1

A novel factor OPT2 mediates exposure of phospholipids during cellular adaptation 1

to altered lipid asymmetry 2

3

Saori Yamauchi, Keisuke Obara#, Kenya Uchibori, Akiko Kamimura, Kaoru Azumi, and 4

Akio Kihara# 5

6

Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan 7

8

#Address correspondence to Keisuke Obara 9

Faculty of Pharmaceutical Sciences, Hokkaido University 10

Kita 12-jo Nishi 6-chome, Kita-ku, Sapporo, 060-0812 Japan 11

Tel: +81-11-706-3720 12

Fax: +81-11-706-4900 13

E-mail: obarak@pharm.hokudai.ac.jp. 14

15

#Address correspondence to Akio Kihara 16

Faculty of Pharmaceutical Sciences, Hokkaido University 17

Kita 12-jo Nishi 6-chome, Kita-ku, Sapporo, 060-0812 Japan 18

Tel: +81-11-706-3754 19

Fax: +81-11-706-4900 20

E-mail: kihara@pharm.hokudai.ac.jp. 21

22

Running head: A novel factor for phospholipid exposure 23

24

Word count (except the References): 6180 words 25

26

Key words: lipid asymmetry, plasma membrane, phospholipid, yeast27

© 2014. Published by The Company of Biologists Ltd.Jo

urna

l of C

ell S

cien

ceA

ccep

ted

man

uscr

ipt

JCS Advance Online Article. Posted on 29 October 2014

2

ABSTRACT 28

Plasma membrane lipid asymmetry is important for various membrane-associated 29

functions and regulated by membrane proteins termed flippases and floppases. The 30

Rim101 pathway senses altered lipid asymmetry in the yeast plasma membrane. The 31

mutant lem3Δ cells, in which lipid asymmetry is disturbed due to the inactivation of the 32

plasma membrane flippases, showed a severe growth defect when the Rim101 pathway 33

was impaired. To identify factors involved in the Rim101 pathway-dependent adaptation 34

to altered lipid asymmetry, we performed DNA microarray analysis and found that Opt2 35

induced by the Rim101 pathway plays an important role in the adaptation to altered lipid 36

asymmetry. Biochemical investigation of Opt2 revealed its localization to the plasma 37

membrane and the Golgi apparatus and provided several lines of evidence for the 38

Opt2-mediated exposure of phospholipids. In addition, Opt2 was found to be required for 39

the maintenance of vacuole morphology and polarized cell growth. These results suggest 40

that Opt2 is a novel factor involved in cell homeostasis by regulating lipid asymmetry.41

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

3

INTRODUCTION 42

A common feature of the eukaryotic plasma membrane is the difference in lipid 43

composition between the inner (cytosolic) and outer (extracellular) leaflets, which is 44

called lipid asymmetry (Devaux, 1991; Verkleij and Post, 2000). For instance, 45

phosphatidylserine (PS) and phosphatidylethanolamine (PE) are mostly confined to the 46

inner leaflet, while sphingolipids and phosphatidylcholine (PC) are enriched in the outer 47

leaflet. This lipid asymmetry is generated and maintained by ATP-dependent membrane 48

proteins termed flippases and floppases that mediate inward (flip) and outward (flop) 49

movement of lipids between the leaflets, respectively (Axelsen and Palmgren, 1998; Hua 50

et al., 2002; Ikeda et al., 2006; Pomorski et al., 2003; Seigneuret and Devaux, 1984). 51

Proper lipid asymmetry is required for various biological processes, including generation 52

of membrane potential, establishment of cell polarity, vesicular transport, cytokinesis, 53

blood coagulation, and removal of apoptotic cells (Chen et al., 1999; Emoto and Umeda, 54

2000; Fadok et al., 1992; Furuta et al., 2007; Gurtovenko and Vattulainen, 2008; Saito et 55

al., 2007; Toti et al., 1996). Yeast cells lacking all known flippases are found to be 56

inviable (Hua et al., 2002), suggesting that lipid asymmetry is essential for cell viability. 57

In humans, several mutations in flippases and floppases have been implicated in various 58

diseases including cholestasis, Stargardt macular dystrophy, and Scott syndrome 59

(Allikmets et al., 1997; Bull et al., 1998; Toti et al., 1996). 60

Yeast cells have five phospholipid flippases of the P4-type ATPase family 61

(Dnf1, Dnf2, Dnf3, Drs2, and Neo1), of which Dnf1 and Dnf2 are the main flippases in 62

the plasma membrane and Drs2 functions primarily in the Golgi apparatus (Hua et al., 63

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

4

2002). Each flippase forms a complex with a regulatory subunit from the Cdc50 family 64

for its activity; for example, Dnf1 and Dnf2 require complexation with Lem3 (a member 65

of the Cdc50 family) in order to exit from the ER and to function as the plasma membrane 66

flippase (Saito et al., 2004). It was recently suggested that phospholipids are flipped along 67

the protein-lipid interface of P4-type ATPases and not through their interior (Baldridge 68

and Graham, 2012). As for the phospholipid floppases, two members of the ABC 69

transporter family (Pdr5 and Yor1) are reported in yeast (Decottignies et al., 1998). In 70

addition, Rsb1 has been identified in yeast as a putative floppase/translocase for 71

sphingoid long-chain bases (Kihara and Igarashi, 2002; Kihara and Igarashi, 2004). It is 72

currently unknown as to how these proteins flop lipids across the bilayer. 73

We previously reported that the Rim101 pathway, known as an 74

alkaline-responsive pathway, also senses altered lipid asymmetry caused by the deletion 75

of LEM3 and/or PDR5 (Ikeda et al., 2008). In this pathway, the plasma membrane protein 76

Rim21 acts as the sensor molecule for both altered lipid asymmetry and external 77

alkalization (Obara et al., 2012) and transmits the signal, at the plasma membrane (Obara 78

and Kihara, 2014), leading to the proteolytic activation of the transcription factor Rim101 79

(Peñalva and Arst, 2004). In the alkaline response, the processed Rim101 in yeast induces 80

transcription of alkaline-responsive genes through suppressing expression of 81

transcription repressors such as Nrg1 and Smp1, while in filamentous fungi the processed 82

PacC (a Rim101 homolog) directly induces alkaline-expressed genes (Lamb and Mitchell, 83

2003; Peñalva and Arst, 2002). Several permeases, secreted enzymes, and proteins 84

involved in intracellular pH homeostasis are induced by changes in external pH for 85

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

5

adaptation (Causton et al., 2001; Lamb et al., 2001; Peñalva and Arst, 2002; Peñalva and 86

Arst, 2004). In contrast to the pH-response, the cellular response to altered lipid 87

asymmetry in the plasma membrane remains largely unknown. 88

In the present work, we have comprehensively analyzed the cellular response to 89

altered lipid asymmetry using DNA microarray and identified Opt2 a novel key factor in 90

cellular adaptation to altered lipid asymmetry.91

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

6

RESULTS 92

The Rim101 pathway is involved in adaptation to altered lipid asymmetry 93

To evaluate the importance of the Rim101 pathway in adaptation to altered 94

lipid asymmetry, the RIM21 gene encoding the sensor protein in this pathway was deleted 95

in lem3Δ cells, in which lipid asymmetry is disturbed. The resultant rim21Δ lem3Δ 96

double-mutant cells suffered a severe synthetic growth defect (Fig. 1), indicative of the 97

involvement of the Rim101 pathway in the response to altered lipid asymmetry. Although 98

Rsb1, a putative floppase for sphingoid long-chain bases, is induced by alteration in lipid 99

asymmetry (Kihara and Igarashi, 2002; Kihara and Igarashi, 2004), the growth of rsb1Δ 100

lem3Δ cells was comparable to that of lem3Δ cells, indicating that Rsb1 is not important 101

for the adaptation. 102

103

Altered lipid asymmetry induces expression of genes encoding transporters and 104

sugar metabolizing enzymes 105

In order to comprehensively characterize the cellular response to altered lipid 106

asymmetry, DNA microarray analysis of the yeast genome was performed on wild-type 107

