Traffic 2012; 13: 1335–1350 © 2012 John Wiley & Sons A/S

doi:10.1111/j.1600-0854.2012.01394.x

Dissection of Minimal Sequence Requirements forRhoptry Membrane Targeting in the Malaria Parasite

Ana Cabrera1, Susann Herrmann1,2, Dominik

Warszta1, Joana M. Santos3,4, Arun T. John

Peter5, Maya Kono6, Sandra Debrouver3,

Thomas Jacobs7, Tobias Spielmann6, Christian

Ungermann5, Dominique Soldati-Favre3 and

Tim W. Gilberger1,6,∗

1M.G. DeGroote Institute for Infectious DiseaseResearch, Department of Pathology and MolecularMedicine, McMaster University, Hamilton, ON, Canada2Current address: Department of Microbiology, MonashUniversity, Victoria, Australia3Department of Microbiology and Molecular Medicine,University of Geneva, Geneva, Switzerland4Current address: Department of Molecular Biology,Princeton University, Princeton, NJ, USA5Department of Biology/Chemistry, BiochemistrySection, University of Osnabruck, Osnabruck, Germany6Department of Molecular Parasitology,Bernhard-Nocht-Institute for Tropical Medicine,Hamburg, Germany7Department of Medical Microbiology,Bernhard-Nocht-Institute for Tropical Medicine,Hamburg, Germany*Corresponding author: Tim Gilberger,tgilber@mcmaster.ca

Rhoptries are specialized secretory organelles charac-

teristic of single cell organisms belonging to the clade

Apicomplexa. These organelles play a key role in the

invasion process of host cells by accumulating and sub-

sequently secreting an unknown number of proteins

mediating host cell entry. Despite their essential role,

little is known about their biogenesis, components and

targeting determinants. Here, we report on a conserved

apicomplexan protein termed Armadillo Repeats-Only

(ARO) protein that we localized to the cytosolic face

of Plasmodium falciparum and Toxoplasma gondii rhop-

tries. We show that the first 20 N-terminal amino acids

are sufficient for rhoptry membrane targeting. This pro-

tein relies on both – myristoylation and palmitoylation

motifs – for membrane attachment. Although these lipid

modifications are essential, they are not sufficient to

direct ARO to the rhoptry membranes. Mutational analy-

sis revealed additional residues within the first 20 amino

acids of ARO that play an important role for rhoptry

membrane attachment: the positively charged residues

R9 and K14. Interestingly, the exchange of R9 with a neg-

ative charge entirely abolishes membrane attachment,

whereas the exchange of K14 (and to a lesser extent K16)

alters only its membrane specificity. Additionally, 17 pro-

teins predicted to be myristoylated and palmitoylated

in the first 20 N-terminal amino acids were identified

in the genome of the malaria parasite. While most of

the corresponding GFP fusion proteins were trafficked

to the parasite plasma membrane, two were sorted to

the apical organelles. Interestingly, these proteins have a

similar motif identified for ARO.

Key words: acylation, apicomplexa, armadillo repeats,

malaria, protein trafficking, rhoptry

Received 17 January 2012, revised and accepted for

publication 28 June 2012, uncorrected manuscript

published online 3 July 2012, published online 1 August

2012

The phylogenetic group called Apicomplexa comprisesimportant unicellular pathogens such as Plasmodium,Toxoplasma and Cryptosporidium. Plasmodium spp.causative agent of malaria, infects an estimated 500million people annually, resulting in about 1 million deathseach year (1). A key process for apicomplexan parasitesis the invasion and subsequent multiplication within theirhost. This complex process (2–4) is mediated by threetypes of organelles (micronemes, rhoptries and densegranules) (5,6). The secretion of the proteins stored inthese organelles is essential for host cell invasion and istightly orchestrated and regulated (7–10). Rhoptries arelocated at the apical pole of invasive-stage parasites (e.g.sporozoites and merozoites in Plasmodium spp., or tachy-zoites in Toxoplasma sp.). Toward the end of cell division,rhoptries are first detected as small circular organellesformed by fusion of coated vesicles from the Golgi(so-called pre-rhoptries) located close to the cluster oftrans-Golgi vesicles (11,12). The pre-rhoptries mature withthe formation of ducts toward the schizont surface closeto the developing polar rings (12). It has been proposedthat the pre-rhoptry is an endosome-related organelle(13,14). All secreted rhoptry proteins possess a signalsequence that allows entry into the secretory pathway.

