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Genes to Cells (2005) 10, 409-420. doi:10.1111/j.1365-2443.2005.00847.x
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Mutational analysis of the yeast multidrug resistance ABC transporter Pdr5p with altered drug specificity

Andreea Cristina Tutulan-Cunita, Makoto Mikoshi, Masaki Mizunuma, Dai Hirata and Tokichi Miyakawa*

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Multidrug resistance ABC transporter Pdr5p of Saccharomyces cerevisiae is particularly important due to its ability to export a wide range of unrelated substrates. To clarify its function, we generated Pdr5p mutants by random mutagenesis and screened for mutants with altered drug specificity in vivo by using 5 drug compounds. Nine point mutations that caused significant changes in drug specificity distributed throughout the length of Pdr5p, namely, in the extracellular, transmembrane or cytoplasmic regions of the transporter. We then investigated their effects upon drug resistance, using 36 chemically related or distinct substrates. From this study, overall geometry of the Pdr5p was suggested to contribute in acquiring the enormous range of drug specificity. Based on their ability to inhibit the growth of the mutant strains, the 36 tested drugs were classified into: drugs to which the mutants responded differently (Group 1), drugs to which all the mutants showed sensitivity (Group 2), and drugs to which all the mutants exhibited resistance (Group 3). The ability of the compounds to be partitioned to the plasma membrane seemed an important factor for recognition by Pdr5p.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Biological membranes appeared at a very early stage of evolution, as they were necessary to delimit and maintain the definite composition of the hydrophilic microdomains of the primitive organisms. Biological membranes act as selective boundaries, especially towards hydrophilic molecules that require specialized inward transport systems to access the cytoplasm. On the other hand, as hydrophobic and amphiphilic molecules can easily cross phospholipid bilayers, and as many of them may have harmful effects upon cellular processes, cells need to be provided with specialized systems able to detect, collect, and export xenobiotic compounds that may accidentally diffuse through the membrane.

Among these specialized systems, in the yeast Saccharomyces cerevisiae, PDR5 (STS1/LEM1/YDR1) has been independently identified as a transporter that confers multidrug resistance (Balzi et al. 1994; Hirata et al. 1994), suppresses sporidesmin toxicity (Bissinger & Kuchler 1994), and acts in steroid compounds export (Kralli et al. 1995). Based on its structure, function, and consideration of its phylogeny, Pdr5p was classified in the PDR (pleiotropic drug resistance) family of the ABC (ATP-binding cassette) superfamily (Bauer et al. 1999; van Bambeke et al. 2000). Of the 10 P-glycoprotein-like ABC transporter genes revealed by S. cerevisiae genome project, Pdr5p encoded by the PDR5 gene is the most important in multidrug resistance (for a review, see Bauer et al. 1999). Pdr5p is by far the broadest-range drug transporter, being able to export a myriad of compounds that share no discernable structural similarities, such as anti-fungals, anti-cancer drugs, detergents, ionophores, steroids, etc. (Kolaczkowski et al. 1996, 1998; Conseil et al. 2000, 2001; Golin et al. 2000). Due to its close relatedness to P-glycoprotein of the mammalian cell, which is involved in the resistance of cancer cells to anti-oncogenic drugs (Higgins 1992; Gottesman & Pastan 1993), and also because of the multiple advantages of the yeast cell experimental system, Pdr5p quickly became the yeast model of mammalian multidrug resistance.

The function of P-glycoprotein was dissected by site-directed mutagenesis (Loo & Clarke 1994a, 1994b, 1995a, 1995b, 1996, 1997) and by extensive studies on its single nucleotide polymorphisms (see Ishikawa et al. 2004 for a review) to reveal amino acid residues relevant to its transport ability. Attempts to identify the residues of Pdr5p important for its function were done as well, by either random (Egner et al. 1998) or site-directed (Egner et al. 2000) mutagenesis. Another approach to better understand Pdr5p functioning is to analyze the interaction of the protein with various substrates and effectors (Leonard et al. 1994; Kolaczkowski et al. 1996, 1998; Conseil et al. 2000, 2001; Golin et al. 2000, 2003; Hu et al. 2001) in order to determine which domain of the protein or, alternatively, which characteristic of a certain substrate (e.g., hydrocarbon chain length, ion composition, hydrophobicity, size) is of greatest importance for substrate detection, binding, and export.

In the present study, we have chosen a combined approach, using mutagenesis of Pdr5p in conjunction with the investigation of a quite wide range of chemically related or distinct substrates with the purpose of pointing out new aspects of Pdr5p function.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Pdr5p mutants: Isolation and identification of their mutation sites

For Pdr5p to function, particular amino acid residues of the transmembrane domains may act for the recognition and accommodation of the substrate in the drug chamber, whereas specific amino acids of the nucleotide-binding domains are required for breaking down ATP and releasing the energy necessary for transport (Egner et al. 1998, 2000; Moody et al. 2002; Neyfakh 2002). To identify new residues important for Pdr5p function, we mutagenized p-BluescriptII-borne PDR5 by random hydroxylamine-induced mutagenesis, divided it into four fragments of about equal size using single restriction sites, and chimeras of each fragment with wild-type PDR5 were constructed (see Experimental procedures and Fig. 1). Following transformation in {Delta}pdr5 strain, the mutants were screened for drug sensitivity on solid media containing cycloheximide, fluphenazine, cerulenin, tautomycin or staurosporine. The mutants that failed to grow on all the drugs tested were eliminated, and those that grew on one to four of five drug-containing media were retained.



