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Copyright � 2010 by the Genetics Society of AmericaDOI: 10.1534/genetics.110.117846

The Antidepressant Sertraline Targets Intracellular VesiculogenicMembranes in Yeast

Meredith M. Rainey, Daniel Korostyshevsky, Sean Lee and Ethan O. Perlstein1

Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544

Manuscript received April 16, 2010Accepted for publication May 3, 2010

ABSTRACT

Numerous studies have shown that the clinical antidepressant sertraline (Zoloft) is biologically active inmodel systems, including fungi, which do not express its putative protein target, the serotonin/5-HTtransporter, thus demonstrating the existence of one or more secondary targets. Here we show that in theabsence of its putative protein target, sertraline targets phospholipid membranes that comprise the acidicorganelles of the intracellular vesicle transport system by a mechanism consistent with the bilayer couplehypothesis. On the basis of a combination of drug-resistance selection and chemical-genomic screening,we hypothesize that loss of vacuolar ATPase activity reduces uptake of sertraline into cells, whereasdysregulation of clathrin function reduces the affinity of membranes for sertraline. Remarkably, sublethaldoses of sertraline stimulate growth of mutants with impaired clathrin function. Ultrastructural studies ofsertraline-treated cells revealed a phenotype that resembles phospholipidosis induced by cationicamphiphilic drugs in mammalian cells. Using reconstituted enzyme assays, we also demonstrated thatsertraline inhibits phospholipase A1 and phospholipase D, exhibits mixed effects on phospholipase C, andactivates phospholipase A2. Overall, our study identifies two evolutionarily conserved membrane-activeprocesses—vacuolar acidification and clathrin-coat formation—as modulators of sertraline’s action atmembranes.

SERTRALINE, whose trade name is Zoloft, is a Foodand Drug Administration (FDA) approved drug

that belongs to the pharmacological class of antide-pressants called selective serotonin reuptake inhibitors(SSRIs) (Koe et al. 1983). The putative therapeutic tar-get of SSRIs is the human serotonin/5-HT transporter(hSERT/5-HTT/SLC6A4), an integral membrane pro-tein that mediates sodium-dependent reuptake of themonoamine neurotransmitter serotonin at presynapticnerve terminals in the brain (Tatsumi et al. 1997;Mortensen et al. 2001; Meyer et al. 2004). Indeed,the recent crystal structure of sertraline bound to abacterial leucine transporter, LeuT—a homolog ofmammalian monoamine transporters—has led to theproposal of a corresponding high-affinity binding sitefor sertraline in hSERT (Zhou et al. 2009). Inhibitionof hSERT-dependent reuptake by sertraline is thoughtto underlie its antidepressant effect in people, asdocumented extensively over the last two decades in theclinical psychiatric literature (reviewed in Cipriani et al.2009).

However, there are numerous examples of sertraline’seffects on cellular physiology, including, most curiously,

cytotoxicity in yeast (Lass-Florl et al. 2001a,b), by anunknown mechanism that does not involve hSERT,suggesting the existence of one or more heretoforeunrecognized secondary drug targets that may beevolutionarily conserved (Levkovitz et al. 2007; Reddy

et al. 2008). Those observations, coupled with reportsthat antidepressants can accumulate in the brains ofpeople to concentrations at which ‘‘off-target’’ effectsare seen (Levkovitz et al. 2007), suggest that sertra-line’s interactions with targets besides hSERT maycontribute to its antidepressant and/or ‘‘side-effect’’properties in people. Clues as to the identity of suchtargets emerge when one connects sertraline to the vastliterature on a class of compounds, called cationicamphiphilic drugs, or CADs, to which sertraline andmany other FDA-approved substances belong. CADs aredefined by two physicochemical attributes: (i) a hydro-phobic ring system (in the case of sertraline, a naph-thalene scaffold); (ii) a basic, nitrogen-containinggroup (in the case of sertraline, a secondary amine).In addition to their known and/or putative interactionswith proteinaceous targets, it has long been recognizedthat CADs interact with phospholipid membranes; thisinteraction is traditionally understood through the lensof the decades-old bilayer couple hypothesis.

The bilayer couple hypothesis (Sheetz and Singer

1974) arose from studies of the effects of CADs on theshape of human erythrocytes visualized by scanningelectron microscopy. It specified the following molecular

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.110.117846/DC1.

1Corresponding author: Lewis-Sigler Institute for Integrative Genomics,Princeton University, Carl Icahn Laboratory, Washington Road,Princeton, NJ 08544. E-mail: [email protected]

Genetics 185: 1221–1233 (August 2010)

http://www.genetics.org/cgi/content/full/genetics.110.117846/DC1





mechanism of action: a charged (e.g., cationic oranionic) amphiphile preferentially localizes to a leafletexhibiting the opposite charge, resulting in its asym-metric expansion. Subsequent studies using artificialmembranes have shown that the effects of CADs onmembranes depend on factors like lipid composition,membrane curvature, and pH gradients (Verkleij et al.1982; Eriksson 1987). CADs appear to have pleiotropiceffects on membrane-active processes whether oneexamines these effects on proteins, cells, or even wholeorganisms. For example, CADs modulate the activity ofstructurally and catalytically unrelated proteins whoseproper function depends on being either in, on, or nearmembranes, including mechanosensitive ion channels(Martinac et al. 1990), phospholipases (Sturton andBrindley 1977; Leli and Hauser 1987a), and even theGTPase dynamin (Otomo et al. 2008). Indeed, it haslong been thought that inhibition by CADs of phospho-lipases is responsible for drug-induced phospholipido-sis in mammalian cells (Sturton and Brindley 1977).Phospholipidosis is defined as an accumulation ofmultilamellar vesicles or arrays that leads to disruptionof organellar integrity, altered phospholipid metabo-lism, and in some instances cell death (Anderson andBorlak 2006).

Given the broad swathe of CAD-induced perturba-tions of membrane biophysical properties in vitro andmembrane-active processes in vivo, these drugs havebeen the focus of investigators in diverse fields, in-cluding yeast genetics. Two examples that are especiallypertinent to our present study involve the identificationof fungal genetic determinants of resistance/sensitivityto the oft-studied phenothiazine antipsychotic drugstrifluoperazine (Shih et al. 1988) and chlorpromazine(Thorazine) (Filippi et al. 2007), the latter being per-haps the most well-studied CAD in a variety of experi-ment types and model systems. The older studyshowed that resistance to trifluoperazine in yeast iscaused by mutations in subunits comprising the evolu-tionarily conserved vacuolar ATPase (V-ATPase), whichis composed of two functional units, a soluble V1 andmembrane-associated V0. These subunits assemble intoan ATP-dependent proton-translocating complex thatacidifies the lumens of dynamic organelles like the vac-uole (or lysosome, in mammalian cells), thereby ener-gizing intracellular vesicular transport. The resistance-conferring effects of loss of V-ATPase complex activitywas explained using a lysosomal trapping/basicicity model:because acidified vacuoles/lysosomes act as sinks forweak bases, loss of acidification reduces intracellularCAD accumulation. The more recent study showed thatdeletion of genes involved in vesicle trafficking—anumber of which have mammalian homologs—causeshypersensitivity to chlorpromazine. A similar gene setenriched in processes involved in the intracellularvesicle transport system was found to modulate sensitiv-ity to a diverse collection of psychoactive drugs by a

group using an unbiased chemical-genomic approach(Ericson et al. 2008).