(WT), lem3Δ, pdr5Δ (floppase mutant), and rim21Δ lem3Δ cells. Genes induced by 108

alteration in lipid asymmetry (i.e., genes induced in both lem3Δ and pdr5Δ cells) were 109

extracted; twenty-seven of them were further extracted as Rim101 pathway-dependent 110

genes since their elevated expression was not observed in rim21Δ lem3Δ cells (Table S1). 111

These genes are expected to encode proteins that are inducible through the Rim101 112

pathway and to function in adaptation to altered lipid asymmetry, which include those 113

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

7

encoding transporters, enzymes for glycogen and trehalose metabolism, and an 114

arrestin-related protein. 115

116

Opt2 plays an important role in the cellular response to altered lipid asymmetry 117

Among the 27 genes extracted, we focused on those encoding, or presumable 118

encoding, integral membrane proteins that might function as flippases/floppases to repair 119

altered lipid asymmetry: MUP3, OPT2, SSU1, MAL31, YDR089W, and YJL163C. Each 120

of these genes was then deleted in lem3Δ cells, and only opt2Δ lem3Δ cells were found to 121

exhibit a severe growth defect similar to rim21Δ lem3Δ cells (Fig. 2A). Thus, Opt2 122

appears to have an important role in adaptation to altered lipid asymmetry. Consistent 123

with the results of DNA microarray analysis (Fig. 2B), Opt2 levels were elevated in both 124

lem3Δ and pdr5Δ cells but were reduced in lem3Δ rim21Δ cells (Fig. 2C). In addition, 125

Opt2 levels in rim21Δ cells were comparable to that in WT cells, suggesting that the 126

dependency of Opt2 expression on the Rim101 pathway becomes prominent when lipid 127

asymmetry is altered. Deletion of OPT2 did not affect the activation of the Rim101 128

pathway as monitored by proteolytic processing of Rim101 (Fig. 2D). 129

To further confirm the importance of Opt2 in adaptation to altered lipid 130

asymmetry, Opt2 was overexpressed in rim21Δ lem3Δ and opt2Δ lem3Δ cells from the 131

Rim101 pathway-independent ADH1 promoter. As expected, overexpression of Opt2 132

suppressed the severe growth defect in both cells (Fig. 2A). Since the Rim101 pathway 133

was originally reported as an alkaline-responsive pathway, the involvement of Opt2 in 134

alkaline response was next investigated. The Rim101 pathway-defective rim21Δ cells 135

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

8

were found to be hypersensitive to alkaline pH (Fig. 2E), as others have reported 136

(Castrejon et al., 2006). Interestingly, cells deleted for LEM3 (lem3Δ cells) also exhibited 137

similar sensitivity to alkaline pH. Deletion of OPT2 in WT and lem3Δ cells did not affect 138

alkaline tolerance. Therefore, Opt2 appears to have no significant role in alkaline 139

response. 140

141

Opt2 cycles between the plasma membrane and the Golgi apparatus 142

Intracellular localization of Opt2 was monitored by using the N-terminally 143

GFP-fused Opt2. The GFP-tagged Opt2 (GFP-Opt2) was mainly localized to punctate 144

structures, which were colocalized with the late Golgi marker Sec7-mCherry, with 145

limited localization to the plasma membrane (Fig. 3A). When endocytosis was blocked 146

by transient degradation of the actin assembly factor Las17 using the auxin-inducible 147

degron system (Nishimura et al., 2009; Obara and Kihara, 2014), the GFP-Opt2 signal 148

was detected primarily in the plasma membra ne (Fig. 3B). This result indicates that Opt2 149

cycles between the late Golgi and plasma membrane via endocytosis and secretion of 150

vesicles. 151

152

Opt2 is required for the maintenance of vacuole morphology when lipid asymmetry 153

is altered 154

Since Opt2 is implicated in the regulation of vacuolar morphology (Aouida et 155

al., 2009), we investigated the potential involvement of the Rim101 pathway-dependent 156

response to altered lipid asymmetry in vacuolar homeostasis. In contrast to the normal 157

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

9

WT morphology in lem3Δ, rim21Δ, and opt2Δ cells, the vacuoles in rim21Δ lem3Δ and 158

opt2Δ lem3Δ cells were highly fragmented and formed small spheres attached to each 159

other (Fig. 4A). Overexpression of Opt2 from the ADH1 promoter could however restore 160

the normal vacuole morphology in both double mutant cells. These observations suggest 161

that Opt2 is involved in maintenance of vacuole morphology when lipid asymmetry is 162

altered. 163

Abnormal vacuole morphology is often attributed to defects in vesicular 164

trafficking to the vacuole (Raymond et al., 1992); thus, we examined two known 165

Golgi-vacuole transport pathways (the carboxypeptidase Y (CPY) and AP-3 pathways) in 166

WT, lem3Δ, opt2Δ, and opt2Δ lem3Δ cells. A soluble vacuolar protein such as CPY is 167

transported from the Golgi apparatus to the vacuole via the late endosome (the CPY 168

pathway) (Valls et al., 1987), while a vacuolar protein such as the alkaline phosphatase 169

Pho8 is transported to the vacuole directly from the Golgi apparatus (the AP-3 pathway) 170

(Klionsky and Emr, 1989). These two pathways were monitored by following the 171

processing of CPY and Pho8, respectively, using immunoblot analysis (Fig. 4B). In 172

vps21Δ cells in which the CPY pathway is defective, some of the Golgi CPY (p2-CPY) 173

was secreted to the extracellular medium as reported in the literature (Robinson et al., 174

1988). By contrast, in lem3Δ, opt2Δ, opt2Δ lem3Δ, as well as WT cells, both CPY and 175

Pho8 were processed to their normal vacuolar forms, i.e., the mature form of CPY 176

(m-CPY) and the mature and soluble forms of Pho8 (m- and s-Pho8), respectively. Next, 177

the endocytic pathway was monitored using the lipophilic dye FM4-64, which is first 178

incorporated into the plasma membrane and then transported to the vacuole via 179

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

10

endocytosis. Cells were treated with FM4-64 for 15 min and chased for 10 min. In all the 180

cells, the FM4-64 signal was detected mainly in the vacuolar membrane and partially at 181

the endosome (Fig. 4C), indicating that endocytosis is not affected in opt2Δ lem3Δ cells. 182

Taken together, it can be concluded that Opt2 does not play a role in vesicular trafficking 183

to the vacuole. 184

185

Opt2 is involved in exposure of phospholipids 186

To gain further insight into how Opt2 mediates adaptation to altered lipid 187

asymmetry, we measured the degree of lipid asymmetry in cells lacking or 188

overexpressing Opt2 based on their sensitivity to toxic peptides duramycin and 189

papuamide B, which specifically bind to cell surface PE (Zhao et al., 2008) and PS 190

(Parsons et al., 2006), respectively. In lem3Δ cells, PE and PS, normally confined to the 191

inner leaflet of the plasma membrane, are exposed to the cell surface (the outer leaflet) 192

due to their defective flippase activity; as a result, lem3Δ cells were hypersensitive to both 193

duramycin and papuamide B (Fig. 5A) (Noji et al., 2006; Parsons et al., 2006). 194

Deletion of either OPT2 or RIM21 in lem3Δ cells (opt2Δ lem3Δ or rim21Δ 195

lem3Δ cells, respectively) conferred some tolerance to both peptides, although the growth 196

of the double mutants on plates without the peptides was severely retarded; however, 197

these cells became sensitive again when Opt2 was overexpressed from the ADH1 198

promoter. Thus, Opt2 induced by the Rim101 pathway seems to be involved in the 199

exposure of PE and PS. However, it must be noted that deletion of RIM21 had a greater 200

effect than the deletion of OPT2 on the sensitivity of lem3Δ cells to papuamide B. 201