Although the role of the rhoptries in host cell invasionhas been well established and multiple proteins havebeen localized to these compartments, their biogenesisand membrane biology is still poorly understood (15).Some studies implicate the adaptor protein 1 complex(AP1) in vesicle trafficking to this organelle (16,17). Amore recent study showed a central role of the alveolate-specific dynamin-related protein B (DrpB) in the biogenesisof secretory organelles in Toxoplasma gondii (18). Theunderlying molecular mechanism that enables trans-Golgiderived vesicles to initiate and propagate rhoptry formationand membrane fusion is still unknown. Studies in bothPlasmodium and Toxoplasma localized Rab11A at leasttemporarily to the rhoptries (19,20). Both proteins, AP1and Rab11A, are cytosolic factors that are apparentlytemporarily attached to rhoptry membranes.

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Recently, a protein lacking a signal peptide (PFD0720w)was identified and localized to the apical end of free mero-zoites of Plasmodium falciparum (21). PFD0720w is char-acterized by two features: C-terminal armadillo repeatsand predicted N-terminal acylation motifs. Armadillodomains are 42 amino acids long motifs implicated inprotein–protein interactions (22,23) that consist of threehelixes with two hydrophobic areas (24). PFD0720w hasbeen termed Armadillo Repeats-Only (ARO) protein. Thesecond feature of this protein, acylation, is an impor-tant protein modification involving two types of fattyacids, myristate and palmitate (25). Myristoylation isthe permanent co-translational addition of myristic acidto an N-terminal glycine via an amide bond. This pro-cess is catalyzed by the enzyme N-myristoyl transferase(NMT). Palmitoylation is the reversible post-translationalmodification where palmitic acid is linked to a variablylocated cysteine via thioester bond. This is catalyzed bymembrane-associated palmitoyl acyl transferases (PATs)characterized by the presence of a DHHC motif (26).How membrane specificity of dually acylated proteins isconferred in any system is largely unknown.

Results

ARO is an armadillo-domain containing rhoptry

protein that is expressed late in blood stages

of P. falciparum and is conserved in Apicomplexa

PfARO (Plasmo DB gene accession number PFD0720w) islocated on chromosome 4 in the genome of P. falciparumand comprises 2175 bp with a complex 11-exon structure.Its coding region has a length of 828 bp that results ina protein with a predicted molecular weight of 31 kDa.Transcription is strongly upregulated in late blood stageparasites (27). The gene product is predicted to be involvedin invasion (PlasmoINT) and is localized in the apical end ofmerozoites upon ectopic expression as GFP fusion (21).

Analysis of the primary sequence of PfARO reveals twodomains with homology to armadillo-like repeats predictedby MotifScan. Three additional stretches show somehomology to armadillo domains, and are predicted to foldin a similar way (Figure S1A, Supporting Information).PfARO is one of 20 proteins that contain several of theserepeats. Interestingly, PfARO contains no signal peptide(or recessed hydrophobic patch) as opposed to the vastmajority of known rhoptry or microneme proteins (28), butcomprises myristoylation and palmitoylation motifs at itsN-terminus (Figure 1A). While myristoylation is predictedwith a medium confidence score (Myr: 0.569829; NMT:0.277), palmitoylation of the cysteines C4 and C5 showshigh confidence (CSS-Palm: 3.545 and 4.281, respectively)using the available prediction tools.

The expression and localization of the endogenous PfAROprotein was analyzed using specific antibodies raisedagainst the full-length protein. Western blot analysisusing sorbitol-synchronized parasite material harvested

at different time points throughout the asexual lifecycle revealed that in line with its transcription profile,PFD0720w is exclusively expressed in late stages as anapproximately 30 kDa protein (Figure 1B). Localizationstudies using PfARO-specific antibodies show an apicaldistribution in schizonts and free merozoites (Figure 1C),resembling the distribution of the GFP fusion (21). Co-localization with the rhoptry bulb marker RALP-1 (28)(Figure 1D) and the microneme marker EBA-181 identifyPfARO as a rhoptry protein (Figure 1E).