In an effort to understand why sertraline exhibitscytotoxic effects in a model system lacking the putativeprotein target, we began by combining the unparalleledspecificity of a drug-resistance selection with rapid andcost-effective microarray-enabled polymorphism detec-tion. In other words, we used whole-genome tilingmicroarrays, as opposed to cloning by complementa-tion, to locate rapidly resistance-conferring mutations.The advent of microarray-enabled polymorphism de-tection (Gresham et al. 2006) greatly reduces theexpenditure of cost and time traditionally associatedwith identifying drug resistance-conferring loci, therebymodernizing ad hoc cloning-by-complementation ap-proaches and complementing high-throughput chem-ical genomic screening of engineered deletion libraries(Lehar et al. 2008). And throughout the present study,we also examined the effects of the classic CADchlorpromazine to strengthen the prima facie linkagebetween sertraline and the pleiotropic effects ofcationic amphiphiles on biological membranes.

MATERIALS AND METHODS

Yeast strains and genetic analysis: Standard genetic manip-ulations of Saccharomyces cerevisiae were performed as pre-viously described (Guthrie and Fink 1991). Standard growthconditions were YPD (1% yeast extract, 2% peptone, and 2%dextrose) media at 30�. BY4712 (leu2D0) and BY4716 (lys2D0)were chosen as the parental strains and are congenic to S288C(Brachmann et al. 1998). The chc1-521 temperature-sensitivestrain and isogenic control strain (BY4742) were kindly pro-vided by G. Payne (University of California, Los Angeles)(Bensen et al. 2000).

Chemical compounds: Sertraline hydrochloride (cat. no.S6319) was purchased from Sigma Aldrich, resuspendedin dimethyl sulfoxide (DMSO) to a final concentration of25 mg/ml (�73 mm), and 100-ml aliquots were stored in glassvials at �20� until use and subjected to a maximum of onefreeze/thaw cycle. Chlorpromazine hydrochloride (cat. no.C8138), cycloheximide (cat. no. C7698), gramicidin (cat. no.G5002), and bafilomycin A1 (cat. no. B1793) were alsopurchased from Sigma Aldrich and treated similarly.

Drug-resistance selection and hypersensitivity screen: Weplated 100 ml of unmutagenized overnight cultures inoculatedfrom single colonies of BY4712 and BY4716 on YPD plates (2%agar) supplemented with �45 mm sertraline, which is approx-imately three times the IC50 of sertraline as determined bydose–response experiments in liquid culture. Mutagenesis wasnot performed so as to minimize the background polymor-phism rate. Plates were grown for 3–5 days at 30� until distinctsertraline-resistant colonies appeared. On average, indepen-dent cultures plated on selection plates yielded �12 large-to-medium colonies, and up to 12 microcolonies, depending onthe exact incubation time. We note that the optimal selectiveconcentration range is exceedingly narrow: lowering theselection concentration from �45 to �42 mm resulted, onaverage, in a 10-fold increase in the number of resistantcolonies. A total of 16 sertraline-resistant mutants were colonypurified: 8 each of BY4712 and BY4716. All BY4712 mutantswere backcrossed to the parental BY4716, and vice versa, in adominance test. Copies of the complete homozygous and

1222 M. M. Rainey et al.

heterozygous deletion collections originally purchased fromInvitrogen were screened in 384-well plates in duplicate aspreviously described (Perlstein et al. 2006). P-values associ-ated with GO enrichments were calculated using the GO TermFinder, which is accessible on the Saccharomyces Genomedatabase (www.yeastgenome.org).

Microarray-based polymorphism detection: Genomic DNA(gDNA) was prepared for hybridization to Affymetrix whole-genome S. cerevisiae tiling arrays (1.0 R) as previously described(Gresham et al. 2006). However, the gDNA labeling protocolwas modified: a BioPrime DNA labeling system (Invitrogen)was used to generate labeled gDNA fragments for hybrizida-tion accordingly to manufacturer’s protocol. We validatedSNPscanner predictions by targeted resequencing of PCRamplicons corresponding to the entire open reading frame(ORF) containing a candidate mutation. We then verifiedcausality by showing 2:2 cosegregation of validated mutationswith sertraline resistance in linkage analysis of tetrads. PCR wasperformed using the Accuprime Pfx polymerase kit (Invitro-gen). For the analysis of recessive mutants, inclusion of twoindependent members of the same complementation groupeffectively excluded false positives. For dominant mutants,microarray-based polymorphism detection using a singlehybridization worked reliably enough that independent bi-ological replicates were not necessary. ORFs in which apolymorphism was predicted were amplified using the ‘‘A’’and ‘‘D’’ primers designed by the Stanford Deletion Consor-tium for each ORF in the yeast genome. Resequencing of theimmediate regions surrounding the candidate polymor-phisms was performed with sequencing primers whose se-quences are available upon request.

Growth-related assays of sertraline-resistant strains: Dose–response experiments were performed in 96- or 384-well platesas previously described (Perlstein et al. 2006). Growth-ratetime courses were generated using a TECAN plate reader(Tecan Group); these experiments were performed at twoconcentrations of sertraline in YPD over an �16-hr observa-tion period. We selected 30 mm (high-dose regime) and 7.5 mm

(low-dose regime) because they are approximately twofoldhigher and lower than sertraline’s IC50, respectively. Data wasanalyzed using Prism 4 (GraphPad software). Cytotoxicityassays (n ¼ 3 per strain) were performed as follows: 3 �3 107

wild type, but 1.5 3 107 cup5 and chc1 mutant cells, were treatedwith 120 mm sertraline, quickly pelleted by centrifugation(500 3 g), and then washed three times. Half as many cup5 orchc1 mutant cells were used because these mutants exhibittwofold greater resistance to sertraline compared to wild-typecells. Regardless of strain, high cell densities required moresertraline to achieve growth inhibition comparable to thatobserved at low cell densities. Cell concentration was de-termined by Coulter counter and 200 ml of cells at aconcentration of 103 cells/ml were plated on YPD plates andcolony-forming units were counted after 2–3 days of incuba-tion at 30�. Data were normalized to basal, pretreatmentviability measurements. Micromanipulation of single cells wasperformed using a benchtop dissecting microscope.

Transmission electron microscopy and phospholipaseassays: Samples were prepared as previously described (Banta

et al. 1988). For the high-dose treatment, mid-log phase cells atOD600 0.5 were treated with 120 mm sertraline for 1 hr; for thecup5 comparison, mid-log phase cells at OD600 0.5 were treatedwith 45 mm sertraline for 1 hr. For the 120-mm dataset,quantification of vacuole number was performed usingImageJ on multicell, low magnification fields composed of214 untreated or 77 treated cells; quantification of all otherultrastructural phenotypes was performed on a collection ofsingle-cell, high magnification images containing 71 un-treated or 52 sertraline-treated individual cells. For the

45-mm dataset, quantification of dead cells was performed on57 wild-type cells and 35 cup5 mutant cells. (Cell totals notlisted here are directly listed in results). The following kitsfor assaying phospholipase activity were purchased fromInvitrogen: EnzChek Phospholipase A1 (cat. no. E10219),EnzChek Phospholipase A2 (cat. no. E10217), EnzChekPhospholipase Phospholipase C (cat. no. E10215), andAmplex Red Phospholipase D (cat. no. A12219). Assays wereperformed according to manufacturer’s specifications inclear-bottom, black 384-well plates (Corning). The PiPerPhosphate assay kit (cat. no. P22061) was also purchased fromInvitrogen. Assay plates were read in a SyneryMx multimodespectrophotomer (Biotek). Curve fitting and analysis wereperformed using the Prism 4 (GraphPad software).

