A Role for Ectophosphatase in Xenobiotic Resistance · Robert Dudler, b Alan Lloyd, a and Stanley J. Roux a,3 a Section of Molecular Cell and Developmental Biology, Biological Laboratories

The Plant Cell, Vol. 12, 519–533, April 2000, www.plantcell.org © 2000 American Society of Plant Physiologists

A Role for Ectophosphatase in Xenobiotic Resistance

Collin Thomas,

a,1,2

Asha Rajagopal,

a,1,2

Brian Windsor,

a

Robert Dudler,

b

Alan Lloyd,

a

and Stanley J. Roux

a,3

a

Section of Molecular Cell and Developmental Biology, Biological Laboratories 311, University of Texas at Austin, Austin, Texas 78713

b

Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland

Xenobiotic resistance in animals, plants, yeast, and bacteria is known to involve ATP binding cassette transporters thatefflux invading toxins. We present data from yeast and a higher plant indicating that xenobiotic resistance also involvesextracellular ATP degradation. Transgenic upregulation of ecto-ATPase alone confers resistance to organisms thathave had no previous exposure to toxins. Similarly, cells that are deficient in extracellular ATPase activity are more sen-sitive to xenobiotics. On the basis of these and other supporting data, we hypothesize that the hydrolysis of extracellu-lar ATP by phosphatases and ATPases may be necessary for the resistance conferred by P-glycoprotein.

INTRODUCTION

Multidrug resistance (MDR) is a biological phenomenoncommon to many organisms in which cells have become re-sistant to various toxins. Past studies of MDR have generallyemphasized the role of the chemical transporters involved inthe efflux of toxins. One set of transporters upregulated inthe MDR response are P-glycoproteins, and the genes en-coding P-glycoproteins are capable of conferring drug resis-tance to drug-sensitive cell lines (Kolackzkowski et al.,1996). Competition assays involving reconstituted P-glyco-protein–containing vesicles have shown that toxins are pref-erentially removed from the cell by this class of transporter(Sharom et al., 1993). Members of the ATP binding cassette(ABC) superfamily, P-glycoproteins are encoded by the

MULTIDRUG RESISTANCE 1

(

MDR1

) genes and are nowknown to exist in numerous organisms. P-glycoproteins trans-port various heterocyclic, aromatic, nitrogen-containing com-pounds (Gros and Hanna, 1996), and they do so in an ATP-dependent fashion (Sharom et al., 1993).

Since the

MDR1

gene was first cloned in 1985, severalmodels have been proposed to explain the mode of actionof P-glycoprotein. Most of these mechanisms suggest thatATPase activity is essential for moving drugs across theplasma membrane. In the most widely proposed model,MDR1 acts as a drug pump, utilizing the free energy of ATPhydrolysis to actively transport a toxic substrate outside thecell (Gros and Hanna, 1996). This same mode of transporthas been suggested for a few different types of MDR pro-teins (Chang et al., 1998; Decottignies et al., 1998). Other

models suggest, however, that drug resistance involvesmore than the MDR1 protein acting as a pump. Crystallo-graphic evidence suggests that eukaryotic P-glycoproteinsmay be a chimeric form of pump channel (Welsh et al., 1998)that uses ATP hydrolysis to gate the drug conduit.

Some evidence indicates that P-glycoprotein and possi-bly other ABC proteins can mediate the release of ATP fromcells (Abraham et al., 1993; Roman et al., 1997) and thatATP release from yeast cells (Boyum and Guidotti, 1997a)possibly occurs through indirect action of an ABC trans-porter (Boyum and Guidotti, 1997b). We present data foryeast and plants to indicate that a plant MDR1 homolog maytransport ATP and that extracellular ATP concentrations arekept at a low steady state level by ecto-ATPases. Destroy-ing cell surface ATPase activity, through either chemical in-hibition or genetic deletion, results in the loss of xenobioticresistance both in yeast and in plants. Conversely, upregu-lating extracellular ATPase activity confers the MDR state.Our results indicate that ecto-ATPases are a second compo-nent of xenobiotic resistance and thus present a novel classof targets for understanding and manipulating resistance.

RESULTS

A Plant P-Glycoprotein Gene Expressed in Yeast Promotes ATP Release into the Culture Medium

To test previous claims that

MDR1

is itself involved in re-lease of ATP from cells, we used an

MDR1

homolog clonedfrom Arabidopsis and

known as

atPGP1

(Dudler and Hertig,1992). We chose this gene because the protein it encodesresembles mammalian P-glycoproteins and has been

1

These authors contributed equally to this work.

2

Current address: The Rockefeller University, 1230 York Ave., NewYork, NY 10021.

3

To whom correspondence should be addressed. E-mail [email protected]; fax 512-232-3402.

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520 The Plant Cell

successfully manipulated in previous genetic studies involv-ing Arabidopsis (Sidler et al., 1998).

When we introduced the Arabidopsis

MDR1

cDNA intoyeast by transformation, the ATP concentration in the extra-cellular fluid produced by the

MDR1

-transformed INVSC1strain was greater than in the untransformed yeast, as seenin Figure 1A. Although untransformed yeast cells have been

shown to release ATP into the extracellular fluid (Boyum andGuidotti, 1997a), presumably through protein-mediatedtranslocation (Boyum and

Guidotti, 1997b), our P-glycopro-tein transformants had twice the steady state extracellularATP concentration of wild-type cells, in both the presenceand absence of glucose. Previous evidence has indicatedthat ATP translocation is mediated by ABC proteins in some

Figure 1. Luciferase Assays of Extracellular ATP and Adenosine Pulse–Chase in Experiments with Yeast.

(A) The steady state concentration of ATP in the extracellular fluid was doubled in wild-type yeast overexpressing the Arabidopsis P-glycopro-tein, denoted MDR1. Data shown are the average of four experiments in which samples were taken every hour for 5 hr and averaged (6SEM).Data are reported as an ATP concentration calculated from a relative light units–based standard curve. xATP, ATP found in the extracellular ma-trix; + or – glucose, the presence or absence of glucose in the assay solution, respectively; Mock, the INVSC transformed with vector alone.(B) The yeast ectophosphatase mutant YMR4 accumulated ATP in the extracellular fluid. At bottom, the same yeast strains placed in acid-buff-ered NBT/BCIP show differential extracellular acid phosphatase activity. Activity is indicated by the development of a dark color along the sideof the microcentrifuge tube in which the cells are pelleted. Wt, wild type.(C) The yeast phosphatase mutant YMR4 accumulated six times the amount of extracellular ATP when the plant P-glycoprotein, denoted MDR1,was overexpressed. pvt101 is a mock transformant. Averages (6SEM) of four experiments are shown.(D) Early differential efflux of ATP in cells overexpressing MDR1. The graph plots the ratio of labeled extracellular adenosine to labeled intracel-lular adenosine during a pulse–chase in wild-type (INVSC) or mutant (YMR4) yeast cells transformed with either Arabidopsis MDR1 or pvt101(vector only). Cells were labeled with 3H-adenosine for 20 min before the chase was started. Experiments were performed three times with simi-lar results.

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Ectophosphatase and Xenobiotic Resistance 521

cell types, such as the ocular epithelium (Mitchell et al.,1998), whereas other data suggest that P-glycoproteinfunctions as an ATP efflux channel in mammalian systems(Abraham et al., 1993; Roman et al., 1997). Our data suggestthat the plant homolog functions in ATP efflux in nonmam-malian systems as well.