PfARO is conserved throughout Apicomplexa withorthologs in T. gondii, Cryptosporidium spp., Theileria spp.,Babesia bovis, Eimeria and could also be retrieved fromthe genome of the dinoflagellate Perkinsus marinus usingBLAST searches (Figure S1B). The rhoptry localization ofthe T. gondii homolog TGME49_061440 was confirmed byoverexpression of C-terminal TY1 tagged TgARO and sub-sequent localization in tachyzoites (Figures S2A–E, S3A).

N-terminal acylation motifs are necessary

for peripheral ARO rhoptry membrane attachment

Although ARO does not possess a signal peptide forentering the secretory pathway, it is nevertheless targetedexclusively to the rhoptries, most likely by attachment tothe cytosolic face of the membrane by N-terminal lipidmodifications. To investigate the role of the predictedmyristoylation and palmitoylation motifs, we expressedARO mutants in P.falciparum lacking the predicted sites(ARO�20, AROA2A5A6, AROA2 and AROA5A6, respectively).These PfARO mutants were fused to GFP and localizedin transgenic parasites. Deletion of the first 20 aminoacid residues (removing the entire region containing theacylation motifs), as well as the mutation of either acylationmotif or both of them changed the apical GFP distributionto a cytosolic one (Figure 2A–E). These results suggestthat ARO is attached to the cytosolic face of the rhoptrymembrane using N-terminal acyl moieties. It also revealsthat the armadillo repeats alone do not confer membraneassociation (Figure 2B).

To confirm the topology of ARO, a Proteinase K protec-tion assay was performed. After digitonin permeabilizationof the parasite plasma membrane and degradation of allexposed proteins by Proteinase K, those proteins pro-tected by organelle membranes remain. Following thisscheme, ARO is sensitive to Proteinase K treatment,whereas rhoptry internal proteins were protected, con-firming its attachment to the cytosolic face of the rhoptrymembrane (Figure 3). This topology was also evaluated inT. gondii showing the same results (Figure S3B).

Acylated ARO N-terminus is sufficient for rhoptry

membrane targeting

After establishing the necessity of both acylation motifsfor ARO membrane association and the apparentindependence of the armadillo domains for its targeting,we investigated the minimal sequence requirement for

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A B

C

D

E

Figure 1: ARO is late transcribed

in blood stages of P. falciparum

and co-localizes with the rhop-

tries. A) Domain structure of P. falci-parum ARO (PfARO); red: armadillodomains. B) Stage-specific expres-sion pattern of PfARO using synchro-nized parasite material (0–9, 9–17,18–26, 26–34 and 34–42 h) andanti-PfARO antibodies. Antibodiesdirected against the late expressedrhoptry associated leucine zipper likeprotein 1 (RALP-1) were used as astage-specific control. Anti-GAPDHantibodies were used as loading con-trol. C) Immunofluorescence of glu-taraldehyde/formaldehyde fixed wildtype parasites using anti-PfAROantibodies (green) on schizont (s)and merozoite (m) stage parasites.Co-localization of PfARO with (D)the rhoptry marker RALP-1 (red)and (E) the microneme markerEBA-181 (red). Nuclei stained withDAPI (blue). Enlargement of selectedareas are marked with white squareand referred as Zoom. Scale bar,1 μm.

rhoptry localization. We hypothesized that all necessaryinformation for membrane attachment are encoded withinthe N-terminal residues. To test this, we fused the first20 amino acid residues of PfARO (20AROwt) to GFPand expressed the fusion protein in the parasite. Thisminimal construct showed rhoptry localization like the full-length protein. Hence, the N-terminal residues of PfAROencode sufficient information for its membrane specificity(Figure 4A). This minimal construct was co-localizedwith ARO-specific antibodies to confirm correct organellerecruitment (Figure 4B). Similarly, the first 20 residuesof TgARO were fused to GFP-TY1 (20TgARO-GFPTY)

showing that this minimal construct is primarily targetedto the rhoptries in T. gondii (Figure S2F,G). Additionally,the expression of 20TgARO in P. falciparum results inrhoptry localization like its malaria counterpart (Figure 4C),supporting functional homology in the well-conservedorthologs.

