1 sphingolipids regulate the yeast high osmolarity-responsive hog

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Sphingolipids regulate the yeast high osmolarity-responsive HOG pathway 1

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Mirai Tanigawaa, Akio Kiharab, Minoru Terashimaa, Terunao Takaharaa, and Tatsuya 3

Maedaa# 4

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Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japana, 6

and Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japanb 7

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Address correspondence to Tatsuya Maeda, [email protected] 9

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Running title: Sphingolipids regulate the HOG pathway 11

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Word count 14

Material and Methods: 1,493 words 15

Introduction, Results, and Discussion: 3,695 words 16

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.06111-11 MCB Accepts, published online ahead of print on 14 May 2012

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ABSTRACT 18

The yeast high osmolarity-responsive MAP kinase pathway, the HOG pathway, is 19

activated in response to hyperosmotic stress via two independent osmosensing 20

branches, the Sln1 branch and the Sho1 branch. While the mechanism by which the 21

osmosensing machinery activates the downstream MAP kinase cascade has been 22

well-studied, the mechanism by which the machinery senses and responds to 23

hyperosmotic stress remains to be clarified. Here we report that inhibition of the de 24

novo sphingolipid synthesis pathway results in activation of the HOG pathway via 25

both branches. Inhibition of ergosterol biosynthesis also induces activation of the 26

HOG pathway. Sphingolipids and sterols are known to be tightly packed together in 27

cell membranes to form partitioned domains called rafts. Raft-enriched detergent 28

resistant membranes (DRMs) contain both Sln1 and Sho1, and sphingolipid depletion 29

and hyperosmotic stress have similar effects on the osmosensing machinery of the 30

HOG pathway: dissociation of an Sln1-containing protein complex and elevated 31

association of Sho1 with DRMs. These observations reveal the sphingolipid-mediated 32

regulation of the osmosensing machinery of the HOG pathway. 33

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INTRODUCTION 35

The osmolarity response is a fundamental stress response that is observed in all cells. 36

The high osmolarity glycerol (HOG) MAP kinase (MAPK) pathway in Saccharomyces 37

cerevisiae plays a central role in survival and adaptation under high osmolarity 38

environments (Fig. 1A) (25). In S. cerevisiae, high osmolarity stress induces rapid 39


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activation of the MAPK Hog1 through phosphorylation by the MAPK kinase (MAPKK) 40

Pbs2. The HOG pathway has two osmosensing branches, the Sln1 branch and the Sho1 41

branch, and these two branches independently activate Pbs2. In the Sln1 branch, the 42

transmembrane histidine kinase Sln1, which is a homolog of bacterial two-component 43

signal transducers, serves as the osmosensor. Under normal osmolarity conditions, Sln1 44

forms a dimer and auto-phosphorylates a histidine residue in its own histidine kinase 45

domain. This phosphate group is sequentially transferred to an aspartic acid residue in the 46

Sln1 receiver domain, then to a histidine residue in the histidine phosphotransfer protein 47

Ypd1, and finally to an aspartic acid residue in the response regulator Ssk1. High 48

osmolarity inactivates Sln1, and thereby increases unphosphorylated Ssk1, which, in turn, 49

binds to and activates the redundant MAPKK kinases (MAPKKKs) Ssk2 and Ssk22. 50

Activated Ssk2 and Ssk22 then phosphorylate and activate Pbs2 (18, 27, 28, 35). In the 51

Sho1 branch, the recently identified mucin-like proteins Msb2 and Hkr1 interact with, and 52

transmit the signal to the tetraspanning protein Sho1. Sho1 then serves as a facilitator of a 53

signaling module assembly that includes Cdc42, Ste20, MAPKKK Ste11, Ste50, and Pbs2 54

(36, 37). This assembly leads to Ste11 activation by Cdc42-activated Ste20, and to 55

subsequent Pbs2 activation by thus activated Ste11. Although Sln1 is proposed to monitor 56

changes in turgor pressure induced by osmotic stress (29), the exact mechanism by which 57

the osmosensing machinery, especially that of the Sho1 branch, senses and responds to 58

osmotic stress, and by which it is kept inactive under normal osmotic environments, 59

remains to be clarified. To address these issues, we here performed a genetic screen to 60

isolate mutants that exhibit constitutive activation of the HOG pathway under normal 61


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osmotic environments. We found that a mutation in LCB2, which encodes a component of 62

serine palmitoyltransferase (SPT) (19), results in activation of the HOG pathway. Yeast SPT 63

is comprised of Lcb1, Lcb2, and a minor subunit Tsc3, and catalyzes the first step of 64

sphingolipids biosynthesis, condensation of serine with palmitoyl-CoA (Fig. 1B). SPT is 65

essential for yeast growth and is conserved among all organisms that make sphingolipids. 66

Sphingolipids and sterols are tightly packed together with specific proteins in cell 67

membranes, resulting in the formation of partitioned membrane domains called rafts or 68

membrane microdomains (15). Rafts have been suggested to have roles in cell signaling 69

and membrane trafficking through directly changing local membrane structure or by 70

allowing protein-protein interactions to occur with higher affinity or specificity, thereby 71

functioning as platforms for up- or down-regulation of these processes (6, 8). Recent 72

studies have shown that certain proteins are transiently associated with rafts, by either 73

entering or leaving these compartments in response to various stimuli such as ligand 74

binding, heat, or oxidative stress (3, 26, 34). These observations suggest that rafts 75

dynamically control physiological events including stress responses via regulation of the 76

localization of those proteins. While it has been suggested that heat stress affects raft 77

structure through hyper-fluidization of the membrane (20), whether and how other stresses 78

including osmotic stress act on rafts and lead to cellular responses remains unknown. 79

In this study, we found that inhibition of sphingolipids or ergosterol biosynthesis induces 80

activation of the HOG pathway. We also show that both Sln1 and Sho1 are distributed in 81

raft-enriched detergent resistant membranes (DRMs). Furthermore, both sphingolipid 82

depletion and osmotic stress similarly lead to dissociation of an Sln1-containing protein 83


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complex and elevated association of Sho1 with DRMs. Based on these observations, we 84

suggest that rafts are involved in the regulation of the osmosensing mechanisms of the 85

HOG pathway. 86

87

MATERIALS AND METHODS 88

Yeast strains and growth conditions. The yeast strains used are listed in Table 1. All 89

strains used were in the S288C background except where noted otherwise. SHO1, SSK2, 90

and SSK22 genes were disrupted as previously described (17). The STE11 gene in MH273 91

and MH322 was disrupted by transformation with pNC276 (31), and that in MH276 was 92

disrupted by replacement with a loxP-kanMX-loxP cassette that was amplified by PCR as 93

described previously (9). MH293, MH296, MH305, MH302, and MH314 were constructed 94

by replacing ELO1, CSG1, or SUR2 of TM100, CSH1 of TM101, and FEN1 of TM141, 95

respectively, with corresponding kanMX4 cassettes that were amplified with PCR using 96

genomic DNA of the knockout collection strains as templates. MH417 and MH425 were 97

constructed by replacing ERG3 of TM225 and ERG2 of TM141, respectively, with 98

His3MX6 cassettes that were amplified by PCR as previously described (16). MH309, 99

MH311, and MH421 were constructed by crossing MH296 with MH302, TM221 with 5281, 100

and MH280 with MH417, respectively. Epitope-tags and EGFP were attached to the 101

