalyzes the
formation of 3-ketosphinganine from serine and palmitoyl CoA. This is
the first committed step in the synthesis of ceramides and
sphingolipids. This step is also believed to be rate-limiting, making
it likely that regulation of SPT controls the rate of sphingolipid synthesis. For example, treatment of mammalian cells with sphingosine results in down-regulation of SPT activity (1). However, little is
known about how the enzyme is regulated.
Two genes, LCB1/TSC2 and LCB2/TSC1, are required
for SPT activity (2-4). Both genes encode proteins that belong to a
small subfamily of pyridoxal 5'-phosphate-dependent enzymes
that catalyze the condensation of an amino acid and a carboxylic acid
CoA thioester with concomitant decarboxylation of the amino acid. This
SPT also differs from the other members of this enzyme family because
it is membrane-associated. SPT appears to be located on the cytoplasmic
side of the endoplasmic reticulum (9). In comparison to AONS, Lcb1p and
Lcb2p each have amino-terminal extensions that contain potential
membrane-spanning segments. However, it is not known whether these
hydrophobic segments are important for membrane association.
Attempts to increase SPT activity by over-expression of LCB1
and LCB2 have met with limited success (3), raising the
possibility that additional proteins may be required for SPT activity.
Many genes involved in sphingolipid synthesis have been identified through the characterization of suppressors of the Ca2+
sensitivity of csg2 Yeast Strains and Growth--
The yeast strains used in this
study are listed in Table I. The lcb1 Cloning TSC3--
The CJYa16 (tsc3-2) mutant cells
were transformed with a YCp50-based genomic library (17), and
transformants that regained the ability to grow at 37 °C were
selected. Plasmids were recovered from six temperature-resistant
transformants and the genomic fragments on the plasmids were sequenced.
The complementing sequences all contained an overlapping region of
chromosome 2 including the amino terminus of YBR058c, YBR059c, the
carboxyl terminus of YBR060c, and two additional small ORFs between
059c and 058c with the potential to encode an 80-amino acid protein and
an 84-amino acid protein (Fig.
1A).
Subcloning to Identify the tsc3-Complementing ORF and Sequencing
the tsc3 Mutant Alleles--
Subclones were constructed by inserting
the indicated restriction fragments into pRS316 (18) (Fig.
1A). The two potential small ORFs from this region (labeled
ORFa and ORFb in Fig. 1A) were amplified by PCR and cloned
into pRS316. For the 80-amino acid ORFa, a 380-bp
XhoI/BamHI-ended fragment was generated using primers 5'-GGCCCTCGAGGCTCGCAATTTGACAGAA and
5'-GGCCGGATCCTTGCCTCCAGCTTATACTA. For the 84-amino acid
ORFb, a 460-bp PstI/BamHI-ended fragment was
generated using primers 5'-GGCCCTGCAGTAGTATTTAGTATGCCTTC
and 5'-GGCCGGATCCGTAGTGCATCCAGTAGTGG. The subclones were
tested for the ability to complement the tsc3-2 mutant (Fig.
1A). Genomic DNA was isolated from each of the five mutants
in the TSC3 complementation group and used as the template for PCR
amplification of the mutant alleles using the primers for ORFa. The
fragments were sequenced.
Disruption of TSC3, LCB1, and LCB2--
The coding sequence of
TSC3 was deleted and substituted with a TRP1
marker gene. This was accomplished by generating two restriction fragments that contained the flanking sequences of TSC3 by
PCR. The fragment containing the upstream flanking sequence was
KpnI/XhoI-ended and extended from about 200 bp
before the start codon to 30 bp past the start codon, and the
downstream flanking fragment was XhoI/EcoRI-ended
and extended from about 40 bp before the stop codon to 300 bp past the
stop codon. These fragments were ligated together and inserted between
the KpnI and EcoRI sites of pUC19, yielding a
plasmid having a XhoI site replacing most of the TSC3 coding
sequence. A PCR-generated SalI-ended TRP1 fragment was ligated into the XhoI site, and the disrupting allele was
liberated by digestion with KpnI and EcoRI. The
disrupting allele of the LCB1 gene was constructed by
replacing the 708-bp MscI fragment (internal to the
LCB1 gene) with a PCR-generated TRP1 marker gene in a pUC19-based plasmid containing a 2050-bp ClaI to
HindIII fragment that includes the entire LCB1
gene. Construction of the disrupting allele of LCB2 has been
described (2).
Preparation of Microsomes--
Cells in early exponential growth
were pelleted, washed with H2O, and resuspended at 2 ml/g
wet cell weight in a buffer composed of 50 mM Tris, 7.5, 1 mM EGTA, 1 mM Assay of Serine Palmitoyltransferase Activity--
SPT activity
was assayed in a 300-µl volume containing 50 mM HEPES,
8.3, 50 mM pyridoxal phosphate, 2 mM serine (20 µCi/ml) 0.2 mM palmitoyl CoA (or as indicated in the
legend to Fig. 3), 1 mM NADPH, 2.4 mM
glucose-6-phosphate, and 10 units of glucose-6-phosphate dehydrogenase.