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

11

Furthermore, overexpression of Opt2 had a milder effect on papuamide B sensitivity in 202

rim21Δ lem3Δ cells than in opt2Δ lem3Δ cells (see Discussion). In rim21Δ cells, double 203

deletion of DNF1 and DNF2, both of which encode phospholipid floppases regulated by 204

Lem3, caused the same effects as deletion of LEM3 gene: the dnf1Δ dnf2Δ rim21Δ cells 205

exhibited slow growth and acquired tolerance to duramycin (Fig. S1A), confirming that 206

the effect of the LEM3 deletion is indeed the result of a defect in flip. 207

The surface-exposed PE was then detected by PE-specific 208

biotinylated-Ro09-198 (Bio-Ro) and visualized by FITC-conjugated streptavidin 209

(Iwamoto et al., 2004) (Fig. 5B). The structure of Ro-09-198 closely resembles that of 210

duramycin (Noji et al., 2006). As described in the literature (Iwamoto et al., 2004; Saito et 211

al., 2007), PE was highly exposed in lem3Δ cells but little in WT cells. Notably, the 212

exposed PE in lem3Δ cells was significantly reduced by deletion of OPT2 (opt2Δ lem3Δ 213

cells), and overexpression of Opt2 was found to enhance the surface exposure of PE in 214

opt2Δ lem3Δ cells (Fig. 5C). We confirmed that the levels and localization of Drs2 and 215

Dnf3 were comparable in these mutant cells and in WT cells (Fig. S1B and C) and thus 216

eliminated the possibility of increased levels of Lem3-independent flippases causing the 217

reduction of exposed PE in opt2Δ lem3Δ cells. Interestingly, levels of Pdr5, the plasma 218

membrane floppase, were significantly elevated in opt2Δ lem3Δ cells. These findings 219

substantiate the involvement of Opt2 in the exposure of PE. 220

The flip-flop-mediated transfer of fluorescence-labeled phospholipids 221

(NBD-PE, NBD-PC, and NBD-PS) was next assessed in cells overexpressing Opt2. After 222

back-extraction of NBD-phospholipids from the outer leaflet with BSA, the increase in 223

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

12

flop activity was estimated by the decrease in levels of NBD-phospholipids in the inner 224

leaflet using flow cytometry. Overexpression of Opt2 resulted in an approximately 25% 225

decrease in incorporation of NBD-phospholipids into the inner leaflet (Fig. 5D), 226

indicative of the direct or indirect involvement of Opt2 in phospholipid flop. 227

228

Opt2 is involved in polarized cell growth 229

The yeast bud grows apically during the early phase of budding, during which 230

PE is localized to the outer leaflet at the tip of the elongating bud. When the apical growth 231

switches to an isotropic growth, the exposed PE is flipped back to the inner leaflet. This 232

local alteration in lipid asymmetry regulates polarized cell growth; hence, the 233

flippase-defective lem3Δ cells have an elongated morphology (Saito et al., 2007). We 234

now investigated if Opt2 plays any role in apical growth by measuring the long- to 235

short-axis ratio of the cell. The ratio of lem3Δ cells was significantly larger than that of 236

WT cells as predicted, while opt2Δ cells were found to have a smaller ratio than WT cells 237

(Fig. 6), thus being more spherical than WT cells. In addition to its role in adaptation to 238

altered lipid asymmetry, Opt2 appears to play an important role in apical growth.239 Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

13

DISCUSSION 240

Opt2 plays an important role in cellular response to altered lipid asymmetry 241

We have identified Opt2, originally reported as a member of oligopeptide 242

transporter family (Lubkowitz et al., 1998), as a novel factor required for the Rim101 243

pathway-dependent adaptation to altered lipid asymmetry. Although the Rim101 pathway 244

is known as an alkaline-responsive pathway (Peñalva and Arst, 2004), we have 245

demonstrated that Opt2 is not involved in the alkaline response (Fig. 2). It is also known 246

that its gene OPT2 is not induced by external alkalization (Causton et al., 2001; Lamb et 247

al., 2001; Serrano et al., 2002). Interestingly, only one putative gene (YHR214W-A) 248

among the 27 genes extracted (Table S1) is reported as an alkaline responsive gene. Thus, 249

although both external alkalization and altered lipid asymmetry activate the Rim101 250

pathway, the set of genes induced by each perturbation is not likely to be determined 251

solely by the Rim101 pathway, but rather is determined in cooperation with other 252

signaling pathways. 253

Both trehalose and glycogen are known to accumulate in yeast during stress 254

(Francois and Parrou, 2001). This seems to be also the case in response to altered lipid 255

asymmetry, since most of the genes encoding enzymes for trehalose and glycogen 256

metabolism were induced in cells with altered lipid asymmetry (lem3Δ and pdr5Δ cells) 257

(Fig. S2), of which three genes (TPS2, GSY1, and GIP2) were induced in a Rim101 258

pathway-dependent manner (Table S1). The ART2 and ART4 genes encoding 259

arrestin-related proteins were also induced by altered lipid asymmetry (Fig. S3) with the 260

induction of ART2 being Rim101 pathway-dependent (Table S1). Arrestin-related 261

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

14

proteins are involved in endocytic turnover of plasma membrane proteins such as nutrient 262

transporters and receptors. In this regard, it is interesting that several genes encoding 263

transporters (or putative transporters) were upregulated in lem3Δ and pdr5Δ cells in a 264

Rim101 pathway-dependent manner, including MUP3, OPT2, MAL31, and SSU1 (Table 265

S1). It is an interesting subject to investigate the endocytic turnover of plasma membrane 266

proteins and the involvement of arrestin-related proteins in adaptation to altered lipid 267

asymmetry. It should be noted that the expression of ART9 (the gene encoding the 268

arrestin-related protein Art9, also known as Rim8 and essential for the Rim101 pathway) 269

was downregulated in lem3Δ cells and upregulated in lem3Δ rim21Δ cells (Fig. S3), 270

which is consistent with the fact that its expression is negatively regulated by the Rim101 271

pathway (Lamb and Mitchell, 2003). 272

273

Opt2 is a novel type of protein involved in exposure of phospholipids 274

We have provided evidence for the involvement of Opt2 in phospholipid 275

exposure on the extracytoplasmic leaflet. One simple explanatory hypothesis is that Opt2 276

is a floppase that directly mediates trans-bilayer movement of phospholipids. In this case, 277

Opt2 would represent a novel type of floppase, because it does not belong to the ABC 278

transporter family to which all known floppases (except for Rsb1) belong. An alternative 279

possibility is that Opt2 mediates exposure of phospholipids indirectly, e.g., by inhibiting 280

or activating unknown flippases or floppases, respectively, that function during 281

adaptation to altered lipid asymmetry. It is also possible that Opt2 regulates the 282

localization of unknown flippases/floppases. Therefore, one of the most important 283

