From the Department of Biology, Utah State
University, Logan, Utah 84322 and the § Department of
Biochemistry and the Lucille P. Markey Cancer Center, University of
Kentucky, Lexington, Kentucky 40536
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ABSTRACT |
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The Saccharomyces cerevisiae gene SYR2, necessary for growth inhibition by the cyclic lipodepsipeptide syringomycin E, is shown to be required for 4-hydroxylation of long chain bases in sphingolipid biosynthesis. Four lines of support for this conclusion are presented: (a) the predicted Syr2p shows sequence similarity to diiron-binding membrane enzymes involved in oxygen-dependent modifications of hydrocarbon substrates, (b) yeast strains carrying a disrupted SYR2 allele produced sphingoid long chain bases lacking the 4-hydroxyl group present in wild type strains, (c) 4-hydroxylase activity was increased in microsomes prepared from a SYR2 overexpression strain, and (d) the syringomycin E resistance phenotype of a syr2 mutant strain was suppressed when grown under conditions in which exogenous 4-hydroxysphingoid long chain bases were incorporated into sphingolipids. The syr2 strain produced wild type levels of sphingolipids, substantial levels of hydroxylated very long chain fatty acids, and the full complement of normal yeast sphingolipid head groups. These results show that the SYR2 gene is required for the 4-hydroxylation reaction of sphingolipid long chain bases, that this hydroxylation is not essential for growth, and that the 4-hydroxyl group of sphingolipids is necessary for syringomycin E action on yeast.
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INTRODUCTION |
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Syringomycin E is a member of a family of cyclic lipodepsipeptides produced by strains of the plant bacterium Pseudomonas syringae pv. syringae (1). Traditionally regarded as a virulence factor in a variety of bacterial necrotic diseases of plants (2), syringomycin E and its analogs also possess antifungal properties, and it has been suggested that these metabolites are fungal antagonists that aid survival of the producing bacteria on plants (3, 4).
How these compounds produce their toxic effects is unknown, but past physiological studies have shown that syringomycin E targets primarily the plasma membrane (1, 5, 6). To further investigate the molecular mechanisms of action of this bioactive compound, resistant mutants of Saccharomyces cerevisiae were isolated to identify genes that encode proteins necessary for growth inhibition by syringomycin E (7). Several of the mutants were deficient in sterols, and one group was complemented by the gene SYR1 (identical to ERG3), which encodes sterol C-5,6 desaturase of the ergosterol biosynthetic pathway (8). These findings, when combined with results from binding (9) and lipid bilayer (10) studies, indicate that sterols influence the interaction of syringomycin E with the target plasma membrane.
Syringomycin E action in yeast was more recently shown to require a second, nonsterol biosynthetic gene, SYR2 (11). SYR2 is identical to SUR2, which was identified in a screen for mutants that suppress the impaired recovery of rvs161 strains from nutritional starvation (12). Syringomycin E-resistant syr2 mutants showed altered glycerophospholipid levels, and the SYR2 gene product was localized to the endoplasmic reticulum (11). Nevertheless, the precise function of Syr2p was unclear from these studies.
In addition to sterols and glycerophospholipids, sphingolipids are major lipid components of the plasma membrane (13). Ubiquitous in eukaryotic cells, sphingolipids all possess a sphingoid long chain base with mainly, in fungi and plants, a hydroxyl group at the C-4 position (phytosphingosine) or, in animals, a double bond at the C-4,5 position (sphingosine). Sphingolipids serve numerous roles, including mediating cell-cell interactions, anchoring membrane proteins, acting as enzyme co-factors (14), and serving as receptors for Escherichia coli verotoxin (15-17). In addition, sphingolipids are becoming recognized as significant players in the control of cell growth, differentiation, and response to stress through the second messenger action of sphingolipid metabolites sphingosine, sphingosine-1-phosphate, and ceramide (18-20). Despite their importance, numerous gaps remain in the knowledge of sphingolipid metabolism, including the nature of the enzymes directly responsible for phytoceramide or ceramide formation from the presumed immediate precursor dihydroceramide (Fig. 1) (21).
