1 2 3 The Transmission Interface of the Yeast Multidrug Transporter

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The Transmission Interface of the Yeast Multidrug Transporter Pdr5: Val-656 4 Located in ICL2 Plays a Major Role in Modulating Drug Resistance 5

6 Marianne T. Downes,a Jitender Mehla,a Neeti Ananthaswamy,a Adina Wakschlag,a 7

Micheala Lamonde,a Elliot Dine,a Suresh V. Ambudkar,b and John Golina# 8 9 10

aDepartment of Biology, The Catholic University of America, Washington, DC 20064 11 bLaboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, 12 National Institutes of Health, Bethesda, MD 20892 13

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Running Title: ICL2 of Pdr5 Modulates Drug Resistance 18 19 20

21 22 23 24 25 26 27

# Corresponding author: 28 John Golin 29 Tel: 202-319-5722 30 Fax: 202-319-5721 31 [email protected] 32

33 34 35 36 37 38 39 40 41 42 43 44 45 46

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.02133-12 AAC Accepts, published online ahead of print on 17 December 2012

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Pdr5 is a major ABC multidrug transporter regarded as the founding member of a 50

fungal subfamily of clinically significant efflux pumps. When these proteins are 51

overexpressed, they confer broad-spectrum ultraresistance. To better understand 52

the evolution of these proteins under selective pressure, we exposed a yeast strain 53

already overexpressing Pdr5 to a lethal concentration of cycloheximide. This 54

approach gave mutations that confer greater resistance to a subset of transport 55

substrates. One of these mutations, V656L, is located in intracellular loop 2 (ICL2), 56

a region predicted by structural studies with several other ABC transporters to play 57

a critical role in the transmission interface between the ATP hydrolysis and drug 58

transport domains. We show that this mutation increases drug resistance, possibly 59

by altering the efficiency with which the energy from ATP hydrolysis is used for 60

transport. Val-656 is a conserved residue and an alanine substitution creates a 61

nearly null phenotype for drug transport, as well as reduced ATPase activity. We 62

posit that despite its unusually small size, ICL2 is part of the transmission interface 63

and that alterations in this pathway can increase or decrease resistance to a broad 64

spectrum of drugs. 65

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The World Health Organization lists antimicrobial resistance as a major global concern 71

that threatens advancements in medicine, national security, and economic development 72

(http://www.who.int/mediacentre/factsheets/fs194/en/index.html). Multidrug resistance 73

remains a major clinical problem in the treatment of cancer and pathogenic infection (1). 74

This phenomenon is attributable, in part, to the overexpression of ATP-binding cassette 75

(ABC) efflux pumps that were initially identified and characterized in mammalian cells 76

(2-5). Multidrug resistance was observed in yeast in 1973, when it was attributed to a 77

single-gene alteration (6), but the cloning and characterization of the efflux pumps 78

mediating this phenomenon occurred much later (7-12). 79

80

Pdr5 is a highly promiscuous ABC transporter. Overexpression leads to hyperresistance 81

for all known substrates (13-15). This efflux pump has a basic structure found in full-82

length members of this superfamily. It has two nucleotide-binding domains and two 83

transmembrane domains (TMDs) made up of six alpha helices, each of which is 84

connected by intra- and extracellular loops. In several significant ways, however, Pdr5 85

departs from the canonical architecture of ABC transporters. It has a deviant ATP-86

binding site that lacks an obvious catalytic residue in the Walker B motif; it also contains 87

deviant sequences in the Walker A, signature region (C-loop), and Q-loop motifs. 88

Furthermore, by contrast to most ABC transporters, the intracellular loops (ICLs) are 89

very short. ICL2 and ICL4 are predicted to have only 10 and 7 residues, respectively 90

(16). Important studies with Tap1/Tap2 antigen transporter (17) and P-gp (18) using 91

cross-linking of cysteine-modified residues provide evidence that the X-loop, Q-loop, and 92

intracellular loops are critical for establishing a transmission interface suggested by the 93


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atomic structure of the Sav1866 multidrug transporter (19). In vivo evidence for a similar 94

interface was established in Pdr5 by using suppressor mutations (20, 21). In this 95

transporter, a signal transmission–deficient mutation in transmembrane helix 2 (TMH2) 96

(S558Y) was suppressed by second-site mutations in ICL1 (S597T, S597I) and the Q-97

loop region (N242K, E244G, D246∆). These residues were implicated in intradomain 98

signaling from the Sav1866 atomic structure. 99

100

Here we present new observations on the role of ICLs in the evolution of a highly 101

asymmetric transporter; this investigation arose from a seemingly different issue 102

concerning the evolution of ABC efflux pumps. Although overexpression of transporters 103

creates hyperresistance to xenobiotic substrates, increasing the concentration beyond the 104

IC50 will kill even overexpressing cells. However, mutations can be selected that show 105

even greater resistance than the original overexpressing strain. It is of great interest to 106

understand how this occurs. We sought to determine whether such changes are limited to 107

further increases in expression or to changes in the drug-binding pocket. We isolated 108

mutants with increased resistance in an overexpressing strain on plates containing an 109

otherwise lethal (5.4 μM) dose of cycloheximide (cyh). One of these mutations, V656L, 110

is in ICL2 and confers increased resistance to Pdr5 substrates of diverse structure and 111

function. We present evidence suggesting a novel mechanism of increased drug 112

resistance in which a mutation in the transmission interface increases the efficiency by 113

which the energy of ATP hydrolysis is used to power drug efflux. We also show that Val-114

656 is a conserved residue and that a V656A mutation has a phenotype characterized by 115

markedly reduced transport and ATPase activity. 116


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MATERIALS AND METHODS 117

Chemicals. All chemicals were purchased from Sigma Aldrich (St. Louis, MO) except 118

for 5-fluororotic acid (5-FOA) and G-418, which we obtained from Research Products 119

International (Mt. Prospect, IL), aprotinin which we purchased from Fisher Scientific 120

(Waltham, MA), and climbazole, cyprodinil, cerulenin, cyproconazole, tebuconazole, 121

and imazalil, which we obtained from LKT laboratories (St. Paul, MN). All chemicals 122

were dissolved in DMSO except for cyh, which was dissolved in water, and 5-FOA and 123

G-418, which were dissolved in sterilized media. IAAP was purchased from PerkinElmer 124

Life Sciences (Waltham, MA). 125

Yeast strains and plasmids. All yeast strains used in this study are isogenic and are 126

derived from R-1, a stock lacking all plasma membrane ABC transporters. It also 127

contains a PDR1-3 mutation which results in overexpression of PDR5 when the gene is 128

inserted into its chromosomal location following transformation. This strain offers other 129

advantages for genetic and biochemical analyses, which are described in detail elsewhere 130

