Báo cáo khoa học: ISC1-encoded inositol phosphosphingolipid phospholipase C is involved in Na+/Li+ halotolerance of Saccharomyces cerevisiae pptx

ISC1-encoded inositol phosphosphingolipid phospholipase C
is involved in Na
+
/Li
+
halotolerance of
Saccharomyces cerevisiae
Christian Betz
1
, Dirk Zajonc
1
, Matthias Moll
2
and Eckhart Schweizer
1
1
Lehrstuhl fu
¨
r Biochemie and the
2
Lehrstuhl 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 0.5
M
NaCl or 0.01
M
LiCl. In con-
trast, K
+
tolerance and general osmostress regulation
were unaffected. Isc1D mutant growth with 0.9
M
KCl and

glycerol accumulation in the presence of 0.9
M
NaCl or
1.5
M
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 Mg
2+

-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 Ca
2+
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 low pH 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:
Abbreviations: (N-)SMase, (neutral)sphingomyelinase; HOG, high-

osmolarity glycerol; BSM, BODIPYÒFL-C
5
N-(4,4-difluoro-5,7-
dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingomyelin;
B-ceramide, BODIPYÒFL 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)
Eur. J. Biochem. 269, 4033–4039 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03096.x
pathway 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 Ca
2+

/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.
EXPERIMENTAL PROCEDURES
Yeast strains, plasmids, chemicals and media
The yeast strains used in this study were JS91-15.23 (Mata,
ura3, trp1, his3, can1)andtheISC1 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 HIS5 gene [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. cerevisiae chromosomal 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 vector,
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, BODIPYÒFL C5-sphingomyelin
(BSM) and BODIPYÒFL 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 m
M
Tris/HCl pH 7.4, 10% glycerol, 50 m
M
KCl, 1 m

M
dithiothreitol, 1 m
M
phenylmethanesulfonyl fluoride, 1 l
M
pepstatin A, 10 l
M
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 lL10m
M
Mes/KOH
buffer pH 6.0 containing 400 m
M
KCl, 200 l
M
BSM,
10 m
M
MgCl
2
,30m
M
2-glycerophosphate. After 1 h incu-
bation at 30 °C, 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 D
600
of 0.8, from normal YPD media to YPD containing
0.9
M
NaCl. After 2 h growth at 30 °C, cells were harvested
by centrifugation, washed twice with isotonic saline at 4 °C
and then placed into boiling 0.5
M
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 1.5
M
sorbitol. For subsequent cell
extraction, two alternative methods were used. Method A:
cells were permeabilized by incubation for 15 min at 95 °C.
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
840 °C for 6 h. The residue was dissolved in 0.1
N
HCl and
atomic absorption measurements were performed as
described under method A.
RESULTS
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
4034 C. Betz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
cell 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 30 °C
but also at elevated temperature (37 °C) or low pH (pH 3.5)
stresses (Fig. 2A). However, in the presence of 0.4–0.9
M
NaCl, growth of the mutant was differentially reduced
(Fig. 2B) and wild-type cells rapidly overgrew the mutants
(Fig. 2A). After eight generations in 0.9
M
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 (0.4–0.5
M
) NaCl concentration,
was further confirmed and, in addition, a similar toxicity
was established for 0.01
M

LiCl (Fig. 3). In contrast, 0.8
M
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 1.5
M
sorbitol but also
with 0.9
M
NaCl.
ISC1

is involved in Na
+
and Li
+
salt-induced expression
of
ENA1
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 0.8
M
KCl, 0.8
M
NaCl and 0.25
M
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 lgmembraneproteinwereapplied
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 +pDZ6was 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)andisc1D (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.
Ó FEBS 2002 Salt-stress signalling in yeast (Eur. J. Biochem. 269) 4035
under these conditons was either negligible (LiCl) or
significantly ( 70%) lower (Fig. 4). With 0.8
M
KCl, both
therateandthelevelofb-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
0.8
M
KClorwithNaClandLiClintheisc1D mutant
probably corresponds to the HOG-dependent portion of
ENA1 regulation 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 0.8
M
NaCl
and 0.25
M
LiCl, 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 °Con
theindicatedSCDandYPDsolidmedia
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
YPD media 1 3
+ 1.5
M
sorbitol 16 15
+ 0.9
M
NaCl 38 41
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
mediatoanOD
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 °Cfor
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.
4036 C. Betz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
activity, 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).
DISCUSSION
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 1.5
M
sorbitol. 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 low pH 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 0.4
M
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 follow a different mechanism. According to
current knowledge, a prominent route of ENA1 induc-

tion involves the Ca
2+
/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 °Cforthe
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
hadbeengrowninYPDmediatoOD
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.
Ó FEBS 2002 Salt-stress signalling in yeast (Eur. J. Biochem. 269) 4037
phosphatase 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.Asanalternative,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 Ca
2+
homeostasis [40]. For instance, raising the
intracellular Ca
2+
level is expected to stimulate calcineurin
activity and, thus, ENA1 expression. In mammalian cells
various glycerophosphoinositide-specific phospholipases C
function in Ca
2+
signalling pathways by generating inositol
1,4,5-triphosphate as a second messenger [41]. This messen-
ger subsequently releases Ca

2+
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 Ca
2+
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 Ca
2+
levels and activate
calcineurin-signalling pathways. The transient accumulation
of Ca
2+
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 0.8
M
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.
ACKNOWLEDGEMENTS
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.
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