RESULTS

Sertraline-resistant mutants target clathrin andV-ATPase function: We selected BY4712 and BY4716as parental strains. A total of 16 spontaneous sertraline-resistant (sertR) mutants were isolated: 4 of the mutantsare dominant; the remaining 12 are recessive. A com-plementation test revealed that 8 of the 12 recessivemutants belong to a single complementation group; twoof the remaining 4 mutants comprise a second comple-mentation group; the other two mutants are orphans,i.e., were not assigned to any complementation group.We hybridized fragmented and labeled genomic DNA(gDNA) derived from a subset of sertR mutants toAffymetrix whole-genome tiling arrays. The resultinggenome-wide hybridization intensity measurementswere then analyzed by the SNPscanner predictionalgorithm (Gresham et al. 2006) to identify candidateresistance-conferring mutations, which we expected toreside in protein-coding regions. SNPscanner predictedmutations in all cases save one orphan recessive BY4716sertR mutant, which we did not analyze further. Acomplete catalog of 16 (15 single mutants and onedouble mutant) sertR strains is presented in Table 1. Werecovered mutants affecting two evolutionarily con-served membrane-active processes: clathrin-coat forma-tion and the vacuolar ATPase (V-ATPase) complex.

The largest complementation group corresponds tothe gene SWA2 (YDR320C), which encodes the yeasthomolog of auxilin, a clathrin-binding protein withcochaperone function that mediates uncoating ofclathrin assemblies through activation of the Hsp70chaperone (Gall et al. 2000). We independently ob-served twice a nonsense mutation (C603STOP) thatresults in truncation of the critical C-terminal J domainthat is strongly conserved from yeast to human, andwithout which Swa2p cannot activate the clathrin-remodeling function of Hsp70p (Xiao et al. 2006).Deletion of SWA2 in yeast (Xiao et al. 2006) or RNAi-knockdown of the SWA2 homolog auxilin in mamma-lian cells (Hirst et al. 2008) prevents the uncoatingreaction from disassembling clathrin-coated vesicles,which shifts the equilibrium of clathrin from thedisassembled state to the assembled state.

Sertraline Mechanism of Action in Yeast 1223

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Unexpectedly, one of the mutants that we originallyassigned to the swa2 complementation group actuallyencodes wild-type SWA2, as borne out by targetedresequencing. In other words, this mutant failed tocomplement sertraline resistance when mated to a swa2mutant. Upon closer inspection, this mutant harbors apartially dominant missense mutation (E292K) inCHC1/SWA5 (YGL206C), which encodes clathrin heavychain (Lemmon et al. 1991). The residue affected by themissense mutation resides in the N-terminal domain ofChc1p, which is thought to interact with clathrin adaptorcomplexes, but is not directly involved in trimerization ofclathrin heavy chain molecules into triskelia, the buildingblock of clathrin coats. The fact that we only isolated onechc1 mutant indicates that our selection was not per-formed to saturation. To our knowledge, neither SWA2nor CHC1 has been isolated as a mutant in the context ofdrug-resistance screening or selection, though mutantalleles of both loci were previously shown to be synthet-ically lethal with deletion of ARF1, which encodes a smallsoluble GTPase that initiates clathrin-coat formation atthe Golgi (Chen and Graham 1998).

The second complementation group corresponds tothe gene CUP5/VMA3 (YEL027W ), which encodes the csubunit of the V0 sector of the V-ATPase. Interestingly,the four dominant sertR mutations occur in two otherV-ATPase complex subunits: TFP1 and VMA9. TFP1(YDL185W ) encodes the A subunit of the eight-subunitV1 peripheral membrane domain. VMA9 (YCL005W-A)encodes the e subunit of V0 sector. As expected,phenotypic analysis of sertR mutants affecting V-ATPasecomplex activity reveals that they exhibit phenotypescharacteristic of Vma� mutants: sensitivity to highextracellular calcium and inability to grow in alkaline

(pH 8) media (Figure S1). TFP1 is noteworthy becausedominant alleles of it were previously shown to conferresistance to the phenothiazine antipsychotic trifluo-perazine (Shih et al. 1988).

Next we tested whether the potent and specificchemical inhibitor of the V-ATPase, bafilomycin A1,which binds to the c subunit encoded by CUP5/VMA3,suppresses sertraline-induced cytotoxicity in wild-typecells. This is indeed the case (Figure S2). Furthermore,the proton ionophore gramicidin also suppresses sertra-line-induced cytotoxicity in wild-type cells. Mutations inCUP5/VMA3 that convert the V0 subunit into a passiveproton channel would have the same effect as gramici-din, which inserts into membranes and forms proton-conducting channels. These results reinforce thelysosomal trapping/basicicity model. We further as-sessed the role of acidification by testing the effect ofenvironmental pH on sertraline-induced cytotoxicity inwild-type cells. Just as was observed in a recent study ofchlorpromazine resistance (Filippi et al. 2007), in acidicYPD media (pH 4.0) sertraline does not exhibit anyantifungal activity, while in alkaline YPD media (pH 8.0)sertraline’s antifungal activity is exacerbated (FigureS2). Thus the protonated form of sertraline is inactivebecause it may be impermeable to cells. These resultsreinforce the notion that vacuolar acidification andexternal pH are common determinants of cellularresponse to CADs in yeast.

V-ATPase and clathrin mutants confer partialresistance nonadditively: In addition to the known role ofthe V-ATPase in shaping the cellular response to CADs,our drug-resistance selection uncovered a novel mech-anism of resistance: dysregulation of clathrin function.To characterize further the two mechanisms, we quan-tified the half-maximal inhibitory concentration (IC50)of sertraline in the wild-type strain, BY4716, and twosertR mutants, one from each of the two resistant classes:CUP5R46I and SWA2C603STOP. These experiments yieldedthree important observations. First, the steepness of thedose–response curves of all three strains suggests thatsertraline exhibits cooperative binding at its sites ofaction. Second, the IC50 of sertraline in BY4716 is 15 mm,and this value increases only twofold in all sertR strains(Figure 1A). Partial resistance demonstrates that thebinding sites for sertraline are not completely extin-guished by mutation. Third, sertR strains exhibit cross-resistance to chlorpromazine (Figure 1B), but are notcross-resistant—if anything the clathrin pathway mu-tants are more sensitive than wild type—to the structur-ally and mechanistically unrelated translation inhibitorcycloheximide (Figure 1C).