In order to investigate and provide experimental evidenceof the predicted palmitoylation of the ARO N-terminus, weused a biotin switch assay modified for P. falciparum (29).The glideosome-associated protein GAP45 that, like ARO,is predicted to be palmitoylated (as well as myristoylated)

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A

B

C

D

E

A1

A2

Figure 2: N-terminal lipid modification motifs are essential

for Pf ARO rhoptry membrane association. Schematic repre-sentation PfARO-GFP fusion protein and localization in unfixedparasites (schizonts and merozoites) of (A) wild type, (B)PfARO�20 mutant, (C) PfAROA2A5A6 (double acylation) mutant,(D) PfAROA2 (myristoylation) mutant and (E) PfAROA5A6 (palmi-toylation) mutant. Nuclei stained with DAPI (blue). Enlargementof selected areas are marked with white square and referred asZoom. Scale bar, 1 μm.

was used as control. This assay involves the substitution ofacyl groups by biotin and affinity purification of biotinylatedproteins. We show that 20AROwt gets biotinylated andis enriched in the fraction that is expected to contain allS-acylated proteins assayed by this method (Figure 4D).Of note, in contrast to GAP45 that is exclusively detectedin this fraction, some background binding of 20AROwtto the matrix could be shown also in the untreated,non-biotinylated one. This might be explained by someunspecific binding of the GFP moiety of the fusion proteinto the NeutrAvidin matrix.

Figure 3: ARO is attached to the cytosolic face of the

rhoptry membrane. Topology of PfARO-GFP using ProteinaseK (PK) protection assay on ARO-GFP permeabilized withdigitonin (D). PfARO-GFP was detected with anti-GFP antibodies.Antibodies against a luminal rhoptry protein (anti-RhopH3) andagainst the cytosolic protein GAPDH (anti-GAPDH) were used ascontrols. First lane: control, untreated (D− PK−), second lane:permeabilization control (D+ PK−), third lane: permeabilized andproteinase K cleaved (D+ PK+).

Requirements within the first 20 amino acids

for rhoptry targeting

The rhoptry membrane specificity cannot be solelydependent on acylation motifs given that other P. falci-parum proteins reveal a very similar N-terminal sequenceand transcription profile, but are localized to othermembranes. For instance, this is the case for PF14_0578(MG2XXC5CXX) that is localized to the inner membranecomplex (IMC), a membranous system, which underliesthe plasma membrane (21,30). In order to dissect thedual function of the N-terminal residues of PfARO –membrane attachment and membrane discrimination– an alanine scan was performed. First, the N-terminalportion of 20AROwt was analyzed (residues 1–10,MGN3N4CCAG8R9D10). While the substitution of theasparagines (20AROA3A4) did not interfere with rhoptrymembrane targeting (Figure 5A1) the exchange of aminoacid residues 8–10 (20AROA8A9A10) resulted in a cytosolicdistribution of the fusion protein (Figure 5B1). This changein the localization pattern is supported by solubility assays:The rhoptry-associated 20AROA3A4 can be detected in allthree supernatants (soluble, carbonate and Triton X100fraction) but 20AROA8A9A10 is exclusively found in thesoluble fraction (Figure 5A2,B2). This result points towarda role of one (or all) of amino acids 8–10 in the acylationprocess of PfARO. Secondly, amino acid residues 11–20were exchanged by alanine (20AROA11-20). Although thissubstitution did not interfere with membrane attachment(Figure 5C2), the specific recruitment to the rhoptrymembrane was disrupted and the mutant was directedto the parasite plasma membrane (PPM) (Figure 5C1) –hence this part of the N-terminus of the proteinappears to be crucial for rhoptry membrane specificrecruitment.

