BWth,m,~uet B~oph)s,ca acta, 1073t1991~241 252 © 1991 ElsevierScien~ Pul'AishelsB.V.0304-a165/91/$03.5o A D O N I S 03044165910~1(~1S

BBAGEN23458

Review

Multiple or pleiotropic drug resistance in yeast Elisabetta Balzi and Andr6 Goffeau Unitd de Biochimie Ph)'~lologJque. Unwersad Cathohque de Louvalrz. Lvuvao~-Ia-Neuce fBelglumJ

(R~eJved 31 January lqqa) (Revised manuscript received 11 July 1 9 9 0 )

Key words: Drug resistant: P-Giyeoprot~in;Genctic;(Ye~t)

Contents I. [L

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pleiotroplcdrug resislant mmants in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. First reports on resistance to different inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B, T h e PDRI locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.

The

PDRJ related mulations

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D. Other P D R loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Nucleo-Cyloplasmicinteraclions as determinants of drug resistance . . . . . . . . . . . . . . . . . . . . . Ill. Mechanismsof muhidrus resistan~ in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. ATP-depcnd~t pcrmca~ ~ determinants of d~g ~sistanee . . . . . . . . . . . . . . . . . . . . . . . . . . . . v. Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . gefe,e.~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

'*Drug rcsistml~ is the most important and challengingtopic in cancer trealmenl researck today", B. Chabner. "The very process that is leadingto the death at the ¢¢1Isis also leading to their resistanceto drugs", R. ~:himke at the Ninth Annual Bristol MyersSymposium on Cancer ae~carch(Washington DC, U.S.A..October [5-16~ t986). I. Introduction Multidrug resistance is one of the major obstacles for cancer therapy. One well documented mechanism underlying drug resistance in cancer cells implicates the overexpression of a membrane protein, the P-glycoprotein, functioning as an ATP-dependcnt extrusion pump [or drugs and physiological substrates (for review see ReLI8). However, the prhnary genetic lesion responsibI~ for altered P-glycoprotdn geae expression in cancer cells remains unclear. In another important field of health sciences, the resistance to drugs developed hy P l a s m o d i u m f a l c i p a r u m

Correspoodeace: A. Goffeau, Usit~ d e BiochimiePhysiologique, Uni. v©rsit~ Catholique de Louvain, Pine© Cr0ix du Sud 2-20 B-1348 Louvain-la-Ne~e, Belgium.

241 242

242 242 244 245 245

245 2d8 249 230 2SO

is becoming a major obstacle for the treatment of malaria. The mechanism responsible for drug resistance in malaria parasites seems analogous to that evoked for mammalian tumor cells. It involves the amplification and overexpression of a family of genes highly homologous to ~he mammalian P-glycoprotein encoding genes [23, 87|. Finally, a recent breakthrough in the understanding of a fatal and widespread hereditary disease was obtained by the isolation of the gene responsible for cystic fibrosis, and by the discovery that it encodes a putative membrane transport protein, remarkably similar to the mammalian multidrug resistance pump, and to its counterparts from other spcci~ [68, 67, 37]. In conclusion, the studies of three major lethal diseases, cancer, cystic fibrosis and malaria, have converged to a common element: the involvement of a m~.mber of a new superfamily of transport proteins, responsible for the ATP-dcpendent cellular efflux of still unknown physiological substrates as well as of drugs, Yeast shares the basic structural and functional organization of higher eukaryotes, and has the advantage of being easier to manipulate. Therefore mecha-

242 nisms of fundamental importance for mammalian cells may sometimes be approached more efficiently by the study of similar yeast functions. In this respect, the recent identification in yeast of genes homologous to the mammalian multidrug resistance gene opens new prospects [48]. One important question raised by this discovery is whether the mammalian multiple drug resistance phenotype may be related to some of the drug resistant mutations which have been studied in yeast for more than twenty years. It is the aim of this re,Aew to gather genetic and biochemical data established on multiple drug resistance in yeast, with a view to the implications that these findings may have for unravelling the mechanism of mulddrng resistance m higher enkaryotes. il. Plaiotrople drug resistant mutants in yeast In yeasL the mt?.~tions responsible for a generalized resistance to a large numb¢- of structurally and functionally unrelated drugs are usually called 'plaiotropie drug resistant' (pdr) whereas the synonymous denomination 'multiple drug resistant" (mdr) has been coined for mammalian cells. In the following paragraphs, we will follow an historical approach and show how our present knowledge of multiple drug resistance in yeast has progressively emerged from an apparently non-related field: that of the study of mitochonddal metabolism. 11-+4. First reports on resistance to different inhibitors Pioneering studies by Linnane and colleagues [42, 9] described a set of mutants conferring resistance to chemically unrelated inhlbitors (chloramphenieol, tetracycUne, erythromycin, carbomycin, oleandomycin and spiromycin) interfering with mitochondrial protein synthesis. Thomas and Wilkie [80] also described mutants resistant to erythromycin and classified diem into two categories: the first, of mitochondrial inheritance, was suggested to result from the alteration of some components of the mitochondrial protein-synthesizing system; the second, of Mendehan inheritance, was thought to involve changes ha either plasma membrane permeability or in mitochondrial membrane composition, or yet in drug inactivation. Several subsequent studies [57, 77, 85,1, 2, 3, 51,15, 60, 40, 26,12] reported on mutants resistant to inhibitors (oligomycin, vcoturicidin synthalin and trialkyl tin salts) of. another mitochondrial function, the oxidative phosphorylation. It was confirmed that both nuclear and mitoehondrial genes control the resistance to mitochnndrial inhibitors. The nuclear mutants often showed a generalized resistance to several different inhibitors, while the resistance of mitoehondrial mutants was more drug-epecific. The mltochondrial mutations were shown to alter specifically the mitochondrial ATP-symhase, while the molec-

utar alteration induced by the nuclear mutations were attributed to general properties of mitochondrial membranes. Avner and (3riffiths [2] isolated a series of mutants resistant to oligomycin. Among them, the so called 'Class 1' mutants, of nuclear heredity, were, despite their monogenie character, cruss-resistant to several other inhibitors. These included not only agents acting on mitoehondrial functions, like energy-transduction reagents (triethylfin and aurevertin), uncoupling agents (carbonylcyanide-ra-chlorophenylhydrazone, tetrachlorotrifluorommhylbenzimidazole and the hexafluorotriacetone derivative 1799), electron transport hahibitors (antimycin A), and mltoehondrial protein synthesis inhibitors (mikamycin, chloramphenicol, spiramycin and erythromycin), but also an inhibitor of cytoplasmic protein synthesis (cycloheximide). Even though the ge~,ctics of "Class I" mutants was not fully understood, evidence was presented for the implication of a nuclear gene possibly interacting with extmchromesomai element(s), responsible for some defect in a membrane component leading to the observed pleiotropic alterations.