The reaction was initiated by adding 0.5-1 mg of microsomal protein,
and after 10 min at 37 °C, it was terminated by the addition of 100 µl of 2 M NH4OH and 0.75 ml of
CHCl3:MeOH (1:2). After vortexing, an additional 0.75 ml of
CHCl3:MeOH (1:2), 1 ml of CHCl3, and 2 ml of
0.5 M NH4OH were added with vortexing after
each addition. After brief centrifugation to separate the phases, the
top layer was aspirated off. The organic phase was washed three times
(or until clear) with 30 mM KCl, and a fixed volume was
dried under N2 and resuspended in scintillation fluid for
counting. SPT activity is expressed in pmol of serine converted per mg
of microsomal protein per min.
Generation of the Anti-Lcb1p and Anti-Lcb2p Polyclonal
Antibodies--
Lcb1p and Lcb2p were expressed in Escherichia
coli using the pET-19b vector (Novagen). For this purpose,
the entire coding sequences of the genes were PCR-amplified using
5'-GCCATGGCATATGGCACACATCCCAGAGGT and
5'-GCTAGCGGATCCTTTATTAGATTCTTGGCAAC as primers
for LCB1, and 5'-GCCATGGCATATGAGTACTCCTGCAAACTA,
and 5'-GCTAGCGGATCCATTAACAAAATACTTGTCGT as
primers for LCB2. The PCR products were digested with
NdeI and BamHI (boldface and underlined), ligated
into the pET-19b vector, and the resulting plasmids were transformed
into E. coli BL21 cells. Induction of the T7 promoter
yielded high level expression of the two proteins, and they
fractionated with the inclusion bodies. The proteins were purified by
preparative SDS gel electrophoresis for use as immunogens. Antibodies
to the recombinant Lcb1p and Lcb2p were generated by Cocalico
Biologicals Inc. (Reamstown, PA).
Epitope Tagging the TSC3 and LCB1 Genes--
Two amino acid
insertions were introduced across the coding sequence of the
TSC3 gene by placing 6-bp NheI sites after codon 12, 28, 54, 69, or 79 using the QuikChange site-directed mutagenesis kit (Stratagene). The mutated alleles were tested for complementation of the ts lethality of the tsc3 Immunoblotting--
Proteins were separated by SDS-PAGE and
transferred to nitrocellulose, and the blots were blocked in 0.1 M Tris, 7.5, 0.15 M NaCl, 0.1% Tween 20, 5%
dry milk. For detecting Lcb1p or Lcb2p, blots were probed with
anti-Lcb1p or anti-Lcb2p rabbit polyclonal antibodies (1/500) followed
by a horseradish peroxidase-conjugated donkey anti-rabbit secondary
antibody (1/2000) (Amersham Pharmacia Biotech). Tsc3p-HA was detected
using monoclonal anti-HA (12CA5, Berkeley Antibody Co., Berkeley, CA)
at 1/200 as the primary antibody, and goat horseradish
peroxidase-conjugated anti-mouse (Amersham Pharmacia Biotech) at 1/2000
as the secondary antibody. The bound antibodies were detected by with
the ECL Western blotting detection system (Amersham Pharmacia Biotech).
Immunoprecipitation--
The microsomes were solubilized at 1 mg/ml with 2 mM sucrose monolaurate (Roche Diagnostics,
Indianapolis, IN) for 10 min, and the high speed (1 × 105 × g, 30 min) supernatant was collected. The
solubilized microsomes (150 µl) were incubated with 3 µl of the
precipitating antibody for 2 h, and then 20 µl of protein
A-Sepharose (125 mg/ml from Sigma) for 2 h. The precipitates were
washed three times with 600 µl of 50 mM HEPES, pH 7.5, and resuspended in 150 µl of SDS loading buffer, and a 10-µl sample
was subjected to SDS-PAGE and immunoblot analysis. In some cases, NaCl
was included during the immunoprecipitation.
Isolation of the tsc3 Mutants and Cloning the TSC3 Gene--
A
collection of suppressors of the Ca2+-sensitive phenotype
of the csg2
The five tsc3csg2
The TSC3 gene was cloned by complementation of the ts
phenotype of the tsc3-2 mutant (see under "Experimental
Procedures"). The complementing genomic fragment contained parts of
YBR058c and YBR060c and all of YBR059c (Fig. 1A). The
TSC3 gene was expected to lie in this region of chromosome 2 based on the genetic linkage of the TSC3 locus with markers
from this region. Subcloning experiments demonstrated that the
intergenic region between YBR058c and YBR059c was responsible for
complementing the tsc3 mutants (Fig. 1A). This
region has two potential small ORFs unannotated in the
Saccharomyces Genome Data Base due to their small
(<100-amino acid) sizes. Fragments containing each of the ORFs were
amplified by PCR and inserted into pRS316 (18). The plasmid containing
only ORFa, (Fig. 1A), which potentially encodes an 80-amino
acid protein, complemented the ts phenotype of the tsc3 mutants.