C-termini of Sho1 and Sln1 using a standard PCR-based gene modification (12, 16). 102

Chromosomal LCB2 was replaced with lcb2-M1 using a standard two-step gene 103

replacement method (33). MH335 and MH337 were constructed by crossing MH329 with 104

MH331. For all experiments except for those shown in Fig. 2A, B and Fig. 6B, cells were 105


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grown to early-log phase in YPD medium at 30 °C. For Fig. 2A and B, the cells were 106

cultured in SC-Trp and SC-Trp Ura, respectively, supplemented with 180 μg/mL Leu 107

medium overnight, and were spotted onto the indicated plates. For Fig. 6B, the cells were 108

cultured in SC-Ura medium to mid-log phase. 109

Plasmids. pMT304 was created as follows. BamHI sites were introduced both upstream 110

and downstream of the HOG1 ORF by PCR, and the fragment was cloned into the BamHI 111

site of pBTM116 (40) to create pNS493. pNS493 was digested with SphI, and the fragment 112

containing LexA-HOG1, together with the ADH1 promoter and the ADH1 terminator, was 113

subcloned into the SphI site of YCplac111 to create pMT301. Finally, the 2.7-kb XbaI-PvuII 114

fragment of pMT301 was cloned into the SpeI-HincII sites of pRS414. The NaeI-ScaI 115

fragment containing 8×CRE-lacZ of pKT760 (gift from Dr. Saito, the University of Tokyo) 116

was introduced into the NaeI-ScaI site of pRS415 to create pMH41. To create pMH29, a 117

genomic EcoRI-ApaLI (blunted) fragment containing LCB2 was cloned into the 118

EcoRI-SmaI sites of pRS416. pFP15 (pRS416-SHO1-EGFP) was a gift from Dr. Saito. An 119

SLN1-EGFP fragment containing the SLN1 promoter region was obtained from MH324 by 120

a gap-repair method using pPD2146 (18) as a recipient, and was cloned into pRS426 to 121

create pMH54. 122

Selection for Hog1-activating mutants. TM415 carrying pMT304 was cultivated in 123

SC-Trp supplemented with 180 μg/mL Leu overnight, mutagenized with ethyl 124

methanesulfonate (EMS) as described previously (11) (viability 55%), diluted, and were 125

plated onto SC-Trp His plates supplemented with 180 μg/mL Leu. After 3 days incubation 126

at 30 °C, candidates His+ mutants were selected and retested on the same plates. The 467 127


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His+ mutants thus selected from 2.5 × 105 mutagenized cells were then subjected to 128

Western blotting in order to identify mutants that exhibit elevated Hog1 phosphorylation 129

compared with wild-type cells, and 24 mutants were selected. Among these, mutants that 130

showed temperature-sensitive growth as well as relatively high Hog1 phosphorylation were 131

crossed with TM415α, and were subjected to tetrad analysis to test their monogenic 132

segregation patterns, as well as cosegregation of two phenotypes: Hog1 phosphorylation 133

and temperature-sensitive growth. Only one mutant met these conditions and a healthy 134

tetrad product of the backcross (MH266) was used for subsequent experiments. 135

Treatment of cells with sphingolipids, inhibitors, or filipin. Five or 10 μM 136

phytosphingosine (PHS) (Sigma, St Louis, MO), or 10 μM C2-ceramide (Sigma) was 137

added to the culture of TM141 (wild-type) or MH280 (lcb2-M1) cells, and incubated for 138

1-4 h at 30 °C. Two μg/ml myriocin (Sigma) and 500 ng/ml aureobasidin A (Takara Bio, 139

Shiga, Japan) were added to the culture of TM141 or MH335 (SLN1-9myc SHO1-GFP) 140

cells, which were then incubated for the indicated times at 30 °C. Five μg/ml of filipin 141

complex (Sigma) was added to the logarithmically growing YPD culture of TM141, 142

TM252 (ssk2Δ ssk22Δ), or TM287 (sho1Δ) cells. 143

Detection of phosphorylated Hog1, Fus3, and Kss1. Cells were harvested by 144

centrifugation at 1,200 × g for 30 sec, suspended in Laemmli sample buffer, and 145

immediately boiled for 4 min. Samples were then centrifuged at 9,000 × g for 3 min and the 146

supernatant was subjected to SDS-PAGE. After electrophoretic transfer onto PVDF 147

membranes, the membranes were immunoblotted with the polyclonal anti-phospho-p38 148

antibody D3F9 (Cell Signaling Technology, Danvers, MA), the polyclonal anti-Hog1 149


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antibody yC-20 (Santa Cruz Biotechnology, Santa Cruz, CA), the polyclonal 150

anti-phospho-p44/p42 MAPK antibody 9101 (Cell Signaling Technology), or the 151

polyclonal anti-Kss1 antibody y-50 (Santa Cruz Biotechnology). Membranes were then 152

treated with the appropriate secondary antibody conjugated with horseradish peroxidase, 153

anti-rabbit IgG (NA934; GE Healthcare, Little Chalfont, UK), anti-goat IgG (sc-2056; 154

Santa Cruz Biotechnology), or anti-mouse IgG (NA931; GE Healthcare), and the signal 155

was developed using ECL Plus (GE Healthcare). 156

Quantification of Sphingolipid levels. Yeast cells were precultured in 2 ml YPD 157

medium containing 10 μCi of [9,10-3H]palmitic acid (60 Ci/mmol; American Radiolabeled 158

Chemicals, St. Louis, MO), which had been complexed with 4 mg/ml (final concentration 159

of 0.4 mg/ml) fatty acid-free bovine serum albumin (BSA; Sigma) for 18 hr at 30˚C. Cells 160

were then diluted into 0.5 ml YPD medium containing 2.5 μCi [9,10-3H]palmitic acid 161

complexed with BSA as described above to 0.15 absorbance at 600 nm and grown for 8 hr 162

at 30˚C. After labeling, lipid extraction and alkaline treatment were performed as described 163

previously (13). Lipids were separated by TLC on Silica Gel 60 high performance TLC 164

plates (Merck, Whitestation, NJ) with chloroform/methanol/4.2 N ammonia (9:7:2, v/v) as 165

the solvent system and visualized by autoradiography. Densitometry and calibration of the 166

autoradiogram were performed using the NIH Image J software (http://rsbweb.nih.gov/ij/). 167

Northern blot analysis. Total RNA was isolated from logarithmically growing cells that 168

were treated or not with 0.4 M KCl for 1 hr, or with 2 μg/ml of myriocin for 4 hr. twelve 169

microgram of RNA per lane were separated in formaldehyde gels and transferred to a 170

positively charged nylon membrane (Pall-Life Sciences, Ann Arbor, MI). The GRE2 and 171


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HSP12 probes were amplified from genomic DNA by PCR, and labeled with digoxigenin 172

using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics, 173

Mannheim, Germany), and hybridized with the membrane according to the manufacturer’s 174

instructions. 175

Fluorescence microscopy. Logarithmically growing cells carrying pMH54 176

(pRS426-SLN1-EGFP) or pFP15 (pRS416-SHO1-EGFP) were microscopically observed. 177

Images were captured with the Olympus BX51 microscope (Olympus Optical, Tokyo, 178

Japan) using the U-MNIBA2 filter (Olympus). Image acquisition was performed using a 179