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  		Figure 1  Construction of pdr5 chimeras: mutagenized pdr5 (as described in Experimental procedures) was cut with restriction enzymes with unique cutting site and ligated to wild-type PDR5 cut accordingly.

 
Their mutation points were determined, and the substituted residues and their localization are shown in Fig. 2. The mutation points distributed throughout the length of the protein, and their localization were: three in cytoplasmic loops, two in transmembrane domains, and two in extracellular loops. As the mutant pdr5-6/8 turned out to possess two mutations (E1289K, Y1311S), in order to clarify which mutation was responsible for the phenotype or if there was any additive/synergistic effect, we separated mutation Y1311S by site-directed mutagenesis, generating a new mutant protein (pdr5-8). Interestingly, of the two mutations, pdr5-6 single mutation was obtained independently in the screening procedure. Additionally, to check if cumulation of two mutations leads to increased sensitivity, a new protein containing G1040D and Y1311S mutations was constructed (pdr5-4/8). Mutants pdr5-4/8 and -6/8, as suggested by their names, cumulated mutations 4 and 8, and 6 and 8, respectively, and were designated this way for the sake of clarity.



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  		Figure 2  Localization of the mutation points and an alignment of a conserved region of Walker B motif in the second nucleotide-binding domain of Pdr5p and some related proteins. The alignment was based on sequence comparisons done using WU-BLAST software (Gish 1996–2004) available at Saccharomyces genome database (http://www.yeastgenome.org/) and gene sequences from the same database and from Protein Information Resource (PIR) site (http://www-nbrf.georgetown.edu/pir/).

 
To ascertain that the mutant proteins were indeed expressed, we investigated their expression levels. All the mutants showed levels approximately similar to or slightly increased in comparison to wild-type Pdr5p, except for mutant pdr5-3, which exhibited a reduced level (Fig. 3A). In addition, localization of the mutant proteins was addressed by indirect immunofluorescence assay. No difference in Pdr5p distribution was detected between the mutant strains and wild-type (Fig. 3B). The reduced amount of protein in pdr5-3 mutant strain was not correlated with a decrease in the intensity of the signal, probably due to lack of fine quantitative accuracy of indirect immunofluorescence method.



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  		Figure 3  (A) Expression levels of mutant pdr5 proteins. Protein was isolated as described in Experimental procedures, separated by SDS-PAGE and detected using polyclonal antibody anti-Pdr5p. (B) Cellular localization of wild-type and mutant Pdr5p, by indirect immunofluorescence, as described in Experimental procedures.

 
Drug related-phenotypes conferred by pdr5 mutants

In order to evaluate the effect of the mutations upon the ability of Pdr5p to protect yeast cell against toxic compounds, we examined the phenotype of the mutant strains in the presence of compounds that were cited before to be exported by Pdr5p or derivatives of these compounds (Kolaczkowski et al. 1996, 1998; Conseil et al. 2000, 2001). As we tested a wide range of concentrations, for most of the drugs, the mutants’ phenotypes fell between the sensitivity of the deletion mutant and the resistance of wild-type cells; i.e., at lower drug concentrations, most of the mutants showed a slightly increased sensitivity. This was better noticed in liquid culture assay, where small differences in the sensitivities of the mutant strains, not readily detectable in plate assay, were visible (Fig. 4 shows only three representative drugs). In every case, the results of liquid culture assays were in accordance with the results of the plate assays. At higher-tested drug concentrations, some of the mutants showed markedly increased sensitivity, while others retained resistance in a significant degree. The extent of sensitivity to various drugs was different by the mutants. The order of sensitivity among the mutant strains was preserved at all drug concentrations used, suggesting an alteration of drug specificity of the mutant Pdr5p. Basically similar results were obtained with other drugs (data not shown).



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  		Figure 4  Susceptibilities of wild-type and mutant strains to 3 representative drugs: (A) anisomycin (Group 1); (B) clotrimazole (Group 2); (C) rhodamine 6G (Group 3). Cells from overnight cultures were inoculated on fresh YPD containing 2-fold serial dilutions of drug. OD600 was measured for 50-fold dilutions of drug-containing cultures and plotted against drug concentrations. The concentration used in plate assay is indicated by an asterisk. The inserts show MIC values for each drug. Wild-type (•); {Delta}pdr5 ({circ}); pdr5-1 ({blacksquare}); pdr5-2 ({blacklozenge}); pdr5-3 (*); pdr5-4 ({triangleup}); pdr5-4/8 ( x ); pdr5-6 ({triangledown}); pdr5-6/8 (+); pdr5-8 ({blacktriangleup}); pdr5-9 ({square}). +resistant; – sensitive (as determined by plate assay, shown in Figure 5).

 
This result can be interpreted as a partial loss of function: mutated proteins are able to transport a certain substrate with a reduced efficiency, and the degree of reduction varies among the mutants. Therefore, when cells are exposed to lower concentrations of drug, the function retained by the mutated transporter allows export sufficient for survival; yet, when cells are exposed to higher concentrations, the residual amount of drug remaining in the cells kills them. Hence, parameters such as LD50 or MIC might not fully reflect the real behavior of mutant transporters. Thus, for the purpose of identifying the differences among the phenotypes conferred by the mutant protein, we tested and selected for the concentrations at which these differences were most clearly seen in plate assay. The mutants that grew at these particular drug concentrations were defined as ‘resistant’ (marked with + in Fig. 5), and those that did not, as ‘sensitive’ (marked with – in Fig. 5). The effective concentration for each drug determined in plate assay basically lies between MICs for ‘resistant’ and ‘sensitive’ strains determined in liquid culture assay.