Arginine 9 plays an important role for membrane

attachment

To further analyze the crucial involvement of amino acidsG8R9D10, two point mutants were generated targeting the

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A

B

C

D

Figure 4: The N-terminus of ARO is sufficient for rhoptry association and is palmitoylated. A) Schematic representation of theminimal PfARO construct (20AROwt) encompassimg the N-terminal 20 amino acids fused to GFP and its apical localization in unfixedparasites (S, schizonts and M, merozoites). B) Co-localization in fixed parasites using anti-ARO antibodies. Nuclei stained with DAPI(blue). Enlargement of selected areas are marked with white squares and referred as Zoom. Scale bar, 1 μm. C) Schematic representationand localization of the minimal TgARO construct (20TgARO) fused to GFP (green). Amino acids identical to the P. falciparum counterpartare highlighted or marked with asterisks. Expression and localization of this fusion protein in unfixed malaria parasites (S, schizonts andM, merozoites) showed identical apical distribution as 20AROwt. Nuclei stained with DAPI (blue). D) S-acyl Biotin switch experimentusing 20AROwt expressing parasites. Proteins were detected on western blot using anti-GFP and anti-GAP45 antibodies as control.20AROwt as well as GAP45 is present in the harvested parasite material (input) as well as in the two aliquots (loading, −HA +HA) thatwere incubated with biotinylation reagent with or without hydroxilamine after NEM treatment. After elution from the NeutrAvidin beads,20AROwt is enriched in the hydroxylamine (+HA) treated sample when compared with the untreated sample (−HA), while GAP45 isnearly exclusively detected in the +HA sample.

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AA1

B1

C1

A2

B2

C2

B

C

Figure 5: Sequence requirements for ARO rhoptry association using alanin substitutions within the first 20 amino acids. Eitherthe aparagines N3 and N4 (A, 20AROA3A4), the glycine, arginine and leucine at position 7–9 (B, 20AROA7A8A9) or the entire second half ofthe N-terminus were substituted with alanin (C, 20AROA11 –20). Mutant 20ARO-GFP fusion proteins were localized in unfixed parasites(A1, B1, C1). While 20AROA3A4 is trafficked to the rhoptries (A1), 20AROA7A8A9 reveals a cytoplasmic (B1) and 20AROA11-20 a plasmamembrane (C1) distribution. Nuclei stained with DAPI. Enlargement of selected areas are marked with white squares and referred asZoom. Scale bar, 1 μm. (A2, B2, C2) This change in the localization pattern is supported by solubility assays: The membrane associatedproteins 20AROA3A4 (A2) and 20AROA11 –20 (C2) can be detected in all three extraction fractions (soluble: H2O/SN, carbonate Carb/SNand membrane Triton X100: Tx100/SN) while 20AROA8A9A10 is exclusively in the soluble fraction (B2) (top pannels). Antibodies againstthe cytosolic protein GAPDH were used as control (bottom panels).

charged residues R9 and D10 (G8 was not addressed sinceit has similar characteristics as alanine). The substitutionof the negatively charged aspartic acid (D10) with apositive lysine (20AROD10K) had no impact on localization(Figure 6A1). In contrast, the replacement of R9 by anegatively charged glutamic acid (20AROR9E) completely

abolished membrane attachment (Figure 6B1). This issupported by a changed solubility profile (Figure 6A2,B2).Interestingly, exchange of this positive residue by anothersimilarly charged amino acid, lysine, renders the mutantmembrane attached (Figure 6C2) but to the PPM(Figure 6C1). This argues not only for an important

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B1

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A2

B2

C2

B

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Figure 6: Charged amino acid R9 but not D10 plays a key role in membrane recruitment and specificity of ARO. A) Substitutionof the negative charged aspartic acid D10 with a positive charged lysine (20AROD10K) do not alter rhoptry localization (A1) or membraneattachment (A2). B) Exchange of the positive charged arginine R9 with a negative charge residue (20AROR9E) results in a cytosolicdistribution of the GFP (B1) and completely abolishes membrane association (B2). Nuclei stained with DAPI (blue). C) Exchange of thepositive charged arginine R9 with a positive charged lysine (20AROR9K) results in a plasma membrane distribution of the GFP fusionprotein (C1–2). Nuclei stained with DAPI (blue). Enlargement of selected areas are marked with white square and referred as Zoom.Scale bar, 1 μm.

role of this positive charge but also implies a role ofthe arginine side chain for trapping the protein at therhoptry membranes. To test a positional effect of thearginine, we generated a construct, where G8 and R9were shuffled (20AROGR9RG, substitution of G8/R9 withR8/G9). Interestingly, this construct was not targeted tothe rhoptries but to the PPM, underlining the importanceof this residue (Figure S4A).

