In contrast, specific halotolerance of yeast against extracellular NaCl or LiCl is based on the induction of the ENA1/PMR2 gene designated as ENA1 in the following encoding an enzyme of t
Trang 1ISC1-encoded inositol phosphosphingolipid phospholipase C
Christian Betz1, Dirk Zajonc1, Matthias Moll2and Eckhart Schweizer1
1
Lehrstuhl fu¨r Biochemie and the2Lehrstuhl fu¨r Anorganische und Allgemeine Chemie, Universita¨t Erlangen-Nu¨rnberg,
Erlangen, Germany
In Saccharomyces cerevisiae, toxic concentrations of Na+
or Li+ions induce the expression of the cation-extrusion
ATPase gene, ENA1 Several well-studied signal
transduc-tion pathways are known correlating high salinity to the
transcriptional activation of ENA1 Nevertheless,
informa-tion on the actual sensing mechanism initiating these
path-ways is limited Here, we report that the ISC1-encoded
phosphosphingolipid-specific phospholipase C appears to be
involved in stimulation of ENA1 expression and,
conse-quently, in mediating Na+ and Li+ tolerance in yeast
Deletion of ISC1 distinctly decreased cellular Na+and Li+
tolerance as growth of the Disc1::HIS5 mutant, DZY1, was
severely impaired by 0.5MNaCl or 0.01M LiCl In
con-trast, K+ tolerance and general osmostress regulation
were unaffected Isc1D mutant growth with 0.9MKCl and
glycerol accumulation in the presence of 0.9M NaCl or 1.5M sorbitol were comparable to that of the wild-type ENA1-lacZ reporter studies suggested that the increased salt sensitivity of the isc1D mutant is related to a significant reduction of Na+/Li+-stimulated ENA1 expression Cor-respondingly, Ena1p-dependent extrusion of Na+/Li+ions was less efficient in the isc1D mutant than in wild-type cells
It is suggested that ISC1-dependent hydrolysis of an unidentified yeast inositol phosphosphingolipid represents
an early event in one of the salt-induced signalling pathways
of ENA1 transcriptional activation
Keywords: salt-stress; signaling; sphingolipids; sphingolipid phospholipase C; yeast
The Saccharomyces cerevisiae gene, ISC1, has recently been
shown to encode an inositol phosphosphingolipid-specific
phospholipase C [1] In vitro, the enzyme exhibits the
characteristics of a Mg2+-dependent neutral (N)
sphing-omyelinase (SMase) and, thus, resembles the most
prom-inent member of the SMase family present in mammalian
cells [2,3] According to current knowledge, sphingomyelin
is absent from yeast and, hence, the physiological substrate
of Isc1p is likely to belong to one of the three major classes
of yeast sphingolipids, i.e inositol phosphorylceramides,
mannositol phosphorylceramides, or mannosyldiinositol
phosphorylceramides [4] In mammalian systems, various
intermediates of sphingolipid metabolism act as mediators
of intracellular signalling pathways [5–8] In particular, the
SMase reaction product, ceramide, has been recognized as
a second messenger being induced by a variety of
extracel-lular stress signals [8,9] Subsequent interaction of ceramide with specific protein kinases, protein phosphatases or proteinases induces signalling cascades which finally affect basic cellular functions such as cell cycle progression, cell growth, differentiation, apoptosis or Ca2+ion homeostasis [8,9] In S cerevisiae, sphingolipids represent 20–30% of cellular phospholipids [4] and, thereby, obviously fulfil an important structural function Besides this, they probably contribute to the signal transduction potential of yeast cells, too [10–15] Their vital function is underlined by the lethality of yeast mutants defective in sphingosine base biosynthesis [16] Although sphingosine base-defective mutants may be partly suppressed by the production of C26-fatty acid-containing glycerolipids, these mutants remain sensitive against heat, osmotic and lowpH stresses [4,5,17] From these results, the involvement of sphingoli-pids in distinct stress response pathways of yeast became quite obvious Each one of various different stress responses appears to have its own specific signalling pathway [5] While heat shock induces the biosynthesis
of trehalose [18,19], high extracellular osmolarity either induces the accumulation of glycerol as a compatible intracellular osmolyte [20–22] or, with toxic concentrations
of Na+or Li+ions, extrusion of these cations by induction and activation of the specific, ATP-driven ion pump Ena1p
is initiated [21,23–26] Both pathways of yeast osmoadap-tation have been intensively studied and many of their details are known Non-specific osmostress is exerted by moderate concentrations of various solutes such as NaCl, KCl or sorbitol and induces the high-osmolarity glycerol (HOG) pathway which rapidly raises the intracellular glycerol concentration up to molar levels [20,21] The
Correspondence to E Schweizer, Lehrstuhl fu¨r Biochemie;
Universita¨t Erlangen, Staudtstrasse 5, D-91058 Erlangen, Germany.