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  		Figure 5  Drugs to which various pdr5 mutant strains respond differently: wild-type and mutant strains’ phenotypes and drug formulas. Serial dilutions of cell suspension were applied on YPD medium containing the indicated drugs, in the following concentrations: A23187 [GenBank] (A23) 7 µg/mL, anisomycin (Ani) 50 µg/mL, berberine (Ber) 1125 µg/mL, cerulenin (Cer) 0.2 µg/mL, curvularol (Cur) 6 µg/mL, cycloheximide (Cyh) 0.05 µg/mL, fluphenazine (Fp) 30 µg/mL, ketoconazole (Ket) 0.1 µg/mL, nigericin (Nig) 45 µg/mL, nuarimol (Nm) 300 µg/mL, staurosporine (St) 8.5 µg/mL, tautomycin (Tau) 5 µg/mL (pH 4.5), trifluoperazine (Tfp) 110 µg/mL (phenotype not shown), triton X-15 (T15) 0.75 µL/mL, triton X-114 (T114) 2.25 µL/mL. Plates were incubated for 1–3 days at 30 °C. The cassette summarizes the phenotypes showed in the pictures: + resistant, –sensitive. *The result for tetrazolium red is presented in Figure 8C.

 
Importantly, the double mutant proteins showed a markedly decreased ability to confer drug resistance, a synergistic effect of cumulating two mutations. Figures 4 and 5 illustrate how, in the presence of drugs, each of the double mutant strains grew significantly less than any of the single mutant strains. Actually, the pdr5-8 mutation in itself showed very little effect on the sensitivity to most of the tested drugs.

The drugs covered several categories of chemical structures. According to the differences in their ability to inhibit the growth of the mutant strains, they could be classified into three groups: drugs to which the mutants responded differently (Group 1; 16 chemicals), drugs to which all the tested mutants showed sensitivity (Group 2; 16 chemicals) and drugs to which all the tested mutants exhibited resistance (Group 3; 4 chemicals). The chemical structures of the compounds in Group 1, which inhibited differently the growth of Pdr5p mutants, together with the corresponding phenotypes are shown in Figs 5 and 8C. This group includes: ionophores A23187 [GenBank] and nigericin, protein synthesis inhibitors anisomycin, curvularol, and cycloheximide, alkaloid berberine, sterol and fatty acid biosynthesis inhibitors cerulenin, ketoconazole and nuarimol, calmodulin inhibitors fluphenazine and trifluoperazine, protein kinase inhibitor staurosporine, protein phosphatase inhibitor tautomycin, dye tetrazolium red and detergents triton X-15 and X-114. The Group 2 compounds were drugs that inhibited the growth of all of the investigated mutants; i.e., the drug transport function was lost by all mutants. These drugs were the flavonoid quercetin, the anti-fungals clotrimazole and nystatin, the anti-oncogenic drug daunomycin, the pesticide imazalil, the metal ion chelator 1,10-phenantroline, the ionophores monensin and valinomycin, the detergent SDS and its derivatives (dodecyl aldehyde, dodecyl amine, dodecyl arachidate), zwittergents 3–10 and 3–14 (Fig. 6), rhodamine B (Fig. 8A) and tetrazolium blue (Fig. 8C). Group 3 drugs, which did not affect the growth of any of the mutants (i.e., the drug transport function was not lost by all mutants) included the detergents n-lauroyl sarcosine and brij 35 (Fig. 7) and the fluorescent dyes rhodamine 6G and 123 (Fig. 8A).



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  		Figure 6  Drugs to which all pdr5 mutant strains exhibit sensitivity: wild-type and mutant strains’ phenotypes and drug formulas. Serial dilutions of cell suspension were applied on YPD medium containing the indicated drugs, in the following concentrations: clotrimazole 1 µg/mL, daunomycin 160 µg/mL, dodecyl aldehyde 0.75 µL/mL, dodecyl amine 5 µg/mL, dodecyl arachidate 6250 µg/mL, imazalil 1.5 µg/mL, monensin 5000 µg/mL, nystatin 0.4 µg/mL, 1,10-phenantroline 35 µg/mL, quercetin 6250 µg/mL, SDS 65 µg/mL, valinomycin 65 µg/mL, zwittergent 3–10 2125 µg/mL, zwittergent 3–14 35 µg/mL. Plates were incubated for 1–3 days at 30 °C. Only the result with clotrimazole is shown.

 


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  		Figure 8  (A) Growth of mutant pdr5 strains on rhodamine-containing media and the chemical formula of these fluorescent dyes. Serial dilutions of cell suspension were applied on YPD medium (pH 4.5) containing the indicated drugs, in the following concentrations: rhodamine B (RB) 50 µg/mL, rhodamine 6G (R6G) 10 µg/mL, rhodamine 123 (R123) 150 µg/mL. Plates were incubated for 2 days at 30 °C. (B) Rhodamine accumulation and efflux, as detected by FACS analysis. Loading cells with rhodamine B or 6G as described in Experimental procedures confirmed that while the mutants show no efflux of rhodamine B (RB), they are able to export rhodamine 6G (R6G). Only data for one mutant, pdr5-6 are shown. (C) Growth of all mutants was inhibited by tetrazolium blue (TB, 625 µg/mL), while only two mutants were inhibited by tetrazolium red (TR, 50 µg/mL). Plates were incubated for 1–3 days at 30 °C.