Rank and Bech-Hansen [59] isolated a strain containing a single gene nuclear mutation, oliPgl-1, selected as resistant to oligomycin and c,hloramphenicol and conferring cross-resistances to 10 different drugs: rutamyein, ventaricidin, triethyltin bromide, antimycin A, earbonylcyanide-m-chlorophenylhydrazone, tetra-Nbutylammonium bromide, dibenz'yldimethylammonium chloride, triphenylmethylphoephonium bromide, chloramphenicol, carbomycin and tetracycline. Collateral sensitivity of the same mutant to five other inhibitors, paromomycin, neomycin, dequailnlum chloride, ethidium bromide and acriflavin, was reported. The ofiPR11 phenotype was first suggested to result from the alteration of some component of the milochondrial membrane, since the 15 inhibitors tested interfere with mitochondrial functions. However, shortly later Rank et ai. [61] reported that the single-gene chromosomal mutant OliPttl-I was crees-resistant to cydoheximide, an inhibitor which interferes with cytoplasmic protein synthesis. This cross-resistance was explained by assumhag the establishment of a permeability barrier at the plasma membrane level, altering intracelhalar drugs accumulation. Further evidence supporting this hypothesis was presented by Rank and co-workers [62], showing a reduced ]14C]chloramplienicol transport by resistant cells. In addition, it was shown that, in cells displaying in vivo multiple drug resistan, , both mitochondrlal protein synthesis and respiration were normal, ha terms of in vitro drug sensitivit~

243 A second allele, called oliPal-2 (strain 2-20; Ref. 63), was isolated by growth in the presence of oligomycin and eycloheximide. The oliVR1-2 strain presented a complex phenotype including cross-resistances to antimycin, cerulein, chloramphenicol, tetracycline, triethyltin and triphenylmethylphosphonium bromide, collateral sensitivities to gentamycin, neomycin, thiolutin, paromomycln, dequalininm chloride and inability to grow under adverse physiological conditions, such as elevated pH, temperature or osmolality. It was proposed that this pleiotropic phenotype could result from an alteration of both the plasma and the inner mitochondrial membranes. Meanwhile, a complex inheritance pattern for the mutant strain DRIg, resistant to oligomycin, venturicidin, chloramphenicol, cycloheximide and triethyltin and hypersensitive to paromomyein and ethidium bromide was reported to result from the interaction of at least two nuclear genes and two episomal factors, including the 2 itm plasmid [28]. Subsequent studies by Sannders and Rank [71] suggested that the o//PRI-1 mutation, renamed pdrl-l, the allefie oliPR1-2 mutation, renamed pdrl-2, and the "I"8 mulddrug resistant nuclear allele of the strain DRI9, renamed pdrl-3, are all alleles of the same locus, PDRI, controlling cell permeability to a variety of substances. The PDRI locus was mapped 4.7 centiMorgans centrumere-distal to the LEUI gene on chromesome VII [71]. A large number of reverrants have been isolated from the pdrl mutants. Rank et at. [61] first described a total revertant (strain GR350) of the pdrl-1 resistance and collateral sensitivity phenotype, due to a back mutation at the PDRI locus. Other intragenie reversions were identified as the mutations pdrl-4 (strain D RI9/ T S R1) and pdrl-5(strtfm DRI9/T8-R2), corresponding to reversals of the pdrl-3 drug resistance/hyporsensitivity phenotype [71]. A partial pdrl-I reversion (strain GR361) was attributed to an extragenic mutation at a single locus, sur, suppressing chloramphenicol and eyelobeximide, but not olignmyein, resistance [61]. The sur mutation was shown to suppress also the pdrl-2 and pdrl-3 alleles, us well as the pdr3 mutation of the strain DRI9/T'/. In addition, sur was found to enhance the sensitivity of wild-type strains to chloramph~col, cycloheximlde and trichodermin [71]. Another locus responsible for partial reversion of pdrl-related resistance phenotypes has been identified on chromosome XV and designated scr [13]. Finally, Rank and Sbeard [66] isolated nearly 3000 revertants from the pdrl-2 mutant and classified them into different groups, displaying either pseudo wild.type or pseudo mutant phenotypes, loss of cross resistance or of collateral sensitiv~,'y or yet loss of different aspects of the original resistance phenc~ type. In conclusion, the isolation of pdrl revertants supported the concept of a single nuclear gone control

F~s. t. The PDRt putative transcription regulator. Scheme for the transcriptional control carried out by the PDRI protein on the pl~moters lopen bars) of targetgenes (filledbars)encoding proteins from plasma (PM). irtitochondria (M) and possibly (dotted lin~) other (V = vacuolar. N ~ nuclear) permeability barriers. Among the putative targets, the STE6,PDRS. ADPI. PDR4 and ATRI proleins may be ~asidered (see t~xt).

by the PDRI locus on both plasma and milocho{~dria{ membrane functions [66] hut also reflected the complexity of the ploiotropio drug resistance system, which is controlled by several nuclear and mitochondrial genes [131. The PDRI gone has been more recently isolated [4]. Transformation of pdrl multidrug resistant strains with a multicopy plasmid-berne wild-type PDRI gone, restores normal sensitivity to drugs in the resistant mutants. Disruption of the PDRI gone confers hypersensitivity to cycloheximide. The PDR1 gone encodes a 121 kDa polypcplide resembling DNA binding proteins involved in the regulation gene transcription. A model has been proposed in which the PDR] protein would control the transcription of several different genes, whose products would be components of various cell membranes (Fig. 1). These components would be directly involved in the transport of drugs and physiological substrates across plasma, mitochondria and possibly other permeability barriers. H-C PDRI related mutations In addition to the pdrl-1 to 1-5 alleles, a series of independently isolated mutations, either directly or indirectly related to drug resistance phenotypes, have been mapped in the region of the PDR1 locus on chromosome VII. Saanders and Rank [71] proposed that the drug resistance mutations anti, cyh3, t:ll, AMYI, BOR2 and AXEI could be alleles of the PDRI locus (Fig. 2A). The mutations till [53] and cyh3 [86,

Chromosome VII A (1980)

n lt989l f~

trR.5

/

f ~

I

~ '

T~:gX,, "" ....

i

I

I ....

F'OR5

~I',,

" ...... i

~,,"0

io.

Fig. 2. The multidrus resistance cluster of chromosome VII. The genetic map of the eentromefic regionof chromosome VII presented by Mortimer and Schild [54] is shown in (A). "lhe corresponding molecular map of the g~nesrecently identifiedin tiffsgenomic region [4-7] is depicted in (BL

52] were isolated as resistant to thioisoleucine and cycloheximide respectively and showed fight linkage to leul. The anti mutant described by Cohen and Eaton [14], was shown to be cross-resistant to olignmycin, rhodamine 6G, tetracycline, chloramphanicol and cycloheximide. Genetically, antl was mapped 3.3 cemiMorgans centsomere-distal to leul, proposed to be allelic to pdrl-I and pdrl-3, and suggested to require interactions with mitochondrial genetic factors for determining drug resistances. AMYI [43] is a group of 22 semi-dominant mutants displaying resistance to antimycin A, an inhibitor of the respiratory chain, and showing slight cross-resistance to cycloheximide. Mapped 3.3 ceatiMorgaas ccmromere-distal to leul, AMYI was suggested to be allelic to antl and cyh3. No alterations of mitochondrial structures and functions were observed in these mutants, which were suggested to be modified in cellular drug uptake or in some unidentified cytoplasmic functions, such as drug inactivation or activation of alternative respiratory chains. The BOR2 mutations [55] have been isolated as resistant to borralidin, a specific inhibitor of the