Fax: +49 9131 8528254, Tel.: +49 9131 8528255,
E-mail: eschweiz@biologie.uni-erlangen.de
Abbreviations: (N-)SMase, (neutral)sphingomyelinase; HOG,
high-osmolarity glycerol; BSM, BODIPYFL-C 5
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingomyelin;
B-ceramide, BODIPYFL C 5 -ceramide; YPD, yeast extract,
peptone, dextrose; SCD, synthetic complete, dextrose.
Proteins and enzymes: Ena1 (atn1_yeast; EC 3.6.3.7), Isc1 (isc1_yeast;
EC 3.1.4.-), Gpd1 (g3p1_yeast; EC 1.2.1.12), Gpp2 (gpp2_yeast;
EC 3.1.3.-).
(Received 8 May 2002, revised 26 June 2002, accepted 5 July 2002)
Trang 2pathway comprises a mitogen-activated protein kinase
cascade which finally initiates transcription of the
glycer-ol-3-phosphate dehydrogenase (GPD1) and
glycerol-3-phosphatase (GPP2) genes [20] In contrast, specific
halotolerance of yeast against extracellular NaCl or LiCl
is based on the induction of the ENA1/PMR2 gene
(designated as ENA1 in the following) encoding an enzyme
of the P-type ATPase family This cation-extrusion pump
promotes the efflux of Na+and Li+from the cell ENA1
expression is controlled by various different signalling
pathways [20–29] Salt-stress-dependent induction of ENA1
involves the Ca2+/calmodulin-activated protein
phospha-tase calcineurin [25], the TOR-GLN3 signalling pathway
[27] and possibly also an additional,
calcineurin-indepen-dent mechanism [24] Besides this, the alkaline response
regulator Rim101p [28] as well as glucose repression and
the HOG pathway contribute to ENA1 expression [20–
22,26,29] While many details, mostly of the downstream
parts of these pathways, have been elucidated, little is
known about the sensing mechanisms and the signalling
molecules involved Since in mammalian systems,
N-SMase has been recognized as a prominent effector of
sphingolipid-dependent stress responses [8,9,30], we were
interested to study whether, in yeast, the N-SMase
homologue, Isc1p, possibly serves as a stress signalling
mediator too Here, we report that ISC1 is required for the
development of yeast halotolerance against Na+and Li+
ions by means of HOG-independent induction of ENA1
expression
E X P E R I M E N T A L P R O C E D U R E S
Yeast strains, plasmids, chemicals and media
The yeast strains used in this study were JS91-15.23 (Mata,
ura3, trp1, his3, can1) and the ISC1 deletion strain DZY1
derived from it (MATa, Disc1::HIS5, ura3, trp1, his3, can1)
The HIS5 insertion cassette used for ISC1 disruption was
isolated, by short flanking homology PCR, from plasmid
pFA6a-HIS3MX containing the Schizosaccharomyces
pombe HIS5gene [31] The cassette exhibits, at both ends,
40 nucleotides of homology to positions 477–517 and 911–
951 of the ISC1 gene, respectively The ENA1 ORF was
isolated by PCR amplification of two adjacent regions of
S cerevisiaechromosomal DNA representing base pairs 1–
1182 and 1129–3273 of the ENA1 DNA sequence The two
fragments were ligated by means of two overlapping,
terminal BamHI sites and subsequently inserted, as a PvuII/
XhoI fragment, between the ADH1 promoter and
termina-tor regions of the multicopy yeast expression vectermina-tor,
pVT100-U [32] The resulting plasmid was pCWB20
Plasmid pDZ6 contained the ISC1 reading frame fused to
the MET25 promoter in the multicopy yeast expression
vector, p425MET25 [33] Plasmid pFR70 containing an
ENA1-lacZ promoter–reporter fusion was obtained from
Prof Rodriguez-Navarro, Madrid, Spain Bacillus cereus
sphingomyelinase (SMase) was purchased from Sigma The
fluorescent probes, BODIPYFL C5-sphingomyelin
(BSM) and BODIPYFL C5-ceramide (B-ceramide) were
from Molecular Probes Inc Complex (YPD) and synthetic
complete (SCD) yeast media as well as the appropriate SCD
omission media were prepared according to standard
protocols [34]
Sphingomyelinase assay The assay followed essentially the procedure described by Ella et al [35] Yeast cells suspended in 1 vol lysis buffer (20 mMTris/HCl pH 7.4, 10% glycerol, 50 mMKCl, 1 mM
dithiothreitol, 1 mMphenylmethanesulfonyl fluoride, 1 lM
pepstatin A, 10 lM leupeptin) were disrupted with glass beads Unbroken cells were removed by 5 min centrifuga-tion at 4000 g Total membranes were collected from the supernatant by centrifugation at 100 000 g for 1 h The fluorescent substrate, BSM, was subsequently used in a semiquantitative SMase assay Briefly, 55 lL of the mem-brane suspension were mixed with 45 lL 10 mMMes/KOH buffer pH 6.0 containing 400 mM KCl, 200 lM BSM,