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

15

directions for future work would be establishment of an in vitro system that allows direct 284

evaluation of Opt2-mediated translocation of phospholipids. 285

Our results demonstrate that Opt2 is involved in exposure of phospholipids, 286

especially in lem3Δ cells, in which phospholipid flip in the plasma membrane is largely 287

impaired. In this regard, it is reasonable to predict that induction of Opt2 would 288

exacerbate the phenotype of lem3Δ cells; however, contrary to this assumption, we 289

showed that induction of Opt2 is an important process in Rim101 pathway–mediated 290

adaptation to altered lipid asymmetry caused by LEM3 deletion. These apparently 291

paradoxical results may be explained follows. Disturbance in lipid asymmetry likely 292

affects events throughout the cell, because lipid asymmetry is involved in a wide range of 293

processes including generation of membrane potential, vesicular trafficking, 294

establishment of cell polarity, and cytokinesis. Therefore, the cellular response to altered 295

lipid asymmetry must be fairly complex. Regulated local activation/suppression of 296

flip-flop may be essential for a wide range of cellular events, and several factors involved 297

in flip-flop (including unknown factors) are likely to be induced, activated, or repressed 298

when lipid asymmetry is altered. Indeed, in some cases, NBD-PS is flipped more in 299

lem3Δ cells than in WT cells (Saito et al., 2004), suggesting that some unknown 300

compensatory mechanism is induced in these cases. Therefore, it is unlikely that effects 301

caused by a reduction in flip (in lem3Δ cells) can be cancelled by a simultaneous 302

reduction in flop. Opt2 is likely to be induced as an important factor in such global 303

responses. 304

Overexpression of Opt2 completely counteracted the duramycin resistance of 305

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

16

lem3Δ rim21Δ cells (Fig. 5A). By contrast, overexpression of Opt2 in WT cells caused a 306

rather small reduction in the incorporation of NBD-phospholipids into the inner leaflet of 307

the plasma membrane (Fig. 5D). These somewhat inconsistent results might be due to 308

presence of active plasma membrane flippases, Dnf1 and Dnf2, in WT but not in lem3Δ 309

rim21Δ cells. Alternatively, the seeming inconsistency between the results of the two 310

assays may be due to the fact that Opt2 is primarily localized to the Golgi apparatus, with 311

only a minor fraction to the plasma membrane (Fig. 3), together with the dramatic 312

difference in the incubation times used for each assays: 20 min for the NBD-phospholipid 313

transport assay vs. 50 h for the duramycin-sensitivity assay. Thus, the results from the 314

NBD-phospholipid assay reflect low levels of Opt2 in the plasma membrane; in contrast, 315

during the longer incubation period, the Opt2-mediated lipid asymmetry generated at the 316

Golgi apparatus could travel to the plasma membrane via secretory vesicles, making the 317

cells highly sensitive to duramycin. It could also be possible that if Opt2 functions as a 318

subunit of the floppase complex, the majority of overexpressed Opt2 would not fully 319

function since the other subunit is absent. 320

The effect of overexpression of Opt2 in rim21Δ lem3Δ cells was found to be 321

much greater on the duramycin sensitivity than the papuamide B sensitivity, and opt2Δ 322

lem3Δ showed much weaker tolerance to papuamide B than rim21Δ lem3Δ cells (Fig. 5A). 323

Therefore, Opt2 may not be the sole factor that determines the exposure of PS during 324

adaptation to altered lipid asymmetry; instead, some other key protein(s) might be 325

induced by the Rim101 pathway. 326

327

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

17

Opt2 is involved in the maintenance of vacuole morphology and polarized cell 328

growth 329

We have observed vacuole fragmentation in opt2Δ lem3Δ cells, indicative of 330

the possible involvement of Opt2 in the maintenance of vacuole morphology when lipid 331

asymmetry is altered (Fig. 4). Since Opt2 shuttles between the Golgi apparatus and the 332

plasma membrane (Fig. 3), it is conceivable that the lipid asymmetry generated by Opt2 333

at the Golgi apparatus and the plasma membrane may be propagated to the vacuole 334

membrane via the vesicular transport pathway. The fragmentation of vacuoles could also 335

be the result of the improper localization and activity of proteins in opt2Δ lem3Δ cells 336

required for vacuole maturation such as those involved in the homotypic fusion of 337

vacuoles. However, the latter possibility is less likely since opt2Δ lem3Δ cells did not 338

affect the maturation of vacuolar proteins such as CPY and Pho8, which is known to be 339

impaired in cells defective in the homotypic fusion of vacuoles (Wada et al., 1992). In 340

addition, the morphology and distribution of vacuoles appear to be different between 341

opt2Δ lem3Δ and homotypic fusion mutant cells: small spherical vacuoles attached to 342

each other (opt2Δ lem3Δ cells) vs. much smaller discrete vacuoles dispersed throughout 343

the cytoplasm (cells lacking the Rab-type small GTPase Ypt7 essential for the homotypic 344

vacuole fusion) (Wada and Anraku, 1992). 345

We have also demonstrated that Opt2 is involved in the apical growth of yeast 346

cells. This finding is supported by the previous analysis of yeast cell morphology (Ohya 347

et al., 2005) that revealed the smaller long- to short-axis ratio of opt2Δ cells than that of 348

WT cells at all growth stages investigated (for details, refer to the Saccaromyces 349

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

18

Cerevisiae Morphological Database (http://scmd.gi.k.u-tokyo.ac.jp/datamine/)). 350

Interestingly, the larger ratio was reported for cells deleted for either PDR5 or YOR1 both 351

encoding the phospholipid floppase. It seems that Opt2 may be specifically involved in 352

PE flop at the bud tip during apical growth. 353

Future studies should be aimed at direct in vitro examination of the flop activity 354

of Opt2, identification of proteins interacting with Opt2, and elucidation of the regulation 355

of Opt2 function, which would greatly deepen our understanding of cellular adaptation to 356

altered lipid asymmetry.357

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

19

MATERIALS AND METHODS 358

Yeast culture and media 359

Saccharomyces cerevisiae strains used in this study are listed in Table 1. Yeast cells were 360

grown at 30 ˚C to log phase in YPD (1% yeast extract, 2% bactopepton, and 2% 361

D-glucose) or synthetic complete (SC) medium (2% D-glucose and 0.67% yeast nitrogen 362

base without amino acids) with appropriate supplements. Alkaline treatment was 363

performed by adding 1 M Tris-HCl (pH 8.0) to culture medium at a final concentration of 364

100 mM. A 500 mM stock solution of 3-indoleacetic acid (IAA; Nacalai Tesque, Kyoto, 365

Japan) was prepared in ethanol and added to the medium at a final concentration of 500 366

μΜ. 367

368

Genetic manipulation and plasmid construction 369

Gene disruption was performed by replacing the entire coding region of the gene with a 370

marker gene. Chromosome fusion of mCherry or myc to the C-terminus of Sec7 or Opt2, 371

respectively, was performed using PCR-based gene disruption and modification 372

(Longtine et al., 1998). The sequence encoding mCherry or myc, the ADH1 terminator, 373

and a marker sequence was amplified by PCR from the pFA6a vector series (Longtine et 374

al., 1998) with a primer set containing the homologous region of each gene. For the 375

chromosomal fusion of GFP to the N-terminus of Opt2, the sequence encoding a marker 376

sequence, the ADH1 promoter, and the GFP tag was amplified by PCR from the 377

pYM-N9 (Janke et al., 2004) vector with a primer set containing the homologous region 378

of OPT2. Amplified cassettes were inserted directly into the chromosome by 379

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

20

homologous recombination. Integration of PADH1-HA-OPT2 gene into URA3 or TRP1 380

locus was performed as follows. The PADH1-HA-OPT2-TOPT2 sequence was amplified by 381

PCR from genomic DNA of YOK3260 (PADH-HA-OPT2) to have the Sac I and Xho I 382

sites at the 5’ and 3’ ends, respectively. The amplified fragment was cloned into the Sac 383

I/Xho I site of pRS306 or pRS304 (Sikorski and Hieter, 1989) to generate pOK563 or 384

pOK571, respectively. The pOK563 and pOK571 were linearized by Stu I and Hind III, 385

respectively, and inserted at the URA3 and TRP1 loci, respectively. The plasmid for the 386

expression of HA-Rim101 (pFI1) was a gift from Prof. T. Maeda (University of Tokyo, 387

Japan). 388

389

Immunoblot analysis 390

Proteins were separated by SDS-PAGE and transferred to an ImmobilonTM 391

polyvinylidene difluoride membrane (Millipore, Billerica, MA) as described previously 392

(Yamagata et al., 2011). The membrane was incubated with anti-HA (16B12; Covance, 393

Princeton, NJ), anti-CPY (Molecular Probes, Eugene, OR), anti-GFP (598; Medical & 394

Biological Laboratories, Nagoya, Japan), anti-myc (PL-14; Medical & Biological 395

Laboratories), anti-Pho8 (a gift from Prof. Y. Ohsumi, Tokyo Institute of Technology, 396