threonyl-tRNA synthetase. They were described as dominant mutations, mapped 3.1 centiMorgans centromere-distal to leul and not affecting threonyl-tRNAsynthetase activity. Saunders and Rank [71] suggested that the BOR2 mutations could be alleles of the PDRI locus, possibly affecting ceil permeability. The AXEI mutants [75] were isolated as resistant to the protein synthesis inhibitor axenomycin D. Some of them were found to he cross-resistant to different inlfihitoss of the large ribosomal subunit (eycloheximide, amicetyn, sparsomycin, ricin). The protein synthesis carried out by the ribosomes isolated from these mutants was resistant to axenomycin. The AXEI mutation was mapped less than 0.36 centiMorgans centrumere-proximal to leul. Another group of mutations attributed to the PDRI locus are star2, dominant, semidominant and recessive alleles isolated by resistance to the herbicide sulfometuron methyl and cross-resistant to cycloheximide and olignmycin [19, 20]. Also attributed to the PDRI locus is the NRA2 mutation, selected as failing to accumulate the lysosomottopic vital stain neutral red, and observed to confer resistance to neutral red, cycloheximide, tetracycline and chloramphehicol [58]. Finally two other mutations related to drug resistance were localized very close to leul, and thus PDRI. The atel mutation, localized 2 centiMorgans centromere-distal to leul, causes a deficiency in arginyl-tRNA ~ protein transferase activity [72]. The awl mutant displays resistance to cycloheximide particularly at 37°C (Ulaszewski, S., personal communication). Lastly, the S C L I - ! mutation [46], a dominant suppressor of temperaturedependent lethality and partial suppressor of cycloheximide resistance of crl3, was mapped less than 1 centiMorgans distal to leuL We have recently established a precise genetic and molecular map of the PDRI region (Fig. 2B) by the isolation, suheloning and complementatinn study of a ~ 40 kb DNA fragment from this tract of chromosome VII [4, 7]. The pleiotropie drug resistance gene PDRI, located 4 kb centromcre-distal to LEUI and suggested to encode a transcription regulator, has been correlated to the mutations p d r l - l , pdrl-2, pdrl-3 and BC)R2XI, renamed pdrl-6. The arel mutation has been assigned to a different gene, ATE1. located 14 kb ccntromere-distal to LEUI and shown to encode the arginyl.tRNA ~ protein transferase [6], The scll gene has been isolated in a position 1 kb ccntromere~distal to LEUI and found to encode a 30 kDa putative subunit of the protcasome complex [5]. Finally, evidences have been presented indicating that the AXEI-2 mutation would affect the PMAI gene, encoding the plasma membrane H+-ATPase and located 1 kb centrumereproximal to LEUI, rather than the drug resistance gene PDRI [7]. In conclusion, a cluster of genes controlling different aspects of drug resistance flanks the PDRI locus on chromosome VII (Fig. 2).

245

Falco and Dumas [19] have isolated mutants resistant to snifometuron methyl, an herbicide acting on the isoleucine-vuline biosynthesis enzyme acetolactate synthase, and to unrelated drugs, such as cycloheximide and oligomycin. Some of these mutations, designated star3, were mapped on chromosome XV at a new locus designated PDR2 [20, 47]. The PDR3 locus was defined by Subik et al. [79] as involved in resistance to mu~idin, an inhibitor of the mitochondrial electron transport and cross-resistance to unrelated drugs. PDR3 was localized on the left arm el chromosome II, centromere-linked, and shown to include the two mutations pdr3-1 (former designation mac PR, Ref. 78) and pdr3-2 (former designation DRI9TT, Ref. 29). Reminiscent of the situation of the PDR1 locus, two other drug resistance mutations, AMY2 (resislance to antimycin) and eyhl (resistance to cyclohexlmide) map in the same region of chromosome II [54]. They were suggested to be either alleles of PDR3 or to affect neighbor genes functionally related to PDR3 [79]. Another pleiotropie drug resistance locus was identified, by the group of Jones [581 as the site of the nra5 mutation, which confers resistance to neutral red, cycloheximide, chloramphenienl and tetracycline. This PDR locus was localized on the right arm of chromosome 11, linked to the eentromere, and was suggested to be allelic to the cyhlO mutation. It was originally designated PDR4, but should in our opinion he renuw bered (PDR7) in order to avoid ennfus~on with the PDR4 Incus defined by Leppert et at. [41]. The PDR4 and PDR5 loci have been recently isolated and characterized [41]. These genes are analogous to the mammalian multidrug resistance genes in that they confer resistance to different inhibitors (sulfometurun methyl and cyclolieximide but not oligomyein) following amplification of the wild.type gene. Disruption of the PDR5 gene results in hypersensitivity to cycloheximide [41], similar to ~he phenotype observed after disruption of PDR1 I41. PDR4 was mapped on the right arm of chromosome XIII, 9 centiMorgans from the centromere. PDR5 was mapped to the fight arm of chromosome XV, hetween the nmrkers ADE2 and HI$3 and tightly linked to, but separable from, two other genes involved in drug resistance, SMR3 (specific sulfometuron methyl resistance) and PDR2. Interestingly, in a position equally finked (16 eentiMorgans) to the ADE2 marker on chromosome XV, Cohen [131 had mapped two allelle partial reversion mutations (scrl and scr2) of the multidrug resistance mutation anti (suggested allele of PDR1). Finally, a PDR6 gene has been recently identified closely linked (4 kb centromere-distal) to PDRI, and described as involved in resistance to different cytoplasmic inltibitors, such as cycloheximide, borrelidin

and hygromycin B [7]. The nucleotide sequence of PDR6 has been deterlnined and shown to encode a 123 kDa polypeptide, whose function remains unknown [1 l]. In conclusion, it can be noted that three genetic regions on the three chromosomes VII, II and XV show a clustering of independent drug resistance genes and/or mutations. Table I is a ct.mpilation of the yeast pleiotropic drug resistance and related loci, reported up to date. H-E. Nucleo-cytoplasmic interactions a~ determinants of drug resistance Several studies have established that interactions may occur between nuclear genes and cytoplasmic genetic factors, in the inheritance of drug resistance characters of yeast. Two lines of interpretations attributed respectively a mitochondrial [51, 31, 15, 69, 14, 70, 71] or espisomal [32, 3, 28, 29, 40, 10, 38, 81 nature m the cytoplasmic determinants interacting with the nucleus in establishing drug resist~nee. The cpisomal theory has not been confirmed today. In contrast, in a case of complex nnclenr-cytoplasmic interactions recently elucidated by Mennier and Colson [49, 50], it was found that the complex gcaaetic segregation of some diuron resistance traits results from the cumulative effects of two or three nuclear and mireehondrial mutations. Furthermore, the recent elucidation of the nature of the PDRI gene has confirmed an interference between this nuclear gene and mitoehondfial functions. Indeed, the reduced growth on glycerol media of the pdrl-2 mutant was complemented by the PDRI gene on a multicopy plasmid [4]. This observation led to the suggestion that the PDRI gene product controls the transcription of nuclear-encoded mitocliondrial components in addition to plasma membrane ones. III. Mechanisms d mullidrug resistance in yeast Modifications of membrane permeabilities are usually considered to he responsible for generalized resislance to structurally and functionally unrelated agents. Early studies by Linnane and co-workers [42] showed a decreased cellular drug uptake in chloramphenieel and tetracycline resistant mutants. Later, Rank et at. [621 showed that in pdrl-I mutants the cellular uptake of ehloramphenienl was decreased. However, the viscosity and lipid composition of the plasma membrane were not altered [651, The pdrl-2 allele displays features, such as conditional growth on ethanol medium and incapacity to polymerize amidoimidazole rlboside at 37°C, indicating a deficiency in the mitochondrial inner membrane function [631. Some physiological modifications in both plasma and mitochondrial membranes, including slig,htly decreased ATPase activities, were as-

Yeast genetic ~

TABLE 1

related to pleiotropie drug reMstanee

Gene producl

120 kDA putative transcription regu~'~r

H +-ATPase plasma membrane

?