 


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  		Figure 7  Drugs to which all pdr5 mutant strains exhibit resistance: wild-type and mutant strains’ phenotypes and drug formulas. Serial dilutions of cell suspension were applied on YPD medium containing the indicated drugs, in the following concentrations: brij 35: 4250 µg/mL, n-lauryl sarcosine (NLS) 1250 µg/mL. Plates were incubated for 1–3 days at 30 °C. Only the result with NLS is shown.

 
Significantly, the rhodamine derivatives fell into the two extreme groups. Rhodamine B classified in Group 2 carries an ionizable carboxyl group; apparently, the specificity toward this drug was easily lost by mutation. In contrast, the specificity toward rhodamine 6G and rhodamine 123, which have an esterified carboxyl group and were classified in Group 3, was not lost by mutation (Fig. 8A). To see if the resistance and sensitivity to rhodamines reflected the difference in the substrate export ability, we addressed it by FACS analysis: wild-type cells loaded with rhodamine B and were able to efflux it in 45 min, while {Delta}pdr5 or pdr5-6 cells showed no significant decrease in fluorescence. As far as rhodamine 6G was concerned, wild-type cells loaded poorly and showed a rapid decrease in fluorescence intensity, while {Delta}pdr5 strain showed unchanged load over 45-min period. pdr5-6 strain loaded similarly to {Delta}pdr5 mutant, but appeared to be able to efflux the dye, though at a slightly reduced rate in comparison with wild-type cells. This difference in efflux as detected by flow-cytometric analysis could not be observed in plate assay, but was visible in liquid culture, where pdr5-6 showed a slightly reduced growth. All the mutants showed a similar behavior, thus only the result for pdr5-6 is shown (Fig. 8B). These results demonstrated that rhodamine B, which possesses an ionizable group, is a poorer substrate of Pdr5 mutant proteins compared to rhodamine 6G or 123, which possess no ionizable groups. This finding may imply that hydrophobicity/hydrophilicity of a compound is an important determinant of substrate specificity.

Another two related compounds showed a behavior similar to that of the rhodamines: only two out of nine mutants (pdr5-6, -6/8) showed growth inhibition by the slightly more hydrophobic tetrazolium red (Group 1), whereas all nine mutants ceased to grow on medium containing its more hydrophylic derivative, tetrazolium blue (Group 2) (Fig. 8C).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Altered drug resistance in mutant Pdr5p

There are several hypotheses and models of drug binding and transportation by ABC transporters, most of them the result of studies on bacterial proteins (Borges-Walmsley & Walmsley 2001; Higgins & Linton 2001; van Veen et al. 2001; Davidson 2002). Polyspecificity, the most peculiar characteristic of Pdr5p, seems to be best explained by a model proposing that the transmembrane segments of the transporter form a large intramembrane chamber into which the substrate presumably enters from the inner leaflet of the membrane. Specific substrate characteristics are not an essential requirement, so that an amphiphilic or hydrophobic molecule that can fit into the site without disrupting it would become a substrate. Inside the chamber, the substrate establishes a number of van der Waal's interactions with hydrophobic residues in the transmembrane segments and/or electrostatic attractions with particular charged residues. Binding of ATP induces conformational changes that close the side openings and simultaneously, the affinity of the protein for its substrate may be reduced. Consequently, the substrate is released on the outward side of the membrane. ATP hydrolysis reverts the protein to its original conformation (as reviewed by Neyfakh 2002).

Yet, as many data indicate, point mutations that alter specific residues alter drug specificity of the transporters as well. However, data concerning drug binding and efflux are still scarce and confusing, in contrast to the better understood ATPase activity of these transporters (Schneider & Hunke 1998; Moody et al. 2002). Pdr5p, although intensely studied, has not revealed many details of its mechanisms of action. Even whether it functions as a monomer or as a dimer is still an open question (Ferreira-Pereira et al. 2003). To date, Egner et al. (1998, 2000) have identified several amino acid residues that alter Pdr5p substrate specificity, sensitivity to inhibitors or localization, but no other studies have addressed this aspect of Pdr5p functioning.

In our set of mutant proteins, as far as the drug-related phenotype is concerned, both mutations in transmembrane domains (pdr5-2 in TMD4 and pdr5-6 in TMD9, substitutions involving non-similar amino acids) led to an altered pattern of resistance, conferring resistance to 8 and 6, respectively, out of the 16 chemicals in Group 1. The only difference in the phenotypes of these mutants is that the first one also conferred resistance to staurosporine. The influence of the amino acid residues in the transmembrane domains upon the pattern of resistance is in agreement with previous reports (Egner et al. 1998, 2000).