Membrane specificity is mediated by charged

residues within amino acids 11–20

Although rhoptry membrane recruitment depends ona functional myristoylation and palmitoylation motif,including R9 within the first 10 amino acids, this sequenceis not sufficient to direct GFP to the rhoptries (Figure 5C,20AROA11-20). To further dissect additional determinants,we targeted two features in the second part of the 20

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amino acids stretch (L11L12YK14NK16LQE19F): a putativehelical structure and charged residues. First, leucinesin positions 11 and 12, crucial for helix formation,were exchanged with glycines (Figure 7A1). This mutant(20AROG11G12) showed no impaired rhoptry membraneattachment (Figure 7A2) and therefore argues againstthe involvement of this secondary structural feature indefining membrane specificity. Next, the involvement ofadditional positively charged residues within this aminoacid stretch was analyzed. Initially, K14 and K16 weresubstituted with glutamate (20AROK14,16E). Interestingly,this mutation localizes the protein to the IMC (Figure 7B1),revealing their involvement in the recruitment of thisprotein to the rhoptry membrane. The IMC is, like therhoptries, a Golgi derived compartment and consists offlattened vesicles underlying the plasma membrane. Itshows a unique dynamic during schizogony includingring-like formations (21,30–32) as depicted by thisconstruct.

This phenotype was further analyzed by a set ofsingle point mutants. 20AROK14E showed IMC localization(Figure 7C1) like the double mutant 20AROK14,16E.Rhoptry localization could be restored by the introductionof a different positively charged residue 20AROK14R(Figure 7D1). The same set of mutants was generated toinvestigate the role of K16. The point mutant 20AROK16Erevealed a mixed phenotype with partial rhoptry and partialPPM association (Figure S5A1,A3,A4). Rhoptry localizationremained unchanged by the exchange of K16 with anotherpositively charged residue 20AROK16R (Figure S5B1). Thisargues for a somehow subsidiary but facilitating functionof the positively charged residue K16 to the essential roleof K14 for rhoptry membrane recruitment. In contrast,mutation of negatively charged glutamate 19 with thepositively charged lysine did not alter rhoptry membranetargeting (20AROE19K, Figure S5C1).

Moreover, the substitution of all amino acids withinresidues 11–20 except K14 and K16 (20AROA11–20K14K16)resulted in a protein that was predominantly targetedto the rhoptries (Figure S6A1,A3 and A4). Finally, wesubstituted amino acid residues 12–18 with an unrelatedsequence (20AROK12NSKSNI) that comprised, like the AROwild type, two positive charges but at a different position(K12 and K15). The localization of this mutant to therhoptries suggests that the relative position of thecharges downstream of R9 is not crucial (Figure S6B1).This data was supported by matching solubilities of thecorresponding mutant proteins (Figures S5A2,B2 and C2and S6A2,B2).

Predicted dual acylation in the first 20 amino acids

confers trafficking of GFP to either the apical

organelles, IMC or PPM

We extended our investigations by testing the membrane-specific targeting of an additional 17 unrelated sequences.These sequences were found on PlasmoDB using theprotein motif pattern: M1GX0-17C and further testing their

predictions of being myristoylated and palmitoylated inthe first 20 amino acids (Figure 8). Expression as GFPfusion proteins in late stages and subsequent localizationrevealed three distinct phenotypes. First, the N-terminusof PF08_0062 and PFL1119c traffics GFP to the apicalpole of nascent merozoites, reminiscent of the rhoptrylocalization of ARO. Second, the 20 amino acids ofPF14_0578 and PF10_0107 results in a phenotype that ishighly characteristic for IMC proteins as previously shown(21,30). Third, the majority of sequences (PFI0675w,PFB0815w, Mal13P1.51, Mal13P1.310, PFI1500w,Mal8P1.109, Mal13P1.44, PFL1090w, PF11_0307,PF14_0354, PF08_0064, PFI1005w) resulted in GFPthat was targeted to the PPM (Figure 9). Only onefusion protein (Mal7P1.300) resulted in cytoplasmic GFP(data not shown). Taken together, the first 20 aminoacids of proteins predicted to be dually acylated confersmembrane association. At the same time, this N-terminuscan also contain sufficient information for membranediscrimination.