Despite the fact that the yeast cells transformed with

MDR1

and the plasmid-transformed wild type both effluxATP to some degree, neither strain seems to accumulate in-creased ATP concentrations in the medium over time. It hasbeen suggested that the concentration of extracellular ATPat steady state is kept very low through the action of ecto-ATPases and ectophosphatases (Boyum and Guidotti,1997a). To test whether the very low extracellular ATP atsteady state in yeast is the result of ATP hydrolysis outsidethe cell, we examined cell culture ATP accumulation in ayeast strain that is deficient in extracellular phosphatase ac-tivity. The yeast mutant YMR4 fails to produce the two se-creted acid phosphatases encoded by the

PHO3

and

PHO5

genes (Vogel and Hinnen, 1990) and has almost no ecto-phosphatase activity at pH 5.0, as evidenced by the failureof nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phos-phate (NBT/BCIP) to precipitate on the surface of YMR4cells (Figure 1B, bottom). If phosphatase activity regulatesthe concentration of extracellular ATP, then a yeast deficientin two extracellular acid phosphatases could be expected toaccumulate more extracellular ATP than would the wild type.

Figure 1B (top) shows that the steady state extracellularATP content for the INVSC1 cell line is

,

10 pmol per 3

3

10

6

cells, whereas that for the YMR4 cells is as much as 10times that amount. Although INVSC differs from YMR4 ge-notypically, we have used INVSC as a background for com-parison with YMR4 because the amount of extracellular ATPseen in the INVSC cells,

z

3

3

10

2

18

mol per cell, is nearlythe same as that previously measured for the parent cell lineused to make the YMR4 null, 2.7

3

10

2

18

mol per cell(Boyum and Guidotti, 1997a). When the YMR4 strain istransformed with

MDR1

, the cell line accumulates extracel-lular ATP more quickly by an order of magnitude than doesINVSC (Figure 1C). The YMR4/MDR1 transformants, releas-ing ATP at a greater rate through their P-glycoprotein, alsofail to hydrolyze that ATP; with ATP turnover blocked, theirextracellular concentrations of ATP rise.

To ensure that the measured extracellular ATP was notthe result of cell death, we conducted viability assays of allcells used, and all cell cultures were found to contain atleast 95% viable cells. Cell death itself is unlikely to havebeen the major source of ATP because variances in viabilitydid not always correlate with extracellular ATP abundance.This observation seems counterintuitive because a simplecalculation reveals that only 180 pmol of ATP could be gen-erated per lysed cell. The 600 pmol of transport measuredfor the cells without extracellular phosphatase activity ex-pressing the plant MDR1 is actually more ATP than wouldbe available by lysis alone. Thus, implied in this observationis the potential to have more ATP outside of the cell than

inside.

That idea is easily accommodated if the cells arecontinuously generating ATP. A series of pulse–chase ex-periments seemed to validate this observation.

The previous experiments allowed us to measure externalATP over a period of hours; to learn about the flow of ATPthrough the plasma membrane over a much shorter period,we performed a series of pulse–chase experiments. Yeastcells were grown in a medium containing tritiated adeno-sine, and ATP efflux was measured as a function of the ac-cumulation of labeled ATP in the cell supernatant over a 40-min chase period. When these pulse–chase experimentswere performed in YMR4 cells transformed with

MDR1

,more than half of the labeled adenylate (presumably ATP)was effluxed in the first 30 min (Figure 1D). At the acidic pHof the media, the acid phosphatase mutants would haveminimal ectophosphatase activity and would not be able toparticipate in the ATP dephosphorylation steps necessaryfor internalizing the purine (Che et al., 1992). The phos-phatase mutants therefore would accumulate extracellularATP—and hence label.

In the first 40 min of the efflux assay, more labeled adeno-sine is outside of the cell than inside. Given that the YMR4cells are not energy or nutrient limited when growing in com-plete supplemented medium plus 2% glucose, and giventhat the extracellular ATP measured represents an hourlyaccumulation, perhaps it is possible to have more extracellularATP than the amount that would be instantaneously avail-able when lysis is complete. Similar calculations using theATP efflux data in YMR4 from Boyum and Guidotti (1997a)yielded 3.4

3

10

–18

mol of ATP per cell, whereas we found3.3

3

10

–17

mol of ATP per cell in YMR4/pvt101. The differ-ence in our measurements is probably related to the differ-ence in procedures used: their freon extraction followed byHPLC versus our luminometry of raw media. Irrespective ofthe differences in measurement, our data corroborate thelarge

amounts of ATP in the extracellular matrix first re-ported by Boyum and Guidotti. In contrast to YMR4, wild-type lines are able to hydrolyze the ATP and internalize theadenosine; their extracellular ATP concentrations weretherefore correspondingly lower in our pulse–chase experi-ments. Indeed, after chasing in media for 3 hr, we detectedvery little radioactive adenosine in the extracellular fluid ofwild-type cells. The adenosine had apparently been re-couped by the yeast because nearly all the label wasdetected in the cell pellets and had presumably been incor-porated into nucleic acids. These results suggest that aplant MDR1 can efflux ATP across a yeast plasma mem-brane and that ectophosphatases dissipate the accumula-tion of ATP in the extracellular fluid.

ATP Efflux and Ectophosphatase Activity Correlate Positively with the MDR Phenotype

If ATP efflux is essential to the xenobiotic-extruding activi-ties of cells, then we would expect a strong correlation

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522 The Plant Cell

between the ability of a cell to efflux ATP and its resistanceto xenobiotics. Furthermore, we would expect that cells un-able to regulate extracellular ATP accumulation might like-wise show altered xenobiotic tolerance. For that reason, wewould expect to see some correlation between the resis-tance phenotype and ectophosphatase activity.

To determine whether a cell’s ability to efflux, and subse-quently degrade, ATP is functionally related to xenobiotic re-sistance, we tested the various yeast cell lines for theirresistance to cytotoxic compounds. Cycloheximide and ni-gericin were the toxic agents chosen, both being substratesof a functional

MDR1

homolog in yeast (Kolaczkowski et al.,1996; Decottignies and Goffeau, 1997). Cycloheximide is apotent translation poison; nigericin is a membrane-targetedprotonophore. Replication-targeting toxins were not used,which allowed us to obviate the difficulties encountered withmutagenic agents, such as topoisomerase poisons, thatmight increase mutation frequencies enough to make quan-tification of engineered MDR difficult. The results of experi-ments with these two toxins revealed a strong correlationbetween resistance and ATP efflux and subsequent ATP hy-drolysis.

The YMR4 mutant that was mock-transformed withoutthe plant

MDR1

had the lowest rates of ATP efflux and ex-tracellular ATP hydrolysis; it was also the most sensitive tocycloheximide and nigericin. As shown in Figure 2, the ecto-phosphatase mutant took nearly 5 days of continuous cul-ture in cycloheximide to develop resistance, compared with2 to 3 days for the wild type. When the YMR4 cell line wastransformed with

MDR1

, however, its resistance to the tox-ins increased, as did its measured rate of ATP efflux (Figures2A and 1C). The MDR1-expressing YMR4 cell line was resis-tant whether the toxins were administered separately or to-gether (data not shown). Indeed, its growth rate was mostcomparable to that of the wild-type yeast, for both reachedstationary phase within 2 or 3 days of growth. When thewild-type cell line was transformed with

MDR1

, its ATP ef-flux rate increased (Figure 1A) and so did its resistance totoxins (Figure 2). The wild-type

MDR1

transformant, with itsectophosphatase activity intact and its ATP efflux rate aug-mented, was the line most resistant to cycloheximide andnigericin. Thus, the cell lines were resistant in the same or-der as their increasing MDR1 abundance and ectophos-phatase activity.