There were three mutants carrying alterations in the cytoplasmic loops. The first was pdr5-1, localized in CL3, which exhibited an even increased sensitivity when compared with the substitutions in the TMD mutants (resistant to only 6 out of 16 chemicals). Considering that this mutation did not occur in a conserved region, this result is quite surprising; yet, it is still explainable by a possible effect upon protein architecture. A second mutation, pdr5-3, a replacement in a non-conserved region of NBD1 retained resistance to 11 compounds. Finally and most interestingly, the third mutation, pdr5-4, bore a glycine-to-aspartic acid substitution in the immediate vicinity of the Walker B motif of NBD2, a highly conserved region. Standard Walker B motif is hhhhD (h stands for hydrophobic) (Schneider & Hunke 1998) and is represented by the sequence L1031VFLD1035 in Pdr5p (Balzi et al. 1994). However, the nearby residues, G1040 included, are also highly conserved in full-size transporters of the PDR family of ABC transporters, though not in those of the related families MRP/CFTR (Ycf1p and Yor1p) and MDR (Ste6p) (Fig. 2). The replacement of glycine by aspartic acid seems not to affect the functionality of Pdr5p, as is the case for pdr5-4, which retains resistance to as many as 14 out of 16 compounds.

The alterations in the extracellular loops 5 and 6 had different effects upon mutants’ phenotypes. While pdr5-8 (mutation in EL5) retained most of its resistance (to 14 out of 16 chemicals), pdr5-9 (EL6) lost its resistance to an increased number of chemicals (resistant to only 7 compounds). It is possible that the tyrosine-to-serine change in the smaller EL5 had no considerable effect upon the geometry of the drug chamber or, alternatively, upon drug release. Instead, the replacement of the polar threonine by non-polar isoleucine (T1393I) in EL6 had a more severe effect. Significantly, a similar substitution reported by Egner et al. (1998), T1460I/V1467I retained resistance to rhodamine and partially to azoles, but not to cycloheximide, results that parallel our data.

Substrate characteristics that influence Pdr5p function

An alternative angle by which Pdr5p transport function may be analyzed is what properties of the substrates are important for their detection, binding, and transport by Pdr5p. It can be assumed that a Group 2 substrate, that requires only an intact transporter for resistance, possesses some properties that are important for its recognition by wild-type Pdr5p, but are no longer ‘sensed’ by mutant proteins. This approach may help in pointing out common characteristics of these substrates and in understanding the details of the mechanism of action of Pdr5p. Some of the compounds in Group 2 seem to be tightly associated with the integrity/function of the plasma membrane: the sterol-binding compound nystatin, the sodium ionophore monensin, the potassium ionophore valinomycin, the calcium-activated potassium channel inhibitor clotrimazole, and the detergent SDS. Others are quite hydrophilic: the nucleic acid synthesis inhibitor daunomycin, the flavonoid quercetin, and the fluorescent dye rhodamine B that possesses an ionizing group. Consequently, compounds firmly associated with the membrane, or compounds that partition poorly in the phospholipid bilayer may be more difficult to be recognized, bound and exported by Pdr5p. Moreover, other hydrophilic and/or charged Group 1 substrates (e.g., cerulenin, anisomycin, cycloheximide, zwittergents) are not transported by most of the mutants.

A previous study could not find a straight relationship between Pdr5p substrate and hydrophobicity (Golin et al. 2000), while another one brought evidence that substrate size is of critical importance (Golin et al. 2003). In our small-scale study with closely related compounds we found that the ionizable rhodamine B required a completely intact protein to be exported, in contrast to rhodamine 6G and 123, which do not carry any ionizable group. In the case of the rhodamine derivatives, not their size, but their hydrophilicity (determined as logP, data not shown) seemed to matter. Similarly, in the case of tetrazolium compounds, the same rule seems applicable: the more hydrophilic tetrazolium blue required an intact Pdr5p to be exported, whereas the slightly more hydrophobic tetrazolium red was still transported by the mutant proteins. Yet, this rule is valid only for closely related compounds. When hydrophobicity of all of the investigated compounds was analyzed (data not shown), despite a slight tendency for most hydrophilic substrates to be exported only by wild-type Pdr5p, no straight correlation could be found, implying that other characteristics such as molecule size and/or distance between functional groups are important as well.

Among 10 full-size P-glycoprotein-type ABC transporter genes revealed in the yeast genome, only Pdr5p has the peculiar property of being able to respond to a wide range of drug substrates. The polyspecificity of Pdr5p may be considered to be the result of its evolution, by which the number of compounds to which it can respond was increased; a slight modification of Pdr5p structure by mutations may lead to the alteration of its ability to respond to a given set of compounds. Nevertheless, the answer to the question ‘How does a multidrug transporter protein work?’ is still far from our reach. Obviously, much more data is needed, especially at the structural level, for each specific transporter, in order for a model to be imagined.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Strains and media

S. cerevisiae W303-1 A strain (MAT a ade2 his3 leu2 trp1 ura3 can1-100), and DHR5-4C (pdr5::LEU2, otherwise isogenic to W303-1 A) (Hirata et al. 1994). For plasmid preparation, E. coli DH5{alpha} (supE44 {Delta}lacU169 ({phi}8 0 lacZ{Delta}M15) hsdR17 rec1A endA1 gryA96 thi-1 relA1) was used.

Culture media were prepared as previously described (Kaiser et al. 1994; Sambrook & Russell 2001; Sherman et al. 1979). Yeast transformation was done according to lithium acetate method (Ito et al. 1983). Plasmid isolation was done as described by Sambrook & Russell (2001).