1394-based digital camera (Hamamatsu Photonics, Tokyo, Japan) and the AQUA-Lite 180

software program (Hamamatsu Photonics). 181

Isolation of DRMs and Western blotting. DRMs were isolated essentially as described 182

by Umebayashi and Nakano (38) with the following modifications. All procedures were 183

carried out on a scale three times larger than that of Umebayashi’s protocol. After CHAPS 184

extraction, the lysates were subjected to Optiprep density gradient flotation by 185

centrifugation for 14 h at 170,000 × g in an SW40 Ti rotor (Beckman Instruments, 186

Fullerton, CA) at 4 °C. After centrifugation, nine fractions of equal volume were collected 187

starting from the top. Each fraction was mixed with Laemmli sample buffer, incubated at 188

37 °C for 10 min, and resolved by SDS-PAGE. Western blotting was performed as 189

described above using the following primary antibodies: anti-GFP (Roche, Indianapolis, 190

IN), anti-myc (9E10, ascites, gift from Dr. Hoshikawa, the Cancer Institute of JFCR), 191

anti-Pep12 (2C3, Invitrogen, Carlsbad, CA), and anti-Pma1 (40B7, Abcam, Cambridge, 192

UK). 193


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Detection of membrane protein complexes by blue native PAGE. Logarithmically 194

growing cells cultured in YPD medium were collected, suspended in breaking buffer (50 195

mM Tris-HCl pH 7.5, 1 mM MgCl2, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 40 196

µg/ml aprotinin, 10 µg/ml pepstatin A, 20 µg/ml leupeptin), and then disrupted by 197

vortexing with 425-600 μm glass beads (Sigma). After dilution with an equal amount of 198

breaking buffer, the sediment of glass beads was removed and the supernatant was 199

centrifuged at 1,200 × g for 5 min. After the supernatant was centrifuged at 64,300 × g for 1 200

h, the pellets were washed once with breaking buffer, and were then suspended in 201

solubilization buffer (50 mM imidazole pH 7.0, 50 mM NaCl, 2 mM 6-aminohexanoic acid, 202

1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 40 µg/ml aprotinin, 10 µg/ml pepstatin 203

A, 20 µg/ml leupeptin). Membrane protein complexes were solubilized by addition of 204

digitonin (final concentration 1 %), and the sample was centrifuged at 20,000 × g for 20 205

min. A 1/40 volume of 5 % CBB dye stock (5% CBB G-250, 500 mM 6-aminohexanoic 206

acid) was added to the supernatant, and BN-PAGE was carried out as described previously 207

(41) using a 3-12% acrylamide gradient gel. After electrophoresis, the gel was washed with 208

the Laemmli running buffer for 10 min, and then Western blotting was performed as 209

described above. 210

211

RESULTS 212

Screen for mutants exhibiting constitutive activation of the HOG pathway. To identify 213

novel regulatory components of the HOG pathway, we performed a genetic screen aimed at 214

isolating mutants that exhibit constitutive activation of the HOG pathway under normal 215


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osmotic environments. We utilized the LexA-Hog1 reporter system for this screen (1). It 216

has been shown that phosphorylated Hog1 is recruited to specific osmostress-responsive 217

promoters in the nucleus and induces their expression by facilitating recruitment of the 218

transcription initiation complex (1). Thus the LexA-Hog1 fusion protein can induce 219

transcription of the LexA operator-fused HIS3 reporter when Hog1 is phosphorylated upon 220

osmotic stress, thereby supporting growth of the reporter strain on a histidine-depleted 221

medium (Fig. 2A) (1). The reporter strain was mutagenized using EMS, and mutants were 222

selected that grew on a histidine-depleted medium without osmotic stress. We then 223

identified mutants in which Hog1 was activated by directly examining the phosphorylation 224

status of Hog1. One of these isolated mutants (MH266) showed temperature-sensitive 225

growth (data not shown), and LCB2 was the responsible gene for this phenotype, as well as 226

for the histidine prototrophy (Fig. 2B). MH266 has a mutation in LCB2 (lcb2-M1) in which 227

Ala377 is substituted by Val. Lcb2 is a component of SPT, the enzyme that is responsible 228

for the first committed step in sphingolipid de novo synthesis (Fig. 1B), and is essential for 229

cell viability. To confirm that the observed phenotype was indeed caused by this mutation, 230

and that the phenotype was not specific to the parental strain used for the screen, we 231

introduced the lcb2-M1 mutation into an S288C-derived wild-type strain. The resultant 232

lcb2-M1 cells (MH280) also exhibited elevated phosphorylation of Hog1 (Fig. 2C) as well 233

as temperature-sensitive growth (Fig. 3C). These results indicate that a single lcb2-M1 234

mutation is sufficient to induce activation of the HOG pathway. 235

Inhibition of complex sphingolipid synthesis results in activation of the HOG 236

pathway. To confirm that inhibition of SPT induces activation of the HOG pathway, the 237


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effect of a specific pharmacological inhibitor of SPT, myriocin, was assayed (Fig. 1B). Like 238

lcb2-M1 cells, wild-type cells treated with myriocin exhibited elevated phosphorylation of 239

Hog1 (Fig. 3A). We next tested if activation of the HOG pathway in lcb2-M1 cells could be 240

reversed by rescuing sphingolipid metabolism. It was reported that exogenous addition of 241

some sphingolipid synthesis intermediates including phytosphingosine (PHS) enables lcb1Δ 242

or lcb2Δ cells to grow (4, 19). We therefore examined whether PHS addition can also 243

suppress the activation of the HOG pathway in lcb2-M1 cells. As expected, addition of PHS 244

attenuated the level of Hog1 phosphorylation in lcb2-M1 cells (Fig. 3B), as well as partially 245

rescued their temperature sensitive growth (Fig. 3C). These results indicate that depletion 246

of sphingolipids results in activation of the HOG pathway. 247

Ceramide, a metabolite of sphingolipids, activates protein phosphatases termed 248

ceramide-activated protein phosphatases (CAPPs) in yeast and mammals. We therefore 249

tested if CAPPs are involved in regulation of the HOG pathway. CDC55 encodes a 250

regulatory B subunit of protein phosphatase type 2A (PP2A) and is essential for yeast 251

CAPP activity (22). CDC55-disrupted cells exhibited neither elevated Hog1 252

phosphorylation nor expression of 8×CRE-lacZ, a HOG pathway reporter gene (data not 253

shown). Furthermore, addition of C2-ceramide, which activates yeast CAPP in vitro (22), to 254

wild-type cells did not suppress myriocin-induced activation of the HOG pathway (data not 255

shown). These results suggest that Hog1 phosphorylation induced by depletion of 256

sphingolipids is not mediated by failure to activate known CAPPs. 257

Next, in order to identify which sphingolipid metabolites are necessary for maintaining 258

the HOG pathway in an inactive state under normal osmotic environments, we inhibited 259


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various steps of the de novo sphingolipid synthesis pathway by using a specific inhibitor or 260

by disrupting genes involved in this pathway (see Fig. 1B). Wild-type cells treated with 261

aureobasidin A, an inositolphosphorylceramide (IPC) synthase inhibitor, as well as fen1Δ 262

and sur4Δ cells exhibited elevated Hog1 phosphorylation (Fig. 3D, E). Consistently, fen1Δ 263

and sur4Δ cells induced transcription of Hog1 target genes, GRE2 and HSP12 (Fig. 3F) (5, 264