Japan), or anti-Pgk1 (Molecular Probes) antibody. Immunodetection was performed 397

using Western Lightning ECL Pro system (PerkinElmer Life Sciences, Waltham, MA) 398

with a bioimaging analyzer (LAS4000; Fuji Photo Film, Tokyo, Japan) or X-ray film. 399

400

DNA microarray analysis 401

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

21

Total RNA was prepared from yeast cell homogenates using RNeasy Mini kit (Qiagen, 402

Hilden, Germany). Poly (A)+ RNA was purified using mRNA Purification Kit 403

(Amersham Pharmacia Biotech, Piscataway, NJ). The quality of RNA was verified by 404

electrophoresis using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). 405

Two hundred nanograms of poly (A)+ RNA was labeled with Cy3 using Low Input Quick 406

Amp Labeling Kit (Agilent Technologies), and hybridized with the Yeast (V2) Gene 407

Expression Microarray (Agilent Technologies) according to the manufacturer’s 408

instructions. The microarray was scanned with Agilent G2565BA microarray scanner 409

(Agilent technologies), and the fluorescence intensity for each spot was quantified using 410

Feature Extraction software (Agilent Technologies). RNA from four independent 411

cultures for each strain was subjected to DNA microarray assay and their mean values 412

were analyzed using Subio Platform software (Subio, Kagoshima, Japan). 413

414

Bio-Ro staining 415

Exposed PE was visualized using Bio-Ro as reported previously (Iwamoto et al., 2004; 416

Saito et al., 2007) with slight modifications. A 1-ml culture of cells in log phase was 417

harvested and incubated in 20 μl of YPD medium containing 80 μM Bio-Ro at 4 ˚C for 3 418

h. Cells were washed once with PBS and fixed with 5% formaldehyde in PBS at room 419

temperature for 1 h. Cells were then washed with spheroplast buffer (1.2 M sorbitol, 0.1 420

M potassium phosphate, pH7.4) and resuspended in 100 μl of spheroplast buffer 421

containing 100 μg/ml zymolyase 100T (Seikagaku Kogyo, Tokyo, Japan). After addition 422

of β-mercaptoethanol to the final concentration of 28 mM, cells were incubated at 30 ˚C 423

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

22

for 10 min and washed twice with spheroplast buffer. Spheroplasted cells were attached 424

to poly L-lysine-coated multiwell slides and incubated in PBS containing 0.1% BSA at 425

room temperature for 20 min. Cells were washed three times with PBS, and incubated in 426

PBS containing 5 μg/ml fluorescein streptavidin (Vector Laboratories, Burlingame, CA) 427

at room temperature for 1 h. After five washes with PBS, cells were suspended in 428

ProLong Gold Antifade reagent (Life Technologies, Carlsbad, CA) and subjected to 429

fluorescence microscopy. 430

431

NBD-phospholipid transfer assay 432

NBD-PE 433

(1-myristoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-p434

hosphoethanolamine), NBD-PC 435

(1-myristoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-p436

hosphocholine), and NBD-PS 437

(1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-p438

hosphoserine) were purchased from Avanti Polar Lipids (Alabaster, AL). For each strain, 439

1.0 OD600 cells were harvested and resuspendend in SC medium. After addition of 440

NBD-PE, NBD-PC, or NBD-PS at the final concentration of 50 μM, cells were 441

incubated at 30 ˚C for 20 min, washed with SSA medium (0.67% yeast nitrogen base 442

without amino acids, 2% sorbitol, 0.5% casamino acids, 20 mg/ml tryptophan, 20 mg/l 443

adenine sulfate, 20 mg/l uracil, 0.067% sodium azide), and transferred to a new tube. 444

Cells were further washed twice with SSA medium containing 4% BSA and once with 445

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

23

SSA medium and resuspended in 100 μl SSA medium. Each sample was diluted with 446

SSA medium at 1:100 and analyzed by flow cytometry using FACS Calibur flow 447

cytometer (Becton Dickinson, Franklin Lakes, NJ). For each strain, values were 448

corrected by subtracting the background values for the unstained cells. 449

450

Measurement of the long and short axes of yeast cells 451

Cells were grown in SC medium to log phase, and 0.5 OD600 cells were harvested and 452

resuspended in 400 μl SC medium. Formaldehyde and potassium phosphate buffer (pH 453

6.6) were added at the final concentration of 3.7% and 100 mM, respectively, and cells 454

were incubated at 25 ˚C for 30 min to fix the cells. Cells were collected by centrifugation 455

and treated again with 100 mM potassium phosphate buffer (pH 6.6) containing 4% 456

formaldehyde at room temperature for 45 min. The fixed cells were washed and 457

resuspended in 500 μl of 100 mM potassium phosphate buffer (pH 6.6), stained with 1.6 458

μg/ml FITC-concanavalin A (Sigma, St. Louis, MO) at room temperature for 10 min, 459

and observed under fluorescence microscopy. The long and short axes of each cell were 460

measured using Image J software (National Institutes of Health, Bethesda, MD) and their 461

ratio was calculated. For each strain, 125 individual cells were analyzed. 462

463

Microscopy 464

Fluorescence was visualized using a fluorescence microscope (DM5000B, Leica 465

Microsystems, Wetzlar, Germany) equipped with 100x HCX PL FLUOTAR NA1.30 oil 466

immersion objective. Images were acquired with a cooled CCD camera (DFC365FX, 467

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

24

Leica Microsystems) controlled with LAS AF software (version 2.60, Leica 468

Microsystems) and archived using Photoshop CS3 (Adobe; San Jose, CA). In some cases, 469

a linear adjustment was applied to enhance the image contrast using the level adjustment 470

function of Photoshop. To visualize the vacuole, cells in log phase were stained with 1 471

μM FM4-64 (Molecular Probes) for 30 min, washed and resuspended in the medium, and 472

incubated for an additional 30 min. To monitor the progression of endocytosis, cells in log 473

phase were treated with 1 μM FM4-64 for 15 min, washed and resuspended in the same 474

medium, and incubated for an additional 10 min. After addition of sodium azide to the 475

final concentration of 20 mM, cells were kept on ice until examined by microscopy.476

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

25

Acknowledgments 477

We are grateful to Prof. M. Umeda and Dr. U. Kato (Graduate School of Engineering, 478

Kyoto University) for providing the biotinylated Ro 09-0198 (Bio-Ro), to Dr. T. Maeda 479

(Institute of Molecular and Cellular Biosciences, University of Tokyo) for providing the 480

HA-Rim101 plasmid (pFI1), and to Prof. Y. Ohsumi (Tokyo Institute of Technology) for 481

providing the anti-Pho8 antibody. The template plasmid for N-terminal tagging with GFP 482

(pYM-N9) was provided from the European S. Cerevisiae Archive for Functional 483

Analysis (Euroscarf, Germany) and the AID system was from the National Bio-Resource 484

Project (NBRP) of the MEXT, Japan. We also thank Dr. T. Toyokuni for editing the 485

manuscript. 486

487

Competing interests 488

The authors declare no competing interests. 489

490

Author contributions 491

S.Y., K.O., K.U., A.K. (Akiko Kamimura), and K.A. did the experiments. K.O. and K.A. 492

designed the experiments. S.Y., K.O., and A.K. (Akio Kihara) analyzed the data. K.O. 493

wrote the manuscript. 494

495

Funding 496

This work was supported by a Grant-in-Aid for Scientific Research (C) (25440038) and a 497

Grant-in-Aid for Young Scientists (B) (23770135) to K.O. and a Grant-in-Aid for 498

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

26

Challenging Exploratory Research (25650059) to A.K. from Japan Society for the 499

Promotion of Science (JSPS). 500

501

Abbreviations used in this paper 502

Bio-Ro, biotynylated-Ro09-198; IAA, 3-indoleacetic acid; PC, phosphatidylcholine; PE, 503

phosphatidylethanolamine; PS, phosphatidylserine; SC, synthetic complete; WT, 504

wild-type. 505

506

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

27

References 507

508

Allikmets, R., Singh, N., Sun, H., Shroyer, N. F., Hutchinson, A., Chidambaram, A., 509