The five tsc3 mutants each have nucleotide changes that
alter the coding potential of the 240-bp ORFa providing further
evidence that ORFa encodes Tsc3p. Four of the five tsc3
alleles have mutations in the same stretch of 8 thymidine residues
(codons 61, 62, and 63) in the 240-bp ORF (Fig. 1B). The
tsc3-1 and tsc3-4 mutations have an extra T
resulting in a new reading frame for 39 amino acids prior to
encountering a termination codon. The tsc3-3 mutation has a
deletion of one of the T residues, leading to a frameshift mutation
that alters 4 amino acid residues before causing premature termination.
The tsc3-5 allele has an extra CT at the end of the stretch
of 8 thymidines, also causing a frameshift mutation that alters 5 amino
acids before a premature termination. The point mutation in the
tsc3-2 allele changes the tyrosine codon 39 to a stop codon
(Fig. 1B).
The possibility that the 80-amino acid protein potentially encoded by
the TSC3 gene might not be expressed was considered. For
example, TSC3 could express a regulatory RNA. The single
nucleotide change that converts codon 39 to a stop codon and disrupts
function of the TSC3 gene suggested that the ORF is
translated. To provide further evidence that the encoded 80-amino acid
protein is responsible for complementation of the tsc3
mutants, single nucleotide changes were introduced at codon 7, 24, 50, or 69, converting each to a stop codon. None of these mutated
TSC3 alleles complemented the ts phenotype of the
tsc3 mutants; therefore, it is concluded that expression of
the 80-amino acid protein is necessary for reversing the ts lethality
of the tsc3 mutants. The 80-amino acid protein encoded by
TSC3 has a hydrophilic amino-terminal half and a hydrophobic
carboxyl-terminal half (Fig. 1B). No proteins with
significant homologies to Tsc3p were identified in a BLAST search
against the complete protein sequence data bases (nonredundant data
base, GenBankTM).
The TSC3 Gene Is Essential for Growth at High
Temperatures--
The nature of the tsc3 mutant alleles
suggested that the ts and suppressing phenotypes result from
eliminating, rather than altering, the function of Tsc3p. For example,
the tsc3-2 mutant has a stop codon at residue 39. To
ascertain that expression of the amino-terminal portion of Tsc3p is not
required for viability, a null allele having the TSC3 coding
sequence replaced with the TRP1 gene (see under
"Experimental Procedures"), was used to disrupt the TSC3
gene. The null mutant (tsc3 The TSC3 Gene Is Required for Optimal SPT Activity--
The first
three steps in sphingolipid synthesis are bypassed by exogenous
phytosphingosine (Fig. 2A). Phytosphingosine,
dihydrosphingosine, and 3-ketosphinganine all restore growth of the
tsc3 mutants at 37 °C (Fig. 2). This suggested that SPT
activity might be deficient in the tsc3 mutants, and
therefore, SPT activity in microsomal membranes isolated from
tsc3 and wild-type cells was compared. The in
vitro SPT activity measured in the tsc3 mutants was
reduced more than 30-fold in comparison to the SPT activity measured in cells with the wild-type TSC3 gene (Fig.
3A), making it surprising that
the tsc3 mutants are viable. However, further analysis
indicates that the percentage of reduction in SPT activity is dependent on the palmitoyl CoA concentrations used for the assay. The conditions that give optimal SPT activity when Tsc3p is present are not the optimal conditions for the Tsc3p-independent enzyme activity. As has
been observed previously for the rat enzyme (19), SPT activity
increases sigmoidally with increasing palmitoyl CoA concentrations and
reaches a maximum at about 0.2-0.3 mM, and at higher
palmitoyl CoA concentrations, SPT activity is inhibited (Fig.
3B). The Tsc3p-independent SPT activity is inhibited at
lower (0.05 mM) palmitoyl CoA concentrations than the SPT
activity measured with Tsc3p present (Fig. 3C). Therefore, the 30-fold difference in SPT activity observed at 0.2 mM
palmitoyl CoA is partially related to the difference in substrate
inhibition of the enzyme. At low palmitoyl CoA concentrations, the SPT
activity in the mutant microsomes is only about 3-fold lower than in
the wild-type microsomes.
Tsc3p Is Not Required for Expression, Membrane Association, or
Coimmunoprecipitation of Lcb1p and Lcb2p--
The possibility that
Tsc3p influences SPT activity by affecting the expression of Lcb1p or
Lcb2p was investigated. Polyclonal antibodies to Lcb1p and Lcb2p were
generated (see under "Experimental Procedures") and used for
detecting the Lcb1p and Lcb2p proteins. Although Lcb1p and Lcb2p have
very similar predicted molecular weights (62,196 and 63,115, respectively), Lcb1p migrates with a significantly higher apparent
molecular weight than Lcb2p (Fig. 4A). The calculated pI for
Lcb1p is 5.96, whereas that for Lcb2p is 8.43; it is not unusual for
proteins with acidic pIs to migrate aberrantly on SDS-PAGE gels. The
level of Lcb2p is greatly diminished in lysates prepared from the
lcb1
Tsc3p does not stimulate SPT activity by increasing the levels of the
Lcb1p or Lcb2p proteins because the immunoblot analysis shows that the
proteins are equally abundant in wild-type and tsc3
To investigate whether Lcb1p and Lcb2p are physically associated and
whether their association depends on Tsc3p, immunoprecipitation experiments were conducted. The rabbit polyclonal antibodies to either
Lcb1p or Lcb2p coimmunoprecipitate both Lcb1p and Lcb2p, indicating
that the two proteins are associated (Fig.