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In this report we present evidence that S. cerevisiae SYR2 is required for 4-hydroxylation of sphingoid bases and that this activity is necessary for syringomycin E action. We show that strains mutant in SYR2 produce sphingolipids missing the hydroxyl group at the C-4 position of the long chain base moiety, that supplying such cells with C-4 hydroxylated long chain base suppresses the syringomycin E-resistant phenotype of syr2 strains, and that strains that overexpress Syr2p are enriched in 4-hydroxylase activity.
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EXPERIMENTAL PROCEDURES |
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Yeast Strains and Growth Conditions-- Yeast strains used in this study are listed in Table I. Yeast were grown at 28-30 °C with shaking. YPD, SC-ura, SG-ura (as SC-ura with glucose replaced by galactose), and SC-trp were as described by Kaiser et al. (22). The lcb1 mutants were grown in modified YPD (1% yeast extract, 1% peptone, 4% glucose, 50 mM sodium succinate, pH 5.0, 0.05% Tergitol (U. S. Biochemical Corp.), 25-50 µM phytosphingosine·HCl (PHS)1 or DL-erythro-dihydrosphingosine (DHS) (both from Sigma) or synthetic medium as described by Pinto et al. (23) minus either uracil or tryptophan. Stock solutions (100 mM) of PHS and DHS in 95% ethanol were diluted into 0.5% Tergitol and then added to the media to yield the indicated final long chain base and Tergitol concentrations.
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Long Chain Base Analysis--
Yeast were grown in YPD and
harvested at 1.5 × 108 cells/ml. Methanol-HCl
hydrolysates of wild type (W303C and KZ1-1C) and syr2 cells
(WSYR2
and 13N-F2), as well as C-18 DHS and C-18 PHS
standards were derivatized with the UV-absorbing 4-biphenylcarbonyl chloride by the method of Jungalwala et al. (24) as adapted by Dickson et al. (25). Samples were resolved by HPLC on a
250 × 4.6-mm Alltech Econosil C18 column with a 10-mm guard
column. Elution was isocratic with methanol/water (90:10, v/v) as the solvent at a flow rate of 1.0 ml/min. Effluent was monitored by absorbance at 280 nm. Electrospray ionization mass spectrometry was
performed by the Utah State University Biotechnology Center.
Sphingolipid Analysis--
W303C and WSYR2
cells were inoculated to 5 × 106 cells/ml in modified
YPD containing 0.5 mCi of [3H]inositol (New England
Nuclear Co., 20 mCi/µmol) or 0.5 mCi of [4,5-3H]DHS
(2.56 mCi/µmol) derived by hydrolysis of
[3H]N-acetyl-DHS prepared as described
previously (26). After culture for 18 h at 30 °C the cells
reached a density of 1.9-2.4 × 108 cells/ml, and the
reaction was stopped by adding trichloroacetic acid to a final
concentration of 5%. The cells were processed to deacylate the ester
lipids followed by extraction of the sphingolipids as described
previously (27). To further purify the acidic sphingolipids, the
sphingolipid extract was bound to and eluted from AG4 resin (Bio-Rad)
as described previously (28). The AG4 eluate was dried, dissolved in 1 ml of chloroform/methanol/water (16:16:5, v/v/v), and 3-7-µl
aliquots were subjected to thin layer chromatography on 20-cm silica
gel plates (Whatman HP-K) with the solvent chloroform/methanol/4.2 N aqueous NH4OH (9:7:2, v/v/v). Each lane
contained a mixture of yeast PHS-containing sphingolipids: 2 nmol each
of inositolphosphoryl ceramide (IPC) and mannosylinositolphosphoryl
ceramide (MIPC) species containing mono- and di-OH fatty acids and
mannosyl-di(inositolphosphoryl) ceramide (M(IP)2C) with a
mono-OH fatty acid. Radioactivity was measured with a BioScan
apparatus. The standards were located by charring after spraying with
10% (w/v) CuSO4·5H2O in 8%
H3PO4 followed by heating at 160 °C for 30 min (29). The mannosylated sphingolipids in the deacylated lipid
extract were also detected after thin layer chromatography of larger
aliquots (125 µl) on 20-cm Whatman K5 plates developed with the same
solvent as above. The plate was first treated with orcinol reagent (30)
to detect the carbohydrate containing sphingolipids, MIPC and
M(IP)2C, and then treated with the
CuSO4/phosphoric acid reagent as above.