39). All these conditions under which the HOG pathway is activated show impaired 265

synthesis of complex sphingolipids including IPC, mannosylinositolphosphorylceramide 266

(MIPC), and mannosyldiinositolphosphorylceramide (M(IP)2C). In addition, elevated Hog1 267

phosphorylation and induced transcription of Hog1 target genes were hardly observed in 268

csg1Δ csh1Δ and ipt1Δ cells, suggesting that MIPC and M(IP)2C are not required for 269

maintaining the HOG pathway in an inactive state. Therefore, IPC appears to be the critical 270

sphingolipid for proper regulation of the HOG pathway. It also should be noted that sur2Δ 271

cells exhibited neither elevated Hog1 phosphorylation nor induction of GRE2 and HSP12 272

genes (Fig. 3E, F). Sur2 hydroxylates dihydrosphingosine to form phytosphingosine (Fig. 273

1B). Hence, sur2Δ cells lack mature forms of IPC, but contain immature forms of IPC that 274

lack a hydroxyl group at C-4 position of the long chain base (10). This suggests that IPC is 275

required, but mature forms of IPC are not required, for proper regulation of the HOG 276

pathway. 277

It is unlikely for the following reasons that disturbance of each biosynthetic step results 278

in abnormal accumulation of intermediate metabolites, which, in turn, leads to activation of 279

the HOG pathway. First of all, inhibition of SPT, which reduces the overall synthesis of 280

sphingolipids, causes activation of the HOG pathway. Secondly, although fen1Δ and sur4Δ 281


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cells accumulate PHS (24), wild-type cells treated with 10 μM PHS, which is a toxic 282

concentration for yeast, did not alter the Hog1 phosphorylation level (data not shown). The 283

combined data suggest that it is the abundance of complex sphingolipids, rather than the 284

proper balance of their intermediate metabolites, that appears to be necessary for proper 285

regulation of the HOG pathway. 286

The lcb2-M1 mutation and myriocin treatment caused only moderate Hog1 activation as 287

compared with osmotic stress, estimated at about 1/10 to 1/20 (Fig. 2C and 3A, and data 288

not shown). This can be due to modest effects of these conditions on sphingolipid levels or 289

modest effects of sphingolipid depletion on Hog1 activation. To test these possibilities, we 290

quantified the sphingolipid levels of cells under these conditions. Total sphingolipid level in 291

lcb2-M1 cells was 79% of wild-type cells, where the decrease of IPC was most obvious 292

(70%) among complex sphingolipids (Fig. 3G-H). Total sphingolipid level in 293

myriocin-treated (2 μg/ml) wild-type cells was 74% (2 hr) and 64% (3.5 hr) of untreated 294

cells (Fig. 3I-J). These results suggest that the modest Hog1 activation under these 295

conditions resulted from their moderate effects on sphingolipids levels. 296

SPT inhibition induces activation of Hog1 via both the Sln1 and the Sho1 branches. 297

The HOG pathway has two osmosensing branches, the Sln1 and the Sho1 branches (Fig. 298

1A). We next investigated which of these branches is activated by impaired biosynthesis of 299

sphingolipids. Both lcb2-M1 cells with ssk2Δ ssk22Δ mutations and those with an sho1Δ or 300

an ste11Δ mutation still exhibited Hog1 phosphorylation, while lcb2-M1 ssk2Δ ssk22Δ 301

sho1Δ cells did not (Fig. 4A). These mutants are defective in the Sln1 branch alone, the 302

Sho1 branch alone, and both branches, respectively. In addition, myriocin treatment 303


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induced activation of Hog1 in either ssk2Δ ssk22Δ or ste11Δ cells, but not in ssk2Δ ssk22Δ 304

ste11Δ cells (Fig. 4B). These results indicate that SPT inhibition causes activation of both 305

the Sln1 and the Sho1 branches. 306

Inhibition of ergosterol synthesis results in activation of the HOG pathway. The 307

above observations indicate that depletion of sphingolipids activates the osmosensing 308

machinery of the HOG pathway. Sphingolipids, together with ergosterol, a major sterol in 309

yeast, form detergent insoluble membrane domains called rafts. We therefore hypothesized 310

that depletion of sphingolipids perturb raft structure, and thereby lead to activation of the 311

osmosensors. If this is the case, then depletion of ergosterol may also activate the HOG 312

pathway. To test this possibility, we examined mutants of the ergosterol synthesis pathway. 313

This study showed that erg2Δ, erg3Δ, and erg6Δ cells indeed exhibited elevated Hog1 314

phosphorylation (Fig. 5), supporting the possibility that perturbation of raft structure 315

activates the HOG pathway. In addition, lcb2-M1 erg3Δ double-mutant cells exhibited even 316

higher Hog1 phosphorylation than each single-mutant cells (Fig. 5). This additive effect of 317

two mutations can be due to severer defects they may cause in raft structure than the 318

single-mutants. 319

Sln1 and Sho1 localize in DRMs. We next tested whether Sln1 and Sho1 localize in 320

rafts. To this end, raft-enriched DRMs were separated using Optiprep density gradient 321

centrifugation, and fully functional (29, 30, and data not shown) epitope-tagged Sln1 and 322

Sho1 were probed for by Western blotting. Sln1 and Sho1 were indeed detected in the 323

DRM fraction (Fig. 6A). The distribution of Sln1 into DRMs was not significantly affected 324

by myriocin treatment or by lcb2-M1 mutation, although this result may be due to 325


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incomplete disruption of the raft structure under these conditions, as evidenced by the 326

similar distribution of the Pma1, a typical raft marker (2), under these conditions to 327

non-stressed wild-type cells (Fig. 6A). The distribution of Sln1 was not affected by osmotic 328

stress either. In contrast, the amount of Sho1 distributed in DRMs was increased both in 329

myriocin-treated cells and in lcb2-M1 cells (Fig. 6A). The correlation between the 330

increased distribution of Sho1 in DRMs and activation of the Sho1 branch suggested that 331

translocation of Sho1 to rafts may underlie the activation. We then tested if osmotic stress 332

induces a similar change in Sho1 distribution. The amount of Sho1 in DRMs was 333

moderately, but reproducibly, increased in cells exposed to osmotic stress (Fig. 6A). These 334

results suggest that translocation of Sho1 to rafts may be an essential step in osmosensing 335

by the Sho1 branch. 336

Next, we microscopically observed localization of GFP-tagged Sln1 and Sho1 in living 337

cells (Fig. 6B). These proteins were shown to be functional in previous reports (29, 30). 338

Sln1-GFP localized at the plasma membrane, and Sho1-GFP localized mainly at the bud tip 339

or bud neck in a dot-like pattern, as previously reported (29, 30, 36). Neither lcb2-M1 340

mutation nor myriocin treatment apparently altered the localization of Sln1-GFP. On the 341

other hand, myriocin treatment altered the localization of Sho1-GFP from a punctate pattern 342

to plasma membrane uniformly. This mislocalization of Sho1 seemed to be irrelevant for 343

activation of the Sho1 branch, because lcb2-M1 cells maintained intact localization of 344

Sho1-GFP, despite the fact that the Sho1 branch is similarly activated in both lcb2-M1 cells 345

and myriocin-treated cells. The different localization of Sho1-GFP between 346

myriocin-treated and lcb2-M1 mutant cells might be dependent on the degree of SPT 347