Gerrard, B., Baird, L., Stauffer, D., Peiffer, A. et al. (1997). A photoreceptor 510

cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt 511

macular dystrophy. Nat. Genet. 15, 236-246. 512

513

Aouida, M., Khodami-Pour, A. and Ramotar, D. (2009). Novel role for the 514

Saccharomyces cerevisiae oligopeptide transporter Opt2 in drug detoxification. Biochem. 515

Cell Biol. 87, 653-661. 516

517

Axelsen, K. B. and Palmgren, M. G. (1998). Evolution of substrate specificities in the 518

P-type ATPase superfamily. J. Mol. Evol. 46, 84-101. 519

520

Baldridge, R. D. and Graham, T. R. (2012). Identification of residues defining 521

phospholipid flippase substrate specificity of type IV P-type ATPases. Proc. Natl. Acad. 522

Sci. USA 109, E290-298. 523

524

Bull, L. N., van Eijk, M. J., Pawlikowska, L., DeYoung, J. A., Juijn, J. A., Liao, M., 525

Klomp, L. W., Lomri, N., Berger, R., Scharschmidt, B. F. et al. (1998). A gene 526

encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nat. Genet. 18, 527

219-224. 528

529

Castrejon, F., Gomez, A., Sanz, M., Duran, A. and Roncero, C. (2006). The RIM101 530

pathway contributes to yeast cell wall assembly and its function becomes essential in the 531

absence of mitogen-activated protein kinase Slt2p. Eukaryot. Cell 5, 507-517. 532

533

Causton, H. C., Ren, B., Koh, S. S., Harbison, C. T., Kanin, E., Jennings, E. G., Lee, 534

T. I., True, H. L., Lander, E. S. and Young, R. A. (2001). Remodeling of yeast genome 535

expression in response to environmental changes. Mol. Biol. Cell 12, 323-337. 536

537

Chen, C. Y., Ingram, M. F., Rosal, P. H. and Graham, T. R. (1999). Role for Drs2p, a 538

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

28

P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function. 539

J. Cell Biol. 147, 1223-1236. 540

541

Decottignies, A., Grant, A. M., Nichols, J. W., de Wet, H., McIntosh, D. B. and 542

Goffeau, A. (1998). ATPase and multidrug transport activities of the overexpressed yeast 543

ABC protein Yor1p. J. Biol. Chem. 273, 12612-12622. 544

545

Devaux, P. F. (1991). Static and dynamic lipid asymmetry in cell membranes. 546

Biochemistry 30, 1163-1173. 547

548

Emoto, K. and Umeda, M. (2000). An essential role for a membrane lipid in cytokinesis. 549

Regulation of contractile ring disassembly by redistribution of phosphatidylethanolamine. 550

J. Cell Biol. 149, 1215-1224. 551

552

Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L. and 553

Henson, P. M. (1992). Exposure of phosphatidylserine on the surface of apoptotic 554

lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 555

2207-2216. 556

557

Francois, J. and Parrou, J. L. (2001). Reserve carbohydrates metabolism in the yeast 558

Saccharomyces cerevisiae. FEMS Microbiol. Rev. 25, 125-145. 559

560

Furuta, N., Fujimura-Kamada, K., Saito, K., Yamamoto, T. and Tanaka, K. (2007). 561

Endocytic recycling in yeast is regulated by putative phospholipid translocases and the 562

Ypt31p/32p-Rcy1p pathway. Mol. Biol. Cell 18, 295-312. 563

564

Gurtovenko, A. A. and Vattulainen, I. (2008). Membrane potential and electrostatics 565

of phospholipid bilayers with asymmetric transmembrane distribution of anionic lipids. J. 566

Phys. Chem. B 112, 4629-4634. 567

568

Hua, Z., Fatheddin, P. and Graham, T. R. (2002). An essential subfamily of 569

Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex 570

and endosomal/vacuolar system. Mol. Biol Cell. 13, 3162-3177. 571

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

29

572

Ikeda, M., Kihara, A., Denpoh, A. and Igarashi, Y. (2008). The Rim101 pathway is 573

involved in Rsb1 expression induced by altered lipid asymmetry. Mol. Biol. Cell 19, 574

1922-1931. 575

576

Ikeda, M., Kihara, A. and Igarashi, Y. (2006). Lipid asymmetry of the eukaryotic 577

plasma membrane: functions and related enzymes. Biol. Pharm. Bull. 29, 1542-1546. 578

579

Iwamoto, K., Kobayashi, S., Fukuda, R., Umeda, M., Kobayashi, T. and Ohta, A. 580

(2004). Local exposure of phosphatidylethanolamine on the yeast plasma membrane is 581

implicated in cell polarity. Genes Cells 9, 891-903. 582

583

Janke, C., Magiera, M. M., Rathfelder, N., Taxis, C., Reber, S., Maekawa, H., 584

Moreno-Borchart, A., Doenges, G., Schwob, E., Schiebel, E. et al. (2004). A versatile 585

toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers 586

and promoter substitution cassettes. Yeast 21, 947-962. 587

588

Kihara, A. and Igarashi, Y. (2002). Identification and characterization of a 589

Saccharomyces cerevisiae gene, RSB1, involved in sphingoid long-chain base release. J. 590

Biol. Chem. 277, 30048-30054. 591

592

Kihara, A. and Igarashi, Y. (2004). Cross talk between sphingolipids and 593

glycerophospholipids in the establishment of plasma membrane asymmetry. Mol. Biol. 594

Cell 15, 4949-4959. 595

596

Klionsky, D. J. and Emr, S. D. (1989). Membrane protein sorting: biosynthesis, 597

transport and processing of yeast vacuolar alkaline phosphatase. EMBO J. 8, 2241-2250. 598

599

Lamb, T. M. and Mitchell, A. P. (2003). The transcription factor Rim101p governs ion 600

tolerance and cell differentiation by direct repression of the regulatory genes NRG1 and 601

SMP1 in Saccharomyces cerevisiae. Mol. Cell. Biol. 23, 677-686. 602

603

Lamb, T. M., Xu, W., Diamond, A. and Mitchell, A. P. (2001). Alkaline response 604

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

30

genes of Saccharomyces cerevisiae and their relationship to the RIM101 pathway. J. Biol. 605

Chem. 276, 1850-1856. 606

607

Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, 608

A., Philippsen, P. and Pringle, J. R. (1998). Additional modules for versatile and 609

economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. 610

Yeast 14, 953-961. 611

612

Lubkowitz, M. A., Barnes, D., Breslav, M., Burchfield, A., Naider, F. and Becker, J. 613

M. (1998). Schizosaccharomyces pombe isp4 encodes a transporter representing a novel 614

family of oligopeptide transporters. Mol. Microbiol. 28, 729-741. 615

616

Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. and Kanemaki, M. 617

(2009). An auxin-based degron system for the rapid depletion of proteins in nonplant 618

cells. Nat. Methods 6, 917-922. 619

620

Noji, T., Yamamoto, T., Saito, K., Fujimura-Kamada, K., Kondo, S. and Tanaka, K. 621

(2006). Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating 622

P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region 623

of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast. 624

Biochem. Biophys. Res. Commun. 344, 323-331. 625

626

Obara, K. and Kihara, A. (2014). Signaling events of the Rim101 pathway occur at the 627

plasma membrane in a ubiquitination-dependent manner. Mol. Cell. Biol., in press 628

629

Obara, K., Yamamoto, H. and Kihara, A. (2012). Membrane protein Rim21 plays a 630

central role in sensing ambient pH in Saccharomyces cerevisiae. J. Biol. Chem. 287, 631

38473-38481. 632

633

Ohya, Y., Sese, J., Yukawa, M., Sano, F., Nakatani, Y., Saito, T. L., Saka, A., 634