10 mMMgCl2, 30 mM2-glycerophosphate After 1 h incu-bation at 30C, reaction products were extracted with chloroform/methanol (1 : 1) and separated by TLC on Silica G60 plates (Merck) The plates were developed with chloroform/methanol/water (60 : 35 : 8) and, subsequently, fluorescent spots were visualized and documented by a Fluorescence Binocular (Zeiss, Stemi SV11, 515–565 nm filter) according to the manufacturer’s indications Glycerol determination
Intracellular glycerol was determined enzymatically [36] using a commercial glycerol determination kit (Roche Diagnostics) Briefly, cells from mid-logarithmic phase cultures of wild-type or isc1D strains were transferred, at
an D600of 0.8, from normal YPD media to YPD containing 0.9MNaCl After 2 h growth at 30C, cells were harvested
by centrifugation, washed twice with isotonic saline at 4C and then placed into boiling 0.5M Tris/HCl pH 7.0 for
10 min After removing cell debris by 10 min centrifugation
at 15000 g, the glycerol in the supernatant was determined enzymatically
Measurement of intracellular Na+ and Li+concentrations
Determinations followed essentially the procedure described
by Gaxiola et al [37] In brief, cells were grown in YPD media to the densities indicated for the particular experi-ment After harvesting by centrifugation, cells were washed three times with 1.5M sorbitol For subsequent cell extraction, two alternative methods were used Method A: cells were permeabilized by incubation for 15 min at 95C
Na+and Li+were determined in the cleared extracts using
Na+and Li+specific lamps (L.O.T.-Oriel GmbH, Darms-tadt, Germany) in a Shimadzu AA-6200 atomic absorption flame emission spectrophotometer Method B: cells were washed and lyophilized The dry cells were incinerated at
840C for 6 h The residue was dissolved in 0.1NHCl and atomic absorption measurements were performed as described under method A
R E S U L T S
S cerevisiae ISC1 mutants are sensitive to Na+
and Li+ion stresses According to Sawai et al [1] disruption of the yeast ORF, ISC1, abolishes the in vitro SMase activity of the wild-type
Trang 3cell homogenate The characteristics of the Disc1::HIS5
deletion strain, DZY1, which was constructed in this work
are in accordance with these findings (Fig 1) SMase
activity was efficiently restored in isc1D cells upon
transfor-mation with plasmid pDZ6 encoding the intact ISC1 gene
(Fig 1) Comparable growth rates were observed with
wild-type and isc1D cells in normal YPD media not only at 30C
but also at elevated temperature (37C) or lowpH (pH 3.5)
stresses (Fig 2A) However, in the presence of 0.4–0.9M
NaCl, growth of the mutant was differentially reduced
(Fig 2B) and wild-type cells rapidly overgrew the mutants
(Fig 2A) After eight generations in 0.9M NaCl, the
proportion of isc1D cells had dropped to 2% of the
viable cells, which compares to > 80% isc1D cells surviving
in the absence of NaCl under otherwise identical conditions
(Fig 2A) On solid media, the differential sensitivity of
isc1D cells against elevated (0.4–0.5M) NaCl concentration,
was further confirmed and, in addition, a similar toxicity
was established for 0.01MLiCl (Fig 3) In contrast, 0.8M
KCl had no measurable inhibitory effect on isc1D growth
on solid media (Fig 3)
ISC1 functions independently of the HOG-pathway Adaptation of yeast to high salinity is, according to current knowledge, largely based on two different mechanisms, i.e induction of the HOG pathway responding to nonspecific osmostress [20–23], and induction of the ion extrusion pump Ena1p responding to toxic concentrations of Na+and Li+ ions [21,25–29] According to the data shown in Figs 2 and
3, isc1D cells are specifically sensitive to NaCl and LiCl, but tolerate high osmolarity of other solutes such as KCl (Fig 3) or glucose (data not shown) These characteristics argue against the HOG pathway being affected in the isc1D mutant In agreement with this conclusion, cellular glycerol levels increased to comparable levels in wild-type and isc1D cells upon raising the salinity and osmolarity of the media (Table 1) Thus, the HOG signalling pathway responded normally in the mutant not only with 1.5Msorbitol but also with 0.9MNaCl
ISC1 is involved in Na+
and Li+salt-induced expression