?

?

~tion chJ'omosome, arm, linkage

VII. leR. can-linked

VII, left fen-linked

XV. right, linked to PDR5

II, lull, con-linked

XIIL riuhL fen-linked (9 eM)

D~g resistan~ locus

PDRI

PMAI

PDR2

PDR3

PDR4

multiple drag resistance

multiple drug resistance o r hypersentlvity

multiple drug lesistanfe

- defective ATPase - multiple drag resistance

-

- multiple drug resistance or hypersensensitivity pH-, o~mality-, tempetatuleseasitivitty - respiratory deficiency

Related mutant phenotyp~

DR muc PR

2D

pmal-lOI to pmal-155

AXEI-2 pmal

/

-XI~

/¢P.A2 anrl-I till ~3 AMYI

BO

oliPgl-/ olli vav2

Mutant alleles former name

DRI9/'I'7

6122-13d MG21-29 to - 32

RD35-CR

GR359 2-20 DRI9/P8 DRI9/TU-RI DRI9/TU-R2 BOR2-XI

Mutant strains

28, 71 78

pdr3.1

20. 47

45

75 gl

59] 63 / 28 7] 71 55 19 58 14 53 86 43

References for initial allele designation

pdr3.2

pmnl-2 pma1-1

pdrl

pr~umed

pdrl-I pdrl-2 pdrl-3 pdrl-4 pdrl-5 pdrl-6

new name

79

79

7 c

71 71 71 71

7

71.4

for new allele designation

41

73, 84

4

R:rerences p,~neisolation, sequenang

T h e Saccharora~es cere~isiae loci directly influencing, or related to~ multiple drag r e s i s t ~ am listed. " The A D P I locus is enclosed in this compilation becau.se of its stmel~al homology to the mamma~an muMdrug resistan~ gene MDRI; however, no direct correlation bar. ,een this gene and multiple drug resistance has been reported up Io date. I>The designation "PDR4" has b ~ preliminary suggested for the PDR l ~ s described by Preston et al. (1987); however, a different P D R locus has been r ~ n t l y officially d~ignated PDR4 (Leppert et al., 1990). Glossary of loci designations: P D R = p]eiot topic drag resistance; P M A = plasma membrane ATPase; S T E - sterility; d D P - ATP-depeedent pe~e.&se; scr. ~ = suppressa~.

XV. tight.

ATedependent

XI

IlL tight, linked to pgkl ~d SUF2

STE6

ADPI "

" Van Dijck and Goffeau, personal communication.

putative ATPdependent membr~¢

of a-factor

efnux pump

?

?

?

fink~l to A D E 2 (f6 cM)

sur

scr

?

probably: I11, left, linked to

cpr

HIS4

?

I1, ~ght, c~-n.finked t L 5 cM)

- abnormal sccrczion of a-faclor - ~sist~ncc or hype~ensitivity to valinomycin

d ~ g resistance - eahaneing of wiM-lyp¢ drug sensltbaty

multiple

par/and pc~3

- partial suppression of

drug resistance

suppression of anti multiple

partial

multiple drag resistance or hyper~nsisivity

multiple dins resistance

runcdon unknown

PDR? b

multiple drug resistance

123 k D a polypeptide

VI], left, linked to

PDRI

PDR6

multiple

drag resistance

?

XV, dghL linked to PDR2

PaR5

GR361

RD35-SI DR58-52

CR1

JG334

sur

scr-I scr-2

CPRI-I

nra5

pdrS:in5

6offca=. personal co~um~tlon

Puddle ~ d

7.11

41

248 soeiated to the pdrl-2 mutation [64]. Reduced plasma and mitochondrial membrane activities, accompanied by reduced drug and amino acids uptake, were also reported in multiple drug resistant mutants of the fission yeast Schizosaccharomyces pombe by Johnston and Coddington [34]. These authors suggested that the primary defect of these mutations (eyh3 and cyh4) may be at the level of membrane ATPases, responsible for the proton electrochemical gradient shown to drive the uptake of different compounds [24]. These interpretations have not been confirmed either in S. cereoislae or in S. pombe (Goffeau. A.. unpublished results). On the other hand, mutants affecting the plasma membrane ATPase gene PMA1 both in S. cerevisiae and S. pombe were shown to display resistance to a spectrum of drugs, nantely diguanidines and other positively charged compounds such as the antibiotics Dio-9 and hygromyein [81, 82. 83, 84, 45]. However, the pdr and pma mutants are phenotypically and genotypieally distinct [84]. The correlation of plelotroplc drug resistant phenotypes with modiflcaticn in permeability barriers was also indicated for the pdr3 mutants [78], despite the finding that the diminished ATPase activity often observed in these mutants was not caused by the pdr3 mutation itself [79]. Furthermore, mutants resistant to the protein synthesis inhibitor blasticidin $ were also suggested to be permeability mutants [33]. However, no data on cross-resistances were reported for this mutant. Another level of permeability barrier which has been proposed to be responsible for multiple drug resistance in yeast is the nuclear envelope [8]. An alternative mechanism for yeast multiple drug resistance has been proposed by Dupont et at. [17] in a study on the uncoupler (earbonyleyanide-m-chlorophenylhydrazone)-resistant mutant cpr, displaying different cross-resistances and collateral sensitivities. It was concluded that this resistance is due to a change in the internal pH determining the extent of accumulation of weak acids or bases and moreover influencing drug efficiency. An increased glucose-induced efflax of uncoupler was observed in the mutant and correlated to an intraeenular acidification, which was suggested to result from mutational alteration of the metabolic pathway producing or consuming organic acids. IV. ATP-dependent permeeses as determinants of drug resistance In mammalian cells, the biochemical mechanism of multidrng resistance has been correlated to a defect in active transport across the plasma membrane (for reviews see Refs. 18, 25, 21 and 76), Mammalian multidrug resistance (MDR) appears to be the consequence of decreased intracellular drug zccumulation, due to overexpression of a membrane component, the P-glycoprotein, functioning as an energy-dependent pump for