Hydroxylamine mutagenesis and screening procedure

pBluescriptII-PDR5 plasmid containing a 5.3 kpb fragment including PDR5 ORF was randomly mutagenized using hydroxylamine as previously described (Rose & Fink 1987). Briefly, DNA was incubated with hydroxylamine, at 65 °C for 40–70 min (optimum interval to acquire 5% viability of E. coli cells transformed with the mutagenized plasmid, on ampicilin-containing medium), precipitated and transformed in E. coli. 12 000 colonies were pooled and the resulted mutagenized PDR5 bank was isolated and cut with single restriction site enzymes HindIII, BamHI, BstEII, MluI, or ClaI, which cut only inside PDR5 ORF. The resulted restriction fragments of 0.9 kpb, 1.8 kpb, 1.2 kpb, and 1.4 kpb, respectively, were used to construct chimeras with wild-type PDR5 cut accordingly. Next, the resulted plasmids were transformed in {Delta}pdr5 strain by lithium acetate method (Ito et al. 1983) and the resulted 153 transformants were screened for drug sensitivity using cycloheximide, fluphenazine, cerulenin, tautomycin, and staurosporin. The constructs that rendered colonies sensitive to at most four out of the five drugs were selected for further investigation and the plasmids were then isolated, amplified, and re-transformed into the same yeast strain for confirmation of the phenotype.

Sequencing

Sequencing of mutagenized constructs was done subsequently to their cloning into pUC119, with an ALFredTM Pharmacia Biotech automatic sequencer, by Sanger method (Sanger et al. 1977).

Site-directed mutagenesis

It was done by using Stratagene QuikChange® XL Site-Directed Mutagenesis Kit according to its instruction manual (Stratagene #200517, Stratagene, La Jolla, CA, USA). The template was a pUC19 plasmid modified to include PmeI and MluI restriction sites in its polycloning site, to which PmeI-MluI fragment of PDR5 was ligated. Mutagenic primers were 5'-GCTTGCATGCCTGCAACGCGTTGGATGAAAATCCAATTGG-3' and 5'-CCAATTGGATTTTCATCCAACGCGTTGCAGGCATGCAAGC-3', respectively, purchased from Sawady Technology. Insertion of the mutations was double-checked by restriction with BsaBI and sequencing by Sanger method (Sanger et al. 1977).

Protein isolation and Western blotting

Six x 107 cells from an overnight culture were collected by centrifugation, washed with TEG buffer (50 mM TrisHCl, pH 7.5, 1 mM EDTA, 10% glycerol, 30 mM NaCl) containing a cocktail of inhibitors (1 mM DTT, 10 mM Na3VO4, 50 mM KF, 1 mM PMSF, 2 mg/mL pepstatin, 2 mg/mL leupeptin, 1 mM EGTA), and resuspended into a mixture containing 45 µL of the same buffer-cocktail, 5 µL ß-mercaptoethanol, and 50 µL of 4 x sample buffer (0.25 M TrisHCl pH 6.8, 4% SDS, 40% glycerol, 20%ß-mercaptoethanol, 0.004% bromphenol blue). An appropriate amount of glass beads (GMB-40) was added, and cells were lyzed by vortexing 20 min at 4 °C. A 20 µL volume of the aqueous extract was applied to SDS-PAGE gel after 1 min centrifugation at 9000 g, 4 °C. SDS-PAGE and transfer to membrane were done as described before (Sambrook & Russell 2001). Polyclonal antibody against Pdr5p was a gift from Dr Karl Kuchler. For detection of Cdc28p anti-PSTAIRE antibody (Santa Cruz Biotechnology) was used.

Immunofluorescence microscopy

Immunofluorescence microscopy was performed as described elsewhere (Pringle et al. 1991), with the following modifications. Cells from an exponential culture were harvested and re-suspended in fresh YPD to 8 mL final volume, to which formaldehyde mixture was added (2.8 mL formaldehyde mixed with 1.2 mL 1 M potassium phosphate buffer, pH 6.8), and then incubated for 20 min, at 28 °C. Next, cells were collected by centrifugation, washed and re-suspended in cell wall digestion buffer (100 mM potassium phosphate buffer, pH 7.5, 1.2 M sorbitol) to final volume of 300 µL. To this suspension, a mixture containing 0.7%ß-mercaptoethanol and 7% (w/v) zymolyase T100 in digestion buffer was added, followed by incubation at 37 °C for 7 min. After digestion, cells were re-suspended in PBS, applied on multiwell slides treated with polylysine and dried, after which 10 µL 5% BSA/0.025% Nonidet-40 in PBS was applied on wells for 10 min. Following its removal, 20 µL of a 0.05 polyclonal anti-Pdr5p antibody in 5% BSA/PBS was applied and the slides were incubated at room temperature for 1 h. After thorough washing, same amount of the secondary, anti-rabbit antibody labeled with Cy3 (Sigma) 1% in 5% BSA/PBS was applied and the slides were again incubated at room temperature for 1 h, in dark. After thorough washing, the wells were covered with mounting medium containing 1 µg/mL DAPI and subjected to fluorescent microscopy.

