Supporting InformationHarbut et al. 10.1073/pnas.1216016110SI Materials and MethodsPlasmodium falciparum Culture.Briefly, 3D7parasites were culturedin RPMI 1640 (Life Technologies) supplemented with AlbuMAXII (Life Technologies) at 37 °C under an atmosphere of 5% CO2,5% O2, and 90% N2 (vol/vol). For synchronization, schizont-stageparasites were magnet-purified using a SuperMACS II Cell Sep-aration Unit (Miltenyi Biotech).

Taxon Sampling and Homology Searching. Data were retrieved fromthe following sources: (i) National Center for Biotechnology In-formation (www.ncbi.nlm.nih.gov), (ii) PlasmoDB (http://plasmodb.org/plasmo), (iii) TriTrypDB (http://tritrypdb.org/tritrypdb), and (iv)ToxoDB (http://toxodb.org/toxo). Database homology searchingwas performed using OrthoMCL (www.orthomcl.org), BLASTP,and profile hidden Markov algorithms (http://hmmer.janelia.org).TheHomo sapiens protein was used as an initial query against eachdatabase. Candidate proteins were deemed orthologues based onseveral criteria. All candidate orthologues from the queried ge-nomes that were retrieved with a cutoff E-value of 1 × 10−5 orbetter were subsequently verified by reciprocal BLASTP analysisusing the identified sequences as queries and the use of the can-didate gene as queries against the H. sapiens genome. Candidatesthat retrieved the original query sequence were deemed positive.In addition, candidates were also subjected to manual inspectionto deduce unique aspects of their structure such as putative lo-calization signals, membrane topology, and domain organization.

Parasite IC50 Determination. For IC50 determinations, synchro-nized parasites were plated at 1% parasitemia and 6% hemato-crit (vol/vol) in 96-well plates at a total volume of 50 μL. Serialdilutions of 2× concentration of the respective compound wereadded to the wells to bring the total volume to 100 μL and 0.5%parasitemia and 3% hematocrit (vol/vol). Compounds were as-sayed for a 72-h period, after which an equal volume 2× SYBRGreen (Life Technologies) in PBS solution with 0.25% (wt/vol)Triton X-100 was added for a final concentration of 10 μM andincubated at 37 °C for 30 min. DNA content, as an indicator ofparasitemia relative to uninfected erythrocytes, was analyzed onan Accuri C6 Flow Cytometer with C-Sampler. IC50 curves weregenerated by using Prism software (GraphPad). Compoundswere purchased from Sigma-Aldrich (DBeQ, 16F16, 17DMAG),Cayman Chemical (epoxomicin), and EMDMillipore [(Z-LL)2].

Mammalian Cell IC50 Determination. HepG2 cells were grown inDMEM (Life Technologies) supplemented with 10% (vol/vol)FBS. Cells were plated in 96-well black clear-bottom plates at 1 ×104 per well and treated with serial dilutions of compound, intriplicate, the following day. Cells viability was assessed after 24 hof treatment using CellTiter-Fluor Cell Viability Assay (Promega).Fluorescence as a measure of cell viability was measured ona TriStar LB 941 microplate reader (Berthold).

Endoplasmic Reticulum-Associated Degradation Assay. U-2 OS cellswere grown in DMEM supplemented with 10% (vol/vol) FBS. Cellswere seeded at density of 1 × 104 per well on a 24-well plate. Thefollowing day, 50 pmol of human signal peptide peptidase (SPP;hSPP1) siRNA (Life Technologies) was transfected by using Lip-ofectamine 2000 (Life Technologies). For degradation assays, 500ng of substrate plasmid was transfected per well of a 24-well plate 48h after siRNA transfection. For P. falciparum SPP (PfSPP) com-plementation, 500 ng of plasmid was cotransfected with substrateplasmid. Total transfected DNA was held constant by addition of

empty vector. Assays were conducted the following day. Cyclo-heximide (100 μg/mL) was added to wells with or without SPPinhibitors (Z-LL2 50 μM, LY-411575 10 μM, and NITD731 10μM). After 3 h treatment, cells were harvested and analyzed byWestern blot.