drug-effiux. The P-glycoprotein was found to share structural, and presumably functional, homology to bacterial components involved in the active transport of a wide range of molecules (oiigopeptides, histidine, maltose, ribose, phosphate, vitamin BI2, haemolysin) across the membrane. Homologs of these ATP-dependent transport-proteins have been reported from several different species, including bacteria, yeast, protozoans, insects and mammals. They constitute a superfamily of related import-export proteins conserved throughout evolution (for reviews see Refs. 30 and 35). The most recently discovered members of this MDR-related family include the human C T FR protein responsible for cystic fibrosis [67], the chloroquioe resistance (CQR) protein encoded by pfmdr in Plasmodiam faleiparum [23, 87], the yeast STE6 protein, essential for the export of the a-factor mating pheromone [48, 39], and, with somewhat lower degree of homology, the products of the white and brown genes of Drosophila, involved in the transport of eye pigment precursors [56,16]. The isolation and characterization by MeGrath and Varshavsky [48] and Kuchler et al. [39] of the yeast gene STE6, homologae of the mammalian MDR gene, has established for the first time a normal cellular function of an eukaryotic member of the M DR protein family. The STE6 gene product was shown to have a physiological role in the export of the a-factor yeast pheromone, through a new pathway distinct from the classical, signal sequence-dependent, protein secretion system. The a factor lipopeptide, whose precursor contains neither a hydrophobic signal sequence nor potential glycosylation sites but undergoes post-translational farnesylation rendering it lipophilie, was proposed to diffuse in the lipid bilayer of the plasma membrane, where it would encounter the STE6 glycoprotein, catalyzing an ATP-dependant a-factor efflux. Similarly, in the multidrug-resistant mammalian calls, various hydrophobic eytotoxic compounds share the tendency to partition into the plasma membrane lipid bilayer, where the P-glycoprotein pump catalyzes their extrusion from the cell. By analogy to the a-faetor/STE6 transporter system, the lymphokine interlenkine 1 was proposed to he a physiological substrate for the P-giycoproteinmediated, signal sequence-independent, secretion pathway of mammalian cells [48]. Amplification of the STE6 gene, unlike MDR1, was found not to confer resistance to different eytotoxic compounds, including cyclohcximide, oligomyein, thriendermine and chloramphenicol 148]. This finding, together with the observation that STE6 seems to be a member of a yeast multigene family, was interpreted to reflect a similarity with the mammalian MDR gene family, which comprises different members either related (e.g., human MDR1, mouse mdrl and mdr3) or not (e.g., human MDR2. also termed mdr3, and mouse mdr2) to drug resistatice, despite their high degree of

249 structural homology. However, it has more recently been reported that disruption or amplification of the STE6 geue leads respectively to a decreased or increased resistance to valinomycin, a potassium ionophore collapsing membrane potential [39]. This indicates that the cellular membrane is modified by disruption or overexpression of STE6, either because the STE6 gene product pumps charged molecules or because it modilies the ion conductance of the membrane. Some other MDR-relatcd, ATP-dependem permeases are already described in yeast. A yeast transmemhrane protein, ATR1. putatively capable of binding ATP and involved in specific resistance to aminotriazole, has been described [a6l. By analogy to the bacterial proteins involved in active resistance to tetracycline, cadmium or arsenate, ATRI was proposed to be a membrane efflux-pump responsible for the extrusion of aminotriazole, and possibly toxic amino acid precursors, out of the cell. Recently, a new MDR-reluted yeast gene, ADP1, predicted to be an ATP-dependent permease similar to the white and brown genes of Drosophila [56,16] has been sequenced on chromosome 11I of S. cerevisiae (Purnelle, B. and Goffeau, A., personal communication). V. Parspecfives The model of a PDRI encoded regulator, controlling the expression of various target genes mediating multiple drug resistance in yeast [4], implies the existence of a molecular network including various interactions between regulators and targets, or between regulatory and co-regulatory proteins. The recent discovery in yeast of the genes STE6, PDRS, PDR4, A T R ] and ADPI, whose products may be, structurally or functionally, related to the mammalian multidrug resistance effiuxpump, raises the possibility that some of these genes may be responsible for the cellular efflux of drugs as well as of physiological substrates, and that they may constitute some of the targets of the PDRl.encoded transcription regulator. In agreement with this model, very recent data obtained in our laboratory indicate that the mRNA transcripts of the STE6 and PDR5 genes are overexpressed in some pdrl mutants (unpublished data). These results confirm that in yeast the PDR1 regulator controls the transcription of MDR-related genes and that mutations at the PDR1 transcription regulator can cause the alteration generally believed to be at the basis of multidrug resistance: the overexpression of MDR-related membrane pumps. It is also interesting to note that the null alleles of either PDR1 [11] or S'1"~6 [48, 391 both cause sterility of the a-mating type. Similarly, the null alleles of either PDRI [4] or PDR5 [41] both determine hypersensitivity to cydoheximide. These findings are consistent with the view that the disruption either of a

positive regulatory gene (PDRI), or of one of the regulated target genes ( STE 6/P DR S) , would yield a same phenotype, that is the lack of expression of the functions encoded by the target (extrusion of the a mating factor or of cyeloheximide). Furthermore it has been observed that disruption of the PDR5 gene in a pdrl mutant leads to loss of pdrl-speeified drag resistance, indicating that the PDRI function requires a functional PDR5 gene (J. Golin, personal communication). The existence of other interactions among the PDR genes have been suggested from the eomplememation of pdr3 resistance phenotypes by the wild-type PDR1 gene, or of pdrl mutants by the PDR6 gene [7]. The nature of the interactions among different determinants of multidrug resistance in yeast should now be studied at the molecular level. On the other hand, considering the increasing evidences of similarity between the mammalian and the yeast multidrug resistance systems, it is becoming of primary interest to examine whether regulatory genes like PDRI exist in mammalian cells and control there multiple drug resistance. The involvement of increased gene expression in the establishment of mnltidrug resistance has been proven for the MDRI and other mammalian drug resistance systems (for review see Ref. 44). Although M D R gune amplification is the genetic anomaly most commonly correlated to the overexpression of the P-glyenprotein and to multidrug resistance in mammals, such amplification is not necessarily present in all types of M D R cells. It has been observed that an activation of transcription of the MDR gene can precede its amplification and can be thus an early step in the development of drug resistance [22, 74]. In this line, the possibility should be considered that, analogously to what we observed in yeast, one or several regulatory proteins like PDR1 could control the transcription of the drug-transporter-encoding MDRI gene in mammalian cells. Such model would be in agreement with the fact that no somatic mutations in the structural M D R gene seems to be required to produce a multidrug resistant pheuotype in mammalian cells [27]. Regulatory genes possibly similar to the yeast PDRI could thus be the site of mutations causing the overexpreesion of MDR in mammalian cells. The identification of such regulatory genes, controlling multiple drug resistance in mammals, could yield new possibilities to restore drug sensitivity in tumor cells for instance by acting at the regulatory level, as successfully performed in yeast. Aeknowledganmms We gratefully acknowledge Drs. Piet Borst, Carl Falco, John (]olin, James Haliex, James Mattoon, John McCuskar. John MeGrath, David Perlin, BOa&licte Purnelle. Julius Subik, Slanislaw Ulaszewski and

A l e x a n d e r V a r s h a v s k y for helpful discussions a n d advice a n d fo r h a v i n g p r o v i d e d t h e i r results prior publication. This w o r k was partially s u p p o r t e d b y g r a n t s to A. G o f f c a u f r o m ' L e s Selvices de la Politique Scientifique: A c t i o n Sciences de ]a Vie' a n d f r o m t h e ' F u n d s N a tional p o u r la R e c h e r c h e Scientifique', B e l g i u m .