Drug sensitivity assay

Drug sensitivity of yeast strains was tested by applying serial dilutions (start suspension: 1 x 107 cells/mL) of cell suspensions from overnight cultures, with an applicator, on to solid YPD medium containing different drugs. The plates were incubated for 1–3 days at 30 °C. The drugs, solubilized in adequate solvents (sterile water, DMSO, ethanol, or methanol) were added after the sterilized medium equilibrated at 55 °C. A23187 [GenBank] was acquired from Acros Organics; anisomycin, berberine chloride, cerulenin, clotrimazole, daunomycin, dodecyl aldehyde, dodecyl amine, dodecyl arachidate, fluphenazine, ketoconazole, n-lauroyl sarcosine, monensin, nigericin, nystatin, 1,10-phenantroline, quercetin, rhodamine B, rhodamine 6G, rhodamine 123, SDS, 2,3,5-triphenyl tetrazolium chloride (tetrazolium red), thiazolyl blue tetrazolium bromide (tetrazolium blue), trifluoperazine, triton X-15, valinomycin, zwittergents 3–10 and 3–14, from Sigma; brij 35 from INC Biomedicals Inc.; imazalil, nuarimol from Dr Ehrenstorfer GmbH; cycloheximide and triton X-114 from Nakalai Tesque; curvularol and tautomycin were gifts from H. Osada (RIKEN). Staurosporine was obtained from Kyowa Hakkou Co. Ltd. For rhodamine B and tautomycin, the pH of the YPD medium was adjusted to 4.5 with hydrochloric acid; for all the other drugs, it was left unchanged.

For quantitative assay in liquid culture, cells from overnight cultures were inoculated to the density of 1 x 105 cells/mL in fresh YPD medium containing drugs in two-fold serial dilutions, and grown for 24 h at 30 °C. OD600 was measured for 50-fold dilutions of the cultures and plotted against drug concentration. Minimal inhibitory concentration (MIC) was determined as the lowest drug concentration at which cell growth is nearly completely inhibited (Egner et al. 1998).

FACS analysis

FACS analysis was performed according to Egner et al. (2000) with one modification: dye loading was performed in the presence of 100 mM NaN3, in synthetic medium, for the first hour, and of 10 mM NaN3, in PBS, for the second hour. The procedure was performed using PBS for rhodamine 6G and twice concentrated for rhodamine B. Measurements were made using a Dickinson-Coulter flow cytometer, EXPO32 software for acquisition and analysis.


   Acknowledgements
 
We are grateful to Dr Toshihide Kakizono (Graduate School of Advanced Matters of Science, Hiroshima University) for helpful technical assistance with FACS analysis.

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to TM (no. 10217208) and a Research Grant of the Noda Institute for Scientific Research.


   Footnotes
 
Communicated by: Yoshinori Ohsumi

* Correspondence: E-mail: tmiyaka{at}hiroshima-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Balzi, E., Wang, M., Leterme, S., Van Dyck, L. & Goffeau, A. (1994) PDR5, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator Pdr1. J. Biol. Chem. 269, 2206–2214.[Abstract/Free Full Text]

van Bambeke, F., Balzi, E. & Tulkens. P.M. (2000) Antibiotic efflux pumps. Biochem. Pharmacol. 60, 457–470.[CrossRef][Medline]

Bauer, B.E., Wolfger, H. & Kuchler. K. (1999) Inventory and function of yeast ABC proteins: about sex, stress, pleiotropic drug and heavy metal resistance. Biochim. Biophys. Acta 1461, 217–236.[Medline]

Bissinger, P.H. & Kuchler, K. (1994) Molecular cloning and expression of the Saccharomyces cerevisiae STS1 gene product. J. Biol. Chem. 269, 4180–4186.[Abstract/Free Full Text]

Borges-Walmsley, M.I. & Walmsley, A.R. (2001) The structure and function of drug pumps. Trends Microbiol. 9, 71–79.[CrossRef][Medline]

Conseil, G., Decottignies, A., Jault, J.-M., et al. (2000) Prenyl-flavonoids as potent inhibitors of the Pdr5p multidrug ABC transporter from Saccharomyces cerevisiae. Biochemistry 39, 6910–6917.[CrossRef][Medline]

Conseil, G., Perez-Victoria, J.M., Jault, J.-M., et al. (2001) Protein kinase C effectors bind to multidrug ABC transporters and inhibit their activity. Biochemistry 40, 2564–2571.[CrossRef][Medline]

Davidson, A.L. (2002) Not just another ABC transporter. Science 296, 1038–1040.[Abstract/Free Full Text]

Egner, R., Rosenthal, F.E., Kralli, A., Sanglard, D. & Kuchler, K. (1998) Genetic separation of FK506 susceptibility and drug transport in the yeast Pdr5p ATP-binding cassette multidrug resistance transporter. Mol. Cell. Biol. 9, 523–543.

Egner, R., Bauer, B.E. & Kuchler. K. (2000) The transmembrane domain 10 of the yeast Pdr5p ABC antifungal efflux pump determines both substrate specificity and inhibitor susceptibility. Mol. Microbiol. 35, 1255–1263.[CrossRef][Medline]

Ferreira-Pereira, A., Marco, S., Decottignies, A., Nader, J., Goffeau, A. & Rigaud, J.L. (2003) Three-dimensional reconstruction of the Saccharomyces cerevisiae multidrug resistance protein Pdr5. J. Biol. Chem. 278, 11995–11999.[Abstract/Free Full Text]

Gish, W. (2004) (1996-2004) http://blast.wustl.edu

Golin, J., Barkatt, A., Cronin, S., Eng, G. & May, L. (2000) Chemical specificity of the PDR5 multidrug resistance gene product of Saccharomyces cerevisiae based on studies with tri-n-alkyltin chlorides. Antimicrob. Agents Chemother. 44, 134–138.[Abstract/Free Full Text]

Golin, J., Ambudkar, S.V., Gottesman, M.M., et al. (2003) Studies with novel Pdr5p substrates demonstrate a strong size dependence for xenobiotic efflux. J. Biol. Chem. 278, 5963–5969.[Abstract/Free Full Text]

Gottesman, M.M. & Pastan. I. (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 52, 385–427.[CrossRef]

Higgins, C.F. (1992) ABC transporters: from microorganisms to man. Annu. Rev. Cell. Biol. 8, 67–113.