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inhibition: sphingolipid synthesis was more strongly inhibited by myriocin treatment than 348

by the lcb2-M1 mutation (Fig. 3G-J). Since sphingolipids are known to be necessary for 349

endocytosis, myriocin might cause the delocalization of Sho1 through impaired endocytosis 350

of Sho1 from the plasma membrane. 351

We then tested if sphingolipid depletion alters protein-protein interactions or complex 352

formation of the Sln1 or the Sho1 branch. Cell membranes were solubilized with digitonin 353

and separated by blue native PAGE (Fig. 6C). The lcb2-M1 mutation, as well as osmotic 354

stress, increased the fast migrating species, and decreased the slow migrating species, of 355

Sln1. This result suggested that both sphingolipid depletion and osmotic stress induce 356

dissociation of the Sln1 complex. On the other hand, the migration pattern of Sho1 was not 357

affected by lcb2-M1 mutation or by osmotic stress. The apparent lack of change in Sho1 358

may reflect the transient nature of the interaction of Sho1 with upstream or downstream 359

components. 360

Filipin modulates the activation of the HOG pathway. To further examine the possible 361

relevance of rafts to the regulation of the HOG pathway, we tested the effects of filipin. 362

Filipin is an anti-fungal polyene macrolide that stoichiometrically interacts and thereby 363

forms stable complex with sterol (7, 23). When yeast cells are treated with filipin, the 364

sterol-rich membrane compartment of Can1 (MCC), which appears to represent a specific 365

type of rafts, is immobilized and lateral redistribution of the MCC-resident transporter 366

Can1 is inhibited (7). We treated cells with filipin and then monitored activation of the 367

HOG pathway upon osmotic stress. Filipin pretreatment hindered activation of the Sho1 368

branch (shown in sho1Δ cells), while hardly affected the Sln1 branch (shown in ssk2Δ 369


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ssk22Δ cells) (Fig. 7A). On the other hand, when cells were treated in the opposite order, 370

first with osmotic stress and then with filipin, phosphorylation of Hog1 sustained longer 371

than untreated cells both in ssk2Δ ssk22Δ and sho1Δ cells (Fig. 7B). The sustained 372

phosphorylation of Hog1 in filipin-treated ssk2Δ ssk22Δ cells implies that filipin treatment 373

prolongs activation of the Sho1 branch induced by osmotic stress. In contrast, slow 374

activation of the Sln1 branch induced by filipin treatment may account for the sustained 375

phosphorylation of Hog1 in filipin-treated sho1Δ cells, as filipin treatment alone caused 376

phosphorylation of Hog1 in sho1Δ cells, but not in ssk2Δ ssk22Δ cells, at later time points 377

(Fig. 7C). These results further suggest that raft integrity affects the activation of the HOG 378

pathway. 379

The lcb2-M1 mutation causes activation of Fus3 and Kss1 in a Sho1-independent 380

but Ste11-dependent manner. Finally, to examine whether sphingolipid depletion affects 381

other MAPK pathways, phosphorylation of Fus3 and Kss1 was monitored in lcb2-M1 cells. 382

Fus3 and Kss1 were indeed activated in lcb2-M1 cells in a Sho1-independent but 383

Ste11-dependent manner (Fig. 8). These results suggest that sphingolipids depletion also 384

cause activation of the mating pathway but not the filamentous growth pathway, as 385

activation of the latter of which is strictly dependent on Sho1. 386

387

DISCUSSION 388

In this study, we show that inhibition of sphingolipid biosynthesis results in activation of 389

the HOG pathway. lcb2-M1, fen1Δ, and sur4Δ mutations, as well as myriocin and 390

aureobasidin A treatment, cause activation of the HOG pathway (Figs. 2 and 3). All of these 391


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cells show impaired synthesis of complex sphingolipids. On the other hand, csg1Δ csh1Δ 392

and ipt1Δ mutations hardly activate the HOG pathway, suggesting that IPC is the critical 393

sphingolipid for proper regulation of the HOG pathway (Fig. 3). We conclude that it is 394

depletion of complex sphingolipids, rather than accumulation of minor sphingolipid 395

biosynthetic intermediates, that causes activation of the HOG pathway. It is unlikely that 396

HOG pathway activation is caused by gross general defects in membrane structure, because 397

growth of the lcb2-M1 strains is apparently normal at 30 °C. 398

If this is the case, then how does depletion of sphingolipids result in activation of HOG 399

pathways? One possible explanation is that intact rafts organized with sphingolipids are 400

required for proper regulation of the osmosensors or of other regulatory molecules. Our 401

observations that inhibition of ergosterol biosynthesis also results in activation of the HOG 402

pathway (Fig. 5), and that both Sln1 and Sho1 localize in DRMs (Fig. 6A), support this 403

possibility, although we should keep in mind that association with DRMs is not necessarily 404

an accurate criterion for raft residency, because detergent treatment could cause artificial 405

aggregation of lipids and proteins (14). 406

Sphingolipid depletion, like osmotic stress, activates the HOG pathway via both the Sln1 407

and the Sho1 branches (Fig. 4). However, sphingolipid depletion affects each branch in a 408

distinctive manner. In the case of the Sho1 branch, we observed that both sphingolipid 409

depletion and osmotic stress promote association of Sho1 with DRMs. If this redistribution 410

indeed reflects translocation of Sho1 to rafts, then it should be elucidated whether this 411

translocation has a regulatory role in activation as well as in the osmosensing mechanism of 412

the Sho1 branch. We also found that treatment of cells with filipin, which forms stable 413


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complex with sterols and therefore is suggested to immobilize raft structure, impedes 414

osmotic stress-induced activation of the Sho1 branch (Fig. 7A). This is consistent with the 415

idea that redistribution of Sho1 to rafts is a key step for activation of the Sho1 branch. 416

Furthermore, filipin treatment after exposure to osmotic stress prolonged duration of the 417

Sho1 branch activation (Fig. 7B). This might also be due to that filipin treatment impedes 418

Sho1 from leaving rafts, and thereby sustains the active state of the Sho1 branch. These 419

indirect evidences support the idea that redistribution of Sho1 to rafts has an important 420

regulatory role in the activation of the Sho1 branch although caution for potential toxic or 421

artifactual effects of filipin has to be kept in mind. 422

If translocation of Sho1 to raft is a key step in the activation of the Sho1 branch, how the 423

translocation can be regulated? The structural change induced by osmotic stress in Sho1 424

itself may change Sho1’s affinity to raft vs. non-raft compartments. Alternatively, it is 425

possible that other molecules that are regulated by rafts recruit Sho1 to rafts in response to 426

stress, as Sho1 is known to interact with many components of the Sho1 branch. Cdc42 is a 427

possible target of raft-mediated regulation. Recently, sphingolipids were reported to be 428

necessary for activation of Fpk1/2 (32). Since Fpk1/2 are activators of the 429

aminophospholipid flippase Dnf1/2 (21), which transport phosphatidylethanolamine 430

(PtdEth) and phosphatidylserine (PtdSer) from the external to the cytosolic leaflet of a 431

bilayer membrane, sphingolipid depletion may cause depletion of aminophospholipids from 432

cytosolic leaflet through downregulation of Dnf1/2. As it was also reported that 433

aminophospholipids stimulate the GTPase-activating protein (GAP) activity of Rga1/2 434

toward Cdc42 in vitro (21), sphingolipid depletion may result in elevated activation of 435