1 Avner. P.R. and Grifflths, D.E. (1970) Oligomycin resistant mutants in ','cast. FEBS LelL 10. 202-207. 2 Avner. P.R. and Griffiths. D.E. (1973M Studies on energy-linked reactions. Isolatlon and characterization of oligomycin resistam mutants of Saecharomy~s cerevisia¢. Ear. J. Bi~hem. 32, 301 311. 3 Avner. P.R. and Griffiths, D.E. {1973b) Studies on energy-linked reactions. Genetic analysis of oligomyeln-resistant mutants of Sacchur~yces cerevislae. Ear. J, Biochem. 32. 312 321. 4 Balzi, E,, Chert. W., Ulaszewski. S.. Capieanx, E. and Goffeau, A. (1987l The multid~g resistance gene PDRI from Saccharomyces cerevtsiae. J. Biol. Chem. 262,16871 16879. s Balzi. E,, Chen, W,, Capieaux, E., McCusker, J.H., Haber. J.E. and Gorfcau, A, (19892 The ~uppressor gene sill of Saecharomyces cerevistae is e~ential for growth. Gene 83. 271-279. 6 BalzL E., Choder. M.. Chert. w., Varshavsky. A. and Gol[eau, A. 1990) Cloning and functional analysis o[ the arginyl-tRNA-protein transferase gene ATE/ of Saccharomyc~* ceret~isiae. J, Biol. Chem, 265, 7464 7471. 7 Balzi. E. (1989) Molecular analysis of a pleiotropic drug resistance mutatiom cluster from the yeas1 Seccharoa,yces cerevisiae. Ph.D. thesis. Universil8 Calholique de Lnuvaln {Belgium). 8 Earanowska. H., Polakowska. R. and Putrament. A. (1979} Spantanc~us and induced non-sp~ifi¢ d ~ g resistance in Succharom2'ces cereviMae. Acta Microbiol. Pol. 28,181-201. 9 Bunn, C.L., Mitchell. C.H., Lukins, H.B. and Linnane, A.W. (19702 alugenesls of mitochondria. XVI[I. A new class of CylOplasmicagy determined antlhlollc resistant mutants in Saccharomyces cerevisiae. Prec, Natl. Acad. SOL USA 67, 1233-1240. 10 Carignanl. G., Lancashire. W.E. and Griffiths. D.E. (1977) Eatrachromosomal inheritance of rhodamine 6G resistance in Saccharomyces cerevtsiac. Mulet. Gun. Genet. 151.49 56. 13 Chen. W.. BalzL E.. Capieaux. E.. Choder. M. and Goffeau. A. (19912 The DNA sequencing o[ the 17 kb Hindlll fragment spanmng the LEU! and ATEI loci on chromosome VII from Saccharomyce~ cemuL¢iae reveals the PDR6 gene, a new member of the genetic network controlling pleiolropie drug resistance. Yeast. in press. 12 Claviher. L. (1976) Mitochondrial genetics. Xll. An oligomyeinresistant mutant I~alized at a ne~ mil~bondrial l~us in Sacchuromyces cercvisiae. Genetics 83. 227-2a3. 13 Cohen. J.D, (1977) Genetic analysis of multiple drug cross resistance in Saccharomycea eereuisiae: A nuclear mitochondrial gene interaction. Ph,D. Thesis, City University of New York. 14 Cohen, J.D. and Eaton, N.R. (19792 Genetic analysis of multiple drug cross resistance in Saccharomyces e~reoisJae: a nuclear mitochondrial gene interaction, Genetics 91,19 33. 15 Colson. A.M.~ Galleon. A.. Briquet, M.. Weigel. P. and Matt~n. I {19742 Nucleo-cylop!asmic interaction between ollgomycin resistant mutations in Saccgaromyces cere~tsiae. Molec. Gen. Genet. 13S. 309-326. 16 Drcescn, T.D,, Johnson, D.H. and Henikoff, S. (108g). The brown protein o1 Drosophila melanogaster is similar to the white protein and to components of active transport complexes. Mol. Cell, Biol. 8, 5206-52t 5. 17 Dupont, C.H., Caubet. R., Mazat. J.P. and Guedn, B. (19942

Isolation end characlerlzation of an uncoupler-resistant mutant of

S~cct,~r~,.yc~ c~re~isioe Corr. Ge.~t. S, S0V 516. 18 Endi~lt. J.A. and Ling. V. (t989) The biochemistry nr P-glycoproteln-mediated multidrug resistance. Annu. Rev. Biochem. 28. 137-171. 10 Falcn. g.C. and Dumas. K.S. {1985) Genetic analysis of mutants of Saccharnmyces cerevisiae resistant In the herbicide sulfometut~n methyl. Genedcs 109, 21-38. 20 Falco, S.C., Dumas. K.S.. McOevitt. R.E. and (/olin. J.E, 09861 Mol~ular biology of sulfonylurea herbicide action on Saecharomvces cerevisiae. In: Proceedings of the Upjohn/Laoott Symposium on the biechemislry and molecular biology of industrial y~st, pp. 315 322, 21 Ferro-Luzzl A ~ s , G. {1986) The basis of muttldrug resistance in mammalian cells: homology with bacterial transport. Cell 47, 323-324. 22 Fojn. A.T., Whang-Peng, 2.. Gottesman. M.M. arm Pastan, L (19gs) Amplification of DNA sequences in human multidrug-resistant KB carcinoma cells. Prec. Natl. Acad. Sci. USA 82. 7661 760~. 23 F ~ Ic , SJ., Thompson, J.K., Cowman, A.F. and Kemp, DJ. (19892 Amplification of the multidrug resistance gene in some chloroquine resistant isolates of P. fafciporam. Cell 57, 921-930. 24 Foury, F. and Goffcau, A. {19752 Stimulation of active uptake of nucleosides and amino acids by cyclic adenosine 3':S'-monophosphate in the yeast Schia~accharomyce~ gombe. J. Biol. Chem. 250, 2354-2362. 25 GoResmam M.M. and Pastan, I. (1988) The mulddrug transporter. a double-edged sword. J. Biol. Chem. 263.12163-12166. 26 Griffiths, D.E., Houghton, R.L.. Lancashire. W.E. and Meadows, P.A. 0978) Studies on energy-linked reactions: isolation and properties o[ mitochondrial venturicidin-rmistant mutants of Saccharomyces cerevisiae. Eur. J. Biochem. 51. 393-402. 27 Gins, P., Ben Neriah, Y., Croup, J. and Housman, D. (19862 Isolation and expression of a oomptcmentary DNA that confers muhidmg ~istance. Nature 323, 728-731. 28 Gudrlneau, M,. Slonimski, P.P. and Avner, P.R. (1974) Yeast episome: oligomycin resistance associated with a small covalently closed non mitochondrial DNA. Biochem. Biophys. Res. Commun. 61. 462-469. 29 Gu~dneau, M. Graodehamp, C. and Stunimski, P.P. (19762 Strutlure and genetics o1 the 2/zm circular DNA in yeast. In: Genetic and Biogenesis of ehloropl~ts and mit~hondria. (Th. Bucher ~t al.. eds.), pp. 557-564, Elsevier/North Holland Biomedical Press. Amsterdam. 30 Higglns, C. (1989) Export-import family expands. Nature 340, 342 31 Howell N., Monoy, P.L., Linnane, A.W. and Luldns, H,B, 0974) Biogenesis of mitochondtia 34. The synergistic interaction of nuclear and mit0c'~0ndrial mutaaons to produce resistance to high levels of nfikamycln in $acchar~myees cereoisiae. Mulet. Gun. Genet. 128, 43-44. 32 Hughes, A.R. and Wilkie. D, (19722 Genetic analysis of mitochondrial resist:;nce to tetracycline in Saccharomyces oereuisiae. Heredity 28.117-t27, 33 Ishiguro, J. and Miyagaki, M. (1985) Characterization of blasticidin S-resistant mutants of Sacch¢romyces cerevistae. Curt. Genet. 9,179-181. 34 Johnston, P.A. and Coddington, A. (1983l Muhiple drug r~istance in the fission yeast Scgizmacchar~yces pombe: correlation between drag and amino acid uptake and membrane ATPase activhles. C a m Genec 7, 299-307. 35 Juranka, P.F.. Zastawny. R.L. and Ling. V. (19892 P - g l y ~ proteins: multidrug-resistanc¢ and a superfamily of membrane associated transport proteins. FASEB J. 3. 2583-2592. 36 Kanazawa, S., Ddsc~ll, M. and StruhL K. (19g8l ~fTRI. a Saccharomyees cerevis,ae gene encoding a transmembrane protein required for aminolriazole resistance. Mol. Cell. Biol. g, 664-673.