(20, 21) and include the ability to easily construct stocks with two copies of either wild 131

type (WT) or mutant PDR5 genes thus resulting in more robust biochemical assays. All 132

culturing of strains was carried out at 30°C. The pSS607-integrating plasmid (22) was 133

used for all site-directed mutagenesis, as previously described. This plasmid has a WT 134

PDR5 gene under the transcriptional control of its own upstream region as well as a 135

URA3-selectable marker. Strains containing two copies of either WT or a mutant PDR5 136

gene were used for ATPase and IAAP photoaffinity labeling experiments. The rationale 137

for using and the method for generating these strains were previously described (20). 138


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Yeast transformation. Integrative transformation of pSS607 into the ∆pdr5 strain and its 139

isogenic derivatives was performed with a Sigma-Aldrich lithium acetate transformation 140

kit (Sigma-Aldrich). 141

Determination of IC50s. We determined the IC50s for Pdr5 transport substrates by 142

incubating cells in 2 ml YPD broth containing varying concentrations of drug. Cultures 143

were started with an initial inoculum of 0.5 × 105 cells and incubated at 30°C for 48 h in a 144

shaking water bath. The cell concentration was determined with a spectrophotometer at 145

600 nm. The percent growth relative to the untreated culture was calculated. 146

Isolation of mutants with increased resistance to Cyh. Ten 2-ml cultures of YPD were 147

inoculated with 104 cells of the yeast strain JG2015. This strain was produced by 148

transforming R-1 with the integrating plasmid pSS607, which contains a WT copy of 149

PDR5. The strain also contains a G-418 resistance cassette that replaces the PDR5 orf, 150

although the PDR5 upstream and downstream sequences remain. This arrangement 151

permits several important genetic manipulations, which are described elsewhere (20, 21). 152

The cultures were grown for 48 h at 30°C before plating 107 cells on YPD medium 153

containing 5.4 μM cyh. The plates were incubated 96 h, and resistant colonies were 154

picked for further testing. 155

Site-directed mutagenesis. Mutations were introduced into pSS607 with a Quikchange 156

Lightning site-directed mutagenesis kit (Life Technologies, Grand Island, NY). Mutant 157

primers were designed with a genomics program provided by Agilent Technologies 158

(Santa Clara, CA; www.genomics.agilent.com). The mutant plasmids were introduced 159

into XL-Gold E. coli by transformation, as described in the Quikchange instruction 160

manual. Plasmid DNA was extracted from the transformants with an IBI miniprep kit 161


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(MIDSCI, St. Louis, MO) and sequenced commercially to confirm the presence of the 162

mutation in the plasmid (Retrogen, San Diego, CA). The mutant plasmid DNA was 163

introduced into R-1 with a Sigma Aldrich yeast transformation kit. Following genetic 164

testing to confirm that the construct was correctly inserted (21), genomic DNA was 165

extracted from yeast transformants using a Qiagen Pure Yeast Gene / Bact kit (Qiagen, 166

Valencia, CA) and the pdr5 gene was amplified by PCR using 80–120 ng purified 167

chromosomal DNA per reaction. The PCR product was purified and the entire coding 168

region was sequenced commercially (Retrogen and SeqWright, Houston, TX) to confirm 169

that only the desired alteration was present. 170

Preparation of purified plasma membrane vesicles. Purified plasma membrane (PM) 171

vesicles were prepared from 750 ml of exponentially growing yeast cells containing 172

either two copies of WT or mutant PDR5. Following harvesting by centrifugation, the 173

pellets were weighed and resuspended in homogenization buffer (50 mM Tris pH 7.5, 2.5 174

mM EDTA, and a protease cocktail containing 1 mM phenymethylsulfonate, 10 µl/ml 175

aprotinin, 10 µg/ml TPCK and TLCK, 1 µg/ ml pepstatin and leupeptin) in a 2:1 ratio of 176

buffer volume / pellet weight. The cells were lysed with glass beads in a bead beater 177

(Biospec, Bartlesville, OK) using five 30 second intervals of lysing followed by 30 178

seconds of cooling as directed in the accompanying product manual. Whole cells and 179

glass beads were pelleted at 1000 x g for 7.5 min in a SS34 rotor at 4°C. The supernatant 180

was spun at 100,000 x g in a Ti50 rotor for 1 hr at 4°C to pellet the crude membranes. 181

Following this, the crude membrane pellet was resuspended in 2 ml resuspension buffer 182

(10 mM Tris, 0.5 mM EDTA, 10% glycerol with protease inhibitors) using a 7 ml dounce 183

homogenizer. The resuspension was applied to a discontinous sucrose gradient made up 184


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of an equal volume of 53.5% (wt/vol) and 43.5% (wt/vol) sucrose. Following 185

centrifugation for 5 hr at 100,000 x g in a SW28 swinging bucket rotor, the purified PM 186

vesicles were collected from the interface. The sucrose was diluted out by addition of 187

resuspension buffer and centrifugation for 1 hr at 100 x g in a Ti50 rotor. The pellets 188

were resuspended in 2 – 3 ml of resuspension buffer and 75 µl aliquots were frozen in 189

1.5 ml eppendorf tubes at -80°C. The amount of protein in solubilized vesicles was 190

determined with a BCA protein determination kit (Thermo Scientific, Rockford, IL). 191

Gel electrophoresis and Western blotting.The presence and amount of Pdr5 in PM 192

vesicles were confirmed by Western blotting. Proteins were separated by electrophoresis 193

at 150 volts on 7.5 % tris acetate gels (Life Technologies, Grand Island, NY) before 194

transferring to a nitrocellulose membrane at constant current (400 mA, 1 h). The blots 195

were blocked for 30 min in PBS buffer containing 1% tween 20 and 5% nonfat milk. We 196

obtained antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). The Pdr5 antibody 197

was diluted 1:500; the Pma1 antibody was diluted 1:2000. Following overnight 198

incubation at 4 °C in primary antibodies, the blots were washed 3 times for 15 min in 199

PBS plus 1% tween 20. Following this, the blots were incubated in PBS with nonfat milk 200

and 1% tween containing a 1:10,000 dilution of donkey, antigoat IgG horseradish 201

peroxidase antibody. The filters were washed 3 times for 5 min in PBS with 1% tween 202

20. The chemoluminscent signal was developed by adding 10 ml ECL reagent (Life 203

Technologies) for 1 min before exposing to X-ray film for 0.5–1 min. The ratio of Pdr5 204

to Pma1 signal was calculated with Image J software (rsbweb.nih.gov/ij/) using scans 205

with a resolution of 1200 dpi. 206


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Rhodamine 6G (R6G) transport assay. R6G transport assays were carried out with 5 207

μM R6G. Cells were grown overnight in SD medium supplemented with uracil and 208

histidine to a concentration of ~107 cells/ml. The cells were washed with 0.05M Hepes 209

buffer (pH7.0) and concentrated to one tenth the starting volume in Hepes buffer. For 210

each sample, 106 cells were incubated in 0.05M Hepes containing 5 μM R6G for 90 min 211

at 30°C in a final volume of 100 μl. Following loading, the cells were spun down, the 212

supernatant was removed, and the pellets were resuspended in 0.05M Hepes, 1 mM 213

glucose for 30 min. Following efflux, the cells were pelleted, the supernatant was 214

removed, and the cells were resuspended in 400 μl Hepes for analysis by fluorescence-215

activated cell sorting. R6G fluorescence was measured with a Becton-Dickinson 216

(Franklin lakes, NJ) FACSort with an excitation wavelength of 488 nm and an emission 217

wavelength of 620 nm. The data were analyzed with a CellQuest program. Retained 218

fluorescence is expressed in arbitrary units. 219

ATPase assays.We assayed the Pdr5-specific ATPase activity with purified PM vesicles 220

in an ATPase assay buffer containing 100 mM MOPS (pH 7.4), 50 mM KCl, 5 mM 221

NaN3, 2 mM EGTA (pH 7.0), 2 mM DTT, and 10 mM MgCl2 at 35°C for 8 min as 222

previously described (22). 223

Crosslinking[125I]Iodoazidoarylprazosin (IAAP) to Pdr5. Purified membranes, 224

prepared from double-copy–bearing yeast cells (30 μg protein/125 µl) were incubated 225

with the competing substrate clotrimazole (clo) for 5 min in ATPase buffer, followed by 226

incubation with [125I]IAAP (3.5 nM) at room temperature for 5 min under subdued light. 227

The samples were photocross-linked for 10 min with 365nm UV light in an ice water 228

bath followed by electrophoresis on 7% NuPAGE gels (Life Technologies). The gels 229


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were quantified as described previously (20). The dried gels were also exposed to X-ray 230

film to create an autoradiogram of the samples. 231

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RESULTS 233

The V656L mutation appeared in two distinct genetic screens. We recovered the 234

V656L mutation in two separate genetic screens, the first of which is illustrated in the 235

Supplemental Material (Fig. S1). We suppressed S558Y drug hypersensitivity by a series 236

of second-site mutations, including several near or in the Q-loop, a region required for 237

intradomain communication (20, 21). One of these mutants was N242K. When N242K is 238

not coupled to S558Y, it is also drug hypersensitive (21). Therefore, it can also serve as a 239

target for suppressor isolation. This tactic recovered a V656L mutation that exhibited 240

greater resistance than the wild-type (WT) residue to cyh although it was not extensively 241

characterized (21). 242

243

We carried out the second screen for our present study, exposing cells overexpressing 244

WT Pdr5 (JG2015) to cyh at the minimum inhibitory concentration for the WT strain on 245

solid medium (5.4 µM). The selected mutations were dominant. Therefore, standard 246

complementation testing was not useful for determining whether these lie in PDR5. This 247

was necessary because we expected to observe hyperresistant mutations both in and 248

outside of Pdr5. The genetic approach for distinguishing these possibilities in the JG2015 249

strain was recently described (20, 21). We identified five putative mutants in PDR5 and 250

we estimate that they arose with a frequency of ~10-8. 251

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We recovered the PDR5 gene from these mutants by PCR amplification and sequenced 253

the DNA (Retrogen, San Diego, CA). Each mutant had a single-base-pair substitution 254

resulting in a missense mutation. One of these was V656L. The other mutations were 255

P596L in ICL1, L1367F in TMH12, A670S in TMH5 and I1063M in NBD2. In this 256

study, we focus on Val-656 because of its location in a region strongly implicated in 257

signal transmission by structural and cross-linking studies. We therefore recreated the 258

V656L mutation in pSS607 and placed it into the Δpdr5 strain by yeast transformation. 259

We compared these strains to the original chromosomal mutants. The data in Fig. 1 show 260

that the cyh killing curves for the plasmid and chromosomal mutations are statistically 261

indistinguishable. A two-way ANOVA test comparing each of the mutant curves to WT 262

indicated that the likelihood that they are the same is <0.0001. We used the plasmid 263

reconstruction in all of the subsequent experiments. Fig. 2 shows a representative 264

Western blot for the mutant strains used in this study. The V656L mutant protein is found 265

at WT levels in purified plasma membrane (PM) vesicles. We determined the ratio of 266

Pdr5 to Pma1 signal for all of the strains. This value allowed us to make a quantitative 267

comparison between the WT and mutants analyzed. The amount of Pdr5 protein in 268

V656L was 0.89 + 0.13 that of the WT. Therefore, the increased resistance of the former 269

can’t be attributed to an increased level of the transporter in the PM. 270

Val-656 is one of four conserved residues in ICL2. According to the bioinformatic 271

analysis performed by Rutledge et al. (16) to build their atomic model of Pdr5, ICL2 is 272

remarkable for its relatively short size. It is predicted to contain only 10 amino acids 273

(654-663); by contrast, ICL1 is made up of 21 residues. They also observed two loops 274

with analogous sizes in the second half of Pdr5. 275


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276

We performed a sequence alignment using the Swiss Protein Database alignment tool 277

(www.unitprot.com).We compared Pdr5 to 17 members of the Pdr5 subfamily with 278

amino acid identities ranging from 30.7% to 74.4%. These sequences were initially 279

selected for use by Rutledge et al. (16) as part of an atomic modeling study. The 280

alignment that we obtained is found in the Supplemental Data (Fig. S2) The first and last 281

amino acids in the loop are a highly conserved arginine and either a serine or threonine 282

residue, respectively. The third position has a medium-to-large hydrophobic residue (V, I, 283

L, M, F) followed by a small non-polar one (G or A) in the fourth. All of the residues 284

located at the third position can be interchanged as a result of a single-nucleotide 285

substitution. ICL4 is located in TMD2 and is predicted to comprise only seven amino 286

acids (22), suggesting a correspondence to ICL2 because of its size similarity. In contrast 287

to ICL2, however, none of the residues in ICL4 are conserved. This indicates that ICL2 288

and ICL4 could be functionally distinct. A recent report clearly demonstrates that ICL4 is 289

necessary for drug resistance, although its role is unknown (23). 290

Val-656 is an essential residue for Pdr5-mediated drug transport. To determine 291

whether Val-656 is essential for Pdr5 function, we used site-directed mutagenesis to 292

create a V656A mutation and introduced it into the Δpdr5 strain. Ordinarily, a valine to 293

alanine change is structurally neutral and a single-base-pair substitution can interchange 294

these residues. However, no alanines are found in position 3 of ICL2 in the alignment 295

sequences. This strongly suggested that such a substitution would be unfavorable, as 296

indeed it is. The level of the V656A mutant protein in purified PM vesicles was, if 297

anything, slightly higher (1.3X + 0.1) than WT (Fig. 2). The relative cyh, clo, bifonazole, 298


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tamoxifen, and climbazole resistance of V656A was compared to the isogenic WT 299

(JG2015) and Δpdr5 strains (Fig. 3A–E). The V656A and Δpdr5 mutations were equally 300

hypersensitive to cyh, tamoxifen, bifonazole, and climbazole. Although V656A showed 301

significantly more resistance to clo than Δpdr5, it was considerably more sensitive than 302

WT (Fig. 3A). We also tested the ability of V656A to efflux R6G in a whole-cell assay. 303

These data demonstrate that the V656A and the ∆pdr5 control were equally deficient in 304

R6G efflux (Fig. 4). 305

ATPase activity is significantly reduced in V656A. A hallmark of loss-of-function 306

mutations in the signal transmission interface of Pdr5 analyzed thus far is a reduction—307

but not an elimination—of ATPase activity. This feature does not fully reflect the 308

severity of the mutation. For instance, although the E244G (deviant Q-loop) and S558Y 309

mutants both showed ATPase activity that is 3 to 4 times lower than WT (mutants, ~50 310

nmol/min/mg vs WT, ~200), the E244G mutant retained significant transport capability, 311

and the S558Y mutant was phenotypically null (20, 21). 312

313

We performed similar experiments with V656A (Fig. 5A). Purified plasma membrane 314

vesicles exhibited a reduced but significant level of ATPase activity. The average Vmax 315

values obtained for three independent membrane preparations was 42 nmol/min/mg. This 316

value is similar to those observed with the transmission interface mutants S558Y, E244G, 317

and Q951G. The Km values were 1.6 mM for the WT and 0.8 mM for V656A, within the 318

range seen routinely in our experiments (for example, the WT data in Fig. 9C indicate a 319

Km of 0.9 mM). 320


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V656A ATPase is significantly more resistant to clo than the WT enzyme. Although 321

Pdr5 ATPase is not stimulated by drug substrates, some, such as R6G, clo, and 322

ketoconazole, are inhibitors (22, 24). The residual ATPase activity found in S558Y is 323

considerably more resistant to allosteric inhibition by clo than is the ATPase activity 324

found in the WT (20). This strongly suggested that the inhibitory signal is not being 325

properly transmitted in the mutant. To determine whether ICL2 is required for allosteric 326

inhibition of ATPase, we tested the residual V656A ATPase activity for its susceptibility 327

to clo. The results show that the mutant ATPase exhibits significantly greater clo 328

resistance than does the WT protein (Fig. 5B). The IC50 (clo) observed for the WT in this 329

particular set of experiments was 2.2 μM which is similar to the WT values obtained in 330

the experiments described in Fig. 9 and 10. In contrast, the mutant enzyme had a value of 331

9.9 μM. A two-way ANOVA test indicated that the probability that the two curves are the 332

same was less than 0.0001. These results strongly suggest that both transport and 333

ATPase inhibition are highly dependent on Val-656. 334

V656A drug binding is similar to WT-Pdr5. The crystal structure of mouse P-gp 335

showed that binding of its drug substrates takes place in the TMDs (25). It could be the 336

ICLs play an indirect role in drug binding and that V656A is deficient in this process. To 337

find out, we compared the ability of WT and V656A to bind IAAP in the presence of the 338

competing substrate clo (Fig. 6). Previous work in our laboratory demonstrated that both 339

R6G and clo show a concentration-dependent inhibition of IAAP photoaffinity labeling 340

(20, 26). This is significant because R6G is a competitive inhibitor of clo efflux ( 341

Supplemental Material, Fig. S3). Substrates such as tripropyltin chloride or 342

tritylimidazole that do not block R6G or clo efflux do not inhibit IAAP labeling (26). In 343


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addition, Pdr5 mediates IAAP efflux in a whole cell transport assay (Z. Sauna and J. 344

Golin, unpublished data). In the present experiments, we found that IAAP binds to the 345

WT-pdr5 and V656A and yields a signal of similar intensity. Furthermore, the addition of 346

clo yielded inhibition curves (% IAAP incorporation at a specific dose) that were 347

indistinguishable from each other. In each case, the IC50 was about 15 µM. Thus, it is 348

unlikely that V656A alters the transporter–drug substrate-binding interaction to a degree 349

that would yield the observed null phenotype. 350

The response of V656L to Pdr5 substrates of varied strength. The Western blot in 351

Fig. 2 demonstrates that the increased resistance of V656L to cyh is not attributable to a 352

further increase in the expression of Pdr5. We considered several models to account for 353

the observation that V656L increases resistance to cyh. The first, known as the kinetic 354

selection model was proposed by Ernst et al. (24) to explain the behavior of an H-loop 355

mutation in Pdr5 that reduced R6G transport but exhibited WT resistance to other 356

transport substrates. Pdr5 has a high basal ATPase activity that is not stimulated by drugs. 357

Thus, hydrolysis may well be constitutive and independent of drug substrate stimulation. 358

This model posits that mutations can alter the rate of switching from a drug-binding to a 359

drug-effluxing conformation. While precise measurements of drug-on and off-rates of 360

drugs would be required to rigorously establish or reject the model, it leads to a specific 361

phenotypic prediction. If the Pdr5 pump is altered so that relative to the WT protein the 362

mutant is more frequently in the drug-binding (according to the Sav1866 model, the 363

inward facing conformation) rather than the drug-effluxing state (the outward facing 364

model), modest or poor Pdr5 substrates that normally interact slowly will have a greater 365

opportunity for binding. 366


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The first model makes a specific prediction about substrate behavior: V656L exerts its 367

strongest effect on substrates that interact relatively poorly with Pdr5. We therefore tested 368

the response of V656L to a set of 10 transport substrates (Table 1). We determined the 369

relative strength of each substrate by calculating its IC50 in isogenic PDR5 and ∆pdr5 370

strains and calculating the PDR5/∆pdr5 ratio. These data are summarized in Table 1 and 371

six plots are shown in Fig. 7, where they are arranged according to relative substrate 372

strength. The IC50 indices form a continuum ranging from tamoxifen (1.7) to 373

cyproconazole (106). We determined the IC50s for V656L, WT, and ∆pdr5. V656L 374

conferred greater resistance to eight compounds. These did not cluster according to IC50 375

index, but were evenly distributed. Furthermore, we observed a similar increase in 376

resistance for each one. Thus, V656L is 1.4 to 2.1 times more resistant to the compounds 377

than the WT. These results cast doubt on the kinetic selection model as an explanation for 378

the observed ultraresistance of the V656L mutant, although it remains an important 379

possibility for the H-loop mutant described by Ernst et al. (24). 380

381

We then considered a second and a third explanation for the behavior of the V656L 382

mutant. The second asserts that Val-656 affects the level of Pdr5 ATPase. Increased 383

resistance is due to an increased ATPase activity available for transport. A third model is 384

that these mutants alter the transmission interface, causing a more efficient use of energy 385

from rather than an increase in ATP hydrolysis. 386

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Both of these models predict that if a transport substrate such as clo eliminates this 388

activity before or at the WT IC50 for growth, V656L will not increase resistance. We 389

therefore looked at the effect of substrates on ATPase activity. 390

391

The effect of Pdr5 transport substrates on ATPase activity. We tested the effect of the 392

same set of compounds on the WT Pdr5-mediated ATPase activity (Fig. 8A). Three of 393

these, clo, bifonazole, and cyprodinil, caused strong (>50%) inhibition of WT ATPase 394

activity. Although bifonazole and clo are similar in structure, cyprodinil is much smaller 395

and it is not an imidazole derivative. The V656L mutant did not show an increase in 396

resistance to cyprodinil or clotrimazole. The remaining transport substrates had a weak (< 397

30% inhibition) effect on ATPase activity or none at all. It would appear, then, that strong 398

substrate inhibition of ATPase activity is seen with only a minority of the transport 399

substrates. In some instances, an extremely weak stimulation of ATPase occurred at very 400

high transport substrate concentrations. The V656L mutant showed increased resistance 401

to all of the substrates that leave ATPase activity largely unaffected. However, it also 402

exhibited increased resistance to bifonazole which shows substantial inhibition of 403

ATPase activity. Though this was initially unexpected, we observed that the IC50 for 404

killing in WT whole cells is 3.7 µM while that of V656L (5.7 μM) is very close to the 405

IC50 for ATPase inhibition by this drug (5.3 μM). This suggests that as long as the IC50 406

for allosteric inhibition is above the IC50 for killing of WT cells, the V656L mutation can 407

promote increased resistance. An exact correlation between inhibition in vesicles and 408

killing in whole cells can’t be made, however, without determining the effective 409

intracellular concentration of the drug in question. Our observations are thus consistent 410


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with either the second or third model, which invoke either an increase in or a more 411

efficient utilization of ATPase activity. We next attempted to distinguish these 412

possibilities. 413

The ATPase activity of the V656L gain-of-function mutation. Suppressor mutations 414

located very near (N242K) or in a Pdr-specific residue defining the Q-loop that were 415

isolated for their ability to restore resistance to the transmission interface–deficient 416

mutant S558Y do not increase ATPase level. Rather, they apparently improve the 417

efficiency with which the signal from ATP hydrolysis is transmitted, and they restore 418

noncompetitive inhibition of the Pdr5 ATPase by clo. Presumably, if V656L were to 419

increase drug resistance without a concomitant increase in ATPase activity, we would 420

have additional evidence that Val-656 and therefore ICL2 play a role in transmitting the 421

signal from the nucleotide-binding domains to the TMDs. We compared the ATPase 422

activity of V656L to WT Pdr5 in purified plasma membranes (Fig. 8B). The Vmax of the 423

V656L mutation (~200 nmol/min/mg) fell well within the range of WT values (216 424

nmoles/min/mg in these experiments) but was certainly not greater. The Km values for the 425

WT and mutant were 1.6mM and 1.2 mM, respectively. The increased resistance that we 426

observed is therefore not attributable to greater ATPase activity. 427

428

The ATPase activity of V656L is slightly more sensitive to clo than is the WT (Fig. 8C). 429

According to a two-way ANOVA test, the two curves are probably not equivalent (p = 430

0.007); however, the IC50 values are nearly the same. Any effect of V656L is therefore 431

very small. The effect of climbazole, tamoxifen, and cyproconazole on V656L ATPase 432

activity was also tested. Climbazole and cyproconazole had identical effects on V656L 433


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and WT ATPase activity (Fig. S4, Supplemental Materials). The V656L ATPase activity, 434

however, was significantly more sensitive to tamoxifen than the WT (Fig. 8D). The IC50 435

was about 32 μM and the inhibition leveled out to about 70%. In V656L cells, the IC50 436

(tamoxifen) was about 18 μM (Table 1); a value not terribly different considering that the 437

comparison is between measurements in PM vesicles and whole cells which may not be 438

equivalent. This observation may explain why the enhancement in resistance observed in 439

V656L vs. WT (~1.4X) was less than that obtained with some of the other compounds. 440

The V656L and V656A mutations therefore create opposite phenotypes that are 441

consistent with altered signal transmission. The V656L mutation causes increased 442

resistance to many drugs, but increased sensitivity to allostery at least in the case of 443

tamoxifen. In contrast, V656A is markedly drug hypersensitive, but more resistant to 444

allosteric inhibition by clo. 445

V656L also suppresses E244G, a mutation in the residue defining the pdr-specific Q-446

loop.These data are consistent with the idea that Val-656 is part of the transmission 447

interface. To test this idea further, we created a E244G, V656L double mutant to detect 448

increased drug resistance and/or ATPase activity. E244G is the deviant Q-loop residue 449

that is completely conserved in the Pdr5 subfamily. Recent work in our laboratory 450

established its role in the transmission interface (20). When E244G is present in an 451

otherwise WT strain, the result is reduced ATPase activity and increased drug sensitivity. 452

The Western blot in Fig. 9A shows that Pdr5 is present at similar levels in the PM 453

vesicles of each strain. The data in Fig. 9B clearly demonstrate that V656L suppresses the 454

significant cyh hypersensitivity of the E244G transmission interface mutant. In fact, the 455

resistance observed in the suppressor strain is similar to that of the WT. The resistance of 456


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the double mutant is about four times that observed with E244G. At issue, however, is 457

whether the suppression is attributable solely to better use of the residual ATPase activity 458

in E244G or to an actual increase in the level of hydrolysis (Fig. 9C). Significantly, the 459

ATPase activity of the double mutant was no different than the activity observed for 460

E244G, which was in the range of 60-75 nmole/min/mg. 461

462

DISCUSSION 463

Multidrug resistance is often caused by overexpression of ABC efflux pumps, and it is a 464

major problem in the clinical treatment of cancers and infection. Furthermore, Pdr5 465

defines an ABC subfamily found only in fungi; many of these are of agricultural 466

significance. It is important, to determine whether a further increase in resistance can be 467

obtained under continued selective pressure in strains that are already overexpressing 468

multidrug transporters. We found that mutations in the Pdr5 gene imparting resistance to 469

cyh are recovered with a frequency of roughly 10-8. The V656L mutation leads to an 470

increase in resistance to a broad spectrum of Pdr5 substrates that is 1.5–2 times that of 471

WT. The enhancement is not dependent on substrate strength. Although the increased 472

resistance may seem modest, numerous chemotherapeutic and antifungal agents are toxic 473

if the dose is doubled. Microevolution often works incrementally but effectively. 474

V656L is located in the very short ICL2 of TMD1. Thus, this residue is not in the drug-475

binding pocket, as might be expected, nor does this alteration result in an increased 476

steady-state expression level of Pdr5 in purified PM vesicles. Rather, Val-656 is one of 477

four conserved residues in ICL2. An alanine substitution has an effect that is entirely 478

different from V656L. The V656A mutation is nearly phenotypically null to all Pdr5 479


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substrates tested, although its drug-binding capability is intact. It is plausible that 480

although valine and alanine are biochemically similar, the larger size of the former is 481

required for interaction between ICL2 and the NBD(s). In fact, the behavior of the 482

V656A mutant resembles the transmission-deficient mutant S558Y which lies in TMH2. 483

As was the case with S558Y, the noncompetitive inhibition of Pdr5-specific ATPase by 484

clo is significantly diminished in V656A. These data show that the signals for drug 485

transport and allosteric ATPase inhibition both use ICL2. V656L's suppression of the 486

E244G and N242K signaling deficiencies in the Q-loop region also strongly implies a 487

role for Val-656 and ICL2 in the transmission interface. This is further supported by the 488

observation that although V656L increases resistance either in a WT or E244G 489

background, it does so without raising ATPase activity. These observations indicate that 490

in the WT transporter, the energy from hydrolysis is not used to full capacity. From the 491

standpoint of evolution, mutations that improve the efficiency of energy utilization 492

should have a strong selective advantage in the presence of a xenobiotic agent. Thus, 493

multidrug transporters can evolve to greater resistance by manipulating signal 494

transmission. This is a novel mechanism of multidrug resistance not previously 495

described. In the case of V656L, this results in additional resistance to a broad spectrum 496

of Pdr5 substrates that appears limited only in cases where ATPase activity is reduced by 497

allosteric inhibition. The behavior of mutants in this study further emphasize that it is 498

impossible to link ATPase activity levels with the resistance phenotype in Pdr5. 499

500

The high resolution structure of the Sav1866 homodimer shows physical contact between 501

ICL2 and the X-loop and Q-loop regions of the opposite monomer. Crosslinking studies 502


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with other eukaryotic transporters also lend additional credence to a transmission 503

interface arrangement in a trans orientation (17, 18). Sav1866 and P-gp were used 504

heavily by Rutledge et al. (22) to construct a homology model of Pdr5. As a result, the 505

Pdr5 interface is modeled in a trans conformation with ICL2 in close proximity to the Q-506

loop of NBD2. This model was extremely challenging to build, however, because there 507

are big structural differences and relatively low amino acid identity between Pdr5 and 508

these transporters. In Table S1 (Supplemental Material), we list the residues of the Pdr5 509

transmission interface as defined by suppressor mutations. ICL1, ICL2, and the Q-loop 510

region are all represented in the collection. All of these lie in the same half of the 511

transporter. Our original collection of suppressors restored resistance to a transmission-512

deficient S558Y mutant. Ser-558 is located at the extracellular end of TMH2 which is 513

admittedly a good distance from the NBDs so the interaction between these residues 514

could be very indirect. The V656L and E244G residues, however, are presumably in 515

much closer proximity to each other and their observed interaction strengthens our 516

contention that unlike other eukaryotic ABC transporters, ICL2 of Pdr5 (represented by 517

Val-656) interacts with the cis Q-loop. We do not mean to imply that Val-656 and Glu-518

244 are necessarily a physically interacting pair in the WT as their chemistry makes that 519

implausible. It is possible that V656L and E244G create a new hydrophobic interaction 520

that restores the signal to the interface. Alternatively, these mutant residues may never be 521

in physical contact although they clearly are part of the same transmission pathway. 522

523

Although the E244G (Q-loop NBD1), Q951G (Q-loop NBD2), and V656A substitutions 524

are deficient in intradomain communication, the Q-loop deficiences show some drug 525


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specificity. For instance, E244G exhibits almost WT clo resistance (20). We also 526

observed that E244G has R6G transport capability that is indistinguishable from the WT 527

(J. Mehla, unpublished results). In contrast, a double E244G, Q951G mutant exhibits 528

strong hypersensitivity to clo and cyh (20). Therefore, the Q-loop residues are 529

overlapping in function. The broad, striking hypersensitivity of the V656A mutant and 530

the absence of an obvious consensus sequence in ICL4 or ICL3 suggest the possibility 531

that there is no equivalent residue in TMD2. 532

533

Acknowledgements 534

This work was supported by PHS grant GM07721 and NSF grant 1048838 to JG and by 535

the Intramural Research Program of the NIH, National Cancer Institute, Center for 536

Cancer Research. This paper is dedicated to the memory of Seth Areyh Golin Weisman 537

(1981-2012) who isolated the first mutants with increased resistance in the summer of 538

2003 and continued to insist on the importance of their characterization. 539

540

REFERENCES 541

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Isolation and characterization of DNA sequences amplified in multidrug resistant 547

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9. Hirata, D., Yano, K., Miyahara, K., and Miyakawa, R. 1994 Saccharomyces 565

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a strong size dependence for xenobiotic efflux. J Biol Chem 278: 5963-5969. 584

15. Rogers, B., Decottignies, A., Kolaczkowski, M., Caravajal, E., Balzi, E., and 585

Goffeau, A. 2001 The pleiotropic drug ABC transporters from Saccharomyces 586

cerevisiae. J. Mol Microbiol Biotechnol 3: 207-214. 587

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understanding of the mechanism of action of the yeast multidrug 589

resistance transporter Pdr5p: a molecular modeling study. J Struct Biol \ 590

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18. Zolnerciks, J. K., Wooding, C., and Linton, K.J. (2007) Evidence for a 596

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19. Dawson, R. J., and Locher, K. P. 2006 Structure of a bacterial multidrug ABC 599

transporter. Nature 443: 180-185. 600

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L., Xia, D., Ambudkar, S. V., and Golin, J. 2008 Mutations define cross 602

talk between the N- terminal nucleotide-binding domain and transmembrane- 603

helix 2 of the yeast multidrug transporter Pdr5. J Biol Chem 283: 35010-35022. 604

21. Ananthaswamy, N., Rutledge, R., Sauna, Z. E., Dine, E., Nelson, E., Xia, D., and 605

Golin, J. 2010 The signaling interface of the yeast multidrug transporter Pdr5 606

adopts a cis conformation and there are functional overlap and equivalence of the 607

deviant and canonical Q-loop residues. Biochemistry 49: 4440-4449. 608

22. Golin, J., Kon, Z. N., Wu, C.P., Martello, J., Hanson, L., Supernavage, S., 609

Ambudkar, S. V., and Sauna, Z. 2007 Complete inhibition of the Pdr5p 610

multidrug efflux pump ATPase activity by its transport substrate clotrimazole 611

suggests GTP as well as ATP may be used as an energy source. Biochemistry 612

46: 18109-18119. 613

23. Guo, X., Li, J., Wang, T., Liu, Z., Chen, X., Li, Y., Gu, Z., Mao, X., Guan, 614

W.,and Li, Y. 2012 A mutation in intracellular loop 4 affects the drug efflux 615

activity of the yeast multidrug transporter Pdr5p. PLoS One 7 (1): e29520 doi: 616

10.1371. 617


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24. Ernst, R., Kueppers, P., Klein, C. M., Schwarzmueller, T., Kuchler, K., and 618

Schmitt, L. 2008 A mutation of the H-loop selectively affects rhodamine 619

transport by the yeast ABC transporter Pdr5. Proc Natl Acad Sci (USA) 105: 620

5069-5074. 621

25 . Aller, S., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R., Harrell, P. M., 622

Trinh, Y. T., Urbatsch, I. L., and Chang, G. 2009 Structure of P-glycoprotein 623

reveals a molecular basis for polyspecific drug binding. Science 323: 1718-1722. 624

26. Hanson, L., May, L., Tuma, P., Keeven, J., Mehl, P., Ferenz, M., Ambudkar, 625

S. V., and Golin, J. 2005 The role of hydrogen bond acceptor groups in the 626

interaction of substrates with Pdr5, a major yeast drug transporter. 627

Biochemistry 44: 9703-9713. 628

629

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642

TABLE 1 Enhancement of drug resistance in V656L is independent of substrate strength 643

________________________________________________________________________ 644

Compound WT/∆pdr5 IC50 WT [μM] IC50 V656L [μM] V656L/WT IC50 ATPase [μM] 645

(ratio IC50 s) (ratio IC50s) 646

Tamoxifen 1.7 13 18 1.4 Incomplete 647

Cyprodinil 2.0 140 140 1.0 200 648

Cerulenin 10 14 28 2.0 No inhibition 649

Imazilil 10 4.3 7.7 1.8 Incomplete 650

Cycloheximide 17 7.5 16 2.1 No inhibition 651

Bifonazole 19 3.7 5.7 1.5 5.3 652

Climbazole 40 14 28 2.0 Incomplete 653

Clotrimazole 75 9.7 9.7 1.0 2.5 654

Tebuconazole 80 6.2 9.5 1.5 No inhibition 655

Cyproconazole 106 7.5 15 2.0 No inhibition 656

______________________________________________________________________________ 657

658

659

660

661

662

663

664

665


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666

667

Figure Legends 668 669 FIG. 1. V656L increases resistance to cyh.. We constructed V656L in pSS607 and placed 670

it in R-1 (Δpdr5) by yeast transformation. Following this, the original chromosomal 671

mutation and the plasmid reconstruction were compared to WT and Δpdr5 isogenic 672

controls for their relative resistances to cyh in liquid culture as described in the 673

Materials and Methods. All plots in this study were made using GraphPad PRISM (San 674

Diego, CA). In this study, n = 3. ▲ = Δpdr5, ■ = WT, ♦ = chromosomal V656L 675

mutation, ◊ = plasmid reconstructed V656L mutation. 676

677

FIG. 2. The expression of the V656L and V656A mutants is similar to WT. PM vesicles 678

were solubilized in SDS-PAGE sample buffer at 37°C for 30 min. We used 10 μg of 679

protein to produce the Western blot. Each is a representative example from three 680

independent experiments. Lane 1, WT; lane 2, Δpdr5; lane 3, V656A; lane 4, V656L. 681

The values are the ratio of Pdr5 signal to Pma1 signal computed from Image J software as 682

described in the Experimental procedures. 683

684

FIG. 3. The V656A mutant exhibits substantial drug hypersensitivity. The sensitivity of 685

V656A (▼, blue line) was compared to isogenic controls (□, red = Δpdr5, ▲, green = 686

WT, ♦, violet = V656L) for relative resistance to A, clo ; B, cyh; C, bifonazole; D, 687


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tamoxifen; and E, climbazole in liquid culture as described in the Experimental 688

procedures. All concentrations are in μM units. The error bars denote standard error, n 689

=3. 690

691

FIG. 4. R6G transport is impaired in V656A. A. Histogram plots from a single 692

experiment using two independent cultures of Δpdr5 and V656A and a single culture of 693

the isogenic WT strain (JG2015). The assay is described in the Materials and methods. B. 694

Representation of data pooled from the experiment in A and an additional independent 695

experiment 696

FIG. 5. The ATPase activity of V656A. ATPase was measured as described in the 697

Experimental procedures using 16 μg of PM vesicle protein from double copy strains (see 698

23). Reactions with a final volume of 100 μl were initiated by adding ATP and incubated 699

at 35°C for 8 min before terminating with 2.5% (final volume) SDS. The non-specific 700

activity (< 5%) present in the Δpdr5 strain is subtracted prior to calculating the activity. 701

A. Activity as a function of ATP concentration. The data represent the averages of at 702

least two independent vesicle preparations per strain and 3 independent experiments were 703

performed. B. Activity in the presence of clotrimazole (3.1- 50 μM). Standard ATPase 704

conditions were used. PM vesicles were incubated at room temperature for 5 min in the 705

presence of clo before initiating the reactions with the addition of ATP to 3 mM . In these 706

experiments, ■ = WT, ▲ = V656A and n = 3. 707

FIG. 6. V656A exhibits WT drug-binding behavior. We carried out photoaffinity 708


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labeling with [125IAAP] in the presence and absence of clo as described in the Materials 709

and Methods. A. Each lane contains 10.8 μg of solubilized PM vesicle protein. After 710

electrophoresis, the gels were dried and exposed to an X-ray film. The radioactivity 711

incorporated into the pdr5 band was quantified using a phosphorimager and analyzed 712

using the GraphPad PRISM. The autoradiogram above shows the IAAP binding to the 713

WT-pdr5 and the mutant-pdr5 in the presence and absence of clo. The isogenic strain 714

lacking Pdr5 (_pdr5) was used as a control in the experiment. The WT and V656A 715

mutant strains were analyzed in the same experiments. B. Plots of the experiment shown 716

in Panel A. In these experiments, ■ = WT, ▲ = V656A. We performed this experiment 717

an additional time with nearly identical results. 718

FIG. 7. The resistance of V656L to Pdr5 transport substrates.We show plots of growth 719

versus substrate concentration for six of the ten compounds tested. The conditions for 720

testing substrates in liquid culture are found in the Materials and Methods. These vary in 721

their substrate strength. Panels A-C show data from substrates ranging from weak 722

(tamoxifen) to strong (cyproconazole) to which V656L (purple plots) confers increased 723

resistance. The strength is determined by comparing the ratio of the IC50s of the WT 724

(green) and Δpdr5 (red) plots. Panels D-F show data from substrates ranging from weak 725

(cyprodinil) to strong (clo) that cause significant (> 50%) inhibition of ATPase activity 726

(see Fig. 9). All concentrations are in μM units. In these experiments, n = 3. 727

FIG. 8. The effect of substrates on ATPase activity.A. We performed assays of ATPase 728

activity with purified PM vesicles from the WT double-copy strain in the presence of 729

transport substrates as described in Figure 5 using 3 mM ATP in each reaction. Plots for 730


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each compound are shown. At least two curves were generated for each compound. B. 731

The ATPase activity of V656L.ATPase activities were determined as described in Fig. 5. 732

The data represent determinations from two independent PM vesicle preparations from 733

each strain. A. ATPase activity as a function of ATP concentration (0-3 mM). In these 734

experiments, n = 3 and error bars represent standard error. C. The effect of clo (1.78-12.5 735

μM) on ATPase activity is shown using 3 mM ATP in each reaction. In both sets of 736

experiments ■ = WT, ▲ = V656L. The inhibition curves are the average of 3 737

independent experiments including two different PM preparations of V656L. D. The 738

effect of tamoxifen on V656L ATPase. The conditions are identical to those described in 739

panel C except that tamoxifen was added to the reaction mixes. 740

FIG. 9. V656L suppresses the cyh hypersensitivity of E244G. A. Colloidal blue –stained 741

gel (30 μg of solubilized protein) and Western blot (10 μg of solubilized protein) of 742

E244G (lane 1) and the E244G, V656L double mutant (lane 2) prepared as described in 743

Fig. 3. B. Plots of growth versus cyh concentration. Experiments were performed in 744

liquid culture as described in the Materials and Methods. The conditions for testing 745

strains in liquid culture are found in the Materials and Methods. In these experiments,● = 746

Δpdr5 (red), ■ = WT (green), □ = E244G (orange), ♦ = V656L (violet), and ▼= V656L, 747

E244G (gray). C. The ATPase activity of the E244G, V656L double mutant was 748

compared to the single E244G mutant and isogenic control strains using the assay 749

conditions described in Fig. 5. In the figure, ■ = WT, □ = E244G, ♦ = V656L, and ▼= 750

V656L, E244G. 751


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: % c

Tamoxifen [mM] Climbazole [mM]


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A B

600

rary

uni

ts

B

WT ∆pdr5V656A

300

400

500

scen

ce: a

rbitr

dr5 WT 6A0

100

200

edia

n flu

ores

PdrΔ

WTV65

6AM

Fluorescence intensity


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200

250

/ mg 100

ontr

olA B

50

100

150

mol

es /

min

/

50

75

% u

ntre

ated

co

0 1 2 3 40

50

ATP (mM)

nm

0 5 10 15 20 250

25

Clotrimazole [μM]

ATP

ase:

%


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JG2004

V656A

0 1 2.5 5 10 25 50 100 µM


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75

100

cont

rol

75

100

cont

rol

75

100

cont

rol

A B C

0.0 2.5 5.0 7.5 10.0

12.5

15.0

17.5

20.0

0

25

50

Gro

wth

: %

0 10 20 30 400

25

50

Gro

wth

: % c

0.0 2.5 5.0 7.5 10.0

12.5

15.0

17.5

20.0

0

25

50

Gro

wth

: %

T if C l h i id C l

Weak Medium StrongD E F

Tamoxifen Cycloheximide Cyproconazole

25

50

75

100

wth

: % c

ontr

ol

50

75

100

wth

: % c

ontr

ol

50

75

100

wth

: % c

ontr

ol

010

020

030

040

00

25

Gro

w

0 1 2 3 4 5 6 7 8 9 100

25

Gro

w

0.0 2.5 5.0 7.5 10.0

12.5

15.0

17.5

20.0

0

25

Gro

w

Cyprodinil Bifonazole Clotrimazole


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150 bifonazoleated 200g

A B

50

75

100

125

bifonazoleclimbazoletamoxifenclotrimazoleimazalilcyprodinilcycloheximideac

tivity

:% u

ntre

a

50

100

150

ase

nm /

min

/ m

g

0 50 100 150 200 2500

25cycloheximidecerulenincyproconazoletebuconazole

Drug [μM]

ATP

ase

0 1 2 3 40

50

ATP [mM]

ATP

C

50

75

100

ty: %

unt

reat

ed

75

100

% u

ntre

ated

C D

0 1 2 3 4 5 60

25

50

Clo [μM]

ATP

ase

activ

it

0 50 100 150 200 2500

25

50

ATP

ase

activ

ity:

[μ ]Tamoxifen [μM]


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75

100on

trol

A B

1 2 1 2

Pd 5

25

50

Gro

wth

vs.

co←Pdr5

0 10 20 300

Cycloheximide [μM]

G

C

150

200

mol

/ m

in /m

g

C

0 1 2 3 40

50

100

TP h

ydro

lysi

s: P

i nm

0 1 2 3 4ATP [mM]A

T


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Documents

1 2 3 The Transmission Interface of the Yeast Multidrug Transporter