Yeast Strains for SPP Activity Assay. w303 pump mutants (MATacan1-100, his3-11, 15, leu2-3,112, trp1-1, ura3-1, ade2-1, pdr1::kanMX, pdr3::hygMX)were used tomake aΔspp by recombinationusing a pAG304ccdb under a Trp selection. pLZGreLacZ was usedas the reporter strain containing the glucocorticoid receptor with aleu marker. GR526-gpUL40 was made using glucocorticoid re-ceptor amino acids 1 to 526 fused to the TMD domain of humanCMVglycoproteinUL40 (NKFSNTRIGFTCAVMAPRTLILTVG-LLCMTITSLL) by 2×myc tag and inserted into pAG426GALccdb.The pLZGrelacZ and GR526-gpUL40 were transformed in WTw303 and w303, Δspp pump mutant strains to determine en-dogenous ScSPP and Δspp activities. Parasite SPPs [PfSPP (re-codonized for yeast expression), TbSPP, TgSPP] were cloned intopAG423GALccdbHA (his marker) and human SPP was clonedinto pAG423GPDccdbHA vectors using recombination. The vec-tors were transformed into yeast using standard Li/Ac protocol andselected on appropriate (-leu, -ura or –leu, -ura, -his) plates.

Yeast Activity Assays. Briefly, yeast cells were grown overnight inS-raffinose (-leu, -ura or –leu, -ura, -his) media to prevent ex-pression of the substrate. They were then diluted to an OD of ∼0.2and induced with 2% (vol/vol) galactose overnight (∼16 h). Asample of uninduced cell served as a control. Appropriateamounts of chemical compounds were diluted in 500 μL of yeastcells at the time of induction. Because of solubility issues, eachcompound was diluted into 1:1 DMSO:water solution and addedto cells at a nonlethal DMSO concentration of 4.25 μL/500 μL.Cells were then adjusted to an OD of ∼0.3, and 100 μL of cellswere incubated with 100 μL Gal-Screen reagent (Applied Bio-systems) for 1 h at 27 °C on a 96-well plate. The luminescence wasread by using a Berthold microplate luminometer.

Parasite Transfections. P. falciparum vectors expressing PfSPP-HAand PfSPP L333F-HAwere cloned in the piggyBacII vector via NotIand XhoI restriction sites. For transfections, 100 μg of the piggy-BacII PfSPP vector and 50 μg in 50 μL of water and 50 μL of 2×CytoMix were combined with 250 μL of packed red blood cells thathad been previously washed in Cytomix was 3 times in 1 mL ofCytoMix. The solution was brought up to 400 μL with 1× CytoMix(120 mMKCl, 0.15 mMCaCl2, 2 mMEGTA, 5mMMgCl2, 10 mMK2HPO4, 25 mM Hepes, adjusted to pH 7.6 with KOH). The so-lution was electroporated by using a Gene Pulser Xcell II (Bio-Rad), with settings 0.31 kV and 950 μF, in 0.2-mm cuvettetes.Electroporated cells were washed twice in complete media andadded to magnet-purified schizonts in a total of 5 mL completemedia. Selection for transgenic parasites was carried out byapplication of 2.5 nM WR99210 to cultures.

Antibodies. The following antibodies were used: mouse anti-plas-mepsin V (23.1.2; gift from Mike Klemba, Virginia PolytechnicInstitute and State University, Blacksburg, VA); rabbit anti–HA-tag (C29F4; Cell Signaling); goat anti-rabbit IgG (H+L), per-oxidase conjugated (Pierce); and goat anti-mouse IgG (H+L),peroxidase conjugated (Pierce). Rabbit anti-hSPP and rabbitanti-PfSPP were provided by T.E.G.