Fukuda, T., Ishihara, S., Oka, S. et al. (2005). High-dimensional and large-scale 635

phenotyping of yeast mutants. Proc. Natl. Acad. Sci. USA 102, 19015-19020. 636

637

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

31

Parsons, A. B., Lopez, A., Givoni, I. E., Williams, D. E., Gray, C. A., Porter, J., 638

Chua, G., Sopko, R., Brost, R. L., Ho, C. H. et al. (2006). Exploring the 639

mode-of-action of bioactive compounds by chemical-genetic profiling in yeast. Cell 126, 640

611-625. 641

642

Peñalva, M. A. and Arst, H. N., Jr. (2002). Regulation of gene expression by ambient 643

pH in filamentous fungi and yeasts. Microbiol. Mol. Biol. Rev. 66, 426-446. 644

645

Peñalva, M. A. and Arst, H. N., Jr. (2004). Recent advances in the characterization of 646

ambient pH regulation of gene expression in filamentous fungi and yeasts. Annu. Rev. 647

Microbiol. 58, 425-451. 648

649

Pomorski, T., Lombardi, R., Riezman, H., Devaux, P. F., van Meer, G. and Holthuis, 650

J. C. (2003). Drs2p-related P-type ATPases Dnf1p and Dnf2p are required for 651

phospholipid translocation across the yeast plasma membrane and serve a role in 652

endocytosis. Mol. Biol. Cell 14, 1240-1254. 653

654

Raymond, C. K., Roberts, C. J., Moore, K. E., Howald, I. and Stevens, T. H. (1992). 655

Biogenesis of the vacuole in Saccharomyces cerevisiae. Int. Rev. Cytol. 139, 59-120. 656

657

Robinson, J. S., Klionsky, D. J., Banta, L. M. and Emr, S. D. (1988). Protein sorting in 658

Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing 659

of multiple vacuolar hydrolases. Mol. Cell. Biol. 8, 4936-4948. 660

661

Saito, K., Fujimura-Kamada, K., Furuta, N., Kato, U., Umeda, M. and Tanaka, K. 662

(2004). Cdc50p, a protein required for polarized growth, associates with the Drs2p P-type 663

ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae. Mol. Biol. 664

Cell 15, 3418-3432. 665

666

Saito, K., Fujimura-Kamada, K., Hanamatsu, H., Kato, U., Umeda, M., Kozminski, 667

K. G. and Tanaka, K. (2007). Transbilayer phospholipid flipping regulates Cdc42p 668

signaling during polarized cell growth via Rga GTPase-activating proteins. Dev. Cell 13, 669

743-751. 670

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

32

671

Seigneuret, M. and Devaux, P. F. (1984). ATP-dependent asymmetric distribution of 672

spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes. Proc. 673

Natl. Acad. Sci. USA 81, 3751-3755. 674

675

Serrano, R., Ruiz, A., Bernal, D., Chambers, J. R. and Ariño, J. (2002). The 676

transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for 677

calcium-mediated signalling. Mol. Microbiol. 46, 1319-1333. 678

679

Sikorski, R. S. and Hieter, P. (1989). A system of shuttle vectors and yeast host strains 680

designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 681

19-27. 682

683

Toti, F., Satta, N., Fressinaud, E., Meyer, D. and Freyssinet, J. M. (1996). Scott 684

syndrome, characterized by impaired transmembrane migration of procoagulant 685

phosphatidylserine and hemorrhagic complications, is an inherited disorder. Blood 87, 686

1409-1415. 687

688

Valls, L. A., Hunter, C. P., Rothman, J. H. and Stevens, T. H. (1987). Protein sorting 689

in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the 690

propeptide. Cell 48, 887-897. 691

692

Verkleij, A. J. and Post, J. A. (2000). Membrane phospholipid asymmetry and signal 693

transduction. J. Membr. Biol. 178, 1-10. 694

695

Wada, Y. and Anraku, Y. (1992). Genes for directing vacuolar morphogenesis in 696

Saccharomyces cerevisiae. II. VAM7, a gene for regulating morphogenic assembly of the 697

vacuoles. J. Biol. Chem. 267, 18671-18675. 698

699

Wada, Y., Ohsumi, Y. and Anraku, Y. (1992). Genes for directing vacuolar 700

morphogenesis in Saccharomyces cerevisiae. I. Isolation and characterization of two 701

classes of vam mutants. J. Biol. Chem. 267, 18665-18670. 702

703

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

33

Yamagata, M., Obara, K. and Kihara, A. (2011). Sphingolipid synthesis is involved in 704

autophagy in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 410, 786-791. 705

706

Zhao, M., Li, Z. and Bugenhagen, S. (2008). 99mTc-labeled duramycin as a novel 707

phosphatidylethanolamine-binding molecular probe. J. Nucl. Med. 49, 1345-1352. 708

709

710

Figure legend 711

Fig. 1. Sensing of altered lipid asymmetry by the Rim101 pathway is important for 712

cell growth. 713

SEY6210 (WT), YOK2030 (lem3Δ), YOK2027 (rim21Δ), YYS4 (rim21Δ lem3Δ), 714

YOK2349 (rsb1Δ), and YYS7 (rsb1Δ lem3Δ) cells were grown to stationary phase, 715

serially diluted at 1:10, spotted on YPD plates, and grown at 30 ˚C for 24 h. 716

717

Fig. 2. Opt2 plays an important role in adaptation to altered lipid asymmetry 718

mediated by the Rim101 pathway. 719

(A) SEY6210 (WT), YYS5 (lem3Δ), YYS12 (opt2Δ), YYS13 (opt2Δ lem3Δ), YOK2027 720

(rim21Δ), YYS52 (rim21Δ lem3Δ), YOK3260 (rim21Δ lem3Δ PADH-HA-OPT2), and 721

YOK3259 (opt2Δ lem3Δ PADH-HA-OPT2) cells were grown to stationary phase, serially 722

diluted at 1:10, spotted on YPD plates, and grown at 30 ˚C for 28 h. (B) DNA microarray 723

analysis was performed on SEY6210 (WT), YOK2030 (lem3Δ), YOK2368 (pdr5Δ), and 724

YOK2209 (rim21Δ lem3Δ) cells. Expression levels of OPT2 (closed circle) and, to 725

demonstrate uniform use of poly (A)+ RNAs, ACT1 (open circle) are shown as a relative 726

value to that in WT cells. (C) Total lysates were prepared from YYS351 (OPT2-myc), 727

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

34

YYS352 (OPT2-myc lem3Δ), YYS354 (OPT2-myc pdr5Δ), YYS355 (OPT2-myc 728

rim21Δ), and YYS356 (OPT2-myc rim21Δ lem3Δ) cells in log phase and subjected to 729

immunoblotting with an anti-myc antibody. Ponceau S staining was used to demonstrate 730

uniform protein loading. (D) SEY6210 (WT), YOK2027 (rim21Δ), YOK2030 (lem3Δ), 731

YYS12 (opt2Δ), and YYS13 (opt2Δ lem3Δ) cells harboring pFI1 (HA-RIM101) were 732

grown to log phase and exposed to an alkaline medium (pH 8.0) for 20 min. Total lysates 733

were then prepared and imuunoblotted with anti-HA antibody. FL and ΔC denote the 734

full-length and processed Rim101, respectively. (E) SEY6210 (WT), YYS5 (lem3Δ), 735