Next, we tested whether CUP5 and SWA2 function inthe same pathway or in parallel pathways. We generateda cup5 swa2 double mutant by crossing the aforemen-tioned CUP5R46I and SWA2C603STOP strains. Tetrad analysisrevealed that a cup5 swa2 double mutant exhibitsmodest synthetic sickness in the absence of sertraline;

TABLE 1

Sixteen spontaneous sertraline-resistant (sertR) mutants

Background Gene ORF name Allele(s) Dom/Rec

BY4716 CHC1 YGL206C E292K Partiallydominant

BY4712 CUP5 YEL027W R46I RecessiveBY4716 CUP5 YEL027W G115S RecessiveBY4712a SWA2 YDR320C C603STOP RecessiveBY4712 SWA2 YDR320C A438D RecessiveBY4716 SWA2 YDR320C C603STOP RecessiveBY4716 SWA2 YDR320C Q505STOP RecessiveBY4716 SWA2 YDR320C K380STOP RecessiveBY4712 TFP1 YDL185W G604V RecessiveBY4712b TFP1 YDL185W G257S DominantBY4716 TFP1 YDL185W G742D DominantBY4716 VMA9 YCL005W-A S35R DominantBY4712 cup5 CSG2 YBR036C V280STOPc ND

ND, not determined.a Three clones of this allele were isolated.b Two clones of this allele were isolated.c Frameshift mutation at residue 277 results in premature

stop at residue 280.


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some allelic combinations resulted in greater syntheticsickness. Surprisingly, the cup5 swa2 double mutantinitially appeared to be more sensitive to sertraline thaneither single mutant with an IC50 of �6.5 mm (Figure2A). However, if the double mutant was incubated for anadditional 12–24 hr its dose–response assumed anappearance closer to that of either of the single mutants,indicating a kinetic defect in the emergence of re-sistance, as opposed to a diminution of absolute re-sistance (Figure 2B). Interestingly, the dose–response of

a cup5 swa2 double mutant is better fit by a less steepcurve, suggesting that the kinetic defect may affect thecooperativity of sertraline binding at its sites of action.Similar results were obtained using a cup5 swa2 doublemutant comprised of different single mutant alleles. Asa control, we generated a cup5 tfp1 double mutant; thesetwo genes encode interacting subunits in the V-ATPasecomplex. Two cup5 tfp1 double mutants composed ofdifferent single mutant alleles yielded similar results,namely the double mutant is as resistant as the averageof both single mutants (Figure 2A); moreover, there isno change in the steepness of the dose–response curvesor the kinetics of resistance.

To dissect further the resistance-conferring role ofthe V-ATPase, we conducted a second round of drug-resistance selection, but this time we started with theCUP5R46I mutant, as opposed to the parental strain. Weincreased the stringency of selection to �60 mm sertra-line given the twofold increased basal resistance of theCUP5R46I mutant. Using the aforementioned microarray-enabled polymorphism detection approach, we identi-fied and subsequently validated by resequencing andlinkage analysis (data not shown) two point mutations

Figure 1.—Summary of dose–response experiments andcross-resistance test. For all panels the strains are color codedas follows: BY4716, black; CUP5R46I, red; and SWA2C603STOP, blue.Strains were treated with sertraline (A), chlorpromazine (B),or cycloheximide (C). Small-molecule drug concentration isgiven as log [M]. All strains were grown at 30�. ExperimentalOD600 values were normalized to OD600 values of untreatedisogenic controls. Data points are the average of quadrupli-cate OD600 measurements. Points were fit with a variableslope sigmoidal dose–response equation. Error bars indicatestandard deviation.

Figure 2.—Epistasis analysis of cup5 swa2 and cup5 tfp1 dou-ble mutants. (A) IC50 values normalized by the wild-type tet-ratype of three spores derived from a representative tetrad.The three spores are color coded as follows: cup5, red; swa2and tfp1, blue; and cup5 swa2 and cup5 tfp1, purple. (B) Timecourse of cup5 swa2 double mutant (purple circles) and thewild-type strain (black circles). Both strains were treated withsertraline and grown at 30�. Data points are the average of sixreplicates. Error bars indicate standard deviation.


in the gene CSG2 (YBR036C) (Table 1 and Figure S3). Adeletion of a single adenine base in a short run ofadenosines is located three nucleotides upstream of aT/C transition point mutation. The single-base de-letion is predicted to cause a frameshift and subsequentpremature STOP codon at residue 280. CSG2 encodesan integral membrane protein that localizes to theendoplasmic reticulum and is involved in calcium–ionhomeostasis and sphingolipid metabolism (Beeler et al.1994). Interestingly, a cup5 csg2 double mutant waspreviously shown to be more sensitive than a cup5 singlemutant to the calcineurin inhibitor FK506 (Tanida et al.1996). We show that a cup5 csg2 mutant is more resistantto sertraline than a cup5 mutant, while a csg2 mutantexhibits wild-type sensitivity (Figure S3). Consistentwith a role for calcium in determining sertraline sen-sitivity, we also observed that calcium suppresses, whilethe calcium–ion chelator EGTA enhances, sertraline-induced cytotoxicity of wild-type cells (data not shown).Thus, CSG2 is a genetic modifier of CUP5, whichsuggests that modulation of calcium–ion homeostasismay in part be driving the selection of V-ATPasehypomorphs.

Growth-stimulatory effects of sertraline on mutantswith impaired clathrin function: CHC1E292K andSWA2C603STOP mutants exhibit nonallelic noncomple-mentation. Therefore, we posit that these mutationsexert similar effects on clathrin function. As swa2mutants accumulate assembled clathrin (Gall et al.2000), a similar shift in the equilibrium of clathrinwould be predicted to occur in the dominant CHC1E292K

mutant. To examine more closely how dysregulation ofclathrin function engenders resistance to sertraline, weincluded a loss-of-function allele of clathrin heavy chain(chc1-521), which is conditionally assembly incompetent(Bensen et al. 2000). Again, dose–response experimentsyielded three important observations (Figure 3A). First,the CHC1E292K mutant, like the other sertR strains,exhibits a twofold increase in resistance compared towild type. Second, sertraline exhibits concentration-dependent or biphasic effects over its bioactive concen-tration range. We define a sublethal regime and a lethalregime. The lethal regime is any concentration greaterthan or equal to 30 mm and results in maximal growthinhibition of wild-type and all sertR strains. The sub-lethal regime spans 0.1 to 10 mm and results insuppression of the constitutive growth defect of theCHC1E292K mutant that may arise from the failure ofclathrin assemblies to recycle properly, or downstreameffects thereof. Third, the temperature-dependentgrowth defect of the chc1-521 strain is surprisingly alsosuppressed in the sublethal regime. These resultsdemonstrate that sertraline and clathrin exhibit com-plex competition at sertraline’s sites of action.

Interestingly, chlorpromazine also exhibits biphasiceffects, though not as potently as sertraline (Figure 3B).Thus, concentration-dependent rescue of growth de-

fects of mutants with altered clathrin function may be ageneral feature of psychoactive CADs, which is consis-tent with previous observations that some CADs affectclathrin self-assembly in vitro (Nandi et al. 1981;Di Cerbo et al. 1984) as well as clathrin-dependentendocytosis in vivo (Wang et al. 1993). Importantly, theconcentration-dependent effects of sertraline appear tobe specific to clathrin function, because at no concen-tration does cycloheximide rescue the growth defect ofeither mutant with altered clathrin function (Figure3C). The specificity for clathrin function is further

Figure 3.—Biphasic effects of sertraline and chlorproma-zine on mutants with dysregulated clathrin function. For allpanels the strains are color coded as follows: BY4716, black;CHC1E292K, blue; and chc1-521, red. Strains were treated withsertraline (A), chlorpromazine (B), or cycloheximide (C).Small-molecule drug concentration is given as log [M]. Allstrains were grown at 30�, except as indicated. ExperimentalOD600 values were normalized to OD600 values of untreatedisogenic controls. Data points are the average of quadrupli-cate OD600 measurements. Points were fit with a variableslope sigmoidal dose–response equation. Error bars indicatestandard deviation.




supported by the observation that the moderately slowgrowing VMA9S35R mutant strain does not experiencegrowth-rate enhancement in the low-dose sertralineregime (data not shown).

