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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
14 15 16 17
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
232
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
252
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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
387
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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
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Schmitt, L. 2008 A mutation of the H-loop selectively affects rhodamine 619
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reveals a molecular basis for polyspecific drug binding. Science 323: 1718-1722. 624
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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
630
631
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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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75
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75
100
cont
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A B C
0 1 2 3 4 50
25
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Clotrimazole [mM]
Gro
wth
: %
0 1 2 30
25
50
Cycloheximide [mM]
Gro
wth
: %
0.0 0.5 1.0 1.5 2.0 2.50
25
50
Bifonazole [mM]
Gro
wth
: %
Clotrimazole [mM] Cycloheximide [mM] Bifonazole [mM]
75
100
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75
100
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D E
0 1 2 3 4 50
25
50
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25
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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
on February 11, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
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:
%
on February 11, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
JG2004
V656A
0 1 2.5 5 10 25 50 100 µM
on February 11, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
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
on February 11, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
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]
on February 11, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
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
on February 11, 2018 by guest
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Dow
nloaded from