5A). A plasmid carrying a
GST-tagged allele of LCB1 (see under "Experimental Procedures") was transformed into the lcb1
The coimmunoprecipitation of Lcb1p and Lcb2p was not altered in the
tsc3 Tsc3p Is a Microsomal Protein That Coimmunoprecipitates with Lcb1p
and Lcb2p--
An epitope-tagged allele of TSC3 was
generated by placing the triple HA epitope between codons 12 and 13 (see under "Experimental Procedures"). The Tsc3p-HA protein is
functional because it complements the ts phenotype of the
tsc3
The rabbit polyclonal antibodies to either Lcb1p or Lcb2p
coimmunoprecipitated Tsc3p-HA along with Lcb1p/Lcb2p (Fig.
6C), or conversely, the monoclonal anti-HA antibodies
coimmunoprecipitated Lcb1p and Lcb2p along with Tsc3p-HA (Fig.
6D). The association of Tsc3p with Lcb1p and Lcb2p is stable
at 0.3 M NaCl (Fig. 6C). At a higher salt
concentration (0.6 M NaCl), Lcb1p and Lcb2p
coimmunoprecipitated with each other (Fig. 5B), but Tsc3p-HA
was not present in the complex (data not shown). These experiments show
that Tsc3p physically associates with Lcb1p and/or Lcb2p. It has not
been possible to determine whether Tsc3p associates with Lcb1p, Lcb2p,
both proteins, or a complex of the two proteins, because of the
instability of Lcb2p in the absence of Lcb1p and vice
versa.
Tsc3p is a novel membrane-associated protein that binds to the
other proteins required for SPT activity. Tsc3p acts
posttranslationally and through association with Lcb1p and/or Lcb2p to
stimulate the activity of SPT, but it remains to be determined how
Tsc3p modulates SPT activity. Although it is not homologous to acyl-CoA
carrier proteins, Tsc3p may bind palmitoyl CoA. The other Two observations indicate that Tsc3p is required only for interaction
with the SPT enzyme. First, addition of LCBs to the growth medium
reverses the ts phenotype of the tsc3 Using antibodies generated to either Lcb1p or to Lcb2p for
immunoprecipitation experiments, we find that Lcb1p and Lcb2p are membrane-associated proteins that physically associate with one another. This is consistent with the finding of Nishijima and co-workers (21) that antibodies to hamster Lcb2p co-immunoprecipitate both Lcb1p and Lcb2p. We also observe that the level of Lcb2p in
microsomes from the lcb1 SPT catalyzes the committed and apparently rate-limiting step in
sphingolipid synthesis, and sphingolipids are essential for the
viability of all eukaryotic cells. Although there is a great deal of
evidence that the LCBs, and other sphingolipid metabolites, are
important signaling molecules, little is known about how SPT activity
is regulated. Although we have not identified Tsc3p homologs in the
genomes of higher eukaryotic cells, the high degree of sequence
conservation between the yeast and mammalian LCB1 and LCB2 genes suggests that Tsc3p-like proteins may be required
for optimal activity of SPT in mammalian cells as well. Determining how
Tsc3p acts to stimulate SPT activity is likely to provide information
about the regulation of sphingolipid synthesis.
Mutants in the TSC collection identify many ts alleles of the genes
required for synthesis of the long chain bases including the genes for
3-ketosphinganine (TSC1/LCB2, TSC2/LCB1, and
TSC3), dihydrosphingosine (TSC10), and
phytosphingosine (TSC7/SUR2) synthesis (10). The
SCS1/LCB2 and SCS2/LCB1 genes were
also identified in the SCS suppressor screen, but the scs
mutants were isolated as suppressors of the Ca2+
sensitivity of the csg2 In addition to the genes required for LCB synthesis, genes required for
the synthesis and hydroxylation of the very long chain fatty acids have
also been identified through characterization of the TSC suppressor
mutant collection. The continued analysis of the TSC genes
is expected to provide new insights into the regulation of the
biosynthetic pathway and into the functions of sphingolipids.
*
This work was supported by National Institutes of Health
Grant GM 51891 and Uniformed Services University of the Health Sciences Grant 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.
2
K. Gable, E. Monaghan, and T. M. Dunn,
unpublished data.