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Cdc42 through failure in Rga1/2 activation. Our observation that sphingolipid depletion 436

also causes activation of Fus3 and Kss1, which are MAPKs of another Cdc42-controlled 437

MAPK pathway, the mating pathway, in a Sho1-independent but Ste11-dependent manner 438

supports this idea (Fig. 8). As for the Sln1 branch, we have demonstrated that depletion of 439

sphingolipids, as well as osmotic stress, caused partial dissociation of the Sln1 complex, 440

although the DRM distribution of Sln1 and the localization of Sln1-GFP were apparently 441

not affected (Fig. 6A, B, C). It has been suggested that Sln1 is functional as a homo-dimer 442

(18, 28, 35). We therefore propose the model that Sln1 has to be tightly packed in rafts, in 443

which it forms a dimeric complex, in order to efficiently conduct phosphotransfer relay 444

under non-stressed environments, and that osmotic stress or sphingolipid depletion perturbs 445

the raft-mediated packing of the Sln1 complex, resulting in inhibition of phosphotransfer 446

relay and thereby activation of the Sln1 branch. 447

In this study, we have demonstrated a possible link between osmotic stress-sensing and 448

membrane sphingolipids. Sphingolipid depletion and osmotic stress induce similar effects 449

on the osmosensing machinery of the HOG pathway, even though specific effects on the 450

Sln1 and Sho1 branches are distinct. These observations may imply that yeast cells sense 451

osmotic stress as changes in the structural and/or physical properties of rafts. Further study 452

is necessary to solve the precise mechanism by which sphingolipids regulate the 453

osmosensing machinery. 454

455

ACKNOWLEDGEMENTS 456

We thank Takahito Wada for excellent technical assistance in the initial screening. We also 457


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thank Paul J. Cullen for valuable technical comments, Haruo Saito and Beverly Errede for 458

plasmids, Yutaka Hoshikawa for an antibody, and all members of the Maeda laboratory for 459

valuable supports and discussions. 460

This work was supported in part by a Grant-in-Aid for Scientific Research on Priority 461

Areas (KAKENHI 21025008) from the Ministry of Education, Culture, Sports, Science and 462

Technology (MEXT), a Grant-in-Aid for Challenging Exploratory Research (KAKENHI 463

23651233) from the Japan Society for the Promotion of Science (JSPS), grants from the 464

Noda Institute for Scientific Research and the Salt Science Research Foundation (No. 465

11D2) (all to T.M.), and a Grant-in-Aid for JSPS Fellows (to T.T.). T.T. was the recipient of 466

the Research Fellowship of the Japan Society for the Promotion of Science (JSPS) for 467

Young Scientists. 468

469

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37. Tatebayashi K, Yamamoto K, Tanaka K, Tomida T, Maruoka T, Kasukawa E, Saito 571

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582

FIGURE REGENDS 583

FIG. 1. The pathways dealt in this paper. (A) Schematic representation of the HOG pathway. 584

(B) Outline of the de novo sphingolipid synthesis pathway in S. cerevisiae. The target 585

processes of two inhibitors (myriocin and aureobasidin A) are also shown. 586

587

FIG. 2. Screen for mutants exhibiting constitutive activation of the HOG pathway. (A) The 588

parental strain, which has a LexAop-HIS3 reporter gene and a LexA-Hog1 expressing 589


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plasmid, grows on His-depleted medium in an osmotic stress-dependent manner. TM415, 590

harboring the vector (p414-LexA) or the LexA-HOG1 coding plasmid (pMT304), were 591

spotted onto SC-Trp (SC), SC-Trp His (-His), and SC-Trp His supplemented with 1 M 592

sorbitol (-His +1M sorbitol) plates and incubated for 2 days at 30 °C. (B) The lcb2-M1 593

mutant cells grow on the -His plate in the absence of osmotic stress. Wild-type (TM415, 594

WT) and lcb2-M1 (MH266) cells, harboring pMT304 and a vector (pRS416) or an LCB2 595

plasmid (pMH29), were spotted onto SC-Trp Ura (SC) and SC-Trp His Ura (-His) plates 596

and incubated for 2 days at 30 °C. (C) lcb2 mutant cells exhibit elevated Hog1 597

phosphorylation. Phosphorylated Hog1 in wild-type (TM141) and lcb2-M1 (MH280) cells 598

was detected by Western blotting using an anti-phospho-p38 specific antibody. Wild-type 599

cells treated with 0.4 M KCl for 3 min (WT osm) were used as a positive control. 600

601

FIG. 3. Inhibition of the sphingolipid biosynthetic pathway induces activation of the HOG 602

pathway. (A, B, D, E) Hog1 phosphorylation was assayed by Western blotting for phospho- 603

and total Hog1. (A) Myriocin treatment induces phosphorylation of Hog1. Wild-type 604

(TM141) cells were treated, or not, with 2 μg/ml of myriocin, for the indicated times, or 605

with 0.4 M KCl for 3 min (osm stress), followed by Western blotting. (B) Exogenous 606

addition of PHS attenuates elevated Hog1 phosphorylation in lcb2-M1 cells. Wild-type 607

(TM141) and lcb2-M1 (MH280) cells were cultivated with YPD in the absence or presence 608

of 5 μM PHS for 4 h, and were then subjected to Western blotting. (C) Temperature 609

sensitive growth of lcb2-M1 cells is partially rescued by exogenous addition of PHS. 610

Wild-type (TM141) and lcb2-M1 (MH280) cells were spotted onto YPD plates with or 611


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without supplementation with 5 μM PHS, and were incubated at the indicated temperature 612

for 3 days. (D) Inhibition of IPC synthase by aureobasidin A treatment induces Hog1 613

phosphorylation. Wild-type (TM141) cells were treated with 500 ng/ml aureobasidin A or 2 614

μg/ml myriocin for the indicated times, and were then subjected to Western blotting. 615

lcb2-M1 cells were used as a positive control. (E-F) Mutant cells in which complex 616

sphingolipid synthesis is inhibited show activation of the HOG pathway. Wild-type 617

(TM141), lcb2-M1 (MH280), elo1Δ (MH293), fen1Δ (MH314), sur4Δ (MH311), sur2Δ 618

(MH305), csg1Δ csh1Δ (MH309), and ipt1Δ (4007) cells were tested. (E) Hog1 619

phosphorylation of the indicated strains was examined by Western blotting. (F) Transcript 620

levels of GRE2 and HSP12 in the indicated strains were monitored by Northern analysis. 621

The ethidium bromide-stained rRNA was shown as a loading control. Wild-type cells were 622

treated or not with 0.4 M KCl for 1 hr (osm), or with 2 μg/ml of myriocin for 4 hr 623

(myriocin) before the cells were collected. (G) Decreased sphingolipid levels in lcb2-M1 624

mutant cells. Wild-type (TM141) and lcb2-M1 (MH280) cells were cultured in YPD 625

containing [3H]palmitic acid for 26 hr at 30˚C. Labeled lipids were extracted, treated with 626

alkaline to hydrolyze ester linkages in glycerolipids, separated by TLC, and detected by 627

autoradiography. (H) Quantification of sphingolipid levels that were labeled as in (G). 628