Harbut et al. www.pnas.org/cgi/content/short/1216016110 1 of 6

Microscopy. For indirect immunofluorescence assays, washed, in-fected erythrocytes were fixed with 4% paraformaldehyde/0.008%glutaraldehyde (vol/vol) at specified time points. Cells were per-meabilized with 0.1% (wt/vol) Triton X-100 for 10 min and thenblocked in 5% (wt/vol) BSA in PBS solution for 1 h, then incubatedfor 1 h with rabbit anti-PfSPP (1:500), anti-HA (1:500), and, incolocalization experiments, mouse anti-PM V antibody 23.1.2(undiluted hybridoma supernatant). Following a 1-h incubation,cells were washed three times with PBS solution and then in-cubated with Alexa 488-conjugated goat anti-rabbit IgG (1:1,000)secondary antibody and Alexa 568-conjugated goat anti-mouseIgG (1:1,000 in 5% (wt/vol) BSA in PBS solution; Life Technol-ogies). Hoechst (1 μM) was added for nuclei staining. Parasiteswere imaged via fluorescence microscopy on a Leica DMI6000 Bmicroscope, and image processing was performed by usingImageJ.

Labeling of Parasite Lysates with (Z-LL)2 Activity-Based Probe. Forparasite labeling, mixed-stage parasites were harvested and re-leased from erythrocytes with 1% (wt/vol) saponin, followed bycentrifugation at 1,500 × g for 5 min and three washes in cold PBSsolution. Parasite lysates were prepared by freeze/thaw in the pres-ence of 1% (vol/vol) 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) in 25 mM Pipes-NaOH,pH 6.5, 150 mMNaCl, 5 mMMgCl2, 5 mM NaCl2, and proteaseinhibitor mixture (Roche). Membrane and cell debris wasclarified by centrifugation at 16,000 × g for 10 min at 4 °C. La-beling was performed with indicated concentrations of the activity-based probe for 1 h at 37 °C followed by UV crosslinking (365 nm)for 1 h on ice after dilution of lysate to bring CHAPSO to 0.25%(wt/vol). Competition labeling was carried out by preincubatinglysates with specified inhibitor for 30 min at 37 °C. For immuno-precipitation, lysates were passed through 7K MWCO desaltingcolumns (Pierce) after UV crosslinking, then incubated overnightwith streptavidin UltraLink Resin (Pierce). Proteins were visual-ized by standard Western blotting.

Resistant Parasite Generation. Parasites were treated with sublethalconcentration of inhibitor, increasing as resistance increased. In-hibitor resistance was generated for a period of 4 mo. Resistantparasites were cloned by limiting dilution. Parasites were saponin-treatedand spundown, andRNAwasextractedbyusing anRNeasyKit (Qiagen).RNAwasconverted tocDNAbyusingaSuperScript IReverse Transcriptase Kit, and cloned into pAG423ccdbHA using

SpeI and XhoI restriction sites. The cDNA from the parasite lineswere subjected to three independent PCR reactions and then se-quenced by using primers to pfspp. PfSPP L333F was made bystandard site-directed mutagenesis techniques using the WT re-codonized PfSPP as a template.

Whole-Genome Sequencing and Data Analysis. Genomic DNA fromNITD731r was paired-end–sequenced on an Illumina HiSEq 2000for 50 bp per read plus one 7-bp index read using Illumina v3chemistry. Genomic DNA from theDd2 parental clone was single-end–sequenced on an Illumina Genome Analyzer IIx for 50 bpplus one 7-bp index read using Illumina v3 chemistry. Base callswere made using Illumina RTA (version 1.12) software. Data foreach sample sequenced in this study is available in the NationalCenter for Biotechnology Information Sequence Read Archive.Fastq files obtained from sequencing were aligned to the 3D7reference (PlasmoDB version 9.1) using BWA (version 0.5.9) withsoft clipping of bases with quality scores of 2 and below (1). PCRduplicates were next identified and removed by using Picard(version 1.51) MarkDuplicates. Aligned reads were then realignedaround indels and areas of high entropy usingGATK (version 2.0+)IndelRealigner, and the base quality scores of realigned readswere then recalibrated using GATK BaseRecalibrator (2). Afterrealignment and recalibration, the samples were considered“clean” and ready for use in downstream analysis.Genome-wide coverage and loci covered to a certain per-

centage were calculated by using GATKDepthOfCoverage (2).For all GATKDepthOfCoverage analyses, the minimummappingquality (mmq) was set to 29 and the minimum base quality (mbq)was set to 20.Single nucleotide variant discovery was conducted by using