The abbreviations used are:
SPT, serine
palmitoyltransferase;
LCB, long chain base;
AONS, 8-amino-7-oxononanoate synthase;
ts, temperature sensitive;
bp, base pair(s);
PHS, phytosphingosine;
ORF, open reading frame;
PAGE, polyacrylamide gel electrophoresis;
GST, glutathione
S-transferase;
HA, hemagglutinin;
PCR, polymerase chain
reaction;
SCS, suppressor of the Ca2+-sensitive phenotype
of the csg2
Tsc3p Is an 80-Amino Acid Protein Associated with Serine
Palmitoyltransferase and Required for Optimal Enzyme Activity*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxoamine synthase family required for serine
palmitoyltransferase activity. The other -oxoamine synthases are
soluble homodimers, but serine palmitoyltransferase is a
membrane-associated enzyme composed of at least two subunits, Lcb1p and
Lcb2p. Here, we report the characterization of a third gene,
TSC3, required for optimal 3-ketosphinganine synthesis in
Saccharomyces cerevisiae. S. cerevisiae cells lacking the
TSC3 gene have a temperature-sensitive lethal phenotype
that is reversed by supplying 3-ketosphinganine, dihydrosphingosine, or
phytosphingosine in the growth medium. The tsc3 mutant
cells have severely reduced serine palmitoyltransferase activity. The TSC3 gene encodes a novel 80-amino acid protein with a
predominantly hydrophilic amino-terminal half and a hydrophobic
carboxyl terminus that is membrane-associated. Tsc3p
coimmunoprecipitates with Lcb1p and/or Lcb2p but does not bind as
tightly as Lcb1p and Lcb2p bind to each other. Lcb1p and Lcb2p remain
tightly associated with each other and localize to the membrane in
cells lacking Tsc3p. However, Lcb2p is unstable in cells lacking Lcb1p
and vice versa.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxoamine synthase subfamily includes 8- amino-7-oxononanoate
synthase (AONS), 5-aminolevulinate synthase, 2-amino-oxobutyrate CoA
ligase, and SPT. Although these enzymes share low overall sequence
identity, the recently reported crystal structure of AONS reveals that
several functionally important residues are highly conserved (5). The residues that are involved in pyridoxal phosphate binding, including the lysine that forms a Schiff's base with pyridoxal phosphate, are
conserved in Lcb2p but not in Lcb1p. Therefore, although the AONS
enzyme is a symmetrical homodimer with the active site at the subunit
interface, Lcb1p and Lcb2p may form a heterodimer because both proteins
are required for SPT activity. The yeast and mammalian Lcb2ps are more
similar to each other than they are to the yeast and human Lcb1ps (2,
6, 7). Likewise, the yeast and mammalian Lcb1ps are more similar to
each other than they are to their Lcb2p counterparts (7, 8).
mutants (2, 10-14). Csg2p is
required for mannosylation of the sphingolipid intermediate
inositolphosphorylceramide; therefore, cells lacking Csg2p
overaccumulate inositolphosphorylceramide, which in turn confers
Ca2+ sensitivity (15). The tsc (for
temperature-sensitive suppressors of
Ca2+ sensitivity) mutants were isolated by
first selecting pseudorevertants of the csg2 mutant that
could grow on medium containing 50 mM CaCl2 at
26 °C. In a secondary screen, the subset of mutants with suppressing
mutations that conferred temperature-sensitive lethality (inability to
grow at 37 °C) was identified (10). Here, we report the
characterization of the TSC3 gene and show that it is
required for optimal SPT activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
lcb2, and tsc3 null mutants (with their
respective genes deleted) were constructed in TDY 2039. Media were
prepared and cells were grown according to standard procedures (16). The lcb1 and lcb2 mutant cells were grown
in medium containing phytosphingosine (PHS) at 10-25 µM,
which was added to autoclaved medium containing 0.2% tergitol from a
25 mM PHS stock prepared in ethanol.
View larger version (27K):
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Fig. 1.
The TSC3 gene lies between
YBR058c and YBR059c. A, the complementing genomic
library plasmids contained the indicated region of chromosome 2. Subclones were constructed and tested for complementation of the ts
phenotype of a tsc3 mutant. A + symbol denotes that the
subclone complements, and a - symbol denotes that the subclone fails
to complement. B, the 240-base pair open reading frame and
the encoded 80-amino acid Tsc3p protein are shown. The stretch of eight
thymidine residues where four of the tsc3 mutations reside
and the Tyr codon 39 that is mutated to a stop codon in the
tsc3-2 allele are italicized and
underlined.
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin A. Glass beads were added to just below the meniscus, and cells were lysed
by six cycles (60 s each) of vortexing with cooling on ice between
cycles. Unbroken cells, beads, and debris were removed by
centrifugation (5000 × g for 10 min), and the low
speed supernatant was centrifuged at 4 × 104 × g for 40 min to provide the microsomal pellet. The pellet
was resuspended at 1 ml/g wet cell weight (~10 mg/ml protein) in the same buffer containing 33% glycerol and stored at 
80 °C.
mutant, and it was found
that two amino acids insertions after either codon 12 or codon 28 (but not at the other sites) resulted in functional TSC3 alleles.