251 37 Kerem, B.. Rommens. J.M, Buehman, JA., MarkiewieT~ D., Cox, T,K., ChaI:ravarli. A,. Buchwald, M. and I z u i . L. (198')) Idcntlficalion of the cystic fibrosis gene: genetic analysis. Science 245, 1073 1080. 38 Kruszewska, A and ~zczesniak, B (19781 Janus Green resistance in Saecharomytes cerevlsi,w: Interaetinns of nnclear and e y t ~ plasmie factors. Molec. Gen. Genel. 160. 171 J81 39 Kuchler. K., Sleme. R . E and Thorner. 2 11989) SacU,a ,ogles ,'eret.siae STE6 gene product: a no~el pathway for prolein exporl in eukaryofie cells EMBO J. 8, 3973 3984 4O Lancashire, W . E and Griffiths, D.E. (I9751 Studies on energy-linked reacbons: isolation, characterizalion and genetic analysis of trialkyl-tin-resistant mutants of Saceharomv~s cerevis,ae. E u r J. B i ~ h e m . 51, 377-392. 41 Leppert, G., McDevitt, R., Falco. S.C., Van Dyk, T.. Ficke, M. and Gotin, J. (19901 Cloning hy gene amplification of two k~i conferring multiple dru B resistance in Saccgaronn'ces cere,,isfae Genetics 125:13 20. 42 Linnane. A W . , Lamb. AJ.. Christodoalou. C. and Lukins, H.B. (19681 The biogen~is of mit~hondria, VL Bi~hemical basis of the resistance or Saccharom)~es rerevisiae toward antibiotics which specifically inhibit mitoch0ndrial protein synthesis. Proe Natl. acad. ScL USA 59. 1288-1293. 43 Luc~chini. G.. Carbone. M.L.. C ~ e i . M. and St:lsi, M.L. 119791 N u c l ~ r inheritan~ of ~sistance to anlimycin A in Satchan,ng"ees rerevisiae. Molec. Gen. Oenet. 177. 139-143. 44 Marx, J.L. 11986) DrUB resistance of canner cells probed. Science 234, 318 820. 45 M¢Cusker. J,H.. Perlin. D. and Haber. J.E. (1087) Pleiotropie plasma membrane ATPase mutations of Sacc6aron~vces cereris~ae. Mot. Cell. Biol. 7. 4082 4088. 46 McCusker, J.H. and Haher, 3,E. 11988) Cycloheximide-r¢sistanl t emperaturc-sensitive lethal mutations of Sacehuromyces ~ret,isfue. Genetics 119, 303-318. 47 McDe~tt, R,E., Golin, J.E. Van Dyk, T.K. and Falco, S.C. 11986) Plelolroplc drug resistance by gene amplification in ye~t. Yeast 2. S237. 48 McGrath, J,P. and Varshavsky, A, (1989) ]'he yeast STE6 gent encodes a homologue of the mammalian maltidrug ~ s i s t ~ c c P-glyeoprotein. Nature 34O. 4OO 4O4. 49 Meunier, R a n d ColsolX A.M. (19g9a) Two nuclearly inherited loci conferring increased diuron resistance to N A D H oxidase in Sa~h~omyees ce~uisiae. Curr. Genet. 15.31-33. 50 Meunier, B. and Colson. A.M. (1989b1 Increased diuron resistance ,o ih0 joi.1 eXocPlessi°n ~ h mutati°ns ~lst~°cate , ~ a , ed ~at the DIU2. DIU3 a n d D/U4 I of Sac aromv~ . C u r l Genct. 18, 121-127. 51 Mitchell, C.H., Bunn, C.L.. Lukins. H.B. and Linnane, A.W. 119731 Biogenenesis o{ mitochondfia. 23. The biochemical and genetic cheractedstics of two different oligomycin resistant mutams of Sa~haromy~s cereuisiae under the influence of cyloplasmin genetic modification. Bioenergetics 4.161 177. 52 Mortimer, R.K. and Hawthorne, D.C. (1973~ Genetic mapping in yeast. Methods C e l l Binl. I I, 221-233. 53 Mortimer, R.K, and Schild, D. (19801 Genetic m a p of Saccharomyces cerevisiae. MictobioL Rev. 44, 519 571. 54 Mortimer, R.K. a n d Schild, D. [19881 Genetic map of Sacchar~lyees cemuisiae, Edition 9. MierobinL Rev. 49, 181-212. 55 Mass, G. a n d Pcralls. K. (1976) G e n e t i ~ of bor~lidin resistant mutants of Saccharomyces cereulsfae and properties of their Ihreonyl-tRNA-synthet~e. Mol~. G e m Genet. 147. 39-43. 56 O'Hare, K.. Murphy. C., Levis, R. and Robin. G.M. 11984) D N A sequence of the white locus of D~ophaa mel~ogmler. J. Mol. Biol. 180, 437-458. 57 Parker, J.M,. Tdmbl¢, l.g. and MaRoon. J.R. 11968) Oligomycm resistan~ in n o d a l and m u t a n t yeast. B i ~ h e m . Biophys. Res. C o m m u n . 33, 59O-595.

