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Eur. J. Biochem. 269, 4033–4039 (2002)  FEBS 2002 doi:10.1046/j.1432-1033.2002.03096.x ISC1-encoded inositol phosphosphingolipid phospholipase C is involved in Na+/Li+ halotolerance of Saccharomyces cerevisiae Christian Betz1, Dirk Zajonc1, Matthias Moll2 and Eckhart Schweizer1 1 Lehrstuhl für Biochemie and the 2Lehrstuhl für Anorganische und Allgemeine Chemie, Universität Erlangen-Nürnberg, 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 transduction pathways are known correlating high salinity to the transcriptional activation of ENA1. Nevertheless, information on the actual sensing mechanism initiating these pathways is limited. Here, we report that the ISC1-encoded phosphosphingolipid-specific phospholipase C appears to be involved in stimulation of ENA1 expression and, consequently, 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 contrast, 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. Correspondingly, 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. 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) sphingomyelinase (SMase) and, thus, resembles the most prominent 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 low pH stresses [4,5,17]. From these results, the involvement of sphingolipids 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 osmoadaptation 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 für Biochemie; Universität 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, highosmolarity glycerol; BSM, BODIPY
FL-C5 N-(4,4-difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingomyelin; B-ceramide, BODIPY
FL C5-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) Keywords: salt-stress; signaling; sphingolipids; sphingolipid phospholipase C; yeast.  FEBS 2002 4034 C. Betz et al. (Eur. J. Biochem. 269) pathway comprises a mitogen-activated protein kinase cascade which finally initiates transcription of the glycerol-3-phosphate dehydrogenase (GPD1) and glycerol-3phosphatase (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 phosphatase calcineurin [25], the TOR-GLN3 signalling pathway [27] and possibly also an additional, calcineurin-independent 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) 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 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 terminator 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, BODIPY
FL C5-sphingomyelin (BSM) and BODIPY
FL 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 mM Tris/HCl pH 7.4, 10% glycerol, 50 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 lM pepstatin A, 10 lM leupeptin) were disrupted with glass beads. Unbroken cells were removed by 5 min centrifugation 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 membrane suspension were mixed with 45 lL 10 mM Mes/KOH buffer pH 6.0 containing 400 mM KCl, 200 lM BSM, 10 mM MgCl2, 30 mM 2-glycerophosphate. After 1 h incubation 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 D600 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 experiment. 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, Darmstadt, 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  FEBS 2002 Salt-stress signalling in yeast (Eur. J. Biochem. 269) 4035 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 Fig. 1. SMase activity in wild-type (JS91-15.23) and ISC1-disrupted yeast cells. From each strain  550 lg membrane protein were applied to the fluorescent SMase assay as described in Experimental procedures. 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 was a transformant of the isc1D mutant with the ISC1 containing plasmid, pDZ6. 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 transformation with plasmid pDZ6 encoding the intact ISC1 gene (Fig. 1). Comparable growth rates were observed with wildtype 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 Fig. 2. Differential growth rates of wild-type and isc1D cells under different stress conditions. (A) One-to-one mixtures of wild-type (JS9115.23) 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-omission 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 containing 0.4 M NaCl. Identical cell counts were used for inoculation of the two strains. 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 transformed 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  FEBS 2002 4036 C. Betz et al. (Eur. J. Biochem. 269) 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 ADH1promoter control. Transformed and nontransformed 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. Glycerol content (g/L) Growth conditions Wild-type isc1D YPD media + 1.5 M sorbitol + 0.9 M NaCl 1 16 38 3 15 41 under these conditons was either negligible (LiCl) or significantly ( 70%) lower (Fig. 4). With 0.8 M KCl, 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 measurements (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 KCl or with NaCl and LiCl in the isc1D 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. 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 OD600 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 concentrations. Subsequent incubation was at 30 C for 4.5 h (B) or for the varying time periods indicated in (A). After harvesting by centrifugation, cells were permeabilized according to Gaxiola et al. [37] and b-galactosidase measurements were performed as described by Miller [44]. Solutions were cleared by centrifugation before photometric measurement.  FEBS 2002 Salt-stress signalling in yeast (Eur. J. Biochem. 269) 4037 Fig. 5. Na+ (A) and Li+ (B) concentrations in wild-type (JS91-15.23) and isc1D cells. (A) Wild-type, isc1D and pCWB20 transformed 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 grown in YPD media to OD600 of 0.8 (A) and 0.3 (B). In (B) only Li+ concentrations 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. activity, expression of ENA1 was stimulated by transformation 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 nontransformed 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 mammals [8,9]. In mammalian systems, sphingomyelinases and their product, ceramide, are particularly important effectors 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 nevertheless 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 phosphatases which control cellular functions ranging from proliferation and differentiation to growth arrest and apoptosis [8,9]. In particular, stimulation of protein phosphatase 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 characterized 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 induction involves the Ca2+/calmodulin-dependent protein  FEBS 2002 4038 C. Betz et al. (Eur. J. Biochem. 269) 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. 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 messenger subsequently releases Ca2+ from intracellular stores. Although a homologue to the respective mammalian receptor 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 representing 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 dihydrosphingosine-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 complexity 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 ISC1defective 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 posttranscriptional 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. REFERENCES 1. Sawai, H., Okamoto, Y., Luberto, C., Mao, C., Bielawska, A., Domae, N. & Hannun, Y.A. 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