A SpeI-ended triple-hemagglutinin (HA)-containing cassette
was generated by PCR and ligated in-frame into the NheI site
between codons 12 and 13. A plasmid containing the TSC3-HA
allele (confirmed by DNA sequencing) was found to be functional on the
basis of complementation of the ts phenotype of the tsc3
mutant. An Lcb1p-GST fusion protein was constructed using a similar
strategy. First, an AvrII site was introduced into the
LCB1 gene by changing codons 9 and 10 from CCCAAA to CCTAGG
by QuikChange mutagenesis, and then a SpeI-ended
GST-containing fragment was generated by PCR and ligated into the
AvrII site. The construct was confirmed by DNA sequencing,
and the LCB1 allele containing GST between codons 9 and 10 was demonstrated to be functional on the basis of complementation of
the lethality of the lcb1 mutant and restoration of SPT
activity to membranes prepared from the rescued null mutant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant is being characterized. Each of these
suppressor mutants (the TSC collection) has a single mutation that
suppresses Ca2+ sensitivity and confers
temperature-sensitive (ts) lethality (10). The five mutants in the TSC3
complementation group have phenotypes indicative of defects in SPT
activity, and therefore, we undertook a study of the TSC3
gene. The suppressing and ts phenotypes of one of the mutants in the
TSC3 complementation group are shown in Fig.
2B.
View larger version (28K):
[in a new window]
Fig. 2.
Mutations in the TSC3 gene
suppress the Ca2+ sensitivity of csg2
mutants and confer temperature-sensitive lethality. The
temperature sensitivity is reversed by phytosphingosine. A,
the pathway for synthesis of the long chain bases is shown.
B, the growth phenotypes of wild-type (TDY 2039),
csg2 (TDY 2040), and tsc3-2 csg2 (CJYa16)
mutant cells are compared. Serial dilutions of the cells were
transferred from the wells of a microtiter plate to YPD agar plates
(YPD) with or without 50 mM CaCl2
(Ca), and with or without 20 µM PHS. The
plates were incubated at either 26 °C (3 days) or 37 °C (2 days).
C, the ts growth phenotype of all five tsc3
mutants is rescued by PHS. Growth on PHS is shown, but
3-ketosphinganine and dihydrosphinganine also reverse the ts
phenotype.
mutants (Table
I) were mated with the original
csg2 single mutant. The resulting tsc3/TSC3
csg2/csg2 diploids were both ts and
Ca2+-sensitive, indicating that both the suppressing and
the ts phenotypes of the tsc3 mutant were recessive.
Following sporulation and tetrad analysis, half of the products of
meiosis were found to be ts and resistant to Ca2+, and the
other half were temperature-tolerant and Ca2+-sensitive,
demonstrating that a single mutation caused both the suppressing and
the ts phenotypes. During tetrad analysis, genetic linkage between the
TSC3 locus and the LYS2 gene was fortuitously observed. Therefore, the linkage distance between TSC3 and
three other genes in that region of chromosome 2 (CSG2,
LYS2, and SEC18) was measured. The
TSC3 locus mapped to a 30-kilobase region on chromosome 2 in
the vicinity of YBR058c (data not shown).
S. cerevisiae strains
) displayed the same phenotype as the originally isolated tsc3 mutants; it was viable at
26 °C but unable to grow at 37 °C. Therefore, the ts phenotype
does not result from the temperature-dependent loss of
function of Tsc3p at 37 °C; rather, the ts phenotype arises from the
requirement for Tsc3p at high temperature.
View larger version (19K):
[in a new window]
Fig. 3.
The tsc3 mutants are
deficient in SPT activity. A, microsomes were prepared
from each of the tsc3 mutants (Table I), and SPT activity
was assayed at 0.2 mM palmitoyl CoA (see under
"Experimental Procedures"). SPT activity is reported in pmol of
serine converted to 3-ketosphinganine per mg of microsomal protein per
min. The SPT activity in wild-type (B) and
tsc3 mutant (C) microsomes was measured at
varying palmitoyl CoA concentrations at serine concentrations of either
2 (
), 6 (
), or 18 (
) mM.
mutant (Fig. 4A). The levels of the
LCB2 mRNA are unaltered in the lcb1 mutant
(data not shown), but the rate of translation of Lcb2p in the
lcb1 mutant has not been tested. The genetic and
immunoprecipitation data (discussed below) indicate that Lcb1p and
Lcb2p are subunits of SPT, and the subunits of a multimeric protein
complex are often unstable in the absence of the interacting partners.
Furthermore, although Lcb1p is present at normal levels in microsomes
prepared from the lcb2 mutant, Lcb1p is unstable in the
absence of Lcb2p after the microsomes are solubilized (discussed
below). Therefore, it is likely that Lcb2p is synthesized at normal
levels but is unstable without Lcb1p.
View larger version (25K):
[in a new window]
Fig. 4.
Tsc3p is not required for expression,
stability, or membrane association of Lcb1p or Lcb2p.