Discussion

All apicomplexan parasites possess secretory organellesin their apical region that secrete their contents uponinvasion. The detailed underlying molecular machinery thatcontrols and coordinates their biogenesis and maturationhas yet to be elucidated.

In this study, we characterized in detail the rhoptrytargeting of PFD0720w, a novel and conserved rhoptry-associated protein containing two predicted armadillo-domains termed ARO. ARO is attached to the cytosolicface of the rhoptry membrane. This membrane associationis mediated by the means of its N-terminal myristoylationand palmitoylation motifs. Both modifications are knownto confer membrane attachment for various parasiteproteins like the protein kinases PKG (33) and CDPK1(34) or the glideosome protein GAP45 (35). These motifsare known to be essential for membrane attachment(34,36–38) and are also required for PfARO. It ispredicted that this protein undergoes co-translationalmodification by myristic acid at the glycine at position2, followed by post-translational palmitoylation of oneor both cysteines at positions 4 and 5. Using a biotinswitch experiment on 20AROwt, that expresses the first20 amino acids of ARO as a GFP fusion, we providedirect evidence that palmitoylation takes place in vivo.Further, to differentiate the function of the individualcysteines C5 and C6, point mutants were expressed andlocalized within the parasite (Figure S4B,C). Interestingly,while the alanine substitution of C5 does not interferewith rhoptry membrane association the exchange of C6relocates the fusion protein to the PPM. This not onlypoints toward C6 as the cysteine involved in rhoptrymembrane association, but also underlines the importanceof the spatial relationship between the acylated cysteineand the arginine at position 9.

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AA1

B1

C1

A2

B2

C2

B

C

D1

D2

D

Figure 7: ARO rhoptry membrane specificity is defined by positive charges within amino acids 11–20. A) Secondary structureprediction reveals a helical propensity within amino acids 11–20 (A1). Disruption of this structural feature by substitution of the leucines(L11 and L12) by glycine (20AROG11G12) does not interfere with localization of the GFP to the rhoptries (A2) and membrane association(A3). B) The exchange of the positively charged lysines (K14 and K16) by glutamic acid (20AROE14E16) distributes the GFP fusionprotein to the membrane of the IMC (B1) but do not alter the solubility profile of the protein (B2). The apical orientated ring structuresare indicative for the nascent IMC (B1). C) Mutation of the lysine 14 (20AROK14E) resulted in the same IMC phenotype but (D) theexchange of lysine with the positively charged arginine (20AROK14R) do not alter rhoptry membrane localization. Nuclei stained withDAPI. Enlargement of selected areas are marked with white square and referred as Zoom. Scale bar, 1 μm.

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Figure 8: Predicted N-terminal (20 amino acids) pamitoylated and myristoylated proteins in P. falciparum. Predictedmyristoylation sites are markes in light blue, palmitoylation sites are marked in green, positive charged amino acids are highlighted inpink negative charged ones in blue. The probability scores using different prediction tools (Myr, NMT and CSS-Palm) are shown in thelast row. Sequences are arranged according to their localization in the late stages of the parasite: Apical (red), inner membrane complex(yellow), plasma membrane (grey) and cytosolic (white).

Although these lipid modifications are essential, theyare not sufficient to direct PfARO to the rhoptry mem-branes. Mutational analysis revealed additional residueswithin the first 20 amino acids of PfARO that play animportant role for rhoptry membrane targeting: the pos-itively charged residues R9 and K14. Interestingly, theexchange of the positive charge at position 9 with anegative charge in PfARO abolishes membrane attach-ment altogether, whereas the exchange of K14 (and toa lesser extent K16) alters its membrane specificity. Theessential role of R9 could be explained by either interfer-ence with the myristoylation process (essential for PfAROmembrane attachment) or by abrogation of any enzyme-mediated palmitoyl transfer. Following this scenario, R9(in conjunction with the other positive charges) might playa role for mediating interaction with negatively chargedmembrane phospholipids and therefore provide additionalmembrane affinity for PAT activity. Since approximately30% of the total phospholipids in eukaryotic cell mem-branes contain negatively charged head groups (39), the

importance of positive charges for additional membraneaffinity is obvious and was shown for lysines within theN-terminal sequences of the Src family members that,together with the myristoyl moiety, promotes membranebinding (40,41). Therefore, the exchange of the positivecharge at position 9 with a negative charge in ARO couldlead to inadequate membrane attachment and conse-quently abrogate interaction with a PAT, assuming thatmembrane association promoted by the preceding myris-toylation is insufficient for protein palmitoylation. In agree-ment with this, the substitution of R9 with K does not leadto the loss of membrane attachment (Figure 6C). Never-theless, the exchange modifies the membrane specificityof the fusion protein suggesting a specific role of thearginine side chain for instance as a recognition motif fora rhoptry membrane specific PAT.