Fatty Acid Analysis-- Fatty acids from an acidic sphingolipid fraction (prepared as described above without the addition of radioisotopes) or whole cells were liberated by saponification and converted to UV-absorbing phenacyl derivatives that were resolved and quantitated by reverse phase HPLC as described previously (25).
Assay of 4-Hydroxylase Activity-- The previously constructed SYR2 overexpression plasmid, pYSYR2, placed a 5'-truncated SYR2 gene under the control of the galactose-inducible promoter GAL1 (11). For this work the truncated SYR2 insert was removed and replaced with an AccI-SphI fragment containing the entire SYR2 coding region. Expression of Syr2p from this construct, pYSYR2a, was confirmed by observation of galactose-inducible complementation of syringomycin E resistance of a syr2 strain.
W303C containing pYSYR2a or the control plasmid pYES2 was grown overnight in SC-ura. Cells were washed with sterile water and diluted into 300 ml of SG-ura (8 × 106 cells/ml) to induce Syr2p expression. Cells were harvested after an additional 16 h of growth and washed with water. WConstruction of lcb1 Disruptant Strains--
A disrupted
LCB1 allele, lcb1-3, was constructed by
replacing in pTZ18-LCB1 (32), a 1.5-kilobase pair
SalI/BamHI fragment that contains the C-terminal
80% of the LCB1 gene, with a 1.4-kilobase pair
TRP1-containing fragment to yield plasmid
pLCB1-
3. Plasmids were propagated in E. coli
DH5
. 3 µg of pLCB1-
3 were digested with restriction
endonuclease NdeI, the fragments were separated on a 0.8%
agarose gel, and the 3.1-kilobase pair fragment containing the
lcb1-
3 allele was isolated using an Elu-Quik DNA
Purification Kit as directed by the manufacturer (Schleicher & Schuell). The DNA fragment was transformed into diploid strain W303-1A
by electroporation and plated onto SC-trp medium. Trp+
colonies were isolated and sporulated, and the resultant tetrads were
dissected onto modified YPD plates containing 25 µM PHS. One Trp+ long chain base auxotrophic colony was selected
and designated W
LCB1. Replacement of the LCB1
allele with lcb1-
3 was confirmed by Southern blotting
using enhanced chemiluminescence detection (Amersham Pharmacia
Biotech). The double disruptant, W
LCB1
SYR2, was produced by crossing W
LCB1 with
W
SYR2a and selecting for Trp+
Ura+ long chain base auxotroph progeny. Standard genetic
procedures were as described by Kaiser et al. (22).
Syringomycin E
Treatment--
WLCB1
SYR2 cells were grown
overnight in modified YPD containing either 50 µM DHS or
50 µM PHS. W
SYR2
and W303C were grown in
modified YPD with no long chain base addition. Cells were washed once
with sterile water and then transferred to modified YPD minus Tergitol
and long chain base and with the indicated amounts of syringomycin E,
prepared as described previously (33). Final cell densities were
A600 = 0.2 as measured in a Shimadzu UV-1201 spectrophotometer (1 A600 unit equals
approximately 3.7 × 107 cells/ml). Incubation was
continued at 28 °C. After 1 h Tergitol was restored to 0.05%,
and DHS or PHS was restored to 50 µM as appropriate to
prevent starvation for long chain base. Syringomycin E was found to be
ineffective if added directly to medium containing working
concentrations of Tergitol and long chain base. 16 h after syringomycin E addition, aliquots were removed and diluted 10-fold to
measure growth by turbidity at A600.