A, microsomes were prepared from TDY 2039 (wt) or
from the lcb1 , lcb2, and tsc3
mutant cells, which are isogenic to TDY 2039, but with the designated
gene disrupted. Ten µg of the microsomal proteins were resolved on
8% SDS-PAGE, and immunoblotted with antibodies to Lcb1p (left
panel) or antibodies to Lcb2p (right panel). Apparent
molecular masses of marker proteins and the bands corresponding to the
Lcb1p and Lcb2p proteins are indicated. B, cell lysates were
centrifuged at low speed (4000 × g for 10 min) to
remove unbroken cells, and the supernatants were then centrifuged at
high speed (4 × 104 × g for 30 min). The
proteins from the total cell lysates (total) and the
supernatants (sup) and pellets of the high speed
centrifugation were analyzed by immunoblotting with antibodies to
either Lcb1p (top panel) or Lcb2p (bottom
panel).
mutant cells (Fig. 4A). As mentioned earlier, the mechanism of association of SPT with the membrane is unknown. The predominantly hydrophobic carboxyl-terminal 40 amino acids of Tsc3p suggested that it
might serve to anchor SPT to the membrane. However, the membrane
association of Lcb1p and Lcb2p is not disrupted in the tsc3 mutant (Fig. 4B). These experiments
demonstrate that Tsc3p is not required for the expression or membrane
association of Lcb1p and Lcb2p.
mutant. The
Lcb1p-GST fusion protein restores SPT activity (data not shown) and
coimmunoprecipitates with Lcb2p (Fig. 5A, lanes 6 and
7). Whereas Lcb1p was present in the microsomes from the
lcb2 mutant (Fig. 4), following solubilization of the
microsomes and the immunoprecipitation protocol, Lcb1p was not detected
in the immunoprecipitates from the lcb2 mutant microsomes
(Fig. 5A, lanes 8 and 9). This is due to
degradation of Lcb1p without Lcb2p because Lcb1p was not present in the
unbound supernatant removed from the protein A-Sepharose beads.
View larger version (37K):
[in a new window]
Fig. 5.
Lcb1p and Lcb2p coimmunoprecipitate in
wild-type and tsc3 mutant cells. A,
microsomes were prepared from wild-type (wt),
lcb1 (with (lanes 6 and 7) or
without (lanes 4 and 5) a plasmid carrying the
LCB1-GST allele), lcb2, and tsc3
mutant cells as described in the legend to Fig. 4. The microsomes were
solubilized with 2 mM sucrose monolaurate, and the 1 × 105 × g (30 min) supernatant was
immunoprecipitated using antibodies to either Lcb1p (1) or Lcb2p
(2). The immunoprecipitated proteins were resolved by 8% SDS-PAGE
and analyzed by immunoblotting with the anti-Lcb1p (top
panel) or anti-Lcb2p (bottom panel) antibodies.
Lane 1 contains 10 µg of the microsomes from wild-type
cells to mark the position of Lcb1p and Lcb2p. The Lcb1p-GST fusion
protein (lanes 6 and 7) is larger than Lcb1p. The
dark bands below Lcb1p and Lcb2p are the rabbit
immunoglobulins (IGs). B, the
immunoprecipitations were done using the wild-type and
tsc3 microsomes as described in A except that
the solubilized microsomes were adjusted to 0.6 M NaCl
prior to the immunoprecipitation. Rabbit preimmune serum
immunoprecipitation controls are shown in lanes 2 and
5.
mutant (Fig. 5A, lanes 10 and
11) demonstrating that Tsc3p is not required for the
association of Lcb1p with Lcb2p. The coimmunoprecipitation of Lcb1p and
Lcb2p was unaffected by the absence of Tsc3p even at 0.6 M
NaCl (Fig. 5B). Thus, although it is possible that Tsc3p
influences the interaction between Lcb1p and Lcb2p, the
Tsc3p-independent association of Lcb1p and Lcb2p is stable at high
ionic strengths.
mutant and restores SPT activity. The Tsc3p-HA
protein fractionates with the microsomes (Fig.
6A) and is solubilized
similarly to Lcb1p and Lcb2p with a variety of detergents,
demonstrating that Tsc3p is membrane-associated (Fig. 6B).
The Tsc3p-HA protein has a predicted molecular weight of 13,711, but it
migrates with an apparent molecular mass of approximately 18,000 Da
(Fig. 6A). This may be because Tsc3p-HA has a calculated pI
of 5.49. As was mentioned above, proteins with acidic pIs often migrate
aberrantly in SDS-PAGE.
View larger version (20K):
[in a new window]
Fig. 6.
Tsc3p is a membrane protein that
coimmunoprecipitates with Lcb1p/Lcb2p. A, TDY 2039 cells with (TSC3-HA, lanes 1 and 3) or without
(wt, lanes 2 and 4) a plasmid carrying the
TSC3-HA allele were used. Proteins from cell lysates
(lanes 1 and 2) or from microsomes (lanes
3 and 4) were separated by 15% SDS-PAGE and
immunoblotted with monoclonal anti-HA (12CA5) antibodies. The Tsc3p-HA
protein is a microsomal protein. There is an anti-HA cross-reactive
protein in the soluble fraction of the cells that migrates near the
molecular weight 50,000 marker. B, microsomes (100 µl at 1 mg/ml) from cells containing Tsc3p-HA (as in A) were
incubated for 10 min at 25 °C with the indicated detergents. No
detergent was added to the microsomes in the samples in lanes
1 and 2. The detergents were used at their critical
micelle concentrations: sucrose monolaurate (SML) at 2 mM;
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
(CHAPS) at 6 mM; Triton X-100 (TX100)
at 0.2 mM; tergitol (NP40) at 0.06 mM; deoxycholate (DOC) at 2 mM, and
Mega10 at 6 mM. The samples were centrifuged at 1 × 105 × g for 30 min, and the pellets
(P) and supernatants (S) were subjected to 8%
SDS-PAGE for Lcb1p and Lcb2p and 15% SDS-PAGE for Tsc3p-HA. Lcb1p and
Lcb2p were detected on the same blot (top panel) using the
anti-Lcb1p and anti-Lcb2p antibodies as described in the legend to Fig.