GATK UnifiedGenotyper (2) on the parental strain and the re-sistant strain concurrently with the following options: -stand_call_conf50.0, -stand_emit_conf 10.0, -dcov 50, -baq CALCULATE_AS_NECESSARY, and -nt 4. Variants identified were annotated byusing snpEff (3). GATK VariantFiltration (2) was applied to theresulting variant call set to achieve the highest-confidence singlenucleotide variant set possible with the following options: -window 5-cluster 2, -filter “MQ < 40.0” -filterName “LowMQ”, -filter “QD <5.0” -filterName “LowQD”, -G_filter “GQ < 30” -G_filerName“LowGQ”. Only homozygous genotype calls passing all quality filtersin both sequenced samples were considered in downstream analysis.

1. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheelertransform. Bioinformatics 25(14):1754–1760.

2. DePristo MA, et al. (2011) A framework for variation discovery and genotyping usingnext-generation DNA sequencing data. Nat Genet 43(5):491–498.

3. Cingolani P, et al. (2012) A program for annotating and predicting the effects of singlenucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogasterstrain w1118; iso-2; iso-3. Fly (Austin) 6(2):80–92.

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Functional module

Substrate recognition and processing

HSP70 familyHSP90 family

Reductase

Dithiol IsomeraseCarboxypeptidase

Lectin

ATP-dependent proteaseDislocation

Processing protease

Trafficking

Derlin

RhomboidiRhom

Uiquitin ligation complexes

Substrate extraction

Recruitment factors

Deubiquitylating

Ubiquitin extension

Human

BiP (GRP78)GRP94

ERDJ5 (HSP40)ERFADERO1PDI

CPVLOS9

ERLEC1LONP2

SPPRHDBL4ESYT1ESYT2TRAPDerlin1Derlin2Derlin3

RHBDL4iRhom1

gp78ERLIN1ERLIN2UBAC2TMUB1BRI3BP

Hrd1UBE2G2SEL1L

FAM8A1AUP1HERPRNF5

DOA10

VIMPUBXD2TRAM1

NPLOC4UFD1L

p97SVIPPNG1RAD23DOA1

YOD1/OTU1ATX3

USP13VCIP135UBE4B

P. falciparum

PFI0875wPFL1070cPF11_0099

AbsentPF11_0251MAL8P1.17

AbsentAbsentAbsentAbsent

PF14_0543AbsentAbsentAbsentAbsent

PF10_0317 PF14_0653

AbsentAbsentAbsent

AbsentAbsentAbsentAbsentAbsentAbsent

PF14_0215PFI1030c-bPF14_0462

AbsentAbsentAbsent

PFF1325c Absent

AbsentAbsentAbsent

PFE0380c PF14_0178PFF0940c

AbsentAbsent

PF10_0114 PF13_0335

AbsentPFL1295w PFD0680c

AbsentPF08_0020

T. gondii

TGME49_111720TGME49_044560TGME49_004480

AbsentTGME49_100380 TGME49_011680

AbsentAbsentAbsentAbsent

TGME49_037150AbsentAbsentAbsentAbsent

TGME49_094290 TGME49_017160

AbsentAbsentAbsent

AbsentAbsentAbsentAbsentAbsentAbsent

TGME49_104460 TGME49_002820TGME49_054490

AbsentAbsentAbsent

TGME49_005600 Absent

AbsentAbsentAbsent

TGME49_071440 TGME49_070530TGME49_073090

AbsentAbsent

TGME49_095340TGME49_088210TGME49_077990TGME49_029650TGME49_013870

AbsentTGME49_080490

L. infantum

LINJ_28_1310 LINJ_29_0790

AbsentAbsent

LINJ_16_1630LINJ_26_0630 LINJ_18_0450

AbsentAbsentAbsent

LINJ.29.0990AbsentAbsentAbsentAbsent

LINJ_33_1900LINJ_19_0120

AbsentAbsentAbsent

AbsentAbsentAbsentAbsentAbsentAbsent

LINJ_15_1460LINJ.32.1010 LINJ_07_0650

AbsentAbsentAbsent

LINJ_36_2030 LINJ_27_0120

AbsentAbsentAbsentAbsent

LINJ_36_6780LINJ_36_1420

AbsentAbsent

LINJ_30_3350LINJ_24_1970LINJ_36_6280

AbsentLINJ.29.2410

AbsentLINJ.30.1070

T. cruzi

Tc00.1047053506585.40Tc00.1047053507713.40

AbsentAbsent

Tc00.1047053508837.189Tc00.1047053506559.200Tc00.1047053509695.210 Tc00.1047053506155.99

AbsentAbsent

Tc00.1047053507951.260AbsentAbsentAbsentAbsent

Tc00.1047053509029.110 Tc00.1047053508777.80

AbsentAbsentAbsent

Tc00.1047053509945.10AbsentAbsent

Tc00.1047053506399.60 AbsentAbsent

Tc00.1047053508277.190Tc00.1047053511727.40

Tc00.1047053508543.150AbsentAbsentAbsent

Tc00.1047053507641.236Tc00.1047053508525.30

AbsentAbsentAbsentAbsent

Tc00.1047053504167.40Tc00.1047053509733.170

AbsentAbsent

Tc00.1047053506833.60Tc00.1047053511903.290Tc00.1047053508723.59

AbsentTc00.1047053508153.120

AbsentTc00.1047053509965.250

Fig. S1. Predicted endoplasmic reticulum-associated degradation orthologues in humans and different protozoan pathogens organized by functional modules.

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PM-V HA MergeDIC

PfSPP Hoechst MergeDIC

PM-V PfSPP MergeDIC

Ring

Trophozoite

Early schizont

Lateschizont

Fig. S2. Indirect immunofluorescence assays using an antibody generated to PfSPP reveal ER staining throughout the life cycle of the parasite. The ER stainingis confirmed by colocalization of PfSPP with the ER-localized protease plasmepsin V (Middle). Transgenic parasites expressing HA-tagged PfSPP show similarlocalization.

-7 -6 -5 -4 -30

50

100

log[Thapsigargin], M

% p

aras

ite v

iabi

lity

= 7.0 MIC50

Fig. S3. P. falciparum is sensitive to ER stress produced by the calcium-transporting ATPase inhibitor thapsigargin.

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Fig. S4. Sequence alignment of hSPP1, PfSPP, and Saccharomyces cerevisiae SPP (ScSPP). Asterisks indicate completely conserved residues.

0

100000

200000

300000

400000

500000

600000

700000

Luminescence

Pfspp hspp1 wt S. cerevisiae

Δspp S. cerevisiae

GR526/no substrate

uninduced substrateinduced substrate

Fig. S5. Activity levels of expressed different SPPs. The KO ScSPP shows negligible background activity, and GR526 overexpression without the transmembranedomain results in maximum luminescence.

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0

20000

40000

60000

80000

100000

120000

140000

160000

Luminescence

WT PfSPP L333F PfSPP

uninduced substrateinduced substrate

Fig. S6. Activity levels of WT and L333F PfSPP show equivalent activity levels on the induced substrate.

Table S1. Single nucleotide variants found in the NITD731r line

Chr. Position Ref. Alt.Dd2

parent NITD731r Gene ID Effect bp change aa change

1 159129 T C 1 0 PF3D7_0103500 Intron — —

11 1384736 A T 0 1 PF3D7_1135400 Nonsynonymous/coding aTa/aAa I127K14 2336891 G A 0 1 PF3D7_1457000 Nonsynonymous/coding Ctc/Ttc L333F

Alt., alternative allele; Chr., chromosome; Ref., reference allele.

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