YOK2053 (rim21Δ), YYS4 (rim21Δ lem3Δ), YYS12 (opt2Δ), and YYS13 (opt2Δ 736

lem3Δ) cells in stationary phase were serially diluted at 1:10, spotted on YPD plates, and 737

grown at pH 8.0 and 30 ˚C for 91 h. 738

739

Fig. 3. Opt2 cycles between the plasma membrane and the late Golgi. 740

(A) YYS250 (GFP-OPT2 SEC7-mCherry) cells were grown to log phase and subjected 741

to fluorescence microscopy. Arrowheads indicate GFP-Opt2 co-localized with 742

Snf7-mCherry. Bar, 2 μm. (B) YOK3206 (GFP-OPT2 LAS17-HA-AID) cells were grown 743

to log phase, treated with 500 μM 3-indoleacetic acid (IAA) or ethanol (mock) for 30 min, 744

and subjected to fluorescence microscopy. Bar, 2 μm. 745

746

Fig. 4. Opt2 is involved in the maintenance of vacuole morphology in cells with 747

altered lipid asymmetry. 748

(A) SEY6210 (WT), YYS195 (lem3Δ), YOK2027 (rim21Δ), YYS52 (rim21Δ lem3Δ), 749

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

35

YYS12 (opt2Δ), YYS13 (opt2Δ lem3Δ), YOK3260 (rim21Δ lem3Δ PADH-HA-OPT2), 750

and YOK3259 (opt2Δ lem3Δ PADH-HA-OPT2) cells in log phase were stained with 751

FM4-64 and examined for vacuole morphology under a fluorescence microscope. Inset, 752

magnified image of the region surrounded by the dashed line. Bar, 2 μm. (B) Total lysates 753

were prepared from SEY6210 (WT), YYS5 (lem3Δ), YYS12 (opt2Δ), YYS13 (opt2Δ 754

lem3Δ), and YOk3156 (vps21Δ) cells in log phase and were subjected to immunoblotting 755

with anti-CPY, anti-Pho8 or, to demonstrate uniform protein loading, anti-Pgk1 antibody. 756

Immunoblotting of each extracellular fraction (medium) was also performed with 757

anti-CPY antibody. p, p2, m, and s indicates proform, p2 Golgi form, mature form, and 758

soluble form, respectively. (C) SEY6210 (WT), YYS5 (lem3Δ), YYS12 (opt2Δ), and 759

YYS13 (opt2Δ lem3Δ) cells in log phase were pulse labeled with FM4-64 and examined 760

under a fluorescence microscope. Arrowheads indicate endosomes labeled with FM4-64. 761

762

Fig. 5. Opt2 is involved in the exposure of PE and PS to the outer leaflet of the 763

plasma membrane. 764

(A) SEY6210 (WT), YYS5 (lem3Δ), YYS12 (opt2Δ), YYS13 (opt2Δ lem3Δ), YOK2027 765

(rim21Δ), YYS52 (rim21Δ lem3Δ), YOK3260 (rim21Δ lem3Δ PADH-HA-OPT2), and 766

YOK3259 (opt2Δ lem3Δ PADH-HA-OPT2) cells were grown to stationary phase, serially 767

diluted at 1:10, spotted on YPD plates with or without 5 μM duramycin or 0.25 μg/mL 768

papuamide B, and grown at 30 ˚C for 50 h. (B) SEY6210, YYS5, and YYS13 cells in log 769

phase were collected and the surface-exposed PE was visualized using Bio-Ro and 770

FITC-labeled streptavidin. Arrowheads indicate FITC signal. Bar, 2 μm. (C) The 771

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

36

percentage of cells with FITC signal is presented as the mean ± SD from three 772

independent experiments (*, P < 0.001; Student’s t test). (D) SEY6210 and YYS307 773

(PADH-HA-OPT2) cells in log phase were harvested and subjected to the 774

NBD-phospholipid transfer assay. Values represent the mean ± SD from three 775

independent experiments (*, P < 0.05; Student’s t test). 776

777

Fig. 6. Opt2 is involved in apical growth. 778

SEY6210 (WT), YYS12 (opt2Δ), and YYS5 (lem3Δ) cells in log phase were harvested, 779

fixed, and stained with FITC-concanavalin A. The ratio of the long- to short-axis of the 780

cell was calculated. For each strain, 125 individual cells were analyzed and the value for 781

each cell (open circle) and the average value (white bar) are shown. *, P < 0.001; 782

Student’s t test. 783

784

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

37

Table 1. Yeast strains used in this study 785

Strain Genotype Source

SEY6210 MATα his3 leu2 ura3 trp1 lys2 suc2 (Robinson et al., 1988)

YYS4 SEY6210, rim21Δ::NatNT2 lem3Δ::KanMX4 This study

YYS5 SEY6210, lem3Δ::NatNT2 This study

YYS7 SEY6210, rsb1Δ::NatNT2 lem3Δ::KanMX4 This study

YYS12 SEY6210, opt2Δ::KanMX4 This study

YYS13 SEY6210, opt2Δ::KanMX4 lem3Δ::NatNT2 This study

YYS52 SEY6210, rim21Δ::KanMX4 lem3Δ::HIS3 This study

YYS195 SEY6210, lem3Δ::HIS3 This study

YYS211 SEY6210, DRS2-GFP::TRP1 This study

YYS213 SEY6210, DNF3-GFP::TRP1 This study

YYS219 SEY6210, DRS2-GFP::TRP1 opt2Δ::KanMX4 This study

YYS221 SEY6210, DNF3-GFP::TRP1 opt2Δ::KanMX4 This study

YYS223 SEY6210, DRS2-GFP::TRP1 opt2Δ::KanMX4 lem3Δ::NatNT2 This study

YYS225 SEY6210, DNF3-GFP::TRP1 opt2Δ::KanMX4 lem3Δ::NatNT2 This study

YYS250 SEY6210, PADH-GFP-OPT2::NatNT2 SEC7-mCherry::KanMX6 This study

YYS283 SEY6210, opt2Δ::KanMX4 lem3Δ::HIS3 This study

YYS307 SEY6210, PADH-HA-OPT2::URA3 This study

YYS328 SEY6210, DRS2-GFP::TRP1 lem3Δ::NatNT2 This study

YYS345 SEY6210, DNF3-GFP::TRP1 lem3Δ::NatNT2 This study

YYS346 SEY6210, PDR5-GFP::TRP1 This study

YYS347 SEY6210, PDR5-GFP::TRP1 lem3Δ::NatNT2 This study

YYS348 SEY6210, PDR5-GFP::TRP1 opt2Δ::KanMX4 This study

YYS349 SEY6210, PDR5-GFP::TRP1 opt2Δ::KanMX4 lem3Δ::NatNT2 This study

YYS351 SEY6210, OPT2-MYC::TRP1 This study

YYS352 SEY6210, OPT2-MYC::TRP1 lem3Δ::NatNT2 This study

YYS354 SEY6210, OPT2-MYC::TRP1 pdr5Δ::URA3 This study

YYS355 SEY6210, OPT2-MYC::TRP1 rim21Δ::NatNT2 This study

YYS356 SEY6210, OPT2-MYC::TRP1 rim21Δ::KanMX4 lem3Δ::NatNT2 This study

YOK2027 SEY6210, rim21Δ::KanMX4 (Obara et al., 2012)

YOK2030 SEY6210, lem3Δ::KanMX4 This study

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

38

YOK2053 SEY6210, rim21Δ::NatNT2 (Obara et al., 2012)

YOK2209 SEY6210, rim21Δ::NatNT2 lem3Δ::KanMX4 This study

YOK2349 SEY6210, rsb1Δ::NatNT2 This study

YOK2368 SEY6210, pdr5Δ::KanMX4 This study

YOK3156 SEY6210, vps21Δ::NatNT2 This study

YOK3206 SEY6210, PADH-OSTIR1-MYC::URA3 LAS17-HA-AID::KanMX6

PADH-GFP-OPT2::URA3

This study

YOK3259 SEY6210, opt2Δ::KanMX4 lem3Δ::HIS3 PADH-HA-OPT2::URA3 This study

YOK3260 SEY6210, rim21Δ::KanMX4 lem3Δ::HIS3 PADH-HA-OPT2::URA3 This study

YOK3562 SEY6210, rim21Δ::NatNT2 dnf1Δ::HIS3 dnf2Δ::KanMX4

HA-RSB1::TRP1

This study

KHY612 SEY6210, dnf1Δ::HIS3 dnf2Δ::KanMX4 HA-RSB1::TRP1 (Kihara and Igarashi, 2004)

786

787

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t