4. Tsc3p-HA (bottom panel) was detected as in A. C, the microsomes from the cells containing Tsc3p-HA
(A) were solubilized with 2 mM sucrose
monolaurate as in B and centrifuged at 1 × 105 × g for 30 min, and NaCl was added to the
supernatant to the indicated concentration. The supernatant was
immunoprecipitated using either rabbit preimmune serum (lanes 2, 6, and 10), polyclonal antibodies to Lcb1p (lanes
3, 7, and 11), Lcb2p (lanes 4, 8, and 12), or monoclonal antibodies to HA
(lanes 5, 9, and 13). The immunoprecipitated
proteins were resolved on 15% SDS-PAGE and immunoblotted with the
anti-HA antibodies. The major dark immunoreactive bands in the anti-HA
immunoprecipitates are the mouse immunoglobulins. Lane 1 contains 10 µg of the total microsomal protein. D, the
immunoprecipitated proteins (same samples as in C, lanes
2-5) were resolved by 8% SDS-PAGE and immunoblotted with the
Lcb1p (top panel) or Lcb2p antibodies (bottom
panel). Lane 1 contains 10 µg of total microsomal
protein. The dark immunoreactive bands below Lcb1p and Lcb2p are the
immunoglobulins (IGs).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxoamine
synthases are soluble proteins that utilize short acyl-CoAs, whereas
SPT is membrane-associated and uses palmitoyl-CoA, which itself
partitions into the membrane. Therefore, Tsc3p may be required for the
appropriate interaction of the acyl CoA substrate with the enzyme. The
possibility that Tsc3p is required for the proper interaction of
palmitoyl CoA with SPT may provide an interpretation for our data
showing that Tsc3p-independent SPT activity is more sensitive to
inhibition by palmitoyl CoA. For example, palmitoyl CoA might bind the
enzyme nonproductively without Tsc3p. The observation that Lcb1p and Lcb2p are stable and tightly associated in the solubilized microsomes from the tsc3 mutant suggests that the increased
sensitivity to palmitoyl CoA in the absence of Tsc3p is not due to a
trivial detergent effect of palmitoyl CoA. The ability to detect and
immunoprecipitate Tsc3p will provide the opportunity to investigate
whether Tsc3p binds a photoaffinity labeling analog of palmitoyl CoA
(22).
mutant. Second, we
have isolated and characterized pseudorevertants of the
tsc3 mutant that can grow at
37 °C.2 Forty-nine
suppressor mutants have been analyzed, and all of them have single
amino acid changes at one of eight positions in the LCB2
gene. The SPT activity is increased 6-10-fold in these tsc3 mutants as a result of the suppressing mutation in
Lcb2p. Determining why the SPT activity in these Lcb2p variants has a reduced dependence on Tsc3p is expected to provide information about
the role of Tsc3p. Based on these results, it is concluded that Tsc3p
is not required for any essential process besides 3-ketosphinganine synthesis.
mutant is greatly reduced,
apparently due to instability of Lcb2p in the absence of the
interacting Lcb1p. Therefore, the observation that there is no SPT
activity detected in microsomes prepared from the lcb1
mutants of Saccharomyces cerevisiae (or in the CHO cell line
with a ts mutation in LCB1 (21)) does not necessarily mean
that Lcb1p is required for catalysis. The absence of SPT activity may
reflect instability of Lcb2p when Lcb1p is missing. The level of Lcb2p
present in the membranes prepared from the CHO mutants that lack
detectable Lcb1p has not been reported (21).
mutant at 37 °C (2), and
therefore, the TSC3 gene (essential at 37 °C) was not
represented in the SCS suppressor collection. The screen for
LCB-requiring mutants that identified the LCB1 and
LCB2 genes was conducted at 30 °C (20), and the
tsc3 mutants only display the LCB-requiring phenotype at
high temperatures (e.g. 37 °C), which probably explains
why the TSC3 gene was not found in that screen. The
tsc3 mutant is viable at 26 °C despite the decreased
SPT activity, but the mutant cannot grow at elevated temperatures. This
indicates that the requirement for sphingolipids is greater at higher
growth temperatures and explains why such a high percentage of the
suppressor mutants display the associated ts phenotype.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Uniformed Services University of the Health
Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814. Tel.:
301-295-3592; Fax: 301-295-3592; E-mail: Tdunn@usuhs.mil.
ABBREVIATIONS
mutant.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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