Clathrin and V-ATPase mutant exhibit distinctkinetics of resistance: Because dose–response experi-ments capture the endpoint of the cellular response tosertraline, we quantified the kinetics of resistance tosertraline using an automated plate reader that incu-bates a 96-well plate at constant temperature withagitation. We first assessed the baseline growth rate ofthe wild-type and several representative sertR strains, aswell as the chc1-521 mutant. SWA2C603STOP, CHC1E292K, andthe dominant V-ATPase mutant VMA9S35R grow slowly inthe absence of sertraline, indicating that a fitnesspenalty sometimes accompanies sertraline resistance.On the other hand, the recessive CUP5R46I mutant growsat rate comparable to wild type. Next we tested twoconcentration regimes of sertraline: one twofold above(30 mm) and a second twofold below (7.5 mm) the IC50 ofwild-type cells.

At 30 mm sertraline, the CHC1E292K mutant grows atthe same rate in the presence or absence of sertraline,while the CUP5R46I mutant exhibits a reduced growthrate relative to its steady state that is still faster thanwild type, whose growth rate is completely inhibited(Figure 4, A–C). At 7.5 mm sertraline, we observerescue of the growth defects of the CHC1E292K andchc1-521 mutants (Figure 4, C and D), consistent withthe results of dose–response experiments. These

results support the notion that the CUP5R46I mutantappears to exhibit reduced uptake of sertraline com-pared to wild type. However, the behavior of theCHC1E292K mutant is more complex, and appears notto involve uptake given the growth-stimulatory effectsof sublethal doses of sertraline. Rather, the down-stream, cytotoxic effects of sertraline accumulation aresuppressed, suggesting that the affinity of membranesfor sertraline is reduced.

Sertraline induces all-or-none cytotoxicity: The afore-mentioned experiments all involve populations of cells.To verify that the observed changes in absolute re-sistance or the kinetics of resistance occur at the level ofsingle cells, we assessed cellular response to sertralineusing colony-forming unit assays. These experimentsunequivocally show that sertraline-induced cytotoxicityis all-or-none and irreversible. Viability of wild-typeBY4716 cells after 100 min of sertraline treatmentfollowed by wash out is 56% of untreated isogeniccontrols; after 250 min is 28% of untreated isogeniccontrols (Figure 5). The rate of cell death seems to be�50% of the population per cell cycle at the celldensities and sertraline concentration tested, i.e., inthe linear range of this assay, with the caveats that theseare asynchronous batch cultures and that increasingdrug concentration while holding cell density constantaccelerates the rate of cell death ultimately to �100%.Also, the surviving clones do not exhibit a growth lagafter wash out, indicating negligible aftereffects ofsertraline treatment.

Figure 4.—Kinetic time-course experiments with select sertR strains grown in YPD supplemented with 0 mm (blue circles),7.5 mm (green circles), or 30 mm (red circles) sertraline reveal two modes of resistance. (A) Baseline and sertraline-treated growthcurves of the wild-type BY4716. (B) Baseline and sertraline-treated growth curves of the CUP5R46I mutant. (C) Baseline and sertraline-treated growth curves of the CHC1E292K mutant. (D) Baseline and sertraline-treated growth curves of the chc1-521 temperature-sensitive mutant. Cells were grown in 96-well plates and incubated with shaking at 30� in a TECAN plate reader except for D,which was grown at 37�. The amount 104 sec equals �2.8 hr. All OD595 measurements represent the average of six experimentalreplicates. Error bars indicate standard deviation.


As a control, we used micromanipulation to physicallyisolate individual cells posttreatment and wash out, andindependently calculated loss of viability as a function oftime. We observed nearly identical results using bothmethods. Following micromanipulation of single cells,55% (53/96) yielded colony-forming units after 100 minof sertraline treatment followed by wash out; 23% (22/96)yielded colony-forming units after 250 min; untreatedcells yielded 99% (94/96) colony-forming units. Fur-thermore, visual inspection revealed that single cellsthat failed to form colonies were terminally arrested atthe one-cell stage but showed no evidence of lysis. As anegative control, wild-type cells treated with cyclohexi-mide, which is cytostatic, and then washed, recover withnearly no loss of viability. These results suggest thatthere is a nonuniform distribution of drug uptake ordownstream compensatory mechanisms or both in indi-vidual cells.

Next, we assayed the viability of CUP5R46I andCHC1E292K mutants under the same assay conditions(Figure 5). The CUP5R46I mutant demonstrably losesviability in response to sertraline treatment but at 60% ofthe wild-type rate. Viability of CUP5R46I cells after 100 minof sertraline treatment followed by wash out is 80% ofuntreated isogenic controls; after 250 min it is 56% ofuntreated isogenic controls. On the other hand, nearlyno loss of viability was measured for the CHC1E292K

mutant. In fact, colony-forming units increase to 130%of untreated control levels after exposure of CHC1E292K

cells to sertraline, though this increase in viability onlybecomes apparent after 350 min of sertraline treatment.Taking all growth-based assays together, we hypothesizethat the V-ATPase and clathrin modulate resistance tosertraline in two fundamentally different ways. On theone hand, loss of V-ATPase complex activity appearsto lower but not extinguish the probability that in-dividual clones accumulate the minimum-required

lethal dose of sertraline. On the other hand, dysregula-tion of clathrin function appears to raise uniformly theminimum-required lethal dose of sertraline across thepopulation,

Chemical-genomic screening also implicates clathrin-coat formation: To complement the genetic landscapedetermined by an examination of de novo variation, weperformed two chemical-genomic screens. The firstinvolved identifying strains in the complete homozy-gous knockout collection (�4600 strains) that arehypersensitive to sertraline. This type of synthetic-lethalscreen has been used to identify nonessential cellularpathways that function in parallel to the pathway(s)affected by a small-molecule drug of interest. The sec-ond experiment involved a screen of the complete het-erozygous deletion collection (�6000 strains) for strainsthat are haploinsufficient with respect to sertraline-induced cytotoxicity. Haploinsufficiency screens havebeen used to identify essential protein targets of small-molecule drugs and are much more stringent thansynthetic-lethal screens. Response to sertraline hasalready been assessed using these knockout libraries,though in those experiments pools of deletions mutantswere assayed in bulk as opposed to individually inseparate wells (Ericson et al. 2008).

The top 20 most hypersensitive deletion strains(Table S1) are notably enriched for genes involved inthe process Gene Ontology (GO) term ‘‘vesicle-mediatedtransport’’ (P-value, 1.40e�5), the function GO term‘‘clathrin binding’’ (P-value, 4.54e�5), and the compo-nent GO term ‘‘AP-1 adaptor complex’’ (P-value,4.55e�8). Similar enrichments were reported by theaforementioned chemical-genomic study. The AP-1complex localizes to the Golgi and endosomes, whereit nucleates clathrin assembly. It is composed of twolarge subunits (APL2 and APL4), one of two mediumsubunits (APM1 and APM2), and one small subunit(APS1). There are three known AP complexes in yeast,but we only observe genetic interactions with AP-1subunit deletion strains. Other sertraline-hypersensitivestrains include vps1D, which normally encodes a fungaldynamin-like protein, a GTPase involved in vesiclescission; laa1D, which normally encodes an AP-1 com-plex accessory protein; and mpk1D, which normallyencodes serine/threonine kinase that is regulated bythe PKC1 signaling pathway.

The heterozygous deletion collection yieldedtwo heterozygous deletion strains, PIK1/pik1D andNEO1/neo1D, which are haploinsufficient in sertraline.PIK1, an essential gene, encodes phosphatidylinositol4-kinase, which catalyzes the formation of phosphatidy-linositol 4-phosphate, PI(4)P, a precursor of PIP2 andan effector molecule in its own right that concentratesin the trans-Golgi network (TGN), where it recruits theAP-1 complex in one of the early steps of clathrin-coatedvesicle biogenesis. NEO1, also an essential gene, enc-odes a P-type ATPase and putative flippase of unknown

Figure 5.—Single-cell viability assay shows that sertraline isirreversibly cytotoxic. The percentage of colony-forming unitsis plotted as a function of the duration of sertraline treatmentin minutes. Data correspond to BY4716 (blue circles),CHC1E292K (red circles), and CUP5R46I (green circles).



substrate specificity, and also localizes to the Golgi andendosomes. Both strains exhibit a twofold increase insensitivity to sertraline compared to wild type (FigureS4). NEO1 was originally isolated as a multicopy sup-pressor of neomycin cytotoxicity (Prezant et al. 1996);neomycin is an aminoglycoside antibiotic that has beenshown to bind to the phosphoinositide PIP2. Thesecomplementary genetic and chemical-genomic interac-tion data suggest that sertraline-induced cytotoxicity ismodulated by phosphoinositide metabolism or phos-pholipid asymmetry (or both) at sites of vesicle forma-tion in the TGN. Interestingly, some CADs, includingchlorpromazine, have been shown to affect phosphoi-nositide metabolism at concentrations relevant to thepresent study (Leli and Hauser 1987b).

Sertraline induces ultrastructural hallmarks of phos-pholipidosis: Transmission electron microscopy (TEM)of yeast cells treated with chlorpromazine previouslyshowed evidence of perturbed Golgi- and vacuolar-membrane organization, including the appearance ofso-called Berkeley bodies, or aberrant vesicular struc-tures that take the place of normal Golgi (Filippi et al.2007). We conducted a similar analysis of organellarmembrane ultrastructure in response to sertralinetreatment. First, we examined wild-type cells treatedwith high-dose sertraline for 1 hr. We used a membrane-preserving protocol previously employed to study mem-brane ultrastructure in VPT mutants (Banta et al. 1988).Comparison of fields of untreated and sertraline-treatedcells immediately reveals a vacuolar defect reminiscentof Class C VPT/VPS mutants (Figure 6). Specifically,four changes in membrane ultrastructure followingdrug treatment are noteworthy. First, electron-densevacuoles, which typically number one to three per cell,were not observed. After scoring fields of cells forthe presence or absence of vacuoles, we determinedthat 60% of untreated cells contain one or more vac-uoles, while none of the sertraline-treated cells contain anormal, electron-dense vacuole. Vacuoles that are vis-ible in sertraline-treated cells are much more electron-transparent compared to wild-type vacuoles, and oftenthey contain large inclusions that appear to be in-completely digested autophagosomes (Figure 7, B andD). The increase in vacuolar electron transparency andaccompanying presence of autophagosomal inclusionssuggests a defect in delivery of vacuolar hydrolases, lossof vacuolar acidification, or some combination of both.Second, a surprising number of sertraline-treated cellsalso lack nuclei. Nuclei are visible in 60% of untreatedcells, but only in 30% of sertraline-treated cells. Andwhen the nuclear membrane is present in sertraline-treated cells, it appears swollen and has many gaps. Theloss of nuclei could be a sign of apoptotic death andwould explain the irreversibility of sertraline-inducedcytotoxicity.

Third, every sertraline-treated cell examined con-tained at least one of several classes of aberrant

membrane-enclosed structures, among which 35% con-tain autophagosomal intermediates (e.g, dumbbell-shaped vesicles), indicating elevated rates of autophagy.Upon higher magnification, compacted multilamellarstructures were observed encapsulated by double-membrane structures that are clearly autophagosomes(Figure 7E), indicating active phospholipidosis in ser-traline-treated cells. Although direct comparison be-tween yeast and mammalian cells requires caution, thecompacted, often crescent-shaped multilamellar arrayswe observed in sertraline-treated yeast cells are remark-ably similar to those previously observed in CAD-induced phospholipidosis in mammalian cells. Fourth,in 33% of sertraline-treated cells but never in untreatedcells, we observed 250–500 nm multilamellar bodieswith complex morphology (Figure 7A). Upon highermagnification, some of these multilamellar bodiesappeared highly disordered and contain numerousosmiophilic deposits (Figure 7C). Others display strik-ing organization and resemble kiwi slices: a modestly

Figure 6.—Transmission electron micrographs reveal lossof vacuoles. (A) Representative thin section of a field of un-treated wild-type BY4716 cells. (B) Representative thin sectionof a field of sertraline-treated wild-type BY4716 cells. Bar,2.0 mm.




electron-dense core encircled by a ring of osmiophilicpuncta that is surrounded by a starburst of multilamellarruffles (Figure S5).

As a comparison, we analyzed cup5 mutant cells usingthe same membrane-preserving protocol. At steadystate, cup5 mutant cells appear similar to wild-type cellswith at least one notable exception: the vacuoles of somecup5 mutant cells have a more electron-transparent,mottled appearance consistent with alkalinization dueto inactivation of V-ATPase complex activity (Figure S6).(cup5 mutant cells also appear to have exaggeratedendoplasmic retriculum, but this phenotype was notinvestigated further.) Surprisingly, although all cup5cells harbor the mutant allele, only 56% (23/41) of cellsexhibit the vacuolar defect at steady state. To maximizedifferences between wild-type and cup5 mutant cells,we used a threefold lower dose of sertraline thanwhat was used in the previous TEM experiment. After1 hr of sertraline treatment of cup5 mutant cells,we observed fewer (8.5 vs. 16%) of what appear to bedead or dying cells—cells lacking both nuclei andvacuoles—compared to sertraline-treated wild-typecells. This approximately twofold difference in thenumber of dead cells is consistent with the data

obtained using colony-forming unit assays (Figure 5).Furthermore, these results validate the hypothesis thatsertraline uptake is not uniformly distributed among aclonal population of cup5 mutant cells, with approxi-mately half of the population appearing to requirehigher concentrations of sertraline to achieve the samedegree of cytotoxicity.

Sertraline modulates phospholipase activity in vitro:Inhibition of phospholipases by CADs may be respon-sible for the accumulation of multilamellar bodiesobserved in drug-induced phospholipidosis in mamma-lian cells and may be responsible for sertraline-inducedphospholipidosis we observed in yeast cells. To testwhether sertraline directly affects phospholipases, wescreened a panel of four different phospholipases usingzwitterionic phosphatidylcholine vesicles as substrates.Phospholipase A1 hydrolyzes the ester bond at the sn-1position, yielding a fatty acid and a lysophospholipid;phospholipase A2 hydrolyzes the ester bond at the sn-2position, also yielding a fatty acid and a lysophospholi-pid. Phospholipase C hydrolyzes the ester bond beforethe phosphate group, releasing diacylglycerol and aphosphate-containing head group. Phospholipase D hy-drolyzes the ester bond after the phosphate group,releasing phosphatidic acid and the head group choline.

If, as expected, sertraline intercalates into phospho-lipid bilayers, thereby altering membrane organization,then the substrate availability of individual membranephospholipids might be changed. Indeed, chlorprom-azine is known to affect the activity of reconstitutedphospholipases, exhibiting biphasic or concentration-dependent effects, such that at low doses it stimulatesphospholipase activity but at high doses it inhibitsphospholipase activity (Leli and Hauser 1987a). Forthis reason, we also included chlorpromazine in theseassays.

We screened both compounds across six orders ofmagnitude, from 10�9 to 10�3

m, encompassing theconcentration range in which sertraline exhibits activityin yeast. Our results, summarized in Figure 8, clearlydemonstrate a distinct pattern of activity across thepanel of phospholipases, including stimulation, inhibi-tion, and mixed effects. Phospholipase A1 activity isinhibited equipotently by both sertraline and chlor-promazine above 10 mm. Strikingly, phospholipase A2

activity is stimulated by both drugs to varying degrees.Sertraline’s stimulatory effects begin at 0.1 mm and leveloff at 10 mm, while chlorpromazine’s stimulatory effectsare negligible below 10 mm but increase exponentiallythereafter. Both sertraline and chlorpromazine ap-preciably inhibit phospholipase C activity at or above�50 mm, chlorpromazine is more potent than sertraline,but only sertraline exhibits a modest stimulatory bumpin the 1- to 10-mm range that is not fit by a classicsigmoidal function. Notably, the Hill coefficient forphospholipase C inhibition by both drugs is closer to�2, indicating negative cooperativity. This stands in

Figure 7.—Transmission electron micrographs revealsertraline-induced phospholipidosis. Wild-type BY4716 cellsin mid-log phase were treated with 120 mm for 1 hr. (A) Thinsection of a sertraline-treated cell containing multilamellarbodies. (B) Thin section of a sertraline-treated cell illustratingchange in vacuolar contents and accumulation of inclusions.(C) High magnification image of multilamellar body indi-cated by asterisk in A. (D) High magnification image of dis-tended vacuole-like structure containing undigestedautophagosomes indicated by asterisk in B. (E) High magni-fication image of a representative crescent-shaped multilamel-lar array encapsulated by an autophagosome. Scale bars areincluded in each panel.




contrast to the Hill coefficient for phospholipase A1inhibition by both drugs of �1. Finally, sertraline andchlorpromazine are weak inhibitors of phospholipase Dactivity, achieving 50% inhibition at 100 mm. As a neg-ative control, we used a coupled-enzyme assay for thedetection of inorganic phosphate that does not containlipid in the reaction mixture. The effects of sertralineare indistinguishable from those of vehicle, whilechlorpromazine exhibits nonspecific inhibitory effectsbut only in the high micromolar range (Figure S7).

The following trend emerges from these data withrespect to the modulatory effects of both drugs onenzymatic activity: PLA1 ¼ PLA2 . PLC . PLD. Putanother way, there is a positive correlation between thedistance of the cleaved bond from the head group andthe strength of drug-induced effects. This trend dem-onstrates that both sertraline and chlorpromazine in-tercalate into phospholipid membranes and, notunexpectedly, reside at the interface between the polarhead groups and the apolar/hydrophobic acyl chains.The most striking result, however, is that the sn-1 andsn-2 ester linkages are only angstroms apart yet bothdrugs have opposite effects on their cleavage, suggestinga specific three-dimensional orientation of sertraline inthe bilayer.

DISCUSSION

Upon integrating genetic, ultrastructural, and bio-chemical observations in a model organism that doesnot express its putative protein target, we propose thatthe selective serotonin reuptake inhibitor sertraline(Zoloft) intercalates into the polar/apolar interface ofthe cytoplasmic leaflets of V-ATPase-acidified organellesinvolved in intracellular vesicle transport, e.g., the trans-Golgi network (TGN), endosomes, and vacuole. Themain physiological consequence of a lethal dose of

sertraline is the induction of phospholipidosis, possiblybecause sertraline-intercalated membranes are indigest-ible to phospholipases. Phospholipidosis is correlatedwith membrane ultrastructural changes consistent withirreversible programmed cell death, e.g., increasedautophagy and loss of nuclei. Intriguingly, at sublethalconcentrations, sertraline rescues the growth defectcaused by dysregulation of clathrin function, raisingthe possibility that both sertraline and clathrin consumea limiting reagent in or associated with membranes (peranonymous reviewer’s suggestion).

By exploiting the cytotoxicity of sertraline in a drug-resistance selection, we identified two evolutionarilyconserved membrane-active pathways that confer par-tial resistance to sertraline by completely differentmechanisms. The first membrane-active pathway isvacuolar acidification, which was already known to bea determinant of resistance/sensitivity in yeast to com-pounds with physicochemical and pharmacologicalproperties shared with sertraline. Mutations affectingV-ATPase activity appear to reduce the probability thatan individual clone will uptake the minimum lethal doseof sertraline without changing the underlying affinityof membranes for sertraline. However, the secondmembrane-active pathway is clathrin-coat formation,which was not previously known to affect drug resistancein yeast. Mutations in this pathway appear to reduce theunderlying affinity of membranes for sertraline com-pared to wild type without changing the rate of uptake.

In other words, the rate of sertraline uptake intovesiculogenic membranes and the total binding capacityof these membranes for sertraline can be regulated by atleast two compensatory/protective mechanisms. TheV-ATPase and clathrin may normally function in wild-type cells to alleviate sertraline-induced membranestress but in the absence of mutation these bufferingsystems become overwhelmed. The point at which

Figure 8.—Effects of sertraline and chlor-promazine on enzymatic activity of a panel ofphospholipases. Data corresponding to sertra-line treatment is shown in blue circles; chlor-promazine treatment is shown in red triangles.(A) Dose–response curve of phospholipase A1activity. (B) Dose–response curve of phospholi-pase A2 activity. (C) Dose–response curve ofphospholipase C activity. (D) Dose–responsecurve of phospholipase D activity. All data pointsare normalized to baseline enzymatic activity inuntreated wells and represent the mean of fiveexperimental replicates. Error bars indicate stan-dard deviation.



individual cells succumb varies in a clonal population, asevidenced by the single-cell viability study. Epigeneticfactors that could account for clonal variation in thepenetrance of sertraline-induced cytotoxicity includeintracellular pH, natural cycles of vacuole fission andfusion, or other periodicities associated with vesiculartransport (Bishop et al. 2007).

The V-ATPase has already been shown to play aprotective role in the cellular response to CADs, butclathrin constitutes a novel axis of protection. Byincorporating this new axis, our model improves uponthe drug-accumulation model proffered by previousstudies of cellular response to CADs (Shih et al. 1988,1990; Filippi et al. 2007). With respect to the specificrole of the V-ATPase, our model also rests on a lysosome-trapping/basicicity argument. However, as hintedabove, the results of the epistasis analysis with the cup5swa2 double mutant indicate that a basicicity argumentalone is insufficient. The synthetic sickness of thatdouble mutant in the absence of sertraline, as well asthe kinetic defect in the emergence of resistance, suggestthat the V-ATPase and clathrin chaperones function in acontext-dependent manner to regulate the equilibriumof clathrin assembly/disassembly, a claim for whichthere are precedents in the literature (Forgac et al.1983; Liu et al. 1994). It is also possible that clathrin-mediated transport affects residual proton-translocatingactivity or another aspect of V-ATPase activity.

How might dysregulation of clathrin function alterthe underlying affinity of vesiculogenic membranes forsertraline? To answer this question, we must return tothe aforementioned limiting reagent common to bothsertraline and clathrin function. An attractive candidateis the phosphoinositide PI(4)P, which is generated bythe gene product of PIK1 and is essential for viability.The haploinsufficiency of the PIK1/pik1D heterozygousdeletion strain indicates that PI(4)P levels are limitingduring sertraline-induced membrane stress. ReducedPI(4)P levels may sensitize cells to sertraline by severalmechanisms: (i) PI(4)P may be a ‘‘receptor’’ for sertraline,in which case sertraline binding to it further depletesthe active pool; (ii) the biosynthesis of PI(4)P isparticularly sensitive to sertraline intercalation; and(iii) the presence of PI(4)P is correlated with othermembrane biophysical properties, such as membranecurvature, that favor sertraline intercalation (Mulet

et al. 2008).But what does sertraline intercalation, or CAD in-

tercalation more broadly, entail at the atomic level? Arecent study demonstrating that the s-2 receptoragonist siramesine binds specifically to phosphatidicacid shows that lipids can be specific targets of small-molecules drugs (Parry et al. 2008). Also, studies ofsome CADs and membrane-disrupting antimicrobialcationic peptides reveal two phases of membranebinding. CADs are initially recruited to membranesthrough electrostatic interactions with head groups

before penetrating into the hydrophobic region of thebilayer. It is tempting to hypothesize that the V-ATPasemay affect the rate of the first phase, while clathrin mayaffect the affinity of the second phase.

In conclusion, this study demonstrates that sertraline,an antidepressant developed to have high potency andselectivity at a single protein target, also targets phos-pholipid membranes by a molecular mechanism thatmay be common to many psychoactive cationic amphi-philes, even those that exhibit high degrees of poly-pharmacy at protein targets. Detailed atomic-levelstudies will be required to illuminate the exact natureof drug–lipid interactions. Future work will also berequired to determine the extent to which the V-ATPaseand clathrin shape the cellular response to sertraline inhigher eukaryotes and especially in neurons.

This work was entirely supported by the Lewis-Sigler Institute forIntegative Genomics at Princeton Univeristy. The authors thankP. Bisher (Electron Microscopy Core Facility, Princeton University)for technical assistance; D. Botstein (Princeton University), L. Kruglyak(Princeton University), M. Sheetz (Columbia Uuniversity), and theanonymous reviewers for insightful comments on the manuscript.

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Communicating editor: M. Nonet


Supporting Information http://www.genetics.org/cgi/content/full/genetics.110.117846/DC1

The Antidepressant Sertraline Targets Intracellular Vesiculogenic Membranes in Yeast

Meredith M. Rainey, Daniel Korostyshevsky, Sean Lee and Ethan O. Perlstein

Copyright © 2010 by the Genetics Society of America DOI: 10.1534/genetics.110.117846

M. M. Rainey et al. 2 SI

FIGURE S1.—Summary of phenotypic analyses of sertR tetrads. Each box corresponds to four spores (“a”, “b”, “c”, “d”)

dissected from a representative tetrad generated from a heterozygous diploid strain (labeled below each box). Red indicates that

the spore is sensitive, i.e., gr2owth-inhibited, in a condition; green indicates that a spore is resistant, i.e., not growth-inhibited, in a

condition; gray indicates that a spore was not assayed in a condition.


FIGURE S2.—Intracellular and extracellular pH affects sertraline response. (A) Dose-response curve of wildtype BY4716 cells

treated with 30μM sertraline and two-fold dilutions of bafilomycin A1. (B) Dose-response curve of wildtype BY4716 cells treated

with 30μM sertraline and two-fold dilutions of gramicidin. (C) Dose-response curve of wildtype BY4716 cells in three different

media: unbuffered YPD (yellow); YPD pH 4.0 (red); YPD pH 8.0 (blue). All strains were grown at 30ºC. Experimental OD600 values were normalized to OD600 values of untreated isogenic controls. Data points are the average of quadruplicate OD600

measurements. Data points were fit with a variable slope sigmoidal dose-response equation. Error bars indicate standard

deviation.


FIGURE S3.—Isolation and characterization of mutation in CSG2 that increases sertraline resistance in a cup5 background. (A).

Sequence chromatogram of an PCR product amplified from genomic DNA corresponding to a portion of the CSG2 coding region that contains two polymorphisms predicted by the SNPscanner algorithm. Sequences derived from the csg2 strain

(“mutant”) and the parental strain BY4712 (“wildtype”) are depicted. (B). Final yields of genotyped spores from tetrads derived

from a CSG2/csg2 CUP5/cup5 diploid were measured at optical density 600 nm (OD600). Cells were grown in 96-well plates in

YPD supplemented with 60μM sertraline and observed 24-48 hours post-inoculation. Solid black bars indicate average final yield

values of eight wildtype CSG2 CUP5 spores, four single mutant CSG2 cup5 spores, four single mutant csg2 CUP5 spores, and eight

double mutant cup5 csg2 spores.


FIGURE S4.—Phosphoinositide metabolism implicated in sertraline hypersensitivity. Dose-response curve of wildtype diploid BY4743 (black), PIK1/pik1 (blue), and NEO1/neo1 (green) treated with sertraline. All strains were grown at 30ºC. Experimental

OD600 values were normalized to OD600 values of untreated isogenic controls. Data points are the average of quadruplicate OD600

measurements. Data points were fit with a variable slope sigmoidal dose-response equation. Error bars indicate standard

deviation.


FIGURE S5.—Transmission electron micrograph of sertraline-treated BY4716 cell at high magnification. Asterisk indicates

structure referred to as a kiwi. Scale bar is included.


FIGURE S6.—Transmission electron micrograph of cup5 mutant cell at high magnification. Scale bar is included.


FIGURE S7.—Results of enzymatic assay for the detection of inorganic phosphate. Sertraline (blue-filled circles),

chlorpromazine (red-filled circles) and DMSO (black-filled circles) were tested at the concentrations indicated.


TABLE S1

Top 20 most sertraline hypersensitive haploid deletion strains

Gene ORF name Synthetic lethality score

APM2 YHL019C 0.0464

SLT2 YHR030C 0.0477

LAA1 YJL207C 0.0510

CKB2 YOR039W 0.0534

PDR5 YOR153W 0.0546

HXT12 YIL170W 0.0576

RAM1 YDL090C 0.0626

COS6 YGR295C 0.0675

VPS45 YGL095C 0.0680

ERG6 YML008C 0.0696

LEM3 YNL323W 0.0720

APL2 YKL135C 0.0720

VPS35 YJL154C 0.0731

ARC18 YLR370C 0.0738

APS1 YLR170C 0.0807

ERV14 YGL054C 0.0816

VPS1 YKR001C 0.0833

RCY1 YJL204C 0.0888

CHS5 YLR330W 0.0901

CSM1 YCR086W 0.0997

Synthetic lethality score is calculated by dividing the

sertraline--treated final yield of a given deletion strain by the

DMSO-treated final yield.

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