Higgins, C.F. & Linton. K.J. (2001) The XYZ of ABC transporters. Science 293, 1782–1784.[Free Full Text]

Hirata, D., Yano, K., Miyahara, K. & Miyakawa, T. (1994) Saccharomyces cerevisiae YDR1, which encodes a member of the ATP-binding cassette (ABC) superfamily, is required for multidrug resistance. Curr. Genet. 26, 285–294.[CrossRef][Medline]

Hu, W., Feng, Q., Palli, S.R., Krell, P.J., Arif, B.M. & Retnakaran, A. (2001) The ABC transporter Pdr5p mediates the efflux of non-steroidal ecdysone agonists in Saccharomyces cerevisiae. Eur. J. Biochem. 268, 3416–3422.[Medline]

Ishikawa, T., Tsuji, A., Inui, K., et al. (2004) The genetic polymorphisms of drug transporters: functional analysis approaches. Pharmacogenomics 5, 67–99.[CrossRef][Medline]

Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168.[Abstract/Free Full Text]

Kaiser, C., Michaelis, S. & Mitchell. A. (1994) Methods in Yeast Genetics – a Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Kolaczkowski, M., Kolaczkowska, A., Luczynski, J., Luczynski, J., Witek, S. & Goffeau, A. (1998) In vivo characterization of the drug resistance profile of the multidrug ABC transporters and other components of the yeast pleiotropic drug resistance network. Microb. Drug Resist. 4, 143–158.[Medline]

Kolaczkowski, M., van der Rest, M., Cybularz-Kolaczkowska, A., Soumillion, J.P., Konings, W.N. & Goffeau, A. (1996) Anticancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p. J. Biol. Chem. 271, 31543–31548.[Abstract/Free Full Text]

Kralli, A., Bohen, S.P. & Yamamoto, K.R. (1995) LEM1, an ATP-binding-cassette transporter, selectively modulates the biological potency of steroid hormones. Proc. Natl. Acad. Sci. USA 92, 4701–4705.[Abstract/Free Full Text]

Leonard, P.J., Rathod, P.K. & Golin. J. (1994) Loss of function mutation in the yeast multiple drug resistance gene PDR5 causes a reduction in chloramphenicol efflux. Antimicrob. Agents Chemother. 38, 2492–2494.[Abstract/Free Full Text]

Loo, T.W. & Clarke, D.M. (1994a) Functional consequences of glycine mutations in the predicted cytoplasmic loops of P-glycoprotein. J. Biol. Chem. 269, 7243–7248.[Abstract/Free Full Text]

Loo, T.W. & Clarke, D.M. (1994b) Mutations to amino acids located in predicted transmembrane segment 6 (TM6) modulate the activity and substrate specificity of human P-glycoprotein. Biochemistry 33, 14049–14057.[CrossRef][Medline]

Loo, T.W. & Clarke, D.M. (1995a) Membrane topology of a cysteine-less mutant of human P-glycoprotein. J. Biol. Chem. 270, 843–848.[Abstract/Free Full Text]

Loo, T.W. & Clarke, D.M. (1995b) Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug-stimulated ATPase activity. J. Biol. Chem. 270, 22957–22961.[Abstract/Free Full Text]

Loo, T.W. & Clarke, D.M. (1996) Inhibition of oxidative cross-linking between engineered cysteine residues at positions 332 in predicted transmembrane segments (TM) 6 and 975 in predicted TM12 of human P-glycoprotein by drug substrates. J. Biol. Chem. 271, 27482–27487.[Abstract/Free Full Text]

Loo, T.W. & Clarke, D.M. (1997) Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators. J. Biol. Chem. 272, 709–712.[Abstract/Free Full Text]

Moody, J.E., Millen, L., Binns, D., Hunt, J.F. & Thomas, P.J. (2002) Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding transporters. J. Biol. Chem. 277, 21111–21114.[Abstract/Free Full Text]

Neyfakh, A.A. (2002) Mystery of the drug transporters: the answer can be simple. Mol. Microbiol. 44, 1123–1130.[CrossRef][Medline]

Pringle, J.R., Adams, A.E.M., et al. (1991) Immunofluorescence methods for yeast. In: Method in Enzymology 194: Guide to Yeast Genetics and Molecular Biology (eds C. Guthrie, G. R. Fink, J. N. Abelson & M. I. Simon), pp. 565–602. San Diego: Academic Press.

Rose, M.D. & Fink, G.R. (1987) KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell 48, 1047–1060.[CrossRef][Medline]

Sambrook, J. & Russell. D.W. (2001) Molecular Cloning – a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Sanger, F., Nicklen, S. & Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467.[Abstract/Free Full Text]

Schneider, E. & Hunke, S. (1998) ATP-binding cassette transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains. FEMS Microbiol. Rev. 22, 1–20.[CrossRef][Medline]

Sherman, F., Fink, G.R. & Lawrence, C.W. (1979) Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

van Veen, H.W., Higgins, C.F. & Konings, W.N. (2001) Multidrug transport by ATP binding cassette transporters: a proposed two-cylinder engine mechanism. Res. Microbiol. 152, 365–374.[Medline]

Received: 8 December 2004
Accepted: 24 January 2005




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