ofENA1 Stimulation of ENA1 expression has been recognized as a crucial response of yeast to extracellular high salinity [20– 29] The ENA1 encoded ATPase mediates Na+and Li+ion extrusion from the cell We therefore investigated whether the loss of halotolerance in isc1D cells was due to the failure
of ENA1 induction in the mutant For this, the ENA1-lacZ promoter–reporter construct in plasmid pFR70 was trans-formed into wild-type and isc1D cells The transformants expressing the bacterial lacZ gene under the control of the ENA1 promoter were challenged with 0.8M KCl, 0.8M
NaCl and 0.25M LiCl, respectively In the wild-type transformants, increasing concentrations of NaCl and LiCl caused the expected time- and concentration-dependent, strong induction of b-galactosidase activity (Fig 4) In the isc1D transformants, however, b-galactosidase induction
Fig 1 SMase activity in wild-type (JS91-15.23) and ISC1-disrupted
yeast cells From each strain 550 lg membrane protein w ere applied
to the fluorescent SMase assay as described in Experimental
proce-dures Purified Bacillus cereus SMase (0.1 U) was used in a control
assay The fluorescent sphingomyelin derivative BSM and its SMase
product, B-ceramide were run as references isc1D + pDZ6 w as a
transformant of the isc1D mutant with the ISC1 containing plasmid,
pDZ6.
Fig 2 Differential growth rates of wild-type
and isc1D cells under different stress conditions.
(A) One-to-one mixtures of wild-type
(JS91-15.23) and isc1D cells were inoculated into
SCD media and subsequently incubated under
the following conditions: 30 C (s), 37 C
(.), pH 3.5 (h), with 0.9 M NaCl (d) Both
strains had been precultivated in SCD media
up to mid-log phase At distinct time intervals,
aliquots of each culture were withdrawn and
plated onto SCD media After outgrowth the
cells were replica-plated onto
histidine-omis-sion media The ratio of histidine-positive
isc1D cells to nondisrupted, histidine-requiring
JS91-15.23 cells was then determined for each
sample (B) JS91-15.23 (d) and isc1D (s) cells
were grown separately in YPD media
con-taining 0.4 M NaCl Identical cell counts were
used for inoculation of the two strains.
Trang 4under these conditons was either negligible (LiCl) or
significantly ( 70%) lower (Fig 4) With 0.8MKCl, both
the rate and the level of b-galactosidase induction were
comparable in wild-type and isc1D transformants (Fig 4)
Analysis of ENA1 mRNA by Northern blot analyses
provided additional support to these enzymatic measure-ments (data not shown) Specific b-galactosidase inhibition
in ISC1 mutants was excluded as another reporter construct (INO1¢-lacZ) was expressed normally (data not shown) The basal level of ENA1 promoter activity as is observed with 0.8M KCl or w ith NaCl and LiCl in the isc1D mutant probably corresponds to the HOG-dependent portion of ENA1regulation which is apparently unaffected by ISC1 inactivation
In another series of experiments, intracellular sodium and lithium concentrations were determined by atomic absorption spectrometry upon challenging wild-type, isc1D and pCWB20-transformed isc1D cells with 0.8M NaCl and 0.25MLiCl, respectively It is seen that, after 1.5–4 h incubation, the sodium content in ISC1-defective cells was 25–35% above wild-type levels (Fig 5A) Correspondingly, lithium concentrations were 1.3- and 1.8-fold higher in isc1D than in wild-type cells, when these were exposed to LiCl and NaCl stresses, respectively (Fig 5B) These differences were not increased further by more extended stress exposure periods (data not shown) To demonstrate the correlation between Na+and Li+efflux and Ena1p
Fig 3 Effect of ISC1 and ENA1 gene expression on yeast cell growth under various salt stress conditions Wild-type (JS91-15.23) and isc1D cells were transformed with plasmid pCWB20 containing ENA1 under ADH1-promoter control Transformed and non-transformed cells were grown at 30 C on the indicated SCD and YPD solid media for 2 days.
Table 1 Glycerol content of wild-type and isc1D cells upon osmostress
application Wild-type (JS91-15.23) and isc1D cells were grown in YPD
liquid media to mid-logarithmic phase and subsequently transferred to
YPD media supplemented with 1.5 M sorbitol and 0.9 M NaCl,
respectively After 2 h incubation at 30 C, cells were collected by
centrifugation, washed twice with 0.9% NaCl and their glycerol
content was determined as described in Experimental procedures.