Radioactivity associated with sphingolipids was quantified from autoradiogram. Values 629

represent the mean ± S.D. from three independent experiments. (I) Myriocin treatment 630

results in decreased sphingolipids levels. Wild-type (TM141) cells were cultured in YPD 631

containing [3H]palmitic acid for 26 hr at 30˚C. Myriocin (5 μM) was added 0, 2, or 3.5 hr 632

prior to termination of the labeling. Labeled lipids were detected as in (G). (J) 633


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Quantification of sphingolipid levels that were labeled as in (I). The data were presented as 634

in (H). 635

636

FIG. 4. SPT inhibition induces activation of Hog1 via both the Sln1 and the Sho1 branches. 637

ssk2Δ ssk22Δ is abbreviated as ssk2,22Δ. (A) Wild-type (TM141, WT), lcb2-M1 (MH280), 638

lcb2-M1 ssk2,22Δ (MH317), lcb2-M1 sho1Δ (MH319), lcb2-M1 ste11Δ (MH322), and 639

lcb2-M1 ssk2,22Δ sho1Δ (MH321,) cells were subjected to Western blotting for phospho- 640

and total Hog1. (B) Wild-type (TM141, WT), ssk2,22Δ (TM252), ste11Δ (MH273), and 641

ssk2,22Δ ste11Δ (MH276) cells were treated with 2 μg/ml of myriocin for 3 h, and then 642

subjected to Western blotting as in (A). lcb2-M1 (MH280) cells were used as a positive 643

control. 644

645

FIG. 5. Inhibition of the ergosterol biosynthetic pathway induces activation of the HOG 646

pathway. Wild-type (TM141), lcb2-M1 (MH280), erg2Δ (MH425), erg3Δ (TM537), erg6Δ 647

(TM540), and lcb2-M1 erg3Δ (MH421) cells were subjected to Western blotting for 648

phospho- and total Hog1. Wild-type cells treated with 0.4 M KCl for 3 min (osm) were 649

used as a positive control. 650

651

FIG. 6. DRM-localization and complex formation of Sln1 and Sho1. (A) Sln1 and Sho1 652

localize in DRMs. SLN1-9myc SHO1-GFP (MH335) cells that were treated with 0.4 M KCl 653

for 5 min (osm), or with 2 μg/ml myriocin for 2.5 h (myriocin), and non-treated wild-type 654

(MH335, WT) or lcb2-M1 SLN1-9myc SHO1-GFP (MH337, lcb2-M1) cells were disrupted, 655


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and their membrane fractions were extracted with 20 mM CHAPS and subjected to 656

Optiprep density gradient centrifugation. Nine fractions were collected from the top and 657

were analyzed by Western blotting using anti-myc, -GFP, -Pma1, and -Pep12 antibodies. 658

Pma1 was used as a marker for DRM-associated proteins, and Pep12 as a marker for 659

non-DRM-associated membrane proteins. Fraction 2 and 3 contain DRMs. (B) Cellular 660

localization of Sln1 and Sho1 in sphingolipid synthesis-deficient cells. Wild-type (TM141, 661

WT) and lcb2-M1 (MH280) cells harboring an SLN1-GFP- or an SHO1-GFP-encoding 662

plasmid were grown in SC-Ura. Wild-type cells were treated with or without 2 μg/ml of 663

myriocin for 2.5 h (myriocin) or 0.4 M KCl for 5 min (osm). (C) Detection of Sln1 and 664

Sho1 complexes by BN-PAGE. The membranes of SLN1-9myc SHO1-GFP (MH335, WT) 665

cells and lcb2-M1 SLN1-9myc SHO1-GFP (MH337, lcb2-M1) cells that had been treated 666

with (osm +) or without (osm -) 0.4 M KCl for 5 min were isolated, solubilized with 1% 667

digitonin, and then subjected to BN-PAGE. After electrophoresis, Western blotting was 668

performed using anti-myc and anti-GFP antibodies. An arrow and allowheads indicate the 669

fast- and the slow-migrating species of Sln1, respectively. 670

671

FIG. 7. Filipin treatment affects activation of the HOG pathway. (A) Filipin treatment 672

impedes activation of the Sho1 branch. Wild-type (TM141, WT), ssk2,22Δ (TM252), and 673

sho1Δ (TM287) cells were treated with or without 5 μg/ml of filipin for 5 min, and then 674

treated with final 0.4 M KCl. After 5 min incubation, cells were collected and subjected to 675

Western blotting. (B) Filipin treatment after exposure to osmotic stress sustains activation 676

of the HOG pathway. ssk2,22Δ (TM252) and sho1Δ (TM287) cells were exposed to final 677


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0.4 M KCl and incubated for 5 min (0 min). Then, cells were treated with final 5 μg/ml 678

filipin, incubated for the indicated times, and collected and subjected to Western blotting. 679

(C) Filipin treatment induces activation of the Sln1 branch. ssk2,22Δ (TM252) and sho1Δ 680

(TM287) cells were treated with 5 μg/ml filipin for the indicated times, and subjected to 681

Western blotting. 682

683

FIG. 8. The lcb2-M1 mutation causes activation of Kss1 and Fus3 in a Sho1-independent 684

but Ste11-dependent manner. Wild-type (TM141, WT), lcb2-M1 (MH280), lcb2-M1 685

ssk2,22Δ (MH317), lcb2-M1 sho1Δ (MH319), lcb2-M1 ste11Δ (MH322), and lcb2-M1 686

ssk2,22Δ sho1Δ (MH321) cells were subjected to Western blotting using anti-phospho-p38, 687

which detects phospho-Hog1, anti-Hog1, anti-phospho-p44/p42 MAPK, which detects 688

phospho-Kss1 and phospho-Fus3, and anti-Kss1 antibodies. 689

690


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TABLE 1. Yeast strains used in this study 691

Strain Genotype Reference or Source

TM415 MATa leu2Δ1 trp1Δ63 his3Δ200 ade2 URA3::(lexAop)8-lacZ LYS2::(lexAop)4-HIS3

L40-derived, Lab stock

TM415α same as TM415 except that MATα Lab stock

MH266 same as TM415α except that lcb2-M1 This study

TM100 MATa ura3-52 leu2Δ1 trp1Δ63 17

TM101 MATα ura3-52 leu2Δ1 his3Δ200 18

TM141 MATa ura3-52 leu2Δ1 trp1Δ63 his3Δ200 17

TM221 MATα ade8 Lab stock

TM225 MATα ura3-52 leu2Δ1 his3Δ200 lys2Δ202 11

TM252 MATa ura3-52 leu2Δ1 trp1Δ63 ssk2::LEU2 ssk22::LEU2 18

TM287 MATα ura3-52 leu2Δ1 trp1Δ63 his3Δ200 sho1::HIS3 Lab stock

TM370 MATα ura3-52 leu2Δ1 trp1Δ63 his3Δ200 ssk2::LEU2 ssk22::LEU2 sho1::TRP1 Lab stock

TM537 MATa ura3-52 leu2Δ1 trp1Δ63 erg3::His3MX6 Lab stock

TM540 MATa ura3-52 leu2Δ1 trp1Δ63 erg6::His3MX6 Lab stock

4007 MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 ipt1::kanMX4 Open Biosystems

5281 MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 sur4::kanMX4 Open Biosystems


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MH273 MATα ura3-52 leu2Δ1 his3Δ200 ste11::URA3 This study