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RESULTS |
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Sequence Analysis Suggests That Syr2p Is a Diiron Binding Lipid
Hydroxylase or Desaturase--
BLAST algorithm (34) comparisons of the
deduced amino acid sequence of Syr2p with those in protein data bases
showed significant similarities to endoplasmic reticulum proteins
associated with lipid metabolism. In particular, close similarities
were found with S. cerevisiae SYR1/ERG3 (C-5
sterol desaturase, score 51, p = 0.0098, 33%
identities, 56% positives), Arabidopsis thaliana ERG3 (C-5
sterol desaturase, score 77, p = 0.014, 44%
identities, 68% positives), and yeast ERG25 (C-4 sterol
methyl oxidase, score 65, p = 4.5 × 108, 52% identities, 65% positives). Similar findings
were reported by Bard et al. (35) and Li and Kaplan (36).
Despite the similarity of Syr2p to enzymes of sterol biosynthesis, an
involvement for Syr2p in sterol metabolism could not be found.
Comparisons of sterol profiles of syr2 mutants with
similarly grown wild type strains revealed no differences
(11),2 indicating that Syr2p
functions in some other metabolic pathway.
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Yeast Strains Deficient in SYR2 Lack the Sphingoid Long Chain Base
Phytosphingosine--
Sphingolipids in yeast are normally composed of
the sphingoid long chain base PHS (D-4-hydroxysphinganine),
typically of 18 or 20 carbon chain length (C-18 and C-20,
respectively), an amide-linked very long chain fatty acid, primarily
mono-hydroxy-C-26 chains with lesser amounts of di- and nonhydroxyl
forms, and a phosphoinositol-containing head group (39) (Fig. 1). To
test the involvement of Syr2p in the sphingoid base 4-hydroxylation,
the sphingoid base compositions of mutant strain Wsyr2
and the isogenic wild type strain W303C were determined (25). Reverse
phase HPLC separation of biphenylcarbonyl-derivatized long chain bases
derived from the wild type strain W303C revealed two peaks of
UV-absorbing material, as expected, with retention times of 15 and 25 min (Fig. 3B). Coelution with
a derivatized C-18 PHS standard and electrospray mass spectral analysis
of the material collected from the two peaks verified their identities as C-18 PHS at 15 min and mainly C-20 PHS at 25 min. The derivatized long chain bases from the
syr2 mutant strain also
separated into two peaks, with one again eluting at 25 min but the
other eluting at 42 min (Fig. 3C). Little material with a
retention time of 15 min was apparent (<0.3% of total long chain
base). Authentic C-18 DHS treated in the same manner as the lipid
extracts eluted with a retention time of 25 min (Fig. 3A).
It was not possible to distinguish between the C-20 PHS and C-18 DHS
derivatives by the chromatography system used, but mass spectral
analysis of the
syr2 material eluting at 25 min revealed
a molecular ion mass of 482.3 Da, consistent with its assignment as the
N-biphenylcarbonyl derivative of C-18 DHS. The mass of the
material eluting at 42 min was 510.4 Da, as expected for
N-biphenylcarbonyl-C-20 DHS. Similar results were obtained
with an independently isolated syr2 mutant strain in a
different genetic background (13N-F2 (syr2) and KZ1-1C
(SYR2); data not shown).
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Fatty Acid Analysis of syr2 Strain--
The effect of the
syr2 mutation on hydroxylation of the very long chain fatty
acid component of sphingolipids was also examined as described under
"Experimental Procedures." Hydroxylation of the very long chain
fatty acids was observed in the absence of Syr2p activity. For example,
the percentage of distribution of non-, mono-, and di-hydroxyl fatty
acids for wild type cells was 8, 78, and 14%, respectively, and for
syr2 cells, it was 53, 47, and 0%, respectively. Clearly
syr2 cells hydroxylate the very long chain fatty acid in
sphingolipids. Why the distribution of hydroxylated species differs
from wild type is not known.
