Regulation of pleiotopic drug resistance in yeast

403

Regulation of pleiotropic
drug resistance in yeast

Anna Kolaczkowska,

1,2

Andre Goffeau

1

1

Unite de Biochimie Physiologique, Universite Catholique de Louvain, Louvain-la-

Neuve, Belgium

2

Institute of Biochemistry and Molecular Biology,Tamka 2,

50–137 Wroclaw, Poland

Abstract

This review focuses on the molecular mechanisms

involved in the regulation of multiple drug resistance in the
model yeast Saccharomyces cerevisiae and the pathogenic fungus
Candida albicans. Recent developments in the study of the
transcription factors Pdr1p, Pdr3p and Yap1p are reported.
Understanding the molecular basis leading to multiple drug
resistance is a prerequisite for the development of new
antifungal therapeutics. © 1999 Harcourt Publishers Ltd

INTRODUCTION

rug resistance is an alarming clinical problem in the
treatment of human cancers and infections of bacte-
rial or fungal origin. The most important resistance

mechanism, ubiquitous from bacteria to man, which leads to
multidrug resistance (MDR) is the overexpression of mem-
brane-associated transporters that extrude drugs out of the
cell.The best characterized and clinically the most important
MDR transporters are members of the ATP binding cassette
(ABC) superfamily such as the human P-glycoprotein (P-gp)
and the MDR related proteins (MRPs). A common problem
among immunocompromised patients undergoing long-term
antifungal therapy is overproduction by the pathogenic yeast
Candida albicans

of ABC or MFS (Major Facilitators

Superfamily) transporters which leads to clinical resistance
to antifungals used in chemotherapy treatment.

In Saccharomyces cerevisiae two networks of genes, PDR

(Pleiotropic Drug Resistance) and YAP (Yeast AP-1 like factor)
are involved in multidrug resistance. Overexpression of yeast
multidrug extrusion pumps results from overproduction
genetic alterations (YAP network) and/or spontaneous point
mutations (PDR network) in regulatory factors governing
their expression.

The major determinants of multiple drug resistance in S.

cerevisiae

involve the transcription regulator genes PDR1

and PDR3 which control the expression of ABC pumps, MFS
transporters and other genes (summarized in Fig. 1).
Spontaneous dominant mutations which activate the master
transcription regulators PDR1 or PDR3 result in spectacular
multidrug resistance phenotypes

1,2

which either actively

extrude drugs out of the cell or modify their passive diffu-
sion into the cell by altering the lipid composition of the cell
bilayer.

THE PDR1P AND PDR3P TRANSCRIPTION FACTORS

Pdr1p and its paralog Pdr3p (Table 1) are members of a large
GAL4 family of yeast transcription factors characterized by
Zn

2

Cys

6

DNA-binding motif.

3,4

Pdr1p and Pdr3p bind to the

same consensus sequences

5–7

termed PDRE (Pdr1p/Pdr3p

Response Element), present in varying numbers and combi-
nations in the promoters of target genes (Fig. 2). Despite
their structural (36% identity, 57% similarity over the entire
amino acid sequence) and global functional similarities,
Pdr1p and Pdr3p play distinct roles. Disruption of PDR1 has
a more pronounced effect on drug sensitivity than disrup-
tion of PDR3. However, sensitivity to a set of drugs, such as
rhodamine 6G or diazaborine

8

(Kolaczkowska, personal com-

munication) is more affected by the inactivation of the PDR3
gene.This suggests that Pdr3p and Pdr1p recognize and acti-
vate different subset of genes in vivo what has been recently
confirmed by DNA microarray analysis (DeRisi et al.,
submitted).

THE DNA-BINDING ELEMENTS OF PDR3P AND PDR1P

Pdr1p and Pdr3p, similarly to all Gal4 family members, pos-
sess modular structure (Fig. 3). They contain N-terminally
localized DNA-binding domain with a zinc finger motif
followed by a short linker region and a coiled-coil dimeriza-
tion element.

9

The zinc finger domain binds to CGG triplets

spaced with different number of base pairs. Pdr1p and Pdr3p
are unusual in that respect, because they bind to everted
repeats with no intervening sequence. Absence of interven-
ing sequences between CGG triplets in the PDRE sites could
have consequences on dimer formation and/or on the
involvement of surrounding sequences in DNA binding
specificity and affinity. It has been demonstrated that

D

Fig. 1

The major target genes of PDR1 and PDR3. Represented are the genes for which published data report control by Pdr1p or

Pdr3p as well as a few targets identified by recent DNA-microarray analysis (DeRisi et al, submitted).



1999 Harcourt Publishers Ltd

Drug Resistance Updates (1999) 2, 403–414

DOI: 10.1054/drup.1999.0113, available online at http://www.idealibrary.com on

Kolaczkowska and Goffeau

404

Drug Resistance Updates (1999) 2, 403–414



1999 Harcourt Publishers Ltd

Table 1

Yeast Saccharomyces cerevisiae regulatory factors of the PDR and YAP networks involved in multidrug resistance

Protein

Structure

Target genes

Function

Mutants

Phenotype

Pdr1p

TF, Zn(II)2Cys6, 1063 aa

HXT9 (MFS)

Master regulator in PDR

pdr1–1 to 12

R to a few dozen of

Member of Gal4p family

HXT11 (MFS)

General positive

mitochondrial, cytoplasmic

DNA binding domain,

IPTG1/SYR4 (inositolphospho-

regulator of plasma

and nuclear inhibitors

Acidic activation domain,

transferase 1)

membrane functions

Inhibitory domain

PDR3 (TF)

Regulated by Pdr13p at

Binds PDRE

PDR5 (ABC)

the posttranslational

TPE1–1

Defective in intracellular

Forms homo- and

PDR10 (ABC)

level

(pdr1–11)

accumulation of (M-C

6

-NBD-

heterodimers with Pdr3p

PDR15 (ABC)

PE),Temperature sensitive

Not essential

PDR16 (SEC 14 homologue)

growth on nonfermentable

SNQ2 (ABC)

carbon sources

YOR1 (ABC)

∆

pdr1

HS to many unrelated

∆

pdr3

compounds, including FCZ

Pdr3p

TF, Zn(II)2Cys6, 976aa

HXT9 (MFS)

Involved in PDR

pdr3

chlo

R

, cyh

R

, dia

R

, muc

R

, oli

R

,

Member of Gal4 family

HXT11 (MFS)

Potential “enhancer” in

ner

R

, tet

R

, smm

R

DNA binding domain,

PDR3 (TF)

MDR response by its

Two activation domains,

PDR5 (ABC)

positive autoregulation

tpe2–1

Generalized growth defect on

Inhibitory domain

PDR10 (ABC)

Together with Pdr1p

(pdr3–11)

nonfermentable carbon

Binds PDRE

PDR15 (ABC)

controls plasma

sources,Allele specific defect

Homologous to Pdr1p

SNQ2 (ABC)

membrane functions

in M-C

6

-NBD-PE accumulation

Forms heterodimers with

YOR1 (ABC)

Pdr1p and probably

2

µ

cyh

R

and oli

R

homodimers (mutants)

Not essential

∆

pdr3

Not detectable effect on cyh

S

and rho6G

HS

Growth defect on complete

synthetic medium

Pdr6p

Chr VII, 1081aa,

–

Involved in nuclear

?

?

Member of karyopherin-beta

transport

family

Soluble protein

Pdr13p

Cytoplasmic protein, 572aa

PDR1

Upstream regulator of

2

µ

cyh

R

and oli

R

Member of Hsp70 family

Pdr1p

Increased expression of the

Acts at post-translational

PDR5 and YOR1 genes

Not essential

level

∆

pdr13

cyh

S

, cold-sensitive

Very slow growth at other

temperatures

Yer184C Putative TF, Zn(II)2Cys6,

?

?

?

?

794aa

Activation domain?

Homologue of Pdr3p

Yrr1p

TF, Zn(II)2Cys6

SNQ2

Involved in basal and

yrr1–1

rev

R

, oli

R

, 4-NQO

R

810aa

YOR1

drug-induced activation

Not essential

of SNQ2

∆

yrr1

4-NQO

HS

and loss in drug-

Required for 4-NQO

inducible expression of SNQ2

resistance

Regulation of pleiotopic drug resistance in yeast

405

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1999 Harcourt Publishers Ltd

Drug Resistance Updates (1999) 2, 403–414

Table 1

(Contd.)

Protein

Structure

Target genes

Function

Mutants

Phenotype

Yap1p

TF, bZIP, 650aa

APH1 (alkyl hydro-peroxide

Key determinant in

2

µ

Cd

R

, cyh

R

, dia

R

, flu

R

, 4-NQO

R

,

Member of Yap family

reductase)

oxidative stress tolerance

3-AT

R

MNNG

R

, nna

R

, smm

R

, tre

R

,

Homologue of mammalian

ATR1(MFS)

Controls loci involved in

tra

R

, phe

R

, Zn

R

and Ca

R

AP-1p

FLR1(MFS)

resistance to heavy

∆

yap1

Two activation modules

GLR1 (glutathion reductase)

metals and some drugs

H

2

O

2

HS

, Cd

HS

, diam

HS

, Zn

HS

and

CRD domain at the

GSH1 (glutamyl-cystein synthase) Involved in early general

Ca

HS

C-terminus

PDR5 (ABC)*

stress response

Cold sensitive

Binds to the canonical AP-

SNQ2 (ABC)*

site and YRE

TRX2 (thioredoxin)

Nuclear Export Sequence

YCF1 (ABC)

(NES) -like element

Aryl-alcohol oxido-reductases

Not essential

homologoues

Yap2p

TF, bZIP, 409 aa

YCF1?

Involved in multidrug

2

µ

3-AT

R

, Cd

R

, cyh

R

, phe

R

,

Member of Yap family

resistance,

Zn and Fe chelators

R

Homologue of Yap1p

Not involved in response

CRD domain at the

to superoxide

∆

yap2

H

2

O

2

HS

and oxidative stress

C-terminus

Binds to YAP element

∆

yap1

phe

HS

and oxidative stress

Not essential

∆

yap2

Yap3p

TF, bZIP, 329aa

?

?

2

µ

3-AT

R

Binds to YAP element

Yap4p

TF, bZIP, 295aa

?

Involved in chromosome

2

µ

Increased chromosome

(Cin5p)

Potential repressor

stability

instability, increased sodium

Binds to YAP element

Indirectly affects salt

and lithium tolerance

tolerance

qui

R

, chlq

R

and meq

R

∆

yap4

noc

S

Affects chromosome stability

Yap5p

TF, bZIP

?

?

2

µ

Slightly increased 3-AT

R

245aa

Does not bind to YAP

element

Not essential

Yap6p

Putative TF, bZIP, 1080 aa

?

Indirectly involved in

Increases salt tolerance

salt tolerance

2

µ

Increases ENA1 expression

Not essential

under salt-induced conditions

∆

yap6

Does not affect transcription

from ENA1 promoter

Yap8p

TF, bZIP, 293 aa

ACR2?

Involved in arsenic

2

µ

ars

R

and are

R

when

(ARR1)

Not essential

ACR3?

resistance Putative

simultaneously overproduced

positive regulator of

with Acr2 and Acr3p

ACR2 and ACR3 genes

∆

yap8

ars

HS

and are

HS

3AT-3-aminotriazole, are- arsenate, ars- arsenite, bre-brefeldin A, Ca- calcium, Cd- cadmium, cer-cerulenin, chlo-chloramphenicol, chq- chloroquine,

cyh- cycloheximide, dia- diazaborine, diam- diamide, Fe-iron, FCZ- fluconazole, flu- fluphenazine, itra- itraconazole, keto- ketoconazole, M-C

6

-NBD-PE-

1-myristoyl-2-[6-(NBD)aminocaproyl]- phosphatidylethanolamine, MNNG- N-methyl-N-nitrosoguanidine, meq- mefloquine, muc- mucidine, ner-

neutral red, nna-1-nitroso-2-naphtol, noc- nocadozole, 4NQO-4-nitroquinoline-n-oxide, oli- oligomycin, phe-phenantroline, rev- reveromycin A, rho6G-

rhodamine 6G, smm- sulfometuron methyl, sta- staurosporine, tet- tetracycline, tre- trenimon, qui- quinidine, Zn- zinc. Drug resistance phenotypes are

indicated as HS- hypersensitive, S- sensitive, HR- hyperresistance, R- resistance. Loss-of-function mutations are indicated as

∆

, overexpression of a wild

type genes as 2

µ

.Asterisk (*) indicates putative Yap1p sites identified in the promoter region of PDR5 and SNQ2 genes (Miyahara et al. 1996).

insertion of one nucleotide between the CGG triplets abol-
ishes the binding of Pdr3p.

10

In the case of Gal4p, which

binds to CGGN

5

TN

5

CCG, changes in spacing from 11 to 10

or 12 nucleotides greatly reduces binding in vitro and activ-
ity in vivo.

11

Studies of the DNA binding domains of

Gal4p,

12,13

and other members of the same family such as

Leu3p,

14

Put3p and Ppr1p

9

show that the linker region

(length and composition) determines the inter-triplet dis-
tance of the binding site, hence the DNA binding specificity.
In the case of Pdr1p, Pdr3p and possibly Ume6p,

15

the

absence of spacing between the triplets suggests that a
dimerization element lies just after zinc finger resulting in
very tense dimer geometry. Putative dimerization elements
could also be located more distantly from the Zn(II)

2

Cys6

domain and could modulate flexibility in recognizing the tar-
get sequence.

A single PDRE site is sufficient to trigger the Pdr1p/Pdr3p

response.

6

Bacterially produced Pdr1p and Pdr3p bind to the

canonical PDRE element (5

′

-TCCGCGGA-3). In addition to

this perfect palindrome, other sites degenerated by one base
change (5

′

-TCCGTGGA-3

′

, 5

′

-TCCGCGGGA-3

′

, 5

′

-TCCAC-

GGA-3

′

, 5

′

-TCCGCGCA-3

′

) are protected by Pdr1p. This

indicates that Pdr1p and/or Pdr3p tolerate some sequence
variation without abolishing binding. The ‘degeneration’ of
binding sites could play a role in discrimination between
binding of Pdr1p or Pdr3p and their putative homo- or het-
erodimeric forms, resulting in different activation of certain
PDR genes. Interestingly, one of the two PDRE sites
(5

′

-TCCGTGGA-3

′

) found in the HXT11 promoter does not

bind Pdr1p and/or Pdr3p,

43

whereas the same element found

in the promoters of PDR5,

7

YOR1

16

or PDR10

7

genes

responds to both Pdr1p and Pdr3p. These observations sug-
gest that besides distinct composition of PDRE, other uniden-
tified trans-acting factors affect the net activation by Pdr1p
and/or Pdr3p. For instance, recent data show an interplay of
PDR and YAP regulators as well as with other transcription
factors.

7,8,17

THE STRUCTURE OF PDR1P AND PDR3P

Analysis of the amino-acid sequence of the Gal4p family
reveals the presence of eight variably conserved hydropho-
bic motifs (MI to MVIII in Fig. 3) located in the central part of
the protein and potentially forming a functional domain.

3

The most conserved motifs (IV–V–VI) overlap with the so-
called Middle Homology Region (MHR) defined by Schjerling
and Holmberg.

4

Functional dissection of Pdr1p shows that

the deletion of a large central part of the protein makes it a
constitutive activator (Kolaczkowska et al., submitted). As
reported by the group of Moye-Rowley, a similar centrally
deleted pdr1 mutant does not respond to the upstream

Kolaczkowska and Goffeau

406

Drug Resistance Updates (1999) 2, 403–414

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1999 Harcourt Publishers Ltd

Fig. 2

Pdr1p/Pdr3p target genes contain different

combinations of PDRE sequence elements.

Fig. 3

Localization of gain-of-function mutations relative to the structural regions of Pdr1p and Pdr3p.The mutations are

presented by the abbreviated names of the pdr1 or pdr3 alleles.Amino acid changes corresponding to the appropriate mutation
are in brackets.The conserved hydrophobic motifs MI to MVIII are indicated.The zinc finger motif is denoted as Zn(II)2Cys6,
inhibitory domain as ID and the activation regions as AR.

positively acting modulator Pdr13p. This suggests that the
central part of Pdr1p regulates its transcriptional activity.
Indeed, internal deletions or directed mutagenesis on other
Gal4p family members highlight the functional importance
of the central domain. An example is Leu3p, where deletion
of a large central part converts the protein into a constitutive
activator,

18,19

whereas a single amino-acid alteration in Put3p

leads to loss of inducibility.

20

Removal of a short region of

Pdr1p encompassing the conserved motifs MI–II results in
an hyperactive mutant version (Kolaczkowska et al., submit-
ted).This finding together with a localization of a few hyper-
active pdr1 mutant alleles in that region, indicates the
inhibitory effect of this short domain. Similarly, the localiza-
tion of activating pdr3 mutants (Fig. 3) supports this finding.

Both Pdr3p and Pdr1p can activate transcription when

fused to the heterologous lexA DNA-binding domain.

21

Similarly to Gal4p, the Pdr3 protein contains two separable
activation regions (AR): a weak one, ARI localized near the
N-terminus (61–109 aa), and a second, most important
domain, AR II, encompassing the last 180 residues at the
C-terminus. Pdr1p, however, possess only one activation
region overlapping the acidic and asparagine-rich carboxyl
terminus of the protein (Kolaczkowska et al., submitted).
Many spontaneous pdr1 mutants map to the activation
region (see below).They include pdr1–8,

22,23

pdr1–10

24

and

pdr1–12

.

8

These mutants are improved activators.

The C-terminus of Pdr1p (815–1063aa) and Pdr3p

(815–976aa) interact directly or indirectly with the amino-
terminal 373 amino acids of the co-activator/repressor pro-
tein Ngg1p in the two-hybrid analysis.

25

Other adaptor

proteins, such as Ada2p or Gcn5p, could be involved in this
interaction. As for Gal4p, the adaptor complex inhibits tran-
scriptional activity of Pdr1p.

25

The adaptor proteins are likely

to serve as a regulatory bridge between activators, such as
Pdr1p or Pdr3p and the basal transcriptional machinery,
since they interact with the TATA binding protein.

26

THE MUTANT ALLELES OF THE PDR1 AND PDR3
GENES

It is well established that gain-of-function pdr1 and pdr3
mutant alleles highly up-regulate the expression of the ABC
transporters involved in drug extrusion and thus multidrug
resistance.

22,27–35

Single-point mutations in the PDR1 locus

(pdr1–2, pdr1–3, pdr1–6, pdr1–7, pdr1–8) increase to vari-
ous extents the mRNA level of downstream target genes,
such as PDR5, SNQ2, YOR1, PDR10, PDR15 and the newly
identified PDR16. Similarly, PDR3 mutant alleles (pdr3–2 to
pdr3–10

)

7,34,36

were shown to activate the expression of the

PDR5

, SNQ2, PDR15 and PDR3 genes. Generally, the level of

activation of a given target gene in various pdr1 or pdr3
mutants correlates well with the level of drug resistance
exhibited by those mutated alleles. Mutated Pdr1p and Pdr3p
are thus activated forms of those proteins and are believed to
act as constitutive activators.

The isolated pdr1 or pdr3 mutant alleles leading to hyper-

active alleles are clustered within short regions suggesting
specific functional roles for these domains (Fig. 3).

In addition to single-point mutations, polymorphic forms

of several PDR1 wild-type genes have been reported.

22

Several amino acid differences clearly distinguish PDR1
alleles from American and European origin.The most striking
is the presence of a stretch of 10 or five consecutive
asparagines, respectively, which could contribute to activa-
tion, maintaining the overall structure of the C-terminal
activation region (Kolaczkowska et al., submitted).

The phenotypes of pdr1 and pdr3 mutant alleles are not

restricted to pleiotropic drug resistance. Early reports men-
tion physiological alterations related to pdr1–2 mutant
allele, like inability to grow under adverse conditions such as
high osmolality, elevated pH or increased temperature.

37

Recently isolated PDR1–11 and pdr3–11 alleles show other
phenotypes besides multidrug resistance.

38

The pdr3–11 is

unable to grow on glycerol/ethanol medium or lactate,
whereas the dominant PDR1–11 mutant exhibits tempera-
ture-sensitive growth defect on nonfermentable carbon
sources. Slower grow of the PDR1–11 and pdr3–11 mutant
strains, was observed in synthetic medium. Accordingly, we
have observed that disruptants of PDR3, but not PDR1,
exhibit severe growth defect on complete glucose-contain-
ing synthetic medium in the absence of any drugs.

Moreover, the PDR1–11 and pdr3–11 (also called tpe

mutants) alleles (but not PDR1-2 or pdr3–2) affect the
steady-state distribution of endogenous phosphatidyle-
thanolamine across the plasma membrane.

38

THE GENES REGULATED BY Pdr1p AND Pdr3p

The identification and characterization of the genes that are
under the transcriptional control of Pdr1p and Pdr3p could
provide clues about the true physiological function of the
PDR system.

Many of the known Pdr1p/Pdr3p target genes encode

membrane proteins, which belong to the ABC (Pdr5p,
Pdr10p, Pdr15p, Snq2p, Yor1p) or MF (Hxt9p and Hxt11p)
superfamilies. Pdrlp has also been reported to control the
expression of its homologue, PDR3,

5

the gene IPT1/D4405

whose protein product affects sphingolipid biosynthesis,

1,39

as well as PDR16 affecting phospholipid and sterol composi-
tion of the plasma membrane.

27

The well characterized PDR5, SNQ2 and YOR1 genes

encode ABC-type transporters of extremely broad substrate
specificity including a variety of anticancer drugs, antibi-
otics, antifungals, detergents, ionophores and others.

30,41

Pdr5p which is a functional homologue of human P-gp and
Yor1p which is homologous to mammalian MRP, are involved
in the transport of fluorescent NBD-labeled short chain
phosphatidylethanolamine

28

and steroids.

40–42

These observa-

tions are in agreement with the finding that PDR1–11 and
pdr3–11

mutant alleles are defective in the accumulation of

fluorescent phosphatidylethanolamine.

38

The PDR1–11 and

pdr3–11

mutants along with the pdr1–3 allele are likely to

activate expression of genes encoding proteins decreasing
the steady-state accumulation of phosphatidylethanolamine
by increased efflux or decreased influx.

28,38

In this context, it

is relevant to mention that in 1994 Ruetz and Gros,

43

pro-

posed that the physiological role of mice mdr2 is transloca-
tion of phosphatidylcholine, which was then confirmed and
extended to human mdr2 and mice mdr3.

Regulation of pleiotopic drug resistance in yeast

407

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1999 Harcourt Publishers Ltd

Drug Resistance Updates (1999) 2, 403–414

The precise roles of the other target genes encoding the

ABC proteins Pdr10p and Pdr15p

7

remain to be determined

as well as the involvement in MDR of two hexose trans-
porters, Hxt9p and Hxt11p, whose inactivation leads to
multidrug-resistant phenotype.

45

New target genes for Pdr1p and Pdr3p have been recently

identified by DNA-array analysis (DeRisi et al., submitted).
Pdr1p and Pdr3p were found to have distinct but partially
overlapping specificity. Their activated forms, pdr1–3 or
pdr3–7

, induce expression of different subset of genes; how-

ever most of the targets are affected by both regulators.
Besides known target genes already mentioned newly identi-
fied ones include MFS transporters such as TPO1 encoding a
vacuolar polyamine transport protein,

46

other putative per-

meases such as HXT2, RTA1 and YOR049c, and genes encod-
ing proteins involved in lipid or cell wall metabolism.
Significantly, Pdr1p or Pdr3p were found to activate the
expression of genes encoding stress-defense proteins (trig-
gers: nutrient starvation, osmotic stress, DNA damage).

The cellular function of Pdr1p could be much broader

than originally believed as it activates the expression of at
least one negative regulatory factor (XBP1) (Van Den Hazel,
personal communication). Indeed, Pdr1p or Pdr3p reduces
the expression of several genes. However, there is no recog-
nizable PDRE in their promoters.This suggests a rather indi-
rect role of Pdr1p and activated expression by Pdr1p of
genes encoding repressor proteins. To the group of genes
repressed by Pdr1p or Pdr3p belongs PDR12 which is the
closest SNQ2 homologue. PDR12 codes for a weak-acid-
inducible ABC transporter which confers resistance to sor-
bate,

benzoate and acetate

47

and which transports

fluorescein and preservative anions from the cytosol to the
outside by an energy-dependent mechanism.

48

In single pdr1

and doubly pdr1/pdr3 disruptants, increased sorbate resis-
tance was observed which again argues for a negative action
of Pdr1p on PDR12p.

47

It is however important to be aware that the growth con-

ditions are very likely determining the pattern of expression
of the Pdr1p- or PDR3p-dependent genes.

THE DIFFERENCES IN PDR1P AND PDR3P
TRANSCRIPTIONAL CONTROL

The expression of PDR5, SNQ2 and YOR1 is reduced in the
hypersensitive pdr1pdr3 knockout strain. In that back-
ground, PDR5 expression is severely affected and becomes
undetectable, whereas the expression of SNQ2 and YOR1 is
decreased but maintains an important PDR1/PDR3-indepen-
dent regulatory component. Moreover, deletion of the PDR1
locus has more pronounced effects than that of PDR3.
However, this is not true for all Pdr1p/Pdr3p target genes.

All targets of PDR1 and PDR3 contain at least one PDRE

in their promoter (Fig. 2). DNase I footprinting and muta-
tional analyses performed on the PDR5 promoter region
demonstrate that although a single PDRE is sufficient to
respond to Pdr1p and Pdr3p, at least three PDRE sites are
needed for full promoter activity.

6

Furthermore, PDR5

expression is strictly dependent on the presence of Pdr1p
and Pdr3p.This correlates with steady-state level of the Pdr5
protein in pdr1pdr3 knockout strain and the hypersensitiv-

ity of the doubly disrupted pdr1pdr3 strain or the single
pdr5

knockout derivative to cycloheximide, a drug detoxi-

fied primarily by Pdr5p.

41,42

Accordingly, inactivation of

PDR1

severely decreases the expression of PDR5, whereas

the PDR5 gene is also the most responsive target of the
hyperactive pdr1 mutant alleles.

Analysis of the YOR1 promoter reveals positive and nega-

tive cis-acting elements.

16

One of the two positive elements

corresponds to a single PDRE, which is required for normal
oligomycin tolerance.The Pdr1/Pdr3p independent up-regula-
tion of SNQ2 is mediated by the newly identified Yrr1p
Zn(II)2Cys6 transcription factor (Table 1). In the YRR1–1
mutant the level of SNQ2 transcript is highly elevated.

17

The

Yrr1 transcription factor mediates also 4-NQO-induced activa-
tion of the SNQ2 gene, whereas Pdr1p and Pdr3p are required
mainly for basal expression of that gene. Moreover, higher 4-
NQO sensitivity of the single snq2 knock-out strain than that
of the triply deleted pdr1pdr3yrr1 strain suggests the pres-
ence of another yet unidentified regulatory factor specifically
implicated in SNQ2 mediated 4-NQO resistance.

17

In agree-

ment with those observations is the fact that, despite the pres-
ence of the same number (but different composition) of PDRE
sites, SNQ2 is less responsive to Pdr1p/Pdr3p than PDR5.

32

The newly identified PDR15 gene seems to be more

responsive to Pdr3p than to Pdr1p

7,28

(DeRisi et al., submit-

ted). In the pdr3 null mutant, the mRNA level of PDR15 is
severely decreased, and is almost abolished in a strain lacking
both PDR1 and PDR3 loci. Interestingly, upon inactivation of
the PDR1 locus, the level of PDR15 mRNA is significantly
increased. Since Pdr1p is an activator, its negative effect on
the transcriptional control of PDR15 could be explained by
interaction with other yet unknown co-factors or by the
Pdr1p-activated expression of an unidentified PDR15 repres-
sor. Furthermore, PDR15 is significantly induced by the
pdr3–2

but not by the pdr1–3 mutant allele.

7

This correlates

with the

β

-galactosidase activity driven by this promoter,

which is not highly affected in the pdr1–3 mutant.

28

In con-

trast, the mRNA level of the PDR10 gene is enhanced in the
mutant strain pdr1–3 but remains unchanged in the pdr3–2
background.

7

It is thus clear that it is not the number of

PDRE (Fig. 2) but their sequence composition and interac-
tion with other transcription factor(s) which modulate the
net Pdr1p/Pdr3p activation. It is also noteworthy that in addi-
tion to its dependence to Pdr1p, the PDR15 gene is likely to
be stress-activated in the Msn2p/Msn4p-dependent manner
via the STRE elements present in its promoter region.

49

The Pdr3p regulator has also been reported to control

expression of its own gene via an autoregulatory loop. Two
perfectly palindromic PDRE sequence elements within the
PDR3

promoter region are recognized and are cooperatively

bound by Pdr3p with the predominant role of the upstream
element.

5

The PDR3 promoter fused to lacZ reporter gene

positively responds to increasing doses of either Pdr1p or
Pdr3p.Although direct binding of Pdr1p to PDRE sites in the
PDR3

promoter was not demonstrated, it is likely that Pdr1p

recognizes those regulatory sequences. Decreased PDR3-dri-
ven

β

-galactosidase activity in the absence of PDR1

5

and

induction of the PDR3 promoter by pdr1 mutant alleles
(Kolaczkowska, unpublished results) support this hypothe-
sis. Furthermore, accumulation of PDR5 mRNA in response

Kolaczkowska and Goffeau

408

Drug Resistance Updates (1999) 2, 403–414



1999 Harcourt Publishers Ltd

to cycloheximide shows that the integrity of the two PDRE
elements in the PDR3 promoter is necessary to trigger
induction of PDR5 expression in the absence of PDR1.These
observations, together with that of cycloheximide sensitivity
conferred by mutation of PDRE in the PDR3 promoter, indi-
cate functional involvement of an autoregulatory loop in
drug resistance. It remains, however, to verify the involve-
ment of these PDREs in the regulation of the neighbor gene
YBL006c

of unknown function which is divergently tran-

scribed on the other DNA strand.

OTHER PDR REGULATORS

Pdr1p is post-translationaly regulated by Pdr13p, which is
the unique yeast member of the Hsp70 protein family.

50

Pdr13p regulates the function of Pdrlp but not of Pdr3p.
Overproduction or gain-of-function mutation (S295F) of
Pdr13p elevates Pdr1p wild-type activity.Yeast cells carrying
inactivated PDR13 gene exhibit cold-sensitive growth
defect. Phenotype analysis of the strain lacking the PDR13
gene shows its possible connection with the general stress-
response system. In the pdr13 knock-out strain, the induced
expression of several genes containing STRE elements

50

being under the control of the general stress response fac-
tors Msn2p and Msn4p, is observed. Tested genes include
CTT1

(cytoplasmic catalase), HSP12 (heat shock protein) as

well as CUP1 (metallothionine) Expression of the CUP1
gene is affected by a heat shock transcription factor in
response to oxidative stress.

51

The PDR6/YGL016W gene initially reported as a mul-

tidrug resistance determinant

52

deserves special mention.

That gene has recently been shown to be a member of an
important RanBP7/importin-beta/Cse1p superfamily of Ran
GTP-binding proteins involved in nuclear transport.

53,54

Therefore

PDR6

is a novel candidate protein involved in the

regulation of nuclear transport of transcription factors
involved in multidrug resistance. Since the movement of
many transcription factors, kinases and replication factors
between the nucleus and cytoplasm is an important compo-
nent in regulating their activity, it is tempting to speculate
that the activity of PDR transcription factors is also regulated
at the level of their nuclear import.

Two loci, PDR7 and PDR9, both localized on chromo-

some II, were reported to affect the transcription of the
PDR5

gene.

55

Mutants of both genes, isolated as spontaneous

supressors of the hypersensitive pdr1 knock-out derivative
restore resistance to cycloheximide and sulfometuron
methyl. Since these genes have not yet been identified within
the sequence of chromosome II, it is difficult to predict their
role within the PDR network.

THE YAP NETWORK AND ITS INTERACTIONS WITH
PDR

The Yap network is one of the transcription control systems
activated by stress signals.

56

Yap1p and Yap1p-like regulators

from other yeast species are the major cellular determinants
of the OSR (oxidative stress response) system.

8,57,59

YAP1

encodes a transcription factor of bZIP type, which

is able to activate the mammalian AP-1 response element

(ARE).

60

Yap1p binds to the yeast yAP-1 recognition elements

(YRE), which differ at second position from the AP-1 site (the
AP-1 site is TGA C/G TCA; the preferred yap site is
TTAGTA).

61

Yap1p contributes directly to the transcriptional activa-

tion of a group of proteins synthesized under stress condi-
tions (OSR or MDR) and indirectly modulates the activity of
the Stress Regulated Element (STRE), which is the core of the
general stress-response mechanism.

The Yap1p transcription factor plays also an important

role in increased resistance to variety of drugs and metals
(Table 1). Overexpression or genetic alteration of Yap1p and
its homologue Cad1p leads to multidrug resistance.

62–66

Absence of the YAP1 gene confers cadmium hypersensitiv-
ity. However, loss-of-function mutation does not result in sen-
sitivity to cycloheximide, diazaborine or sulfometuron
methyl, indicating that Yap1p is not a sole determinant of the
observed resistance phenotype and/or affects resistance to
certain drugs indirectly. The YAP1 target gene YCF1 shows
significant sequence similarity to the mammalian ABC trans-
porter-MRP. The YCF1 gene encodes a detoxification pump
which mediates vacuolar and ATP-dependent transport of
gluthatione conjugates.

67,68

It is also essential for cadmium

tolerance in yeast cells.

64–67

Other target genes, such as FLR1 or ATR1, mediate Yap1p-

dependent, multidrug-resistance phenotype. The MFS trans-
porters Atr1p and Flr1p are implicated in 3-aminotriazole,
1,10-phenanthroline and fluconazole, cycloheximide, 4-NQO
resistance, respectively.

69–71

Although direct Yap1p binding to

the promoter of the S. cerevisiae FLR1 gene has not yet been
demonstrated, experimental data on the Yap1p homologue
from C. albicans, Cap1p, show that the mRNA level of FLR1
is enhanced in the strains overproducing Cap1p or its hyper-
active truncated allele lacking the C-terminal part.This analy-
sis shows that the Cystein Rich Domain which is located at
the carboxyl terminus of Yap1p (and Cap1p) is dispensable
for conferring an MDR phenotype to yeast.

70

It is however,

required for mediating resistance to cadmium and is neces-
sary for the oxidative stress response caused by H

2

O

2

.

72

Yap1p seems to be involved in stress-dependent activa-

tion of certain PDR genes, which suggests a functional inter-
play between YAP and PDR networks in S. cerevisiae. The
PDR5

and SNQ2 genes, possess, in addition to PDRE, YAP

response elements in their promoters.

61,73

The presence of a

functional YAP1 gene was reported to be required for the
expression of SNQ2 stimulated by external stress condi-
tions.

7

Thus, at least one PDR target gene, SNQ2, could be

under the control of the general stress response pathway via
Yap1p. On the other hand, a S. cerevisiae strain overproduc-
ing Cap1p, the homologue of Yap1p from C. albicans or its
truncated version, not only shows dramatically enhanced
mRNA level of the MFS transporter-FLR1 but also a slightly
elevated level of the PDR5 transcript. Thus, it seems that
Yap1p could contribute to the control of 4-NQO and cyclo-
heximide resistance via genes encoding ABC transporters,
such as SNQ2 and PDR5 and/or MFS transporters.

The YAP1-mediated diazaborine and 4-NQO resistance

is mainly dependent on the PDR3 gene.

8

Furthermore,

the double pdr1pdr3 knock-out strain is hypersensitive to
diazaborine even when YAP1 is overexpressed, thus

Regulation of pleiotopic drug resistance in yeast

409

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1999 Harcourt Publishers Ltd

Drug Resistance Updates (1999) 2, 403–414

demonstrating a requirement of intact PDR1 and PDR3 loci
for YAP1-mediated resistance to that drug.

8

This, together

with the observation that Yap1p indirectly affects diaza-
borine resistance, suggests the existence of a Yap1p-depen-
dent activation of Pdr3p (but much less of Pdr1p) rather
then direct Yap1p binding to the gene(s) responsible for
multidrug resistance.

The Yap1p-dependent multidrug resistance phenotype of

S. cerevisiae

results from up-regulation of the downstream

MFS target genes FLR1 and ATR1. However, resistance to
diazaborine and 4-NQO drugs depends on the presence of
functional PDR3 and PDR1 genes. This could result from
Yap1-dependent sequential activation of Pdr3p and Pdr1p or
from regulatory interaction between Yap1p and putative
repressor of Pdr1p and Pdr3p. One of the potential repres-
sors could be Ngg1p which was shown to inhibit Pdr1p
activity.

25

REGULATION OF MULTIDRUG RESISTANCE IN
CANDIDA ALBICANS

As recently reviewed by Sanglard et al.

74

C. albicans

contain

Pdr5p-like ABC pumps

75

and multidrug MFS transporters.

76,77

The regulation of their expression is different from that in
S. cerevisiae

. In C. albicans at least two mechanisms lead to

clinical fluconazole resistance: alteration of the drug target
(lanosterol 14-

α

demethylase), as reviewed in Vanden

Bossche et al.,

78

and decrease of drug accumulation associ-

ated with overexpression of multidrug ABC transporters,
such as CDR1,

CDR2

or the MFS transporter

BEN

r

/CaMDR1.

76,77,79

The latter mechanism seems to be

responsible for an early response developing rapidly after
exposure to fluconazole, whereas genetic alterations in tar-
get genes and/or trans-acting regulatory factors are also
observed in long-term treatment.

80

One of the cellular MDR determinants in C. albicans is

the CAP1 gene, encoding an AP-1 like regulator cloned on
the basis of fluconazole resistance in S. cerevisiae.

70

Deletion

of CAP1 in C. albicans results in hypersensitivity to a limited
number of drugs and oxidants, indicating its involvement in
multidrug resistance and oxidative stress response.

72

The

pleiotropic phenotype of Cap1p is likely to result from mod-
ulation of target genes including CaYCF1, CaTRX2, MDR1
(a MFS), CaGSH1, CaGLR1 and possibly the CIP2 gene,
which has a putative YRE element in its promoter.

72,81

The

truncated CAP1 gene lacking the C-terminus, highly overex-
presses the CaYCF1, CaGLR1, CaTRR1 and MDR1 genes and
results in hyperresistance phenotype. The observation that
the hyperactive truncated CAP1 does not display increased
resistance to ketoconazole and fluphenazine, two well-
known substrates of the Cdr1p ABC transporter, suggests that
the CDR1 gene in not likely to be a target for Cap1p.

Cap1p acts as a positive regulator of the FLR1 gene

expression in S. cerevisiae. However, inactivation of CAP1 in
a C. albicans wild-type strain as well as in a fluconazole resis-
tant strain does not affect susceptibility of those cells to
fluconazole and other antifungal agents like cerulenin or
brefeldin A.