MH276 MATa ura3-52 leu2Δ1 trp1Δ63 ssk2::LEU2 ssk22::LEU2 ste11::loxP-kanMX-loxP This study

MH280 MATa ura3-52 leu2Δ1 trp1Δ63 his3Δ200 lcb2-M1 This study

MH293 MATa ura3-52 leu2Δ1 trp1Δ63 elo1::kanMX4 This study

MH296 MATa ura3-52 leu2Δ1 trp1Δ63 csg1::kanMX4 This study

MH302 MATα ura3-52 leu2Δ1 his3Δ200 csh1::kanMX4 This study

MH305 MATa ura3-52 leu2Δ1 trp1Δ63 sur2::kanMX4 This study

MH309 MATα ura3-52 leu2Δ1 trp1Δ63 csg1::kanMX4 csh1::kanMX4 This study

MH311 MATa leu2Δ0 his3Δ1 met15Δ0 sur4::kanMX4 This study

MH314 MATa ura3-52 leu2Δ1 trp1Δ63 his3Δ200 fen1::kanMX4 This study

MH317 same as MH280 except that ssk2::LEU2 ssk22::LEU2 This study

MH319 same as MH280 except that sho1::TRP1 This study

MH321 same as TM370 except that lcb2-M1 This study

MH322 same as MH280 except that ste11::URA3 This study

MH324 MATa ura3-52 leu2Δ1 trp1Δ63 his3Δ200 SLN1-EGFP::His3MX6 This study

MH329 MATα ura3-52 leu2Δ1 trp1Δ63 his3Δ200 SHO1-GFP::His3MX6 lcb2-M1 This study

MH331 MATa ura3-52 leu2Δ1 trp1Δ63 his3Δ200 SLN1-9myc::His3MX6 This study


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MH335 MATα ura3-52 leu2Δ1 trp1Δ63 his3Δ200 SLN1-9myc::His3MX6 SHO1-GFP::His3MX6 This study

MH337 same as MH335 except that lcb2-M1 This study

MH417 MATα ura3-52 leu2Δ1 his3Δ200 lys2Δ202 erg3::His3MX6 This study

MH421 same as MH280 except that erg3::His3MX6 This study

MH425 MATa ura3-52 leu2Δ1 trp1Δ63 his3Δ200 erg2::His3MX6 This study

692


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hyperosmolarity

Sho1

Ypd1

Hog1

Pbs2

Sln1

Ssk1

Ste11 MAPKKK

MAPKK

MAPK

Cdc42Ste20

Ssk2,22

Msb2/Hkr1

palmitoyl-CoA Serine

3-ketodihydrosphingosine

Dihydrosphingosine

Phytosphingosine (PHS)

Dihydroceramide

Phytoceramide

Mannosylinositol-phosphorylceramide (MIPC)

Inositolphosphorylceramide (IPC)

Mannosyldiinositol-phosphorylceramide (M(IP)2C)

C26-CoA

SPT

TSC10

SUR2

SUR2

C26-CoA

Ceramide synthaseLAG1,LAC1

LIP1

Elongation cycle

Malonyl-CoAELO1FEN1SUR4

YBR159WPHS1TSC13

IPC synthase AUR1

CSG1,CSG2,CSH1

IPT1

Ceramide synthaseLAG1,LAC1

LIP1

A B

aureobasidin A

myriocin

Figure 1

LCB1,LCB2,TSC3


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B

A

vec

LexA-HOG1

SC + 1 M sorbitol -His

WT

lcb2-M1vec

LCB2

vec

C WT osm

phospho-Hog1

total Hog1

Figure 2

-His

SC -His

WT lcb2-M1

+ 1 M sorbitol -His


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BA

total Hog1

WT lcb2-M1PHS -

total Hog1os

m st

ress

0 1.5 3.5 hr

myriocin

C

Figure 3

phospho-Hog1phospho-Hog1

+ +-

30 °C37 °C

WT

lcb2-M1

- PHS

D

E

aureobasidin

lcb2-M

1

myriocin

- aureobasidin

myriocin

-

1.5 hr 3 hr

total Hog1

WT lcb2-M1 elo1 fen1 sur4 csh1

total Hog1

phospho-Hog1

phospho-Hog1

sur2 csg1 ipt1

WT elo1 fen1 sur4 csh1

sur2 csg1 ipt1 myriocin

osmlcb2-M

1

GRE2

28S rRNA

F

G H I J

IPC

Cer

palmitic acid

MIPC

M(IP)2C

origin WT

lcb2-M1

***

16

12

8

4Rad

ioac

tivity

(%)

Cer

IPC

MIP

C

M(IP

)2 C

total

20WTlcb2-M1

*

IPC

Cer

palmitic acid

MIPC

M(IP)2C

origin

time (hr) 0 2 3.5

*p<0.05, **p<0.01

16

12

8

4

0

0 hr2 hr3.5 hr

Rad

ioac

tivity

(%)

Cer

IPC

MIPC

M(IP)2 C

total

*****

****

**

**20 **

*p<0.05, **p<0.01

HSP12

28S rRNA on January 30, 2018 by guesthttp://m

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total Hog1

lcb2-

M1

lcb2-

M1

ssk2

,22Δ

lcb2-

M1

sho1

Δ

lcb2-

M1

ssk2

,22Δ

sho

1Δ

lcb2-

M1

ste1

1Δ

WT

A

B

WT ssk2,22Δ ste11Δ ste11Δ ssk2,22Δ

lcb2-

M1

myriocin - - - - -

total Hog1

Figure 4

phospho-Hog1

phospho-Hog1+ + + +


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total Hog1

lcb2-

M1

erg2

Δer

g3Δ

erg6

Δ

WT

Figure 5

phospho-Hog1

osm

lcb2-

M1

erg3

Δ


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A

Pma1

Pep12

Sln1-myc

720

480

242

146

66

kDa

WT lcb2-M1

osm- -

WT lcb2-M1

B

720

480

242

146

kDaosm

Sln1-myc Sho1-GFP

Sho1-GFP

Pma1

Pep12

Sln1-myc

Sho1-GFP

Pma1

Pep12

Sln1-myc

Sho1-GFP

Pma1

Pep12

Sln1-myc

Sho1-GFP

WT myriocin

osm lcb2-M1

DRMtop bottom top bottom

top bottom top bottom

66

Figure 6

Sho1

Sln1

WT lcb2-M1

-

C

-osmmyriocin

+ - - +

DRM

DRM DRM


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ssk2,22Δ

ssk2,22Δ

- 15 30 45 - 15 30 45 min

total Hog1

phospho-Hog1

osmotic stress + + +- - -- - - - - -+ + + + + + filipin

total Hog1

phospho-Hog1

total Hog1

phospho-Hog1

ssk2,22Δ

0 15 30 45

- - - - + + + filipin - - - - + + +

0 15 30 45 min

filipin

Figure 7 A

B

C

WT sho1Δ

sho1Δ

sho1Δ


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phospho-Kss1phospho-Fus3

total Hog1

total Kss1

lcb2-

M1

lcb2-

M1

ssk2

,22Δ

lcb2-

M1

sho1

Δ

lcb2-

M1

ssk2

,22Δ

sho

1Δ

lcb2-

M1

ste1

1Δ

WT

phospho-Hog1

Figure 8


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1 sphingolipids regulate the yeast high osmolarity-responsive hog

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