In Vitro Measurement of 4-Hydroxylase Activity--
Sphingoid base
4-hydroxylase activity was measured in microsomal fractions, because
Syr2p has previously been localized to the endoplasmic reticulum (11).
Microsomes, prepared from SYR2 wild type, overexpression,
and deletion strains, were supplied with substrate, either DHS or
dihydroceramide solubilized in CHAPS, along with NADH and NADPH. Both
NADH and NADPH have been reported to be cofactors for activity of other
putative diiron proteins involved in oxygen-dependent
reactions of hydrocarbon substrates. After 90 min at 25 °C,
sphingoid long chain bases were released by methanol-HCl hydrolysis and
extracted, and their 4-biphenylcarbonyl derivatives were separated and
quantitated by reverse phase HPLC. Hydroxylated product was apparent
when DHS or dihydroceramide were supplied to microsomes from a
SYR2 overexpressing strain, W303C(pYSYR2a). Using
the same amount of protein, 3-4-fold less hydroxylated product was
produced if NADH and NADPH were omitted from the reaction or if the
source of microsomes was a wild type strain, W303C(pYES2), containing
only the chromosomal copy of SYR2 and a control plasmid
(Table II). No 4-hydroxyl products were
detected if microsomes were from the deletion strain
Wsyr2
.
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Growth in the Presence of PHS Suppresses the syr2
Phenotype--
To confirm that the syringomycin E resistance phenotype
of the syr2 mutant is due to a loss of hydroxylase activity
rather than an additional unknown activity of Syr2p, we wished to test whether syringomycin E sensitivity is restored by supplying
syr2 cells with the product of the hydroxylase. It is still
uncertain, however, if the hydroxylase substrate is phytoceramide, PHS,
or both (see "Discussion"). As yeast do not readily utilize
exogenous ceramides but will incorporate exogenous sphingoid long chain bases into the sphingolipid biosynthetic pathway (40), syringomycin E
sensitivity was tested following growth in medium containing hydroxylated (PHS) or nonhydroxylated (DHS) long chain bases rather than hydroxylated or nonhydroxylated ceramides. Also, to ensure that all sphingolipids were built on the exogenous long chain base, the
LCB1 gene was deleted in strain Wsyr2a
(see "Experimental Procedures"). LCB1 encodes a subunit
of serine palmitoyltransferase, the first enzyme in sphingolipid
biosynthesis (Fig. 1). An lcb1 mutation results in
auxotrophy for long chain bases (32).
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Sphingolipid Analysis of syr2 Strain--
The fact that
syr2 mutants are viable, whereas sphingolipids are
essential, raised questions about the mutant lipid composition. The
loss of long chain base hydroxylation could result in the simple
substitution of nonhydroxylated long chain base for 4-hydroxy-long chain base in the sphingolipid pools or, alternatively, in an alteration of the overall levels or types of sphingolipids. The recovery of similar quantities of long chain base from mutant and wild
type strains (Fig. 3) would argue against a large change in total
sphingolipid content, but in order to more directly investigate the
quantity and nature of sphingolipids produced by the
syr2 mutant, wild type and
syr2 cells were cultured overnight
with either [3H]inositol or [3H]DHS to
label the sphingolipids. The cells were processed to deacylate the
ester lipids, such as phosphatidylinositol (PI), and sphingolipids were
extracted. Based on the radioactivities of
[3H]inositol-labeled extracts before and after PI
deacylation, the distribution and amounts of PI and inositol
sphingolipids were about the same in wild type (25.7 × 106 cpm PI, 15.3 × 106 cpm sphingolipid)
and
syr2 cells (30.6 × 106 cpm PI,
14.4 × 106 cpm sphingolipid), confirming that the
mutant strain was able to make as much total sphingolipid as the wild
type.
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DISCUSSION |
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The present study of yeast gene SYR2 reveals that sphingolipids play a key role in the action of the antifungal syringomycin E. It also uncovers a previously uncharacterized activity of the sphingolipid biosynthetic pathway. The lack of PHS-based sphingolipids and accumulation of DHS-based sphingolipids in syringomycin E-resistant syr2 mutants, the greater 4-hydroxylase activity in a SYR2 overexpression strain, the lack of activity in a syr2 mutant strain, and the ability to bypass the syr2 defect with exogenously added PHS provide support for a biosynthetic role of Syr2p in 4-hydroxylation of sphingoid bases. In addition, the sequence similarity of Syr2p to membrane hydroxylases and desaturases and its localization in the endoplasmic reticulum (11), the site of early sphingolipid biosynthetic steps (41), suggest that Syr2p is the enzyme that catalyzes this specific hydroxylation step. An essential catalytic role in fatty acid hydroxylation is not likely because very long chain fatty acid hydroxylation was still detected in the syr2 mutant despite a complete loss of SYR2 transcripts (11). While this report was in preparation, Haak et al. (42) reported that an independent sur2/syr2 mutant produces sphingolipids lacking long chain base 4-hydroxylation, whereas a second gene product, Scs7p, is required for sphingolipid very long chain fatty acid hydroxylation.
Identification of Syr2p as the sphingolipid 4-hydroxylase will pave the way for isolation and characterization of the enzyme. This will, in turn, permit clarification of several issues regarding sphingolipid biosynthesis. One important question concerns the identity of the lipid substrate of Syr2p. The analogous reaction in animal cells, desaturation at the C-4 position, is thought to occur at the level of dihydroceramide (43). In yeast, however, it is not known if 4-hydroxylation occurs before or after long chain base acylation, i.e. if Syr2p converts DHS to PHS, dihydroceramide to phytoceramide, or both (Fig. 1). Inhibition of ceramide synthesis leads to accumulation of both DHS and PHS (44, 45), suggesting a direct conversion from DHS to PHS is possible, at least under conditions of limited ceramide formation. We have shown that incubation of either DHS or dihydroceramide with microsomes from a SYR2 overexpressing strain leads to hydroxylation. With crude microsome preparations, however, enzymes capable of catalyzing acylation or deacylation of the added substrate before hydroxylation may be present and obscure the true nature of the substrate.
Confirmation that Syr2p is in fact an iron-containing oxidative enzyme as predicted from the sequence (Fig. 2) will also be afforded by biochemical analysis. The stimulation of 4-hydroxylase activity by reduced pyridine nucleotide, as shown here, is consistent with Syr2p being a member of this enzyme family. Molecular oxygen has been shown to be the main source of oxygen added to dihydroceramide (46), but little else is known of the mechanism of this reaction. Knowledge about Syr2p will also reveal mechanisms about mammalian sphingolipid biosynthesis. Phytoceramide is produced by certain mammalian tissues (47) as well as by fungi and plants, and these tissues are predicted to contain a Syr2p homolog. The primary mammalian sphingolipids based on ceramide contain the long chain base sphingosine, which has a C-4,5 double bond rather than the 4-hydroxyl group. Recent reports concerning the enzyme in rats that catalyzes this reaction, dihydroceramide desaturase, suggest it also has properties similar to diiron-containing lipid desaturases and hydroxylases (43, 48).
How sphingolipids, and more specifically sphingolipid 4-hydroxylation, allow susceptibility to syringomycin E can only be speculated. One possibility is that 4-hydroxylated sphingolipids directly bind this antifungal compound at the cell surface. The 4-hydroxyl group is expected to influence the degree of sphingolipid exposure on the membrane surface, but it will also affect lipid and protein nearest neighbor interactions in the plane of the membrane. Another possibility is that 4-hydroxylated sphingolipids indirectly influence syringomycin E-cell interaction by modulating sterol or glycerophospholipid compositions or both. Syringomycin E action is influenced by sterols (8, 10), phospholipid bilayers facilitate ion channel formation by syringomycin E molecules (49), and syr2 mutants have lowered cellular glycerophospholipid levels (11). Despite evidence for cross-regulation of the biosynthetic pathways of these various lipid classes in yeast (50), it is difficult to predict precisely how an alteration in the hydroxylation state of sphingolipids could influence cellular sterol and phospholipid composition. Furthermore, 4-hydroxylation could influence the insertion and assembly of lipids as well as proteins into the plasma membrane. Finally, the requirement for sphingolipid 4-hydroxylation may reflect the involvement of phytoceramide-mediated growth inhibition processes in syringomycin E action. Phytoceramide and ceramide, but not dihydroceramide, are reported to mediate cell death in animal cells and growth inhibition in yeast (18, 51, 52), although the phenomenon is not always observed (53). Exposure of yeast cells to syringomycin E may cause increased cellular levels of phytoceramide (perhaps by activating sphingolipid turnover), which in turn may activate specific protein kinases and phosphatases (18, 51, 52), leading to growth arrest. Without the ability to hydroxylate dihydroceramide to phytoceramide and the consequent substitution of dihydroceramide into mature sphingolipids, syr2 mutants would be incapable of undergoing this process.
The observation that SYR2 encodes a nonessential function raises questions about the cellular roles of 4-hydroxylated sphingolipids in yeast growth and survival. Normal SYR2 strains produce sphingolipids that are based almost exclusively on phytoceramide (this study and Ref. 54), but syr2 mutants grow well with dihydroceramide-based sphingolipids. Sphingolipids are indicated to be required for maintaining proton permeability barriers across the membrane or for proton extrusion (55) and for maturation of glycosylphosphatidylinositol-anchored proteins (56). Preliminary observations, however, show that syr2 mutants display wild type growth phenotypes under conditions where proper functioning of these processes may be essential, namely at acidic pHs (4.1), high temperatures (39 °C), and high salt concentrations (0.75 M NaCl).3 Also, Calcofluor staining of chitin, a probe of cell wall structure, was unperturbed in the syr2 mutant, although growth of the syr2 mutant was slightly retarded by Calcofluor.4 The two phenotypes previously associated with syr2/sur2 mutations, resistance to syringomycin E, and suppression of rvs161 mutations can now be said to be associated with a loss of 4-hydroxylation of the long chain base moiety of sphingolipids. The only apparent commonality of these two phenotypes is growth restoration under conditions that inhibit growth of wild type cells. The mechanism(s) responsible for these effects await elucidation, as does a clear definition of the role of 4-hydroxy-sphingolipids in yeast biology and syringomycin E action.
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ACKNOWLEDGEMENTS |
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We thank Gerald Wells and Elizabeth Nagiec for expert technical assistance.
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FOOTNOTES |
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* This work was supported by grants from Eli Lilly, the Utah Agricultural Experiment Station Project 607, and the National Science Foundation Grant 9003398 (to J. Y. T.) and National Institutes of Health Grant GM41302 (to R. C. D. and R. L. L.).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 is Utah Agricultural Experiment Station Journal Paper 6022.
¶ To whom correspondence should be addressed: Dept. of Biology, Utah State University, Logan, UT 84322-5305. Tel.: 435-797-1909; Fax: 435-797-1575; E-mail: takemoto{at}cc.usu.edu.
1 The abbreviations used are: PHS, phytosphingosine; DHS, dihydrosphingosine; HPLC, high performance liquid chromatography; IPC, inositolphosphoryl ceramide; MIPC, mannosylinositolphosphoryl ceramide; M(IP)2C, mannosyl-di(inositolphosphoryl) ceramide; PI, phosphatidylinositol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2 L. Parks, S. Kelly, and M. Bard, personal communications.
3 J. Y. Takemoto, unpublished results.
4 M. M. Grilley and J. Y. Takemoto, unpublished results.
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REFERENCES |
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