The use of additional 17 unrelated protein sequencesencoded in the genome of the parasite and selected onbasis of their predicted myristoylation and palmitoylation

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Figure 9: Localization of the

predicted N-terminal (20 amino

acids) palmitoylated and myris-

toylated proteins in P. falci-

parum. The predicted peptides(indicated above the micrographs)were localized in late stage par-asites using GFP fusion proteins.According to their predominatsubcellular distribution they weregrouped into apical (Api), innermembrane complex (IMC) and par-asite plasma membrane (PPM).Nuclei stained with DAPI. Enlarge-ment of selected areas are markedwith white square and referred asZoom. Scale bar, 1 μm.

motifs further defined the requirements for rhoptry mem-brane association. Interestingly, all apically targeted fusionproteins (Figures 8 and 9) have (i) an arginine downstreamfrom the predicted palimitoylation site that is separatedby two amino acids and (ii) at least one positively chargedlysine in close vicinity (Figure 8). For ARO, we showed thatthe arginine in position 9 (three amino acids downstreamof the palmitoylated cysteine) and the most proximallysine (regardless of position) were essential for rhoptry

membrane targeting (Figures 6B, 7C and S4A). In thecase of the other apically localized N-termini, R7 and K8for PF08_0062, and R8 and K11 for PFL1119c, might havean iso-functional role for membrane specificity. Sequencerequirements for other compartment-specific membraneassociations, like IMC or PPM have to be experimentallyvalidated in future work. From this study, as for proteinsin other organisms (42,43), the PPM emerges as thedefault localization for dually acylated proteins. Although

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the first 20 amino acids of ARO comprise all traffickinginformation for correct rhoptry targeting, other proteinsmight depend on additional information. For instance,full length PFL1090w (GAP45) is an IMC protein;meanwhile, the N-terminal fusion alone is trafficked tothe PPM.

It was previously suggested that palmitoylated proteinsare recruited to their target membranes by substrate-specific PATs within the membrane of the differentorganelles (43–46). While the co-translational lipid mod-ification, for instance by myristic acid, mediates lowmembrane affinity that allow the proteins to cycle on andoff intracellular membranes – only the modification bypalmitate by a specific PAT is responsible for an increasedmembrane affinity and trapping of the protein to a spe-cific compartment. This may also include a compartmentwithin the secretory pathway with subsequent sorting andvesicle-mediated trafficking to other compartments.

The genome of Plasmodium encodes at least 13 PATs (47)of which four are transcribed late in blood stages (TableS2, Supporting Information). While a rhoptry membranebound PAT could mediate PfARO specific targeting, otherPATs within the plasma membrane or the IMC couldtrap PfARO mutants. Alternatively, the specific enzymeresponsible for ARO wild type palmitoylation could belocalized in specialized compartments of the trans-Golgidefining rhoptry-bound vesicles. Of note, the only putativePAT localized in P. falciparum so far shows apparent Golgidistribution in late asexual stage parasites. That studysuggested that its enzymatic activity is responsible fortargeting proteins to the apical organelles (48). It will beinteresting to apply a detailed localization study for all (latetranscribed) PATs and identify the putative rhoptry, IMCor plasma membrane bound enzymes.

Interestingly, Blast searches show that ARO is notonly highly conserved across Apicomplexan parasites butalso retireved homologs in yeast (Vac8,NCBI GenBankNC_001137.3) and the Paramecium (Nd9,NCBI GenBankCAC12829.1) with 26 and 24% identity, respectively. Allthree proteins possess a lipid interacting N-terminus andarmadillo protein–protein interaction moieties at the C-terminus. Different studies have shown that Vac8p isinvolved in vacuole inheritance, homotypic fusion, vacuole-nucleus junctions and cytoplasm-to-vacