CHAPTER 15 – FUNGAL ABC PROTEINS IN CLINICAL DRUG RESISTANCE AND CELLULAR DETOXIFICATION

CHAPTER 15 – FUNGAL ABC PROTEINS IN CLINICAL DRUG RESISTANCE AND CELLULAR DETOXIFICATION CHAPTER 15 – FUNGAL ABC PROTEINS IN CLINICAL DRUG RESISTANCE AND CELLULAR DETOXIFICATION CHAPTER 15 – FUNGAL ABC PROTEINS IN CLINICAL DRUG RESISTANCE AND CELLULAR DETOXIFICATION CHAPTER 15 – FUNGAL ABC PROTEINS IN CLINICAL DRUG RESISTANCE AND CELLULAR DETOXIFICATION

I NTRODUCTION

The genome of baker’s yeast Saccharomyces

cerevisiae contains 30 distinct genes encoding

ATP-binding cassette (ABC) proteins (Bauer

et al., 1999; Decottignies and Goffeau, 1997;

Taglicht and Michaelis, 1998) Expression of

sev-eral yeast ABC proteins is linked to, or causes,

pleiotropic drug resistance (PDR) phenomena

(Wolfger et al., 2001) and certain ABC genes

rep-resent orthologues of mammalian disease genes

S cerevisiae is thus considered an important

model organism to study the function of

evolu-tionary conserved genes, including mammalian

ABC proteins of medical importance The PDR

phenomenon is phenotypically quite analogous

to multidrug resistance (MDR) as it develops

in mammalian cells (Litman et al., 2001),

para-sites, fungal pathogens or even in bacteria MDR

can be described as an initial resistance to a

single drug, followed by cross-resistance to

many structurally and functionally unrelated

compounds (Kane, 1996; Litman et al., 2001).

Baker’s yeast was therefore exploited to dissect

the molecular mechanisms of PDR/MDR

medi-ated by ABC transporters For instance,

cross-complementation studies yielded insights into

the function of mammalian MDR transporters

of the P-glycoprotein (Pgp) family (Kuchler

and Thorner, 1992; Ueda et al., 1993), as well as

the MRP (multidrug resistance-related protein)

family (Raymond et al., 1992; Ruetz et al., 1993;

Tommasini et al., 1996; Volkman et al., 1995).

Importantly, yeast strains lacking endogenous

ABC pumps have been used to identify andclone resistance genes from fungal pathogens

such as Candida and Aspergillus species For example, the Candida genes CDR1 and CDR2,

implicated in clinical azole resistance, were initially identified by virtue of their ability torescue the drug-hypersensitive phenotype of a

mutant S cerevisiae strain (Prasad et al., 1995;

Sanglard et al., 1995, 1997) This chapter is

devoted to a comprehensive discussion of ABCprotein-mediated drug resistance phenomena

as they have been described in model systems

like S cerevisiae as well as in fungal pathogens.

The inventory of S cerevisiae ABC proteins has

been classified into five distinct subfamilies(see also Chapter 14) Several genes of the PDR and MRP/CFTR subfamilies of yeast ABC

proteins (Table 15.1) mediate PDR, as their

expression is tightly linked to compound drugresistance phenotypes These genes are part of

the PDR network (Figure 15.1), which

com-prises several ABC transporters, as well as dedicated regulators controlling the expression

of ABC target genes (Bauer et al., 1999; DeRisi

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CHAPTER

et al., 2000; Wolfger et al., 2001) Moreover, this

network contains at least two permeases of the

major facilitator family (Nourani et al., 1997b),

and several other yeast genes (DeRisi et al.,

2000; Kolaczkowska, 1999) We have not

included these in Figure 15.1, since they

repre-sent non-ABC genes

The major S cerevisiae drug efflux pumps

are Pdr5p, Snq2p and Yor1p, all of which

local-ize to the cell surface (see Chapter 14) These

transporters recognize an amazingly broad

spectrum of xenobiotics and hydrophobic

drugs and extrude hundreds of compounds

to the extracellular space (Egner et al., 1998;

Kolaczkowski et al., 1998; Mahé et al., 1996a).

Thus, PDR arises from expression or induced

overexpression of ABC pumps mediating

cellular efflux of a great variety of differentdrugs or cytotoxic compounds Although drugresistance can also be due to reduced druguptake, target alteration and vacuolar seques-

tration (Figure 15.2), increased efflux through

membrane ABC transporters represents a majorcause of acquired drug resistance phenotypes.Other closely related members of the PDR family include Pdr10p and Pdr15p, sharingabout 70% identity with Pdr5p However, nodrug substrates have been identified and theirexpression and function appears connected to

a cellular stress response (Wolfger et al., in

preparation) Likewise, the function of thePdr12p efflux pump is linked to a stressresponse, but in this case weak organic acidsrather than hydrophobic drugs were identified

TABLE15.1 FUNGALABC TRANSPORTERS AND SOME RELEVANT SUBSTRATES

Non-pathogenic fungi

Saccharomyces cerevisiae

Pdr5p Drugs, steroids, antifungals, 1511 (ABC-TMS6)2 Plasma membrane

phospholipids

Yor1p Oligomycin, reveromycin A, 1477 (TMS6-ABC)2a Plasma membrane

phospholipids Ycf1p GS-conjugates, Cd2⫹, UCB, 1515 (TMS6-R-ABC)2a Vacuole

diazaborine, bile acids

Pathogenic fungi

Candida albicans

Cdr1p Antifungal azoles, rhodamine, 1501 (ABC-TMS6)2 Plasma membrane

drugs, dyes Cdr2p Antifungal azoles, rhodamine, 1499 (ABC-TMS6)2 ?

ABC, ATP-binding cassette; TMS, transmembrane segment; PM, plasma membrane; Vac, vacuole; GS, glutathione S; UCB, unconjugated bilirubin.

aSince Ycf1p and Yor1p belong to the MRP/CFTR family, their membrane topology might be different, displaying an

additional N-terminal transmembrane domain, but this has not been established (Tusnady et al., 1997).

as physiological substrates (Holyoak et al.,

1999; Piper et al., 1998).

A second important mechanism of PDR inyeast involves sequestration into the vacuole

(Figure 15.2) Vacuolar ABC pumps such

as Ycf1p, Ybt1p and Bpt1p, like the plasma

membrane Yor1p, belong to the MRP/CFTR

subfamily, as they are more closely related to

mammalian MRP, and at least to some extent tohuman CFTR Xenobiotics or toxic metabolitescan be sequestered into the vacuole, therebyleading to drug or even heavy metal tolerance

For example, the yeast cadmium factor (Ycf1p) isresponsible for vacuolar detoxification of heavymetals as well as glutathione S-conjugates (GSH

conjugates) (Li et al., 1996; Szczypka et al., 1994).

ABC transporter genes with similar functions

were also discovered in the fission yeast

Schizo-saccharomyces pombe For instance, expression of pmd1 and hba2/bfr1 mediates drug resistance

(Nagao et al., 1995; Nishi et al., 1992; Turi and

Rose, 1995), while Hmt1p is involved in

vacuo-lar sequestration of heavy metals (Ortiz et al.,

1992, 1995)

Because of their medical importance, ABCproteins from fungal pathogens, including

Candida and Aspergillus species, have received

considerable attention in recent years, larly concerning their possible contribution to

particu-clinical antifungal resistance (Table 15.1) To

date, four Candida ABC transporters implicated

in clinical drug resistance have been identified

The CDR1 and CDR2 genes from Candida

albi-cans (Prasad et al., 1995; Sanglard et al., 1995,

1997), as well as PDH1 (Miyazaki et al., 1998) and CgCDR1 (Sanglard et al., 1999) from

Candida glabrata mediate antifungal resistance

both in clinical isolates and in the model system

S cerevisiae For Candida dubliniensis, ABC

trans-porters were also speculated to mediate clinicalfluconazole resistance, and the existence of

CDR1 and CDR2 homologues has at least been

demonstrated by polymerase chain reaction

(PCR) (Moran et al., 1998) Several ABC porters exist in Aspergillus, three of which

trans-confer drug resistance upon overexpression

Aspergillus fumigatus AfuMDR1, when

overex-pressed in a drug-sensitive S cerevisiae strain,

enhances resistance to the antifungal tide cilofungin, although no hyper-resistance to

lipopep-other compounds is observed (Tobin et al., 1997) The expression of the Aspergillus nidulans

atrB and atrD genes is induced by numerous

drugs, suggesting a role in drug resistance

Indeed, deletion of atrD increases drug tivity (Andrade et al., 2000b), and overexpres- sion of atrB in a hypersensitive ⌬pdr5 yeast

sensi-strain confers resistance to various compounds

(Del Sorbo et al., 1997) Unlike for baker’s

yeast, however, the literature contains only alimited amount of information as to the func-tional mechanisms, the regulation or even cel-lular localization of ABC pumps from fungalpathogens

Figure 15.1 The pleiotropic drug resistance

(PDR) network The genes in the center line

represent target genes of dedicated transcriptional

regulators depicted above and below Note, the

cartoon only includes functional drug resistance

genes of the ABC gene family The yeast PDR

network also contains non-ABC genes whose

function is not always established (see text for

details).

Figure 15.2 Principal mechanisms of drug

resistance Drug resistance phenotypes can

arise based on several molecular principles

Pleiotropic or multidrug resistance, which displays

cross-resistance to many structurally and

functionally unrelated drugs, often results from the

induced overexpression of cell surface ABC efflux

pumps causing increased efflux of xenobiotics Each

mechanism on its own or in combination with

another one can cause a drug resistance phenotype

in fungal cells N, nucleus; V, vacuole.

G ENETIC A NALYSIS AND

To study the function of ABC pumps, deletion

and overexpression phenotypes should be

ana-lyzed in the individual cases Thus,

chromoso-mal deletions or disruptions of fungal pump

genes have been generated Remarkably, none

of the yeast drug ABC transporters (Table 15.1)

appears to be essential for viability Hence,

the physiological function of these proteins

must be dispensable in cells growing under

normal conditions However, in the presence

of xenobiotics, including antifungals and

anti-cancer drugs, cells lacking Pdr5p, Snq2p or

Yor1p display marked drug hypersensitivity

phenotypes (Wolfger et al., 2001) Such a

hyper-sensitivity phenotype was exploited for the

cloning of ABC transporters from other fungal

species through functional complementation

(see below) Notably, in cases like Yor1p or

Pdr5p, pump deletion caused hypersensitivity

for some drugs but hyperresistance for others

Such phenomena are difficult to explain at the

moment, but relate to altered drug uptake,

sur-face permeability changes due to pump

dele-tion, the presence of intracellular drug targets or

altered sequestration mechanisms (Figure 15.2).

Apart from hypersensitivity to mutagens like

4-nitroquinoline-N-oxide (4-NQO), deletion of

SNQ2 increases sensitivity to cations such as

Na⫹, Li⫹ and Mn2⫹ (Miyahara et al., 1996).

Notably, deletion of PDR5 in addition to a

⌬snq2 deletion aggravates the effect on

intracel-lular metal ion accumulation and metal

sensi-tivity, suggesting some functional overlap

(Miyahara et al., 1996) Furthermore, a deletion

of PDR5 and SNQ2 strongly increases

preg-nenolone and progesterone toxicity to yeast

cells (Cauet et al., 1999), suggesting an

intracel-lular target for these steroids It has also been

reported that disruption of SNQ2 enhances the

lag phase, while a ⌬pdr5 ⌬snq2 double

disrup-tion influences both lag and log phases,

result-ing in slower growth rates (Decottignies et al.,

1995) Deletion of the YCF1 gene renders cells

hypersensitive to cadmium and completely

abolishes vacuolar uptake of As(GS)3 (Ghosh

et al., 1999; Szczypka et al., 1994) Finally, a loss

of Yor1p causes hypersensitivity to

reveromy-cin A, oligomyreveromy-cin, as well as various organic

anions Moreover, ⌬yor1 cells display cadmium

hypersensitivity, indicating a functional

over-lap of Yor1p and Ycf1p (Cui et al., 1996; Katzmann et al., 1995) As expected, disruption

of the two fission yeast drug transporters, pmd1 and hba2/bfr1, led to a drug hypersensitivity phenotype (Nagao et al., 1995; Nishi et al., 1992;

Turi and Rose, 1995)

Likewise, deletion analysis has been

per-formed for the C albicans transporters Cdr1p and Cdr2p (Sanglard et al., 1996, 1997) While deletion of CDR1 causes hypersensitivity to

azoles, terbinafine, amorolfine and various

other metabolic inhibitors, disruption of CDR2

does not cause obvious hypersusceptibility tothese compounds However, a double dis-rupted ⌬cdr1 ⌬cdr2 strain displays increased

sensitivity when compared to a ⌬cdr1 strain,implying that Cdr2p does play a role in drugresistance Interestingly, spontaneous rever-tants of a ⌬cdr1 strain become resistant by

expressing the second transporter gene CDR2,

which is normally not overexpressed (Sanglard

et al., 1997) Disruption of the C glabrata CgCDR1 gene in a resistant clinical isolate

clearly reduced azole resistance, supporting the

idea that CgCdr1p is the drug pump mediating resistance in this isolate (Sanglard et al., 1999) While a loss of the Aspergillus ABC proteins

atrB and atrD increases susceptibility to drugs,

deletion of atrC did not result in any drug sensitivity phenotype (Andrade et al., 2000a, 2000b) Notably, deletion of atrD also seems to

decrease the secretion of antibiotic compounds

(Andrade et al., 2000b), providing a case

exam-ple for an ABC transporter that effluxes bothphysiological and non-physiological substrates

Fungal ABC pumps and some of their

rele-vant drug substrates are listed in Table 15.1.

SNQ2, which was originally cloned as a gene

conferring resistance to mutagens such as

4-nitroquinoline-N-oxide and triaziquone, was

the first multidrug resistance ABC transporter

identified in S cerevisiae (Servos et al., 1993).

Interestingly, Snq2p also seems to modulateresistance to cations such as Na⫹, Li⫹and Mn2⫹

(Miyahara et al., 1996) Shortly afterwards,

PDR5 was independently isolated by several

groups through its ability to mediate

cyclohex-imide resistance (Balzi et al., 1994), resistance

to mycotoxins (Bissinger and Kuchler, 1994),

cross-resistance to cerulenin and cycloheximide

(Hirata et al., 1994), as well as the transport of

glucocorticoids (Kralli et al., 1995) Finally,

genetic screens for oligomycin and reveromycin

A-resistant yeast cells led to the discovery of

Yor1p, the third plasma membrane drug pump

of S cerevisiae (Cui et al., 1996; Katzmann

et al., 1995).

Extensive studies on the determinants of strate specificity revealed an extremely broad

sub-substrate specificity of fungal PDR

transpor-ters with distinct but considerably overlapping

drug resistance profiles (Egner et al., 1998;

Kolaczkowski et al., 1998; Mahé et al., 1996a; Reid

et al., 1997; Servos et al., 1993) The PDR pumps

mediate extrusion of hundreds of structurally

and functionally unrelated compounds,

includ-ing ions, heavy metals, ionophores, antifungals,

GSH-conjugates, bile acids, anticancer drugs,

antibiotics, detergents, lipids, fluorescent dyes,

steroids and even peptides as well as many

others Notably, Pdr5p and Yor1p may also

transport phospholipids, as demonstrated by

fluorescent phosphatidylethanolamine

accumu-lation in vivo (Decottignies et al., 1998) A similar

role in phosphatidylethanolamine transport has

been speculated for C albicans Cdr1p (Dogra

et al., 1999) The leptomycin B resistance gene

pmd1 from S pombe also confers cross-resistance

to cycloheximide, valinomycin and

stauros-porine (Nishi et al., 1992) The second fission

yeast drug pump, Bfr1p/Hba2p, mediates MDR,

with resistance to brefeldin A, cerulenin and

sev-eral antibiotics (Nagao et al., 1995; Turi and Rose,

1995) In contrast, Ycf1p and Hmt1p are not

involved in drug efflux at the cell surface, but

mediate vacuolar sequestration of heavy metals

and other toxic compounds (Ortiz et al., 1992,

1995; Szczypka et al., 1994) Finally, the Candida

and Aspergillus drug pumps were characterized

mainly on the basis of their ability to cause

resistance to antifungal agents such as azoles

How such a wide variety of xenobiotics can

be translocated by one transporter molecule is

still not understood The best-studied exporters

in this respect are perhaps the drug-transporting

mammalian Pgps, which are extensively

dis-cussed in other chapters of this book

Photo-affinity labeling studies and genetic analysis

indicate that both nucleotide-binding domains

(NBDs) and membrane-spanning domains

(TMDs) somehow contribute to substrate

recognition and transport in mammalian drug

pumps (Gottesman et al., 1995; Zhang et al.,

1995) Transport inhibition studies, mutationalanalyses and genetic studies identified aminoacid residues required for substrate recognitionand binding by Pdr5p and Cdr1p (Egner

et al., 1998, 2000; Kolaczkowski et al., 1996;

Krishnamurthy et al., 1998) The possibility of

genetically separating drug transport frominhibitor susceptibility indicates the existence of

at least two distinct drug-binding sites in Pdr5p

(Egner et al., 1998, 2000), and perhaps in related

transporters such as Cdr1p In addition, the bition of Pdr5p-mediated rhodamine 6G fluo-rescence quenching supports the notion of morethan one drug-binding site in fungal ABC

inhi-pumps (Kolaczkowski et al., 1996) At any rate,

the actual drug transport mechanism and how it

is linked to ATP consumption, the so-called alytic cycle of ABC proteins originally proposed

cat-by Alan Senior (Senior et al., 1995), has not been

established for fungal pumps However, it seemsplausible that fungal ABC pumps may achievesubstrate transport through a mechanism simi-lar to the one described by the catalytic cycle orthe alternating two-cylinder two-piston enginemodel for human Pgp and bacterial LmrA,

respectively (Senior et al., 1995; van Veen et al.,

2000) Extrusion of substrates might be ated by efflux from the cytoplasm to the outside

medi-or, alternatively, they might be recognized andextruded (or flipped) from the inner leaflet of theplasma membrane to the outside through a

‘molecular vacuum-cleaner’ mechanism nally proposed for the human P-glycoproteinMdr1p (Higgins and Gottesman, 1992) Giventhe broad substrate specificity, and the possibleexistence of more than one drug-binding site, one might speculate that the actual trans-port mechanism depends on the substrate to

origi-be transported, and that a single fungal ABCpump can actually function through severalmechanisms

While the transport mechanism has not beenelucidated, the ATP dependence of drug trans-port is established beyond any doubt Pdr5p and Snq2p, albeit highly homologous, displaydifferent pH optima regarding their ATPaseactivity and, interestingly, distinct nucleotidetriphosphate (NTP) preferences The Snq2pATPase activity shows a sharp pH optimum

at 6.0–6.5, while Pdr5p activity remainsunchanged over a broad pH range from 6.0 to 9.0

(Decottignies et al., 1995) As for the NTP

sub-strates, Snq2p is more selective with a preferencefor ATP, whereas Pdr5p also hydrolyzes UTP

and CTP, and to a lesser extent GTP and ITP

(Decottignies et al., 1995) UTP hydrolysis by

Pdr5p and Snq2p is sensitive to vanadate and

Triton X-100 inhibition By contrast, oligomycin

affects only Pdr5p UTPase activity (Decottignies

et al., 1995) Like mammalian P-glycoprotein and

CFTR, Pdr5p and Yor1p can be photolabeled

with the fluorescent ATP analogue

TNP-8-azido-ATP (Decottignies et al., 1998) Regarding pumps

of fungal pathogens, an NTPase activity has only

been shown for the Candida transporter Cdr1p,

which exhibits both vanadate-sensitive ATPase

and UTPase activities (Krishnamurthy et al.,

1998) Likewise, using in vitro uptake assays in

the presence and absence of ATP (Li et al., 1997;

Ortiz et al., 1995), the ATP dependence of the

vac-uolar uptake of heavy metals and glutathione

conjugates via Hmt1p or Ycf1p, respectively, has

also been demonstrated

Some answers to tantalizing questions cerning the molecular mechanisms and catalytic

con-cycles of fungal ABC pumps might emerge

once 3-D crystal structures become available

An important step towards this direction is

the recent elucidation of the Escherichia coli

MsbA high-resolution crystal structure (Chang

and Roth, 2001) MsbA acts as a homodimer,

each subunit consisting of six

transmembrane-spanning ␣-helices, a bridging domain and an

NBD However, despite this fascinating work,

even in this case many mechanistic questions

remain open or lead to ambiguous

interpreta-tions and answers (Higgins and Linton, 2001)

Thus, more structures may have to be solved to

obtain a physiologically relevant model of

drug transport by ABC pumps So far, only

low-resolution structures are available for the

eukaryotic ABC proteins MRP1, Pgp and TAP

(Rosenberg et al., 2001a, 2001b; Velarde et al.,

2001; Chapter 4), but attempts to obtain better

and refined structures are well on their way in

To better understand the molecular basis of

ABC pump function, genetic and mutational

analysis is necessary A detailed mutationalanalysis of Pdr5p permitted the identification

of amino acid residues important for per folding, drug substrate specificity and

pro-inhibitor susceptibility (Egner et al., 1998)

Non-functional mutant proteins were either theconsequence of NBD mutations or caused bymisfolding in the endoplasmic reticulum (ER).For instance, a C1427Y–Pdr5p exchange in thelast predicted extracellular loop 6 betweenTMS11 and TMS12 causes Pdr5p misfoldingand its efficient ER retention, followed by rapidpolyubiquitination and degradation by the

cytoplasmic proteasome (Plemper et al., 1998).

The instability of C1427Y–Pdr5p is perhaps due

to a lack of disulfide bond formation betweencysteines in lumenal loops, which appears as aprerequisite for correct folding and exit from

the ER (Bauer et al., unpublished data).

The structure–function analysis of Pdr5p alsoproduced additional mutant transporters withaltered drug substrate specificity The S1360Fexchange in the predicted TMS10 of Pdr5p isthe most remarkable one This mutation causes

a highly restricted substrate specificity for theantifungal agent ketoconazole, with poor resist-ance to itraconazole and cycloheximide At thesame time, ketoconazole resistance is no longerreversed by the immunosuppressive drugFK506 in S1360F-Pdr5p, while azole transport

of wild-type Pdr5p is completely blocked byFK506 However, when the same residue,S1360, is substituted by alanine instead ofphenylalanine, the resulting S1360A-Pdr5ptransporter suddenly becomes hypersensitive

to FK506 inhibition (Egner et al., 2000) These

studies indicate that TMS10 is a major nant of Pdr5p substrate specificity and inhibitorsusceptibility In addition, these studies allowedthe genetic separation of drug transport frompump inhibitor susceptibility, again suggestingthe existence of more than one drug-bindingsite in certain fungal pumps

determi-While the structure–function relationship ofSnq2p has not been addressed, the MRP/CFTRfamily members Ycf1p and Yor1p have beensubjected to detailed mutational studies

Mutations in YCF1, analogous to the most

prominent mutations in the human CFTR tein were thus constructed Deletions of F713 inYcf1p and F670 in Yor1p, which are the equiva-lents of the ⌬F508-CFTR deletion associatedwith cystic fibrosis, were generated and ana-lyzed Similar to the intracellular traffickingdefect of ⌬F508-CFTR in human cells, ⌬F713-Ycf1p leads to ER retention, together with loss

pro-of cadmium resistance (Wemmie and

Moye-Rowley, 1997) Mutations in NBDs, as well as

in the regulatory (R) domain, produced two

classes of mutants First, those defective in

Ycf1p biogenesis and, second, transporters

causing impaired cadmium tolerance and

glu-tathione S-conjugated leukotriene C4 (LTC4)

transport Interestingly, certain mutations in

the R-domain and in the cytoplasmic loop 4

genetically separate cadmium resistance from

LTC4 transport (Falcon-Perez et al., 1999).

Likewise, a ⌬F670-Yor1p mutant protein was

retained in the ER and thus was unable to

con-fer oligomycin resistance The same effect,

namely an ER retention and loss of resistance,

was achieved by insertion of an alanine residue

at position 652 in NBD1 Notably, replacement

of a basic residue downstream of the LSGGQ

motif (K715M or K715Q), despite a proper

plasma membrane localization of the mutant

proteins, resulted in reduced oligomycin

resis-tance (Katzmann et al., 1999).

A plasma membrane localization has only been

unequivocally demonstrated for Pdr5p, Snq2p

and Yor1p (Decottignies et al., 1995; Egner and

Kuchler, 1996; Egner et al., 1995; Katzmann

et al., 1999; Mahé et al., 1996b), as well as

Candida Cdr1p (Hernaez et al., 1998) Thus, it is

reasonable to assume that the majority of

fun-gal drug transporters are active at the plasma

membrane, mediating extrusion of toxic

com-pounds from within the cell across the plasma

membrane In contrast, transporters

responsi-ble for heavy metal detoxification, such as

Ycf1p and Hmt1p, reside in the vacuolar

mem-brane (Li et al., 1997; Ortiz et al., 1992) This

delimits the main catabolic compartment for

deleterious substances, degradation products

or toxic metabolites

The yeast ABC pumps Pdr5p, Snq2p andYor1p are rather short-lived proteins with ahalf-life ranging from 60 to 90 minutes (Egner

et al., 1995; Katzmann et al., 1999; Mahé et al.,

unpublished data) Trafficking studies revealedthat cell surface proteins such as these trans-porters have to reach the vacuole to undergoproteolytic turnover Yeast mutants defective

in the exocytic and endocytic pathways mulate newly synthesized Pdr5p, indicatingtrafficking by the normal exocytic secretion

accu-machinery (Egner et al., 1995) Using strains

carrying mutations in either one of the majorproteolytic systems represented by the vacuoleand the cytoplasmic proteasome, Pdr5p hasbeen shown to undergo constitutive endocyto-sis and delivery to the vacuole for terminal

degradation (Egner et al., 1995) Interestingly,

Pdr5p (Egner and Kuchler, 1996), Yor1p

(Katzmann et al., 1999) and the related Ste6p

mating pheromone transporter (Kölling andLosko, 1997; Loayza and Michaelis, 1998),

Snq2p (Mahé et al., unpublished data), as well

as several other yeast membrane proteins(Hicke, 1997), are ubiquitinated prior to endo-cytosis However, this ubiquitin attachmentdoes not target the proteins for degradation bythe cytoplasmic proteasome Instead, the ubi-quitin modification, which occurs only at thecell surface (Egner and Kuchler, 1996; Köllingand Hollenberg, 1994) and is limited to a singleubiquitin, acts as an endocytosis signal (Hicke,1997; Laney and Hochstrasser, 1999) A Pdr5pphosphorylation by Yck1p (yeast casein kinaseI) might play a role in Pdr5p trafficking and

turnover (Decottignies et al., 1999), but any

other impact of Pdr5p phosphorylation on thePDR phenotype remains unknown

As outlined above, the physiological Pdr5pturnover requires vacuolar proteolysis but notthe cytoplasmic proteasome However, mis-folded Pdr5p, which may arise from improperfolding in the ER during its biogenesis, requiresthe proteasomal degradation system An exten-sive mutational and genetic analysis of Pdr5pled to the identification of the C1427Y mutation

in the last predicted extracellular loop Thismutation causes the efficient ER retention andrapid degradation of a misfolded Pdr5*p pump

(Egner et al., 1998) by the ER quality control

system The ER-associated degradation (ERAD)

system (Fewell et al., 2001) is devoted to a rapid

removal of secretory membrane proteinsimmediately after or even during their synthesisshould misfolding occur Misfolded Pdr5*p israpidly extracted from the ER membrane

through a Sec61p-dependent retrograde

path-way, becomes polyubiquitinated and

sub-sequently degraded by the cytoplasmic

proteasome (Plemper et al., 1998) Similar

results have been obtained for Yor1p and

Ycf1p, the vacuolar heavy metal resistance

transporter, as well as the a-factor mating

pheromone transporter Ste6p (Loayza et al.,

1998) Since Yor1p and Ycf1p are related to

human CFTR, mutations analogous to the most

frequent cystic fibrosis mutation, ⌬F508, were

constructed in these yeast pumps (see above)

Interestingly, a deletion of F713 in Ycf1p or

F670 in Yor1p yields pump variants which are

efficiently retained in the ER and rapidly

degraded (Katzmann et al., 1999; Wemmie and

Moye-Rowley, 1997) by the ER quality control

machinery (Plemper and Wolf, 1999) These

data indicate that the basic principle of

func-tional folding of ABC proteins is conserved in

mammals and yeast, emphasizing the

impor-tance of yeast as a model system to study the

biology of heterologous ABC proteins of

A number of functional assays to study the

function and substrate transport of fungal ABC

proteins have been established These assays,

which are described below, include standard

resistance assays, photoaffinity labeling and

crosslinking studies, transport studies in vivo

and in vitro using vesicles or proteoliposomes,

and substrate accumulation in whole cells

Perhaps the simplest and most widely usedtests for drug resistance genes are growth inhi-

bition assays on agar plates (Bissinger and

Kuchler, 1994) Susceptibilities of various yeast

strains can be tested qualitatively and

semi-quantitatively by spotting serial dilutions of

yeast cultures on either agar plates containing

various drugs at different concentrations or

continuous drug-gradient plates If both a toxic

substrate and a pump inhibitor are present in

the same plate, even transport inhibition or

drug resistance reversal can be directly

visual-ized by inhibition of cell growth (Egner et al.,

1998) Gradient plates are easy to prepare and

even allow for a semi-quantitative tion of inhibitory substrate concentrations(Koch, 1999) An alternative to plate assays arehalo assays, in which filter disks soaked withdrug solutions are placed onto lawns of testercells, similar to the classical antibiotic agar dif-fusion assay The resulting zone of inhibitionsurrounding the filter disk is a direct quantita-

determina-tive measure of toxicity (Nakamura et al., 2001).

However, these assays may lead to artifacts,particularly when hydrophobic drugs withlimited solubility and diffusibility in agarplates are used Drug susceptibility profiles of

filamentous fungi such as Aspergillus species

can also be tested using a similar type of assay.Mycelial plugs from confluent plates are placedwith the mycelial side down on drug platesand the radial growth is monitored after certain

time periods (Andrade et al., 2000b).

An excellent tool to monitor ABC transporter

function in vivo includes the measurement of

drug efflux or the cellular accumulation ofradiolabeled substrates or fluorescent dyessuch as rhodamine The mitochondria-stainingdyes rhodamine 6G (R6G) and rhodamine 123

(Johnson et al., 1980) have thus been utilized to

study both efflux and energy dependence Dyeefflux is determined either indirectly by fluo-rescence dequenching or directly by measuringthe fluorescence of extruded rhodamine in theincubation buffer To examine the binding ofinhibitors or substrates to multidrug resistanceproteins such as Pdr5p, energy-dependent rho-damine 6G fluorescence quenching has been

applied (Conseil et al., 2001; Kolaczkowski

et al., 1996) This method takes advantage of

the fact that rhodamine 6G fluorescence isquenched upon dye-binding to the transportermolecule Therefore, in the presence of a com-petitor, which could act as an inhibitor or anyother substrate, quenching is reduced and thusfluorescence increases The quenching assayalso provides information, whether the pumpinhibition is competitive and involves the same binding site, or is non-competitive due

to different drug-binding sites This approachshowed that protein kinase C effectors such asstaurosporine analogues are capable of inhi-biting the interaction of rhodamine 6G with

Pdr5p (Conseil et al., 2001) Alternatively, dye

accumulation within yeast cells can be tored using a fluorescence-activated cell sorter(FACS) Such transport measurements wereemployed to determine the activity of Pdr5p

moni-variants (Egner et al., 1998), to screen compounds

for inhibitors of Pdr5p-mediated transport

(Egner et al., 1998; Kolaczkowski et al., 1996) or

as a means to identify overexpressing CDR1

pathogenic Candida strains (Maesaki et al., 1999).

Similarly, the non-fluorescent,

membrane-permeable compound monochlorobimane can

be used to monitor transport of glutathione

S-conjugates, since the glutathione transfer

reac-tion on monochlorobimane results in a highly

fluorescent yet membrane-impermeable

conju-gate Addition of monochlorobimane to yeast

cultures and monitoring the subcellular

local-ization of the fluorescent S-conjugate proved

that Ycf1p is a major factor in the vacuolar

accu-mulation of monochlorobimane-GS (Li et al.,

1996) Because certain yeast pumps such as

Yor1p and possibly Pdr5p may mediate

mem-brane flipping of phospholipids, functional

assays can be used in which the movement of

fluorescent phospholipid analogues such as

C6-NBD-phosphatidylethanolamine (Kean et al.,

1997) is directly followed by time-lapse

fluores-cence spectroscopy (Decottignies et al., 1998).

Another method to study ABC transporteractivity is the use of radiolabeled substrates

For instance, a whole cell in vivo estradiol

accu-mulation assay was developed to demonstrate

that steroid substrates are translocated by

Pdr5p and Snq2p (Mahé et al., 1996a) Since

overexpression of PDR5 and SNQ2 decreases

intracellular estradiol, this approach identified

steroids as new substrates of fungal pumps

These in vivo uptake assays can also be coupled

to steroid/glucocorticoid receptor or steroid/

glucocorticoid response element

(ERE/GRE)-driven reporter systems (Mahé et al., 1996a).

Accumulation of pump substrates such as

mycotoxins and environmental toxins are thus

easy to measure, as these compounds display a

high degree of estrogen activity (Kralli et al.,

1995; Mitterbauer et al., 2000) Moreover, such

systems also elegantly allow for the selection of

mutant transporters and genetic analysis of

ABC-driven substrate transport (Kralli et al.,

1995; Kralli and Yamamoto, 1996; Mahé et al.,

1996a; Tran et al., 1997) Similarly, the

measure-ment of intracellular [3H]-fluconazole has been

used to directly show that antifungal azoles are

extruded from Candida cells by Cdr1p (Sanglard

et al., 1996) In the case of A nidulans, the

accu-mulation of the fungicide [14C]-fenarimol was

measured to indicate a role for atrC and atrD in

drug resistance (Andrade et al., 2000b).

To prove that Ycf1p mediates vacuolarsequestration of organic compounds after their

conjugation to cellular glutathione, in vitro

uptake into vacuolar membrane vesicles has

been measured (Li et al., 1997; Rebbeor et al.,

1998) For these experiments, vacuolar brane vesicles are incubated with various radio-labeled substrate complexes, and accumulation

mem-of substrates within the vesicles is monitored bythe amount of sequestered radioactivity Thisassay revealed that Cd_GS2, but not Cd_GS,transport into the vacuole requires Ycf1p Thistype of assay also allows for the investigation

of transport inhibition or competition by othersubstrates

Finally, since ABC transporters are driven membrane translocators, following theirATP dependence and measuring ATP hydroly-sis is of course an important assay For the

ATP-S cerevisiae transporters Pdr5p, Snq2p and

Yor1p, ATPase activity has been demonstrated

Inhibition by vanadate and oligomycin has also

been reported (Decottignies et al., 1994, 1995,

1998) ATP-binding by Pdr5p and Yor1p wasconfirmed by photolabeling of these proteins

with TNP-8-azido-ATP (Decottignies et al., 1998) The vanadate-sensitive (Rebbeor et al.,

1998) ATP consumption of Ycf1p has beenshown by performing uptake assays into vac-uolar membrane vesicles in the presence and

absence of MgATP (Li et al., 1997) However, in

contrast to mammalian ABC pumps, little isknown about the binding properties of individ-ual yeast NBDs with respect to their interactionwith NTPs/NDPs or the catalytic cycle of yeastdrug pumps

S cerevisiae has always been a valuable model

organism to investigate the function of tionary conserved genes, including ABC pro-teins of medical importance and drug resistancepumps To study functional conservation and

evolu-to clone multidrug transporters, several yotic drug pumps have been functionally

eukar-expressed in yeast For example, the human

Pgp Mdr1p was successfully expressed in an

S pombe strain lacking pmd1 (Ueda et al., 1993)

as well as in baker’s yeast (Kuchler and Thorner,

1992) Although not properly glycosylated, the

human protein was partially functional and

able to confer resistance to valinomycin and

actinomycin D to an otherwise sensitive yeast

strain (Kuchler and Thorner, 1992; Ueda et al.,

1993) To learn more about the mechanism of

action of Ycf1p, its human homologue MRP,

sharing 63% amino acid similarity with Ycf1p,

was expressed in a ⌬ycf1 strain (Tommasini

et al., 1996) Human MRP restores cadmium

resistance to wild-type levels and facilitates

transport of S-(2,4-dinitrobenzene)-glutathione

(DNB-GS) into yeast microsomal vesicles This

was one of the first indirect indications that

Ycf1p, like MRP, is a glutathione S-conjugate

pump In another approach, overexpression of

the A fumigatus MDR1 gene yielded S cerevisiae

cells with increased resistance to the antifungal

cilofungin (Tobin et al., 1997) Certain yeast ABC

pumps have also been successfully expressed in

heterologous systems such as plants For

exam-ple, expression of the PDR5 gene in tobacco

confers increased resistance to the trichotecene

toxin deoxynivalenol (Mitterbauer et al., 2000).

The observation that MDR/PDR arises from overexpression of certain ABC transport-

ers suggested a gene dosage strategy to clone

new drug efflux genes Genomic libraries were

screened for genes which in increased dosage

can confer resistance to various compounds

Taking advantage of the drug

hypersensitiv-ity phenotype of a ⌬pdr5 strain, Candida ABC

transporters have thus been cloned by

func-tional complementation in baker’s yeast

(Prasad et al., 1995; Sanglard et al., 1995, 1997,

1999) A fluconazole and cycloheximide

super-sensitive ⌬pdr5 strain was transformed with

genomic Candida libraries and transformants

resistant to the azole or the antibiotic,

respec-tively, were selected This approach led to the

discovery of the two major C albicans ABC

genes CDR1 and CDR2, as well as CgCDR1

from C glabrata In addition, a gene for a

trans-porter of the major facilitator class, BEN r,

was identified through its ability to confer

benomyl resistance in S cerevisiae (Sanglard

et al., 1995) Likewise, functional

complementa-tion studies verified that the Aspergillus

trans-porter atrB is the orthologue of yeast Pdr5p

(Del Sorbo et al., 1997) Similar approaches

allowed for the identification of the S pombe

genes bfr1/hba2 and pmd1 (Nagao et al., 1995;

Nishi et al., 1992; Turi and Rose, 1995) as typical

immunocompro-of fungal infections is steadily rising (reviewed

in Bastert et al., 2001; White et al., 1998) The

increasing use of antifungal agents in laxis and therapy caused resistance to emerge,and drug resistance has become a significantproblem in health care during the past decade.Several classes of antifungal agents actingeither fungistatically or fungicidally are in clin-ical use to treat local as well as systemic infec-

prophy-tions (Bastert et al., 2001) Polyenes such as the

fungicidal amphotericin B and nystatin fere with ergosterol function in the plasmamembrane, leading to pore formation and leak-age of cellular components (Vanden Bossche

inter-et al., 1994) Flucytosine is minter-etabolized into

5-fluorouracil, which is incorporated into RNAcausing disruption of protein synthesis As

shown in Figure 15.3, other antimycotics also

act via inhibition of the ergosterol biosynthesis,the bulk sterol in the fungal plasma membrane.The Erg1p squalene epoxidase is blocked byallylamines such as terbinafine and naftifine, aswell as by thiocarbamates such as tolnaftate.Morpholines such as amorolfine inhibit boththe Erg24p C-14 sterol reductase and the Erg2pC-8 sterol isomerase The fungistatic azoles,with the imidazoles ketoconazole and micona-zole, and triazoles such as fluconazole, itracon-azole and the newly developed voriconazole,comprise the most widely used class of ergos-terol synthesis inhibitors These azoles inhibitthe Erg11p lanosterol C-14-demethylase, acytochrome P-450 enzyme and the Erg5p C-22-desaturase Because of their good safety profileand relatively high bioavailability, azoles arewidely used to treat fungal infections (White

et al., 1998).

Clinical resistance to these antifungals candevelop through different molecular mecha-

nisms (reviewed in White et al., 1998) These

basic resistance mechanisms, depicted in

Figure 15.2, include reduced drug uptake into

the cell, alterations of the target genes by

muta-tion or induced overexpression, changes in the

ergosterol biosynthetic pathway, as well as

increased drug efflux or facilitated drug

diffu-sion from the cell Next to target alteration, the

induced overexpression of ABC efflux pumps

in clinical strains represents a prime cause of

clinical antifungal resistance A number of

strategies exist through which clinical drug

resistance can be circumvented As for existing

drugs such as azoles, resistance reversal can be

achieved by combination therapy (Ryder and

Leitner, 2001) For new antifungal drugs under

development, one should consider developing

those that are not substrates of ABC pumps like

Cdr1p or Cdr2p Azole resistance may be

man-ageable by reducing prophylactic treatment or

by the use of specific efflux pump inhibitors in

an attempt to reverse antifungal resistance

Nevertheless, the frequency of life-threatening

fungal infections in immunocompromised

patients is still increasing, with Candida and

Aspergillus species representing the major

fun-gal pathogens (Bastert et al., 2001) Funfun-gal

organisms are becoming less susceptible to fungal drugs, and a shift to intrinsically moreresistant fungal pathogens has been observed

anti-(Bastert et al., 2001; White et al., 1998) This

scenario clearly illustrates that there is a need

to better understand the molecular basis of antifungal drug resistance and to developimproved strategies for the treatment of fungalinfections

8,14,24-trienol

4,4-dimethylcholestra- zymosterol

Figure 15.3 The yeast ergosterol biosynthetic pathway This cartoon depicts the biosynthetic pathway

leading to ergosterol synthesis Only the relevant enzymatic steps are shown Antifungal agents that act

via inhibition of some of these enzymes are given, with the relevant targets indicated.