Because resistance seems to correlate with a cell’s abilityto efflux ATP and degrade it extracellularly, we wonderedwhether cells with increased MDR1 and normal ectophos-phatase function would become sensitive if the toxin werecoadministered with exogenous ATP. In other words, couldthe addition of extracellular ATP to the medium affect theability of a cell to extrude toxins? Likewise, could the addi-tion of inhibitors of ecto-ATPase activity result in a diminu-tion of resistance?

Wild-type cells transformed with

MDR1

showed an in-creased sensitivity to toxin when extracellular ATP was in-creased. In fact, all cell lines showed decreased resistance

to cycloheximide in the presence of excess extracellularATP (Table 1). No cell line was able to grow in the presenceof 0.1 M ATP and cycloheximide (OD

660

of 0.00 after 4 daysof continuous culture). However, when grown in 0.1 M ATPalone, all cell lines were able to reach stationary growthwithin 3 days, indicating that ATP alone, even at very highconcentrations, is not toxic to the yeast. This concentrationis at least 25 times greater than the intracellular ATP con-

Figure 2. Toxin Resistance in Yeast.

(A) Resistance to cycloheximide and nigericin in wild-type (INVSC)and in ectophosphatase-deficient yeast strains. At left, strainsgrown in the absence of any added toxin; at center, strains grown inthe presence of nigericin; and at right, strains grown in the presenceof cycloheximide. Clockwise, starting at the top: wild type/MDR1,YMR4/vector, YMR4/MDR1, and wild type/vector. Experimentswere performed at least five times with the same result.(B) Turbidity measurements of the growth in nigericin-containingmedium of the yeast cell lines used in (A). Experiments were per-formed at least five times with similar results.(C) Turbidity measurements of the growth in cycloheximide-contain-ing medium of the yeast phosphatase mutant expressing the plantMDR1 as well as of the same strain transformed with the vectoralone. Experiments were performed at least five times with similarresults.

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centration. At one order of magnitude less ATP, 12 mM, the

MDR1-

transformed YMR4 cell line experienced 91% growthinhibition in the presence of cycloheximide (Table 1),whereas the same concentration of ATP and cycloheximideresulted in only 58% growth inhibition in the INVSC strain. Apriori, it is difficult to estimate what would be a meaningfulextracellular ATP concentration in the yeast periplasm, butthe amount of extracellular ATP required to abrogate resis-tance conferred by

MDR1

appears to be strictly dependenton the presence of extracellular phosphatases; the less ec-tophosphatase activity a cell line has, the more susceptibleits resistance to extracellular ATP is. Nonetheless, the con-centration required to reverse resistance exceeds the ex-pected intracellular ATP concentration.

When cycloheximide was added in the presence of vana-date, an alkaline phosphatase inhibitor, cell lines becamesensitized to the toxin (Table 1). Sensitivities correspondedto the ectophosphatase activity of each cell line: both YMR4cell lines experienced marked reductions in growth, but thewild-type (INVSC) lines were practically unaffected. The highinhibition of growth in the acid phosphatase mutant is mostprobably the result of inhibition of the other ectophos-phatases. The concentration of vanadate used in thesestudies is unlikely to have reversed MDR by directly inhibit-ing MDR1 because the INVSC cell lines transformed with

MDR1

showed minimal growth inhibition in the presence oftoxin and vanadate (Table 1). The wild-type lines were lesssensitive to vanadate because they retained strong acidphosphatase activity. Like the trends for survival on nigericinand cycloheximide, growth inhibition seems to be a functionof MDR1 abundance and ectophosphatase activity. Celllines expressing MDR1 show a greater sensitivity to phos-phatase inhibitors and exogenous applied ATP.

These experiments demonstrate that extracellular ATPconcentrations affect the ability of yeast to extrude xenobi-otics. Yeast cells must carefully regulate the ATP in their ex-tracellular fluid to fully utilize either their endogenous MDRproteins (Kolaczkowski et al., 1996; Carvajal et al., 1997;Nourani et al., 1997) or those introduced by genetic manipu-lation.

Investigations of the Nucleotide Salvage Model

Previous studies have also implicated extracellular nucle-otide hydrolysis in the MDR phenomenon. For example, leu-kemia cells upregulate ecto-5

9

-nucleotidase activity when inthe presence of doxorubicin, even before upregulating P-gly-coprotein activity (Pietkiewicz et al., 1998). These ecto-5

9

-nucleotidases hydrolyze nucleotide monophosphates to theircorresponding nucleosides, a step necessary for transport-ing nucleosides back into the cell. Because MDR tumor cellsextrude ATP as part of their resistance mechanism, perhapsthe increased abundance of the ecto-5

9

-nucleotidase al-lows the cells to replenish their ATP supply by salvagingadenosine (Ujhazy et al., 1994, 1996). If the intracellular ATP

pool of the MDR cell is diminished enough to affect viability,then cells able to salvage their ATP losses in this way wouldhave increased viability. This nucleotide salvage model thuscouples ectophosphatase activity with stabilizing the intra-cellular energy charge of the MDR cell; the model is sug-gested by the observation that AMP added exogenouslywith MDR toxins decreases cell sensitivity to the toxins(Ujhazy et al., 1994). In this model, MDR cells would hydro-lyze their exogenous AMP to remedy their adenylate deficit(Ujhazy et al., 1996).

According to the salvage model, YMR4 cells would bemore sensitive to toxins because the cells are unable to re-plenish their intracellular energy charge. With diminished ec-tophosphatase activity, the YMR4 mutant would not be ableto recoup as much of the ATP effluxed from its active MDRproteins (whether native or transformed), and the cellswould then be less viable in xenobiotic than in wild-typecells. To test this alternative hypothesis, we cultured theYMR4 cell line and its

MDR1

transformant in ATP hydrolysisproducts to determine whether the sensitivities of thesecells would diminish. According to the salvage model, thesehydrolysis products should obviate the need for ectophos-phatase activity and render the YMR4 mutants able to par-ticipate in nucleotide salvage to the same extent as the wild-type strain.

When the cycloheximide-containing medium was supple-mented with adenosine, however, the YMR4 mock transfor-mant still showed diminished growth, and the YMR4

MDR1

transformants did not experience any growth enhancement(Table 2). Likewise, phosphate, whether added alone or inthe presence of adenosine, did nothing to decrease the xe-nobiotic sensitivities of these two cell lines. Controls confirmthat cells are able to grow in medium alone as well as ineach of these ATP hydrolysis products. Therefore, it is un-likely that the ectophosphatase mutants experienced dimin-ished growth in cycloheximide because of a deficiency in anucleotide salvage pathway.

Table 1.

Cycloheximide Sensitivity Conferred by Phosphatase Inhibition or Increased Extracellular ATP

a

Percentage of Growth Inhibitionin Cycloheximide

Cell Line

1

125

m

M Vanadate

1

12 mM ATP

YMR4/pvt101 60 57YMR4/MDR1 85 91INVSC/pvt101 6 65INVSC/MDR1 2 58

a

YMR4 and INVSC cells were transformed with the pvt101 vectorcontaining the Arabidopsis MDR1 or with the vector alone. Cellgrowth was measured at 96 hr in duplicate by optical density at 660nm and expressed as a percentage of the difference in growth of thesame cells in cycloheximide alone, where 100% is complete inhibi-tion.