70–72

Furthermore, disruption of CAP1 in C. albi-

cans

results in up-regulation of the MDR1/BEN

R

gene encod-

ing MFS transporter implicated in fluconazole resistance and

homologous to Flr1p. This indicates that in C. albicans
Cap1p acts as a negative transcription factor of MDR1
expression and that it is not responsible for the stable flu-
conazole resistance phenotype.

72

Interestingly, inactivation of the very recently isolated

C. albicans

Fcr1p, an ortholog of S. cerevisiae Pdr1p,

82

results in fluconazole resistance. The FCR1 C. albicans
knock-out derivative displays increased resistance to flu-
conazole and to drugs known to be transported by Cdr1p
and Cdr2p.This indicates that Fcr1p acts as repressor of PDR
in C. albicans. However, one can not exclude that hyperac-
tive, gain-of-function alleles of Fcr1p (and/or Fcr2p, Fcr3p)
could result in constitutive expression of a set of genes impli-
cated in fluconazole resistance and MDR similarly as in
S. cerevisiae

. Alternatively, other regulatory factors or alter-

ations in cis-acting DNA sequences of the Yap1p target genes
FCR1 or CAP1, could contribute to the development of MDR.
Therefore, detailed characterization of Fcr1p, its target genes
as well as the molecular mechanisms leading to aberrant
up-regulation of MDR1 and CDR1 genes is needed for the
understanding of multidrug resistance in C. albicans and
fluconazole resistance in clinical isolates.

CONCLUSIONS AND PROSPECTS

Figure 4 illustrates our present knowledge (or ignorance) of
the various pathways which could interfere with PDR1 and
PDR3

to control multidrug resistance in S. cerevisiae. We

shall comment only on a few features of this scheme.

No experimental data exist today that indicate the nature

of the factors which modulate the level of expression of
PDR1

and PDR3.The spectacular, dominant gain-of-function

mutations prd1 and pdr3 which are highly selectable in the
presence of drug are likely to act on the activity of Pdr1p and
Pdr3p, rather than on their level of transcription. If present,
the putative induction pathways for increased level of
expression of PDR1 and PDR3 mentioned in Figure 4 have
still to be demonstrated.

Recent experimental data show however, that the activity

of Pdr1p is modulated at least at two levels. Positive modula-
tion by Pdr13p of Pdr1p, but not by Pdr3p, raises the ques-
tion as whether another chaperone acts on Pdr3p. The
participation of Pdr6p in the nuclear transport of unidenti-
fied factors involved in multiple drug resistance is credible. It
is not known however whether such proteins act on Pdr1p
or Pdr3p.Therefore, it is essential to elucidate the exact role
of the chaperone Pdr13p as well as that of the nuclear trans-
port determinant Pdr6p and to search for new putative func-
tional homologues of these two proteins.

Many experimental data suggest a connection of PDR

with the cellular stress-response system. This could be
accomplished by direct or indirect modulation of expression
of some PDR genes by Yap1p (and/or Yap1p homologues)
under stress conditions. Candidate PDR genes modulated by
Yap1p-like factors are the stress genes PDR12, PDR5, SNQ2
containing YRE-like sequences in their promoter regions and
PDR15

, which possesses a STRE element. These genes are

potential targets for stress regulatory factors such as Msn2p
and Msn4p.

83

In particular, it is conceivable that in response

to external stresses, modulation of the permeability barrier

Kolaczkowska and Goffeau

410

Drug Resistance Updates (1999) 2, 403–414



1999 Harcourt Publishers Ltd

occurs by stress-induced activation of Snq2p and Pdr5p
involved in sterol and phospholipid transport and biogenesis
of the cell envelope.

Pdr1p and Pdr3p exert positive and negative effects on

their target genes. Obviously other transcription factors,
some involved in different types of stress-defence, do also act
on the promoters of some of the PDR targets. Elucidation of
the numerous interactions between PDR,YAP and other tran-
scription factors in the promoters of the multidrug resis-
tance genes will probably require exhaustive
DNA-microarray analysis.

Numerous gain-of-function mutations in either PDR1 or

PDR3

regulators result in a variety of phenotypes including

multidrug resistance.The molecular mechanism of action of
those hyperactive Pdr1p and Pdr3p forms is not clear.
Analysis of pdr1 mutants reveals that some of the mutations
improve the efficiency of the activation domain
(Kolaczkowska et al., submitted). This, however, does not
explain the variety of observed phenotypes. It is possible
that different pdr1 or pdr3 mutants control other still
unidentified target genes.

Functional analysis of the major ABC transporters of S.

cerevisiae

will continue to be an important approach for the

study of multidrug resistance.

1,2,84–86

This review shows that a

better knowledge of the regulatory mechanism leading to
the development of the pleiotropic drug resistance in the

Regulation of pleiotopic drug resistance in yeast

411

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1999 Harcourt Publishers Ltd

Drug Resistance Updates (1999) 2, 403–414

Fig. 4

Schematic representation of the different pathways which could regulate either the level of expression of the PDR1 and

PDR3 genes or the level of expression of their target genes.

model yeast S. cerevisiae is also needed. We believe that
analysis of the PDR and YAP networks in S. cerevisiae will
help understand their homologous networks operating in C.
albicans

and that this knowledge will be a prerequisite for

the development of new antifungals.

Acknowledgments

The authors wish to thank Marcin Kolaczkowski for helpful
comments and discussion.Also, they gratefully acknowledge
E. Balzi, A. Delahodde, J. L. DeRisi, C. Jacq for informations
prior to publication. This work was supported in part by
grants from the Service de la Politique Scientifique: Action
Sciences de la Vie and by Fonds National de la Recherche
Scientifique, Belgium.

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