Growth
conditions
Glycerol content (g/L) Wild-type isc1D
+ 1.5 M sorbitol 16 15
Fig 4 Induction of ENA1-lacZ reporter expression in ISC1-positive (JS91-15.23, grey) and ISC1-disrupted (black) cells by NaCl, LiCl
or KCl salt stresses Cells were grown in YPD media to an OD 600 of 0.2 (A) and 0.3 (B), respectively, before NaCl, LiCl or KCl were added from appropriate stock solutions (3–6 M ) to give the indicated final concentra-tions Subsequent incubation was at 30 C for 4.5 h (B) or for the varying time periods indicated in (A) After harvesting by centrifu-gation, cells were permeabilized according to Gaxiola et al [37] and b-galactosidase mea-surements were performed as described by Miller [44] Solutions were cleared by centri-fugation before photometric measurement.
Trang 5activity, expression of ENA1 was stimulated by
transfor-mation of isc1D cells with pCWB20 On the multicopy
yeast plasmid pCWB20, ENA1 transcription is controlled
by a constitutive yeast promoter (ADH1) and is therefore
independent of salt stress In accordance with these
characteristics and with the presumed function of ENA1,
60–90% lower sodium and lithium levels were observed in
the isc1D/pCWB20 transformants even when compared
with the wild-type (Fig 5) As expected from their
increased ENA1 expression, the pCWB20 transformants
exhibited a distinctly higher salt tolerance than
nontrans-formed cells on NaCl- or LiCl-supplemented solid media
(cf Fig 3)
D I S C U S S I O N
The involvement of sphingolipids in cellular stress
responses appears to be conserved from yeast to
mam-mals [8,9] In mammalian systems, sphingomyelinases and
their product, ceramide, are particularly important
effec-tors not only in these but also in other signalling
pathways [6,7] In the present study, we report that the
ISC1-encoded yeast homolog of mammalian N-SMase is
involved in a cellular stress response, too We observed
that mutational inactivation of ISC1 leads to the loss of
cellular salt tolerance and renders the mutants specifically
sensitive to NaCl or LiCl stresses To our knowledge,
these data provide, for the first time, evidence for an
SMase-like activity participating in a stress signalling
pathway of yeast
A comparable sensitivity of the ISC1 mutant was not
observed to increased KCl concentrations or against
osmostress exerted by 1.5Msorbitol Glycerol production
in response to these conditions of general osmostress was
unimpaired indicating that the HOG signalling pathway
functioned normally in the mutant Similarly, ISC1
mutants were not particularly sensitive to high
tempera-ture or lowpH stresses Thus, ISC1-defective cells obviously exert a specifically increased sensitivity against
Na+ and Li+ toxicity In accordance with the known importance of the cation extrusion pump, Ena1p, for maintaining yeast halotolerance [21,25–28], we found that
in ISC1 null mutants expression of ENA1 was distinctly depressed Evidence for this was derived from differential expression studies with ENA1-b-galactosidase reporter constructs in ISC1-defective and isogenic wild-type cells The failure of ENA1 induction in the ISC1 mutant was not absolute but 60% of the wild-type reporter activity These findings agree with the known complexity of ENA1 regulation Clearly, only one of several possible routes of ENA1 activation is affected in the ISC1 mutants The fact that Ena1p activity is reduced but not absent in ISC1 mutants may be responsible for the only moderate increase in intracellular Na+ and Li+ levels: although they were distinctly higher than those in wild-type cells, the differences observed were not dramatic They never-theless correlate fairly well to the differential growth rates
of wild-type and mutant cells in the presence of 0.4M
NaCl (cf Fig 2B) Taken together, the data reported here suggest that in yeast, the ISC1-encoded sphingolipid phospholipase C makes a remarkable contribution to the
Na+/Li+-dependent induction of ENA1
Ceramide is reported to act, as a mammalian second messenger, on distinct protein kinases and protein phos-phatases which control cellular functions ranging from proliferation and differentiation to growth arrest and apoptosis [8,9] In particular, stimulation of protein phos-phatase PP2A by ceramide is conserved in yeast where it mediates the transient growth arrest upon heat stress [9,10,13–15] The induction of salt resistance being charac-terized by ENA1 activation rather than by cell cycle arrest is expected to followa different mechanism According to current knowledge, a prominent route of ENA1 induc-tion involves the Ca2+/calmodulin-dependent protein
Fig 5 Na + (A) and Li + (B) concentrations
in wild-type (JS91-15.23) and isc1D cells.
(A) Wild-type, isc1D and pCWB20
trans-formed isc1D cells were analysed after having
been exposed to 0.8 M NaCl at 30 C for the
indicated periods of time (B) The three strains
(identical symbols as in A) were incubated for
4 h at 30 C with 0.8 M NaCl and 0.25 M LiCl,
respectively Prior to stress application, cells
had been grow n in YPD media to OD 600 of 0.8
(A) and 0.3 (B) In (B) only Li+
concentra-tions were determined independent of the type
of stress Measurements of intracellular Na +
and Li+concentrations were performed
according to either method A (A) or B (B) as
described in Experimental procedures.
Trang 6phosphatase calcineurin [23–25] The target protein of
calcineurin action in yeast is the zinc-finger transcription
factor Crz1p [38,39] Crz1p dephosphorylation initiates its
nuclear import and, subsequently, its binding to the
calcineurin-dependent response element in a variety of
promoters including that of ENA1 As an alternative, a
ceramide-activated phosphatase rather than calcineurin
may be considered to dephosphorylate Crz1p Another
possible mechanism of sphingolipid-dependent ENA1
induction may be connected with the role of sphingolipids
in cellular Ca2+homeostasis [40] For instance, raising the
intracellular Ca2+level is expected to stimulate calcineurin
activity and, thus, ENA1 expression In mammalian cells
various glycerophosphoinositide-specific phospholipases C
function in Ca2+signalling pathways by generating inositol
1,4,5-triphosphate as a second messenger [41] This
messen-ger subsequently releases Ca2+ from intracellular stores
Although a homologue to the respective mammalian
recep-tor is not evident from the yeast genome, an analogous
inositol derivative released by the Isc1p phospholipase C
from an appropriate sphingolipid could fulfil a similar
function As the pathways of sphingolipid metabolism are
highly interconnected, ISC1 and its product, ceramide, must
not be viewed as isolated signalling elements Instead,
ceramide is possibly further metabolized to the true bioactive
effector In this context, a recent report by Birchwood et al
[42] on sphingosine-1-phosphate or related molecules as
stimuli of Ca2+influx and signalling in yeast is of particular
interest The authors report that these compounds
repre-senting intermediates of both sphingolipid biosynthesis and
degradation elevate intracellular Ca2+levels and activate
calcineurin-signalling pathways The transient accumulation
of Ca2+ due to the increase of phyto- and
dihydrosp-hingosine-1-phosphate is well established as a heat shock
response of yeast [12,43] Possibly, a comparable effect is
involved in the salt stress response of yeast
Unlike with heat stress, no alteration of the cellular
ceramide content is observed upon salt stress application to
yeast [5] During heat stress, total ceramides and long-chain
sphingoid base phosphates increase several-fold These
changes are thought to occur as a result of increased
sphingolipid biosynthesis and appear to be required for the
development of thermotolerance rather than for signalling
reactions [5] Due to the abundance and structural
com-plexity of yeast sphingolipids, the breakdown of a single
species or a small percentage of them for the purpose of
signal generation would be difficult to detect Nevertheless,
subtle differences were observed by us (D Zajonc &
E Schweizer, unpublished data) and by Sawai et al [1]
between sphingolipid patterns of wild-type and
ISC1-defective strains Obviously, only a minor fraction of yeast
sphingolipids is susceptible to Isc1p degradation Chemical
characterization of these Isc1p substrates deserves further
investigation
Remarkably, Isc1p activity is detected in extracts of
wild-type yeast even in the absence of salt stress The
activity is not significantly stimulated upon growth in the
presence of 0.8M NaCl (data not shown) Even though
these results need to be reconfirmed once the physiological
substrate of Isc1p is known, the data may indicate that
salt stress-induced activation of Isc1p occurs at a
post-transcriptional level, possibly by its interaction with a
molecule produced further upstream in the signal
trans-duction chain Thus, in vitro and in vivo activities of Isc1p should probably be differentiated, especially as the enzyme
is known to require distinct phospholipid cofactors for full activity [1] Hence, not only the bioactive messenger produced by Isc1p and the mechanism of its action but also the salt-sensing process involving Isc1p activation needs to be studied further
A C K N O W L E D G E M E N T S
This work was supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie We thank Prof Alfonso Rodriguez-Navarro (Madrid) for kindly providing plasmid pFR70.
R E F E R E N C E S
1 Sawai, H., Okamoto, Y., Luberto, C., Mao, C., Bielawska, A., Domae, N & Hannun, Y.A (2000) Identification of ISC1 (YER019w) as inositol phosphosphingolipid phospholipase C in Saccharomyces cerevisiae J Biol Chem 275, 39793–39798.
2 Tomiuk, S., Hofmann, K., Nix, M., Zumbansen, M & Stoffel, W (1998) Cloned mammalian neutral sphingomyelinase: functions
in sphingolipid signaling? Proc Natl Acad Sci USA 95, 3638–3643.
3 Okazaki, T., Bielawska, A., Domae, N., Bell, R.M & Hannun, Y.A (1994) Characteristics and partial purification of a novel cytosolic, magnesium-independent, neutral sphingomyelinase activated in the early signal transduction of 1 alpha,25-dihydroxy-vitamin D3-induced HL-60 cell differentiation J Biol Chem 269, 4070–4077.
4 Dickson, R.C & Lester, R.L (1999) Yeast sphingolipids Biochim Biophys Acta 1426, 347–357.
5 Dickson, R.C & Lester, R.L (1999) Metabolism and selected functions of sphingolipids in the yeast Saccharomyces cerevisiae Biochim Biophys Acta 1438, 305–321.
6 Kolesnick, R.N & Kronke, M (1998) Regulation of ceramide production and apoptosis Annu Rev Physiol 60, 643–665.
7 Hannun, Y.A., Luberto, C & Argraves, K.M (2001) Enzymes of sphingolipid metabolism: from modular to integrative signaling Biochemistry 40, 4893–4903.
8 Mathias, S., Pena, L.A & Kolesnick, R.N (1998) Signal trans-duction of stress via ceramide Biochem J 335, 465–480.
9 Perry, D.K & Hannun, Y.A (1998) The role of ceramide in cell signaling Biochim Biophys Acta 1436, 233–243.
10 Fishbein, J.D., Dobrow sky, R.T., Bielaw ska, A., Garrett, S & Hannun, Y.A (1993) Ceramide-mediated growth inhibition and CAPP are conserved in Saccharomyces cerevisiae J Biol Chem.
268, 9255–9261.
11 Patton, J.L., Srinivasan, B., Dickson, R.C & Lester, R.L (1992) Phenotypes of sphingolipid-dependent strains of Saccharomyces cerevisiae J Bacteriol 174, 7180–7184.
12 Mandala, S.M., Thornton, R., Tu, Z., Kurtz, M.B., Nickels, J., Broach, J., Menzeleev, R & Spiegel, S (1998) Sphingoid base 1-phosphate phosphatase: a key regulator of sphingolipid metabolism and stress response Proc Natl Acad Sci USA 95, 150–155.
13 Jenkins, G.M., Richards, A., Wahl, T., Mao, C., Obeid, L & Hannun, Y.A (1997) Involvement of yeast sphingolipids in the heat stress response of Saccharomyces cerevisiae J Biol Chem.
272, 32566–32572.
14 Wells, G.B., Dickson, R.C & Lester, R.L (1998) Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de novo synthesis J Biol Chem 273, 7235–7243.
15 Nickels, J.T & Broach, J.R (1996) A ceramide-activated protein phosphatase mediates ceramide-induced G1 arrest of Saccharo-myces cerevisiae Genes Dev 10, 382–394.
Trang 716 Wells, G.B & Lester, R.L (1983) The isolation and
character-ization of a mutant strain of Saccharomyces cerevisiae that
requires a long chain base for growth and for synthesis of
phos-phosphingolipids J Biol Chem 258, 10200–10203.
17 Lester, R.L., Wells, G.B., Oxford, G & Dickson, R.C (1993)
Mutant strains of Saccharomyces cerevisiae lacking sphingolipids
synthesize novel inositol glycerophospholipids that mimic
sphin-golipid structures J Biol Chem 268, 845–856.
18 De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T &
Wiemken, A (1994) The role of trehalose synthesis for the
acquisition of thermotolerance in yeast I Genetic evidence that
trehalose is a thermoprotectant Eur J Biochem 219, 179–186.
19 Singer, M.A & Lindquist, S (1998) Thermotolerance in
Saccharomyces cerevisiae: the Yin and Yang of trehalose Trends
Biotechnol 16, 460–468.
20 Rep, M., Albertyn, J., Thevelein, J.M., Prior, B.A & Hohmann,
S (1999) Different signalling pathways contribute to the control of
GPD1 gene expression by osmotic stress in Saccharomyces
cere-visiae Microbiology 145, 715–727.
21 Proft, M & Serrano, R (1999) Repressors and upstream
repres-sing sequences of the stress-regulated ENA1 gene in
Saccharo-myces cerevisiae: bZIP protein Sko1p confers HOG-dependent
osmotic regulation Mol Cell Biol 19, 537–546.
22 Gustin, M.C., Albertyn, J., Alexander, M & Davenport, K.
(1998) MAP kinase pathways in the yeast Saccharomyces
cerevi-siae Microbiol Mol Biol Rev 62, 1264–1300.
23 Ferrando, A., Kron, S.J., Rios, G., Fink, G.R & Serrano, R.
(1995) Regulation of cation transport in Saccharomyces
cerevisiae by the salt tolerance gene HAL3 Mol Cell Biol 15,
5470–5481.
24 Ganster, R.W., McCartney, R.R & Schmidt, M.C (1998)
Iden-tification of a calcineurin-independent pathway required for
sodium ion stress response in Saccharomyces cerevisiae Genetics
150, 31–42.
25 Mendoza, I., Rubio, F., Rodriguez-Navarro, A & Pardo, J.M.
(1994) The protein phosphatase calcineurin is essential for NaCl
tolerance of Saccharomyces cerevisiae J Biol Chem 269, 8792–
8796.
26 Marquez, J.A & Serrano, R (1996) Multiple transduction
path-ways regulate the sodium-extrusion gene PMR2/ENA1 during salt
stress in yeast FEBS Lett 382, 89–92.
27 Crespo, J.L., Daicho, K., Ushimaru, T & Hall, M.N (2001) The
GATA Transcription Factors GLN3 and GAT1 Link TOR to
Salt Stress in Saccharomyces cerevisiae J Biol Chem 276, 34441–
34444.
28 Lamb, T.M., Xu, W., Diamond, A & Mitchell, A.P (2001)
Alkaline Response Genes of Saccharomyces cerevisiae and Their
Relationship to the RIM101 Pathway J Biol Chem 276, 1850–
1856.
29 Alepuz, P.M., Cunningham, K.W & Estruch, F (1997) Glucose
repression affects ion homeostasis in yeast through the regulation
of the stress-activated ENA1 gene Mol Microbiol 26, 91–98.
30 Levade, T & Jaffrezou, J.P (1999) Signalling sphingomyelinases: which, where, how and why? Biochim Biophys Acta 1438, 1–17.
31 Wach, A., Brachat, A., Alberti-Segui, C., Rebischung, C & Philippsen, P (1997) Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae Yeast 13, 1065–1075.
32 Vernet, T., Dignard, D & Thomas, D.Y (1987) A family of yeast expression vectors containing the phage f1 intergenic region Gene
52, 225–233.
33 Mumberg, D., Muller, R & Funk, M (1994) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds Nucleic Acids Res 22, 5767–5768.
34 Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A., Seidmann, J.G & Struhl, K (1987) Current Protocols in Molecular Biology John Wiley & Sons Ltd., NewYork.
35 Ella, K.M., Qi, C., Dolan, J.W., Thompson, R.P & Meier, K.E (1997) Characterization of a sphingomyelinase activity in Saccharomyces cerevisiae Arch Biochem Biophys 340, 101–110.
36 Albertyn, J., Hohmann, S., Thevelein, J.M & Prior, B.A (1994) GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cere-visiae, and its expression is regulated by the high-osmolarity gly-cerol response pathway Mol Cell Biol 14, 4135–4144.
37 Gaxiola, R., de Larrinoa, I.F., Villalba, J.M & Serrano, R (1992)
A novel and conserved salt-induced protein is an important determinant of salt tolerance in yeast EMBO J 11, 3157–3164.
38 Polizotto, R.S & Cyert, M.S (2001) Calcineurin-dependent nuclear import of the transcription factor Crz1p requires Nmd5p.
J Cell Biol 154, 951–960.
39 Matheos, D.P., Kingsbury, T.J., Salma Ahsan, U & Cunningham, K.W (1997) Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae Genes Dev 11, 3445–3458.
40 Beeler, T., Bacikova, D., Gable, K., Hopkins, L., Johnson, C., Slife, H & Dunn, T (1998) The Saccharomyces cerevisiae TSC10/YBR265wgene encoding 3-ketosphinganine reductase is identified in a screen for temperature-sensitive suppressors of the Ca2+-sensitive csg2Delta mutant J Biol Chem 273, 30688– 30694.
41 Rhee, S.G (2001) Regulation of phosphoinositide-specific phospholipase C Ann Rev Biochem 70, 281–312.
42 Birchwood, C.J., Saba, J.D., Dickson, R.C & Cunningham, K.W (2001) Calcium influx and signaling in yeast stimulated by intracellular sphingosine 1-phosphate accumulation J Biol Chem 276, 11712–11718.
43 Skrzypek, M.S., Nagiec, M.M., Lester, P.L & Dickson, R.C (1999) Analysis of phosphorylated sphingolipid long-chain bases reveals potential roles in heat stress and grow th control in Saccharomyces J Bacteriol 181, 1134–1140.
44 Miller, J.H (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 352–355.