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524 The Plant Cell

AtPGP1 Complements a Pdr5 Null

We have thus far demonstrated that the ectophosphatasenull yeast strain cannot become multidrug resistant and thatthe Arabidopsis P-glycoprotein

enhances the ability of themutant to acquire drug resistance. We next wanted to deter-mine whether AtPGP1 functions in yeast analogously to na-tive yeast drug resistance mechanisms. We thereforetransformed the cDNA encoding the plant MDR1 into ayeast strain deficient in an ABC transporter that contributesto the MDR phenotype in yeast. Pdr5p (for pleiotropic drugresistance protein 5) promotes the resistance phenotypewhen overexpressed in yeast cells (Egner et al., 1995). Aplasma membrane–localized protein, Pdr5p has been char-acterized as a drug efflux pump responsible for sporides-min and cycloheximide resistance in yeast (Bissinger andKuchler, 1994). However, like the human MDR1, the yeastPdr5p confers resistance toward several structurally unre-lated compounds, including anticancer drugs such as duan-orubicin, doxorubicin, and tamoxifen (Kolaczkowski et al.,1996). Accordingly,

pdr5

null mutants have increased sensi-tivity to several xenobiotics, including cycloheximide.

When we transformed the MDR1 cDNA into YKKA-7, the

pdr5

null mutant, we found that the MDR1-expressing linegained resistance to cycloheximide, whereas the

pdr5

nullmock-transformed line remained sensitive to the toxin (Fig-ure 3). However, the growth of the

MDR1

-transformed

D

pdr5 line in cycloheximide was comparable to that of thewild-type parent strain YPH499. Unlike the phosphatase nullmutants, the

pdr5

null strain was never able to grow in the

presence of cycloheximide unless complemented with theplant

MDR1

gene. Similar growth trends were seen in thepresence of nigericin (data not shown). When grown in me-dia alone, however, the

pdr5

null mock-transformed straingrew at least as well as the

MDR1

-transformed counter-parts, and both null mutants grew comparably to their par-ent strain (Figure 3).

MDR1 Expression Affects Concentrations of Extracellular ATP

The MDR phenomenon is common to many different organ-isms, and

MDR1

functional homologs are found in plantsand mammals. If ATP efflux and hydrolysis are necessary forresistance, then they should be characteristic of the MDRstate in other organisms as well. To determine whether thepositive correlation between MDR1 overexpression and ex-tracellular ATP accumulation was evident in other systems,we studied Arabidopsis

transformants overexpressing MDR1under a strong constitutive viral promoter (Sidler et al.,1998).

We first assayed the leaf surfaces of MDR1-overexpress-ing plants (Sidler et al., 1998) for extracellular ATP, using thewild-type Arabidopsis as a point of comparison. The assayof extracellular ATP takes advantage of the fact that Arabi-dopsis can be grown in sterile culture under conditions hu-mid enough to suppress cuticle development. On suchplants, a droplet of buffer added to the leaf surface mixeswith the extracellular matrix fluid. When the droplet is re-moved, some fluid from the extracellular matrix is removedwith it, and the droplet can be measured for its ATP content.After assaying the plants in this way, we found that MDR1expression correlated with higher concentrations of extra-cellular ATP. Plants overexpressing MDR1 had as much astwo to three times the amount of extracellular ATP on theleaf surfaces as did wild-type plants (Figure 4), and thisamount was statistically significant in two of five differentlines tested.

These data suggested that the Arabidopsis

MDR1

gene,similar to the human

MDR1

gene, promotes the efflux ofATP. Moreover, because both Arabidopsis and yeast linesonly doubled their extracellular ATP concentrations whentransformed with

MDR1

, we see that both organisms haveseveral highly active extracellular phosphatases maintainingthe steady state extracellular ATP content, albeit at twicethe normal extracellular ATP concentration.

Overexpressed Apyrase or MDR1 Confers Xenobiotic Resistance in Arabidopsis

In yeast, we found that extracellular ATP regulation was es-sential to the xenobiotic-resistant state; cells unable to regu-late their extracellular ATP concentrations lost their MDR.We wondered whether this relationship would be seen in Ar-

Table 2.

Yeast Drug Sensitivity in the Presence of High Levels of ATP Hydrolysis Products

a

Without Cycloheximide With Cycloheximide

Product Addedand Cell Line Turbidity

Product Addedand Cell Line Turbidity

No addition No additionYMR4/MDR1 1.376 YMR4/MDR1 0.937YMR4/pvt101 1.429 YMR4/pvt101 0.001

Phosphate PhosphateYMR4/MDR1 1.351 YMR4/MDR1 0.541YMR4/pvt101 1.341 YMR4/pvt101 0.001

Adenosine AdenosineYMR4/MDR1 1.319 YMR4/MDR1 0.632YMR4/pvt101 1.354 YMR4/pvt101 0.002

Adenosine and Pi Adenosine and PiYMR4/MDR1 0.899 YMR4/MDR1 0.389YMR4/pvt101 1.342 YMR4/pvt101 0.001

a

YMR4 cells were transformed with the pvt101 vector containing theArabidopsis MDR1 and are designated YMR4/MDR1, or YMR4 cellswere transformed with the vector alone and are designated YMR4/pvt101. Cell growth was assayed by optical density at 660 nm andmeasured in triplicate after 96 hr.

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abidopsis

as well. Therefore, we examined for xenobioticsensitivity the plants overexpressing MDR1, and we com-pared their toxin resistance with that in plants overexpress-ing ectoapyrase, an ecto-ATPase with strong hydrolyticcapacity. Most eukaryotes, except for yeast, use ectoapy-rases for the bulk of their ectophosphatase activity. Apy-rases are characterized by their ability to hydrolyze both the

g

-phosphate and

b

-phosphate on ATP and ADP by theirneed for divalent cations, and by their failure to be inhibitedby any known class of phosphatase inhibitors (Plesner,1995). Most apyrases are expressed as plasma membrane–associated proteins, with their hydrolytic activity facing outinto the extracellular matrix (Wang and Guidotti, 1996). Theapyrase we used is derived from pea and localizes in a man-ner consistent with other apyrases (Thomas et al., 1999).

When we transformed a wild-type Arabidopsis line with

the pea apyrase, we observed an increased resistance tocycloheximide, an increase also seen in the MDR1-overex-pressing plants (Figures 5A to 5D). Because neither set oftransgenics had prior exposure to the toxin, their endoge-nous resistance mechanisms had not been upregulated.Therefore, the transgenic plants could have gained resis-tance only as a result of the gene product introduced, thatis, through the addition of MDR1 or the ectoapyrase. Theseresults with Arabidopsis are consistent with our earlier find-ings in yeast. The ATP efflux and ATP hydrolysis were seento be functionally related to xenobiotic resistance. In yeast,mutants lacking ectophosphatase activity became sensitiveto toxins; in plants, transgenics overexpressing ecto-ATPaseactivity became resistant to them.

When Arabidopsis

seeds were exposed to cycloheximide,the germination rate of those overexpressing apyrase was

Figure 3. Complementation of the Yeast Mutant Dpdr5 by the Plant MDR1.

YKKA-7 is the Dpdr5 strain; YPH499 is the wild-type isogenic strain used to make YKKA-7; pvt101 is the empty expression cassette; and MDR1is the plant P-glycoprotein.(A) The growth of strains on 500 ng/mL cycloheximide. Clockwise, starting at the top: YKKA-7/pvt101, YPH499/pvt101, YPH499/MDR1, andYKKA-7/MDR1.(B) Turbidity measurements of the same cell lines grown in cycloheximide. Pdr5p-deficient cells were sensitive to cycloheximide, whereas cellsexpressing the Arabidopsis P-glycoprotein were resistant. Experiments were performed at least five times with similar results.(C) and (D) Growth of each strain in the absence of cycloheximide to control for growth phenotypes related to expression of the plant MDR1 andfailed expression of the yeast Pdr5p.

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526 The Plant Cell

almost double that of seeds overexpressing MDR1 (Figure5D). If ATP efflux is a mechanism used by other, as yet un-characterized, xenobiotic transporters, then overexpressingthe ectoapyrase may have heightened the activity of thesetransporters, thus enhancing their efficacy. Conversely, dis-rupting the ecto-ATPase activity of plant cells served toabolish toxin resistance. When an extracellular ATPaseinhibitor (

a

,

b

-methyleneadenosine 5

9

-diphosphate) wassimultaneously administered with cycloheximide, MDR1-overexpressing plants lost their resistance and became assensitive to the toxin as wild-type plants in a germination-based assay (Figure 5E). Because the ATPase inhibitor itselfdoes not enter the cell, the sensitization of MDR1-express-ing plants was not caused by an abrogation of the MDR1protein ATPase activity. The reversal of resistance was con-sistent with the dissipation of native ecto-ATPase activity.

In separate experiments, both MDR1 and apyrase trans-formants showed increased resistance to toxic concentra-tions of plant growth regulators such as cytokinin. Thecytokinin

N

6

-(2-isopentenyl)adenine (2IP), which itself meetsthe loose chemical criteria for an MDR1 substrate, severelystunts plant growth at high concentrations. Relative to theirgrowth on a hormone-free medium (Figure 6A), wild-type Ar-abidopsis plants showed stunted growth or failed to germi-nate in the presence of 1 mM 2IP (Figures 6B and 6D).Arabidopsis lines overexpressing either MDR1 or ectoapy-rase, however, were much less stunted in their growth (Fig-ures 6B and 6C). Furthermore, at concentrations thatinhibited root elongation in wild-type plants, MDR1-express-ing plants had long roots (Figures 6C and 6E). The resis-tance of the transformed plants to cycloheximide and 2IPwas specific insofar as the overexpressing lines did notgrow any more robustly than the wild type when toxins werenot present.

Taken together, these experiments demonstrate that ex-tracellular ATP hydrolysis may contribute to xenobiotic re-

sistance as does MDR1; indeed, an increase in either canconfer toxin resistance in Arabidopsis. More importantly,studies in Arabidopsis confirm the findings in yeast, sug-gesting that ATP efflux and hydrolysis are part of a moregeneral mechanism of xenobiotic resistance.

Phosphatase Activity Is Itself Crucial in the Development of MDR in Yeast

If extracellular ATP hydrolysis is necessary for establishingcellular toxin resistance, then ectophosphatase activitycould be part of an endogenous mechanism utilized whencells are first introduced to MDR substrates. Using the NBT/BCIP assay for measuring ectophosphatase activity in livingyeast cells (Thomas et al., 1999), we tested whether yeastcells showed increased phosphatase activity when first in-troduced to xenobiotics, as seen in Figure 7. All of the yeaststrains were sequentially tested for phosphatase activity af-ter being cultured in cycloheximide or nigericin for 3 days.Both INVSC mock transformants and INVSC MDR1-expressingcells showed increased extracellular acid phosphatase ac-tivity when cultured in nigericin and in cycloheximide (Figure7A). The YMR4 mock transformants could not be tested be-cause they were unable to grow in cycloheximide. Althoughthe YMR4 MDR1-expressing cells could be cultured in sev-eral xenobiotics, they showed no observable phosphataseactivity, whether cultured in plain medium or in the presenceof toxin. Because the YMR4 line is a

pho3

and

pho5

null mu-tant, this absence of activity is not surprising. In addition tothese cell lines, we tested the

pdr5

null mutants overex-pressing MDR1, their wild-type background strain, and thebackground strain overexpressing MDR1; each exhibited in-creased phosphatase activity after 3 days of culture in thesedrugs (Figure 7B). The

Dpdr5 mock transformants could notbe tested because they did not grow in drug cultures. Whengrown in medium alone, every strain had only slight acidphosphatase activity.

To ensure that the MDR substrates themselves did not af-fect the NBT/BCIP assay, we tested medium containing cy-cloheximide and obtained results identical to those acquiredby the chromogenic assay of medium alone. To ensure thatthe results were not attributable to intracellular phospha-tases released when the drug-grown cells lysed, we vortex-mixed yeast cells with glass beads to release their cellularcontents before assay. These lysed cells showed no moreactivity than normal, medium-cultured cells (data not shown).

DISCUSSION

ATP Gradients

Our results reveal a new role for extracellular ATP in the phe-nomenon of MDR. We see a consistent relationship between

Figure 4. ATP Accumulation on the Leaf Surfaces of Arabidopsis Plants.

Measurement of leaf surface extracellular ATP in wild-type (Wt) andtwo MDR1-overexpressing (OE) lines. Data are reported as an ATPconcentration calculated from a relative light units–based standardcurve. Error bars indicate SE. xATP, extracellular ATP.

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the ability of an organism to degrade extracellular ATP andits ability to acquire toxin resistance. Yeast strains unable tomodulate their extracellular ATP concentrations with ecto-ATPase activity do not grow in the presence of toxin. On theother hand, the yeast strains that do develop toxin resis-

tance have markedly enhanced ectophosphatase activities.Phosphatase inhibitors, such as vanadate, further sensitizeectophosphatase null mutants to toxins and, in the case ofYMR4/MDR1, make an otherwise multidrug-resistant organ-ism unable to grow in the presence of toxin. If yeast cells are

Figure 5. Toxin Resistance in Arabidopsis.

(A) to (C) Resistance to cycloheximide is conferred by the overexpression of either apyrase or Arabidopsis MDR1 in Arabidopsis. (A) Wild-typeseeds failed to germinate in 250 ng/mL cycloheximide. (B) Seeds from lines that overexpress apyrase germinated in 250 ng/mL cycloheximide.(C) Seeds from lines that overexpress the Arabidopsis MDR1 germinated in 250 ng/mL cycloheximide.(D) Histogram showing germination percentages for wild-type (Wt), apyrase-expressing (Apyrase OE), and P-glycoprotein–expressing (MDR1OE) lines sown in 250 ng/mL cycloheximide. Approximately 80 to 100 plants were evaluated for each line.(E) Effects of the ATPase inhibitor methyleneadenosine 59-diphosphate on wild-type (Wt) plants and on plants overexpressing MDR1 (MDR1 OE)in the presence (+) and absence of cycloheximide. Ratios below the photographs denote the percentage of MDR1-expressing plants that germi-nated (numerator) relative to the percentage of wild-type plants that germinated (denominator) for each treatment.

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528 The Plant Cell

cultured in the presence of toxin and nonphysiological con-centrations of ATP, then the cells experience diminishing re-sistance–losses that correspond to both the concentrationof ATP added and the ectophosphatase activity of the cell.More than 50% growth diminution is seen in every cell linethat is cultured in medium containing toxin and ATP at two-fold the expected intracellular ATP concentration.

The sensitivity of ectophosphatase null mutants, the in-creased ectophosphatase activity of drug-cultured yeastcells, and the effect of extracellular ATP on toxin resistanceall suggest that the hydrolysis of extracellular ATP is neces-sary to maintain the resistant state. Our observations indi-cate that the need for this hydrolysis arises from ATPextrusion events associated with the upregulation of theplant MDR1 protein. Whether acting as a channel for ATP it-self or by activating some other ATP pore, the plant MDR1increases the concentrations of ATP both in the periplasm ofyeast and in the apoplasm of Arabidopsis. Our measure-ments of extracellular ATP accumulation are conservative

approximations of the ATP that might actually be found inthe microenvironment of the plasma membrane. Indeed,most of the ATP we measured was that which had escapedectophosphatase activity. Presumably, an important amountis hydrolyzed immediately on the membrane surface if theectophosphatases are intact; conversely, a very largeamount accumulates periplasmically if phosphatases arenot there.

The possibility exists that the extracellular ATP we mea-sured can be accounted for by nonspecific changes inmembrane permeability caused by the overexpression ofMDR1. We find this alternative unlikely. Nonselective in-creases in membrane permeability would not just allow ATPout but would also allow other small molecules in, moleculessuch as methylene blue, tritiated adenosine, and the phos-phate analog NBT/BCIP. However, viability assays usingmethylene blue demonstrate no difference in the INVSCmock-transformed line and the MDR1-transformed line, aresult that would not be obtained if the MDR1-expressing

Figure 6. Resistance to Toxic Doses of Hormone in Arabidopsis Transformed with Ectoapyrase or MDR1.

(A) The growth of wild-type, apyrase-overexpressing, and MDR1-overexpressing plants on untreated culture medium.(B) and (D) Growth of the three types of plants in a culture medium containing the plant hormone 2IP at 1000 mM (B). Toxic effects of 1000 mM2IP on plant growth progression as indicated by leaf expansion (D).(C) and (E) Growth of the same three plant types in culture medium containing 2IP at 100 mM (C). Measurement of toxicity based on root elon-gation averaged (6SEM) for three experiments (E). Data on the left portion of the histogram were collected from plants grown on media alone andon the right from plants grown on 100 mM 2IP.Apy OE, apyrase overexpressing; MDR1 OE, MDR1 overexpressing; Wt, wild type.

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lines were more permeable to the dye. Similarly, the YMR4MDR1-transformed strain exposed to NBT/BCIP showed nomore phosphatase activity than its mock-transformed coun-terpart, a result that would not be possible if the now mem-brane-permeable NBT/BCIP were exposed to intracellularphosphatases in the MDR1-expressing cell line. Last, our tri-tiated adenosine experiments revealed that when the YMR4and INVSC cell lines were incubated with tritiated adenosinefor 40 min, their initial rates of nucleoside uptake were allcomparable; the MDR1-expressing strains did not have agreatly reduced extracellular-to-intracellular ratio for label.Thus, we think it likely that the increased extracellular ATPconcentrations in the MDR1-overexpressing lines resultmore specifically from the channel, or channel-activating,properties of AtPGP1 itself.

Work put forth in the early 1990s demonstrated that P-gly-coprotein is capable of effluxing ATP (Abraham et al., 1993).In that study, MDR1 promoted the electrogenic efflux of ATPfrom transfected mammalian cells at a rate of 4 3 106 mole-cules per sec per cell. More recent estimations have placedthe efflux velocity at 1 to 10 molecules per channel per sec(Abraham et al., 1998). Our data suggest that a population of3 3 106 yeast cells in 1 mL of culture produces a steadystate concentration that is 4 3 105 times less than the intra-cellular ATP concentration of any single cell, given an extra-cellular ATP concentration of 10 nM (our measurement) andan intracellular ATP concentration of 4 mM (Ludin, 1989). Ifthis gradient were to be used to cotransport xenobiotics,then MDR xenobiotics should interact directly with ATP. In-deed, the work of Abraham and colleagues, based on chem-ical electrophoretic mobility shift assays, shows that ATPactually has an affinity for doxorubicin (Abraham et al.,1997). Furthermore, the interaction of zwitterionic xenobiot-ics with ATP is specific to this nucleotide; similarly, there is anotable lack of affinity for several xenobiotics to which MDRcell lines are sensitive.

Our results are consistent with the possibility that ATPand xenobiotics might move together through MDR1. Thespeculation that drugs are symported with ATP also stipu-lates that a very low steady state concentration of extracel-lular ATP must be maintained to form a gradient of ATPacross the plasma membrane. If the gradient is compro-mised either by knocking out the ecto-ATPase activity of thecell or, more simply, by artificially increasing the extracellularATP to twice the intracellular concentration, the result for theresistant cell is the same–death in the presence of a xenobi-otic. If the MDR1 channel cotransports toxins with ATP, thenthe ability of a cell to remain resistant would be contingenton its ability to maintain a steep ATP gradient. Our work withthe YMR4 mutant has revealed that the ATP gradient couldbe diminished simply by destroying extracellular phos-phatase activity. Moreover, the ATP symport model for MDRwould predict that this loss of ectophosphatase activityshould simultaneously decouple cellular resistance to tox-ins, a hypothesis borne out by our observation that theYMR4 cells were the most sensitive to xenobiotics.

If a steep ATP gradient were powering MDR1-mediateddrug efflux, then cells on initial presentation with toxinswould need not only to upregulate MDR proteins to effluxdrugs but also to enhance their ATPase activity to maintainthe gradient. Our studies revealed that cell lines cultured for3 days in cycloheximide, nigericin, or both had markedlyincreased ectophosphatase activities. The fact that all resis-tant yeast strains cultured in toxin take 24 hr longer to reach

Figure 7. Extracellular Phosphatase Activity Increased When CellsWere Exposed to Xenobiotic Agents.

(A) Acid phosphatase activity was visualized by the precipitation ofblue chromogen on the cell surface of yeast incubated in acetate-buffered NBT/BCIP at pH 4.5. Transformed YMR4 and INVSC areshown in the presence (+) and absence of cycloheximide (CHX).(B) Dpdr5 and wild-type cells grown in the presence of CHX or ni-gericin (Nig) and tested for extracellular acid phosphatase activity.

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530 The Plant Cell

growth saturation than do their counterparts cultured innon-toxin-containing media could be explained by the needto upregulate ectophosphatases to energize the availableMDR1-like proteins. Both the 24-hr lag and the correspond-ing increased ectophosphatase activity in toxin are evidentin the INVSC strain, the PDR5 null strain, and YKKA-7 andits parent strain, YPH499.

An ATP symport model would also allow us to explainhow a plant MDR1 cDNA is able to complement both an ec-tophosphatase null mutant and a pdr5 null mutant. The datafrom the pdr5 complementation experiments demonstratethat the plant MDR1 protein is a functional homolog ofPdr5p. However, the fact that the MDR1 gene can also com-plement the drug resistance of a yeast ectophosphatasemutant is suggestive of a mechanistic complementation, acomplementation that implicates ectophosphatases anddrug extrusion pumps in a single and shared mechanism ofachieving toxin resistance. We can imagine that ectophos-phatase mutants, unable to maintain steep ATP gradients,have MDR-like pumps that extrude drugs slowly and ineffi-ciently. The ATP symport model would suggest that by sim-ply increasing the number of drug efflux pumps, theectophosphatase yeast mutant could achieve the same re-sult as a cell line with fewer but more efficiently energizedMDR1-like proteins.

In some sense, the role of ectophosphatase activity in xe-nobiotic resistance was established with the observationthat 59-ectophosphatase upregulation is induced by MDRsubstrates even before MDR1 induction (Ujhazy et al.,1996). In a bacterial system, the LmrA gene, a prokaryoticfunctional homolog of MDR1, is found in the same operonas an alkaline phosphatase gene, which it abuts (van Veenet al., 1996). Although no evidence at present indicates thatthe phosphatase functions with LmrA in MDR, it is conver-gently transcribed with the LmrA gene, a fact highly sugges-tive of a role in MDR. In addition to the evidence frombacteria, results from yeast suggest that ectophosphataseactivity increases resistance to the polygenic antifungal agent,amphotericin B (Ramanandraibe et al., 1998). To date, how-ever, no studies have functionally linked ecto-ATPase activ-ity and xenobiotic resistance.

In Arabidopsis plants, the overexpression of a CD39 ec-toapyrase homolog from pea (Wang and Guidotti, 1996) it-self confers toxin resistance. This provides an example inwhich a multicellular organism has been genetically modi-fied to have MDR addressed with a gene other than the oneencoding an ABC transporter. The fact that increased ecto-ATPase activity can itself confer this seemingly complexphenotype suggests that the activity is pivotal to the translo-cation of xenobiotics across the plasma membrane. The factthat not all intracellular ATP is lost through the ABC proteinattests to the fidelity of the scavenging mechanisms forphosphate (Thomas et al., 1999) and adenosine. It also sug-gests that the MDR1 channel is gated and that its ATP effluxactivity is in some measure controlled.

The ABC proteins found in bacteria are structurally more

reminiscent of pumps, whereas eukaryotic ABC proteinssuch as the cystic fibrosis transmembrane conductanceregulator (CFTR) and MDR1 are channel-like (Welsh et al.,1998). However, the complementation of human MDR1 mu-tants with bacterial transporters (van Veen et al., 1998)unequivocally demonstrates that the pump model is biologi-cally feasible insofar as a prokaryotic pump can functionallysubstitute for a putative eukaryotic channel. We realize thatATP hydrolysis by P-glycoprotein is real and critical to xeno-biotic efflux. However, we speculate that ATP hydrolysismay not be directly coupled to the energetics of transportbut instead could be required for the gating of the ATPchannel. Inferences from structural data suggest that thenucleotide binding domains of the eukaryotic ABC trans-porters may participate in potentiating the MDR “pump-channel” (Welsh et al., 1998). The two pools of ATP, one in-volved in gating and the other in efflux, may be coordinatelyregulated.

Does Extracellular ATP Act Indirectly in MDR?

Because our experiments demonstrated that increased ATPefflux correlated with the xenobiotic resistant state in bothyeast and Arabidopsis, we wondered whether extracellularATP could act as a signaling molecule to promote MDR. Ex-tracellular ATP is known to act as a signaling molecule inseveral systems, for example, as a substrate for P2X recep-tors—a class of ATP gated cation channels—and P2Y re-ceptors—a class of heterotrimeric G protein receptors(North, 1996). In MDR, extracellular ATP has been known toinfluence sodium–proton exchange globally, which explainsthe effect of P-glycoprotein upregulation on plasma mem-brane potential (Weiner et al., 1986; Huang et al., 1992). Ex-trapolating from this idea, Wadkins and Roepe (1997)proposed that extracellular ATP may cause changes inmembrane potential that specifically affect passive xenobi-otic uptake. In this model, MDR would result mainly from xe-nobiotic exclusion.

There is good reason to suspect that ATP may have an in-direct effect on membrane potential because such a mecha-nism has been demonstrated with another ABC ATPconduit, the CFTR. CFTR releases ATP into the extracellularfluid; once there, ATP functions as a signal to regulate out-wardly rectifying chloride channels (Schwiebert et al., 1995).In this scenario, extracellular ATP is released by CFTR(Reisin et al., 1994) and binds to a G protein–coupled ATPreceptor that ultimately activates a cAMP-dependent ki-nase; this process culminates in the opening of an outwardlyrectifying chloride channel (Al-Awaqati, 1995). The activity ofthe chloride channel is dependent on the availability of ATP,which in turn is regulated by ecto-ATPase activity. If ATP ef-flux through MDR1 functions analogously, then a change ofmembrane potential would be expected with the onset ofATP efflux because this extracellular ATP would target puri-nergic receptors that themselves regulate polarization. As

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the membrane potential changes, the partitioning of hydro-phobic xenobiotics into the membrane could be altered insuch a way as to cause xenobiotic exclusion (Wadkins andHoughton, 1995).

Ecto-Apyrases as MDR Reversal Targets

The strongest known ATPases are apyrases, some of whichhave a turnover number of 104 molecules per sec (Handa andGuidotti, 1996). Because ectoapyrases are known to exist inhuman cells (Wang and Guidotti, 1996), we speculate thatthey could be the major ATPases on the cell surface. Theectoapyrase from pea plants, which is encoded by a singlegene, accounts for .60% of the total cell surface ATPaseactivity in roots (Y. Sun and S.J. Roux, unpublished data).Future studies will determine whether the human apyrase,CD39, is itself upregulated in MDR cells in a manner consis-tent with other ATPases. We hypothesize that ectoapyrasewill be an important target for MDR reversal.

METHODS

Expression of AtPGP-1 in Yeast

A full-length 4-kb AtPGP1, or MDR1, cDNA was assembled fromseveral pieces that were obtained by reverse transcription–poly-merase chain reaction (RT-PCR). The 1.9-kb SacI-BamHI insert ofpMDRPCR123 (Sidler et al., 1998) containing the 59 half of theAtPGP1 cDNA was subcloned into the same sites of pUC12 to givepUCcMDR5. A 39 part of the AtPGP1 cDNA that overlapped with the1.9-kb 59 part over 200 bp was obtained by RT-PCR with the oligo-nucleotides 59-GGCTGCTCGAGTCGCAAATG-39 (sense strand; se-quence corresponds to nucleotide positions 2912 to 2931) and 59-cgggatCCAAAGTAGTAAGTACTAAGC (antisense strand; sequencecorresponds to positions 5822 to 5842 [Dudler and Hertig, 1992]);the nucleotides shown in lowercase were added to provide a BamHIrestriction site. The fragment obtained was digested with XhoI andBamHI and was used to replace the z200-bp XhoI-BamHI fragmentof pMDR5 with which it overlapped. The resulting clone was namedpUCcMDR and contained the entire coding sequence of the AtPGP1gene, which we verified by sequencing. The complete 4-kb cDNAfragment was cut out with StuI (which cleaves in the leader se-quence) and SalI (which cuts in the polylinker of pUC12 downstreamof the cDNA insert) and inserted into a EcoRV-XhoI–cleaved pcDNAI/Amp vector (Invitrogen, Carlsbad, CA); from this, it was cut outagain as a BamHI fragment and finally inserted in the correct orienta-tion into the BamHI site of the yeast expression vector pvt101U(Vernet et al., 1987). pvt101 containing MDR1 and pvt101 alone wereeach independently transformed into Saccharomyces cerevisiaeINVSC1 (MATahis3-D1 leu2 trp1-289 ura3-52), YMR4 (MATahis3-11,15 leu2-3 112ura3D5 can Res pho5,3::ura3D1), YPH499 (ura3-52leu2-D1 his3-D200 ade2-101och lys2-801a trp1-D63), and YKKA-7(ura3-52 leu2-D1 his3-D200 ade2-101och lys2-801a Dpdr5::TRP1)by a polyethylene glycol–lithium acetate procedure (Elble, 1992) andselected on uracil dropout medium.

Yeast Growth

Yeast were grown at 308C under conditions of constant selection foruracil auxotrophy. Supplemented YNB (Bio101, Vista, CA) and 2%glucose were used to grow strains having pvt101 constructs. All mediawere at pH 5.5. The nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) assay for cell surface phosphatase activitywas performed as described by Thomas et al. (1999). Cycloheximide(Sigma) was added to liquid medium or spread on solid medium to afinal concentration of 500 ng/mL, unless otherwise stated. Nigericin(Sigma) was added to liquid medium or spread on solid medium to afinal concentration of 0.025 mg/mL. For selection assays on plates,single colonies were streaked; for selection in liquid medium, 0.01mL of saturated culture was added to 5 mL of fresh medium contain-ing the xenobiotic. Plated colonies were grown for 3 to 5 days beforebeing photographed. Yeast selection assays in liquid medium werequantified by turbidity as measured by absorbance at OD660. All se-lection assays were repeated at least five times.

For those experiments testing the salvage hypothesis, all cell lineswere grown in cycloheximide until OD600 5 1.0, at which point 10-mLsamples of each culture were removed and placed in each of the fol-lowing seven media: medium with nothing added; medium plus 3mM potassium phosphate; medium plus 3 mM adenosine; mediumplus 9 mM potassium phosphate and 3 mM adenosine (for controls);medium plus potassium phosphate and cycloheximide; medium plusadenosine and cycloheximide; and medium plus adenosine, cyclo-heximide, and potassium phosphate. Cell cultures were grown foranother 72 hr, and turbidity was measured. In the experiments usingsodium vanadate and ATP in assays of MDR reversal, the vanadateconcentration was 0.125 mM and the ATP concentration was 12mM. Growth was assayed by turbidity at 96 hr and was normalizedfor the same cell line grown in cycloheximide to determine inhibition.

Transgenic Plant Construction

psNTP9 (Pisum sativum apyrase; GenBank accession numberZ32743) was subcloned as a SalI-XbaI fragment into pKYLX71(Schardl et al., 1987). This plasmid was transformed into Agrobacte-rium GV3101 (pMP90) (Koncz and Shell, 1986), which was used to in-fect root calli from Wassilewskija ecotype of Arabidopsis thalianaunder kanamycin selection (Valvekens et al., 1992). The methods forconstruction of overexpressing and antisense-suppressed AtPGP-1(A. thaliana MDR1 gene; accession number X61370) in Arabidopsisare described by Sidler et al. (1998). Homozygous plants for theoverexpressed MDR1 and cDNAs encoding pea apyrase were usedin all experiments.

Plant Growth

Arabidopsis seeds were sown in a solid germination medium con-taining Murashige and Skoog salt (Sigma), 2% sucrose, 0.8% agar,and vitamins (Valvekens et al., 1992). For selection assays, cyclohex-imide was spread on the medium to a final concentration of 250 ng/mL. For cotyledon expansion assays, the cytokinin N6-(2-isopente-nyl)adenine (2IP) was applied to plates to a final concentration of 1mM. For experiments using methyleneadenosine 59-diphosphate,the cycloheximide concentration was 500 ng/mL and the ATPase in-hibitor concentration was 1 mM. Plant growth was measured by thepercentage of plants germinating after 6 days in experiments using

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532 The Plant Cell

cycloheximide alone (Figure 4) and after 10 to 20 days in experimentsusing methyleneadenosine 59-diphosphate or 2IP.

ATP Collection and Luminometry

Yeast cells used in the luciferase assays were grown for 2 days andthen transferred to 5 mL of fresh medium at the time of the assay.From this time forward, the cells were kept at room temperature on arotator. Every hour, a 1-mL aliquot was taken and the cells werecounted with a hemocytometer, checked for viability with methyleneblue, and centrifuged; the supernatant was stored in liquid nitrogenuntil all the aliquots were collected. If viability was ,95%, the exper-iment was ended. For luciferase assays involving plants, Arabidopsisseedlings were grown in sterile culture at 228C under 150 to 200mmol m22 sec21 of continuous light for at least 15 days. Foliar ATPwas collected by placing a single 30-mL drop of luciferase buffer (An-alytical Luminescence Laboratory, Cockeysville, MD) on a leaf and,without making direct physical contact with the plant, immediatelycollecting the droplet and snap-freezing it. For each leaf, the areawas approximated as an integrated area of a two-dimensional imageof the leaf by using National Institutes of Health 1.52 software. An av-erage of at least 10 independent measurements is presented for datapoints in plots of yeast and plant ATP. For luminometry, sampleswere thawed and reconstituted in Firelight buffer (Analytical Lumi-nescence Laboratory) to a final volume of 100 mL. After buffer wasadded, all samples were kept on ice. ATP standards (Sigma) were re-constituted in 100 mL of Firelight buffer, and the standards and sam-ples were loaded onto a 96-well plate, where the absorbance of eachwell was read with an automated luminometer (model mLX; DynexTechnologies, Chantilly, VA). Samples were processed by addition of50 mL of Firelight enzyme, followed by a reading delay of 1.0 sec andan integration time of 10 sec. Output was taken as an average for theintegration time and then as averages for multiple samples. The sam-ple handling time was ,2 hr.

Pulse–Chase Experiments

Yeast were grown to saturation in liquid medium, as described previ-ously, centrifuged, and resuspended in fresh medium containing 1mCi/mL 3H-adenosine (Amersham, Arlington Heights, IL). The cellswere rotated at room temperature for 20 min to allow adenosine up-take. After 20 min, the cells were pelleted by centrifugation. The pel-let was washed twice in ice-cold medium, resuspended in culturemedium at room temperature, divided equally among five tubes (fiveper cell line), and placed on a rotator. Every 10 min, a separate sam-ple-containing tube from each cell line was centrifuged, and the pel-let and the supernatant were placed in separate scintillation vials.The efflux activity was expressed as the ratio of counts in the super-natant to counts in the pellet.

ACKNOWLEDGMENTS

We thank Dr. Guido Guidotti and Dr. Albert Hinnen for help in obtain-ing YMR4. We also thank Dr. Karl Kuchler for the gift of YPH499 andYKKA-7. This work was supported by Grant No. IBN9603884 from

the National Science Foundation to S.J.R., by Grant No. 31-37145.93 from the Swiss National Science Foundation to R.D., andby a National Science Foundation Graduate Fellowship and Univer-sity Fellowship to C.T.

Received December 16, 1999; accepted February 21, 2000.

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A Role for Ectophosphatase in Xenobiotic Resistance · Robert Dudler, b Alan Lloyd, a and Stanley J. Roux a,3 a Section of Molecular Cell and Developmental Biology, Biological Laboratories