58 Preston. R.A,. Mott, P.L., Gl&ger, E.W. and Jones. E W . [19871 Characterization t,f yeast mutants dereelive in vacuolar a~umulalion or neutral red dye. Abslr~ts of the "Meeting on Yeast Genetics and Mtflegalar Biology" June 16-21.1987. San Francisco. 59 Rank. G H . and B e c h - H a n ~ , N.T. (19731 Single nuclear gene inherlted cross resislanee and collateral ~nsitivity to 17 inh~bhors of mit~hondrial function in Saccharnmyces cere~:lsiae. Molec. Gem Genet. 126, 93-102. 60 Rank, G H . 11974) Pleintropy of cytopl~mically and nuclearly inherited resistance to inhibBo~ of mit~hondtial function in S~l,('ha¢ano'ces cerevtsiae. Canad. J. MierobioL 20, 9-12. 61 Rank, G H , Robertson, A and Phillips. K. {1975a1 Modification and inheritance of p!eiotrople c r o ~ - r ~ i s t ~ e e and ~ltateral ~nsitivdv in Saccharomyees ~t,tsiae. Genetics 80, 488-493. 62 Rank. G H . . Robertson. A. and Phillips, K. (1975b) Reduced plasma membrane p e ~ b i b t y in a multiple cross-resistant strain of Sa~':'haromvces cemelrf.c J BacterioL 122, 359 366. 63 Rank, G.H.. Gerlaeh. J.H. and Robert~on, A.J. 11976) Some physiologic,d alterations a~ociated with pleintropi¢ cross resistance and eolluteral scnislivity in Sa.~haromyces ~r~qs~ae. MOIl. Gen. Genet. 14a, 281-288. 64 Rank, G.H.. Roberlson, A J . and Gerlach, J.H. 119771 Single gene Mteration of plasma and mitochondrial m ~ b r a n e [unelion in S.£cha~n~vces cereeisiae Mol~. Gcn. Genet. 152. t3-18. 65 Rank. G.H.. Roberl~n, A.J. and Bu~.sey, H. II9781 The viscosity and lipid composi0on of the p l ~ m a membrane of multiple drug resistant and sensitive y~abt string. Can, J. Bi~hem. 56, 10361041. 66 Rank. G.H. and Sheard, J.W. 11979} Revertants of pleiotropie cross resistance and collateral sensitivity in yeast: A multivariate analysis Mole¢. G ~ , Genet, 167. 809 316. 67 Riordan, J.R., Rommens, J.M., K ~ e m , B., Alon, N . Ro~mahel, R., Grzelezak, Z.. Zielenski. J., Log, S., Plavsic. N., Chou, J,. D r u m m . M.L.. lannuzxi. M C . . Collins, F,S. and Tsui, L. 11989) ]d~tificatlon of the cystic f i b r i l s eerie: cloning and eharacteri~lion of complemenlary DNA. Science 245.1066-1073. 68 Rommens. J.M_ lannuzzi, M.C.. Kercm. K , D r a m m , M.L., Melmer. G., Dean, M,, Rogmah¢l, R., Cole. J . L , Kennedy, D., Hidaka, N., Zsiga. M., Buchwald, M., Riordan, J.R.. Tzul. L.-C. a n d Collins, F.S. 11989) Identification of the cystic fibrosis gene: chromosome walking, and jumping. Science 245,1059-11165. 69 Rotman, A. 119751 Genetics o f a prlmaquin-r~istant yeasl. J. G e m Microbiol. 89, 1-1O. 70 Saunders, G.W., Rank, G.H., Kustermmann*Kuhn, B. and Hollenberg. C.P. 11939) Inheritance of muhidrug resistance in Sacc h a ~ y e e s cemmsf~: fingag¢ to leuJ and analysis ol 2 .am D N A in partial ~vertant~ Mol~. G ~ . G e n t . 175, 45-52. 71 Saunders, G.W. and Rank, G,H. 0982) Allelism or pleiolropic drug resislance in Saceharomyces cerepJsiae. Can. J, Genec Cyloh 24, 493-503. 72 Savage, M., Softer. IL and Leibo~,itL M. 119831 A m u t a n t of Sacchar~yces ce,evisiae defective in arginyl-tRNA-promin trans(erase. Curt. Genet. 7, 285-288. 73 Serrano, R., K i e l l a n d - B ~ d t , M.C. and Fink, G. 09861 Yeast plasma membrane ATPase is essential for growth and has homology with ( N a * / K ÷ ), K * and Cag*-ATPases. Nature 319, 689-693. 74 Shen, D.W., Fojo, A., Chin, J.F-, RoBinson, I.B., Richen, N.. P~tan, I. and Goltesman, M.M. 11986) Human mubidrug, resistant cell lines: incensed mdrl g e n t expr~sion e ~ precede 8cue amplification. Scie~e 232. 643-645. 75 Sofa. S.. Cifecfi. O.. Di Pasqual~ G. ~ d Magni. E. (19801 Gentiles and biochemistry or resistance to axenomyein in S ~ chur~y~s (-erotic. Cuff. Genet. 2. 61-67. 76 Stark, G.R. 113861 Progress in u n d e m t ~ d i n g mulgdrug, resistance. Nature 324, 4O7-4O8.

77 Sluarl, K.D (1970) Cytoplasmic inheritance of oligomycin and rutamycin rcsizlanee in y ~ s t . Bioehem Illophys, R ~ . Commun. 39. 1045 1051. 78 Sublk. J., Kovacova, V. and Takacsova, G. (1977) Mucldin resistance in yeast. Isolalion, characleri~tion and genetic analysis of nuclear and mitc~-hondrial mucldin-resislant mutants of Saccgaromrces cereuisiae. Eur, J. Biochem 73, 2"/5-286. 79 gubik. 2,. Ulas~wski, S and Goffeau, A. (19861 Genetic mapping of nuclear mucldin registan~ mulations in Sa~huromyces ,ere~,rsiae A new pdr I ~ u s on c h r o m ~ o m e II. Curr. Genet. I0. 665 670. 80 Thomas. D Y . and Wilkie. D. (196gl Inhibition of mit~hondrial synthesis in yeasl by erythromycin: cytoplasmic and nuclear factors controlling resistance. Genet. Res. I1.33-41. 81 Ulaszewskl, $.. Grenson, M. and Goffeau, A. (1983) Modified plasma membrane ATP,Ise in mutants of Saccharomyces ce~visiae. Eur, J. Biochem. 130. 235 239. 82 Ulaszewski. S., Coddington. A. and Goffeau. A. (1986) A new mulation for multiple drug resistance and modified plasma mum-

brahe ATPase aelivity in Schizosaccharomycespotage. Curr, Genet. I0. 359-364.

83 Ulaszewski. S.. Van Herck. J.C.. Dufour, g.P., Kulpa. L. Nieuwehuls. B. and Goffeau. A. (1957a1 A single mutation confers vanadate r~istanue to the plasma membrane H +-ATPase from Ihe yeast Schiz~accharomyces pombe. J. Biol. Chem. 262, 223 228. 84 Ulaszewski, S., Balzi, E. and Goffeau, A. (1987b) Genetic and mol~ular mapping of the pmul mutation conferring vanadatc resistance to the plasma membrane ATPase from Sacehuromy~s cerevisiue. Mole¢. Gun. Genet. 207.38-a6. 85 Wakabayashi, K. and Gunge. N. (I9"/0) Extraehromosomal inheritance of oligomycin resistance in yeasl. FEBS L¢lt 6) 302 304. 86 Wilkie, D. and Lee, B.K, (1965) Genetic analysis of aetidione resistance in Sacchuromyces cerevisiae. Genet. Res. Camb. 6 , 1 3 0 138.

87 Wilson. C.M.. Serrano. A,E.. Wasley. A., Bogen Schulz, M.P., Shankar, A.H. and Wirth, D . F (1989) Amplification of a gene related to mammalian mdr genes in d~g-resistant Plusmodlum /alc,pumm. Sciun~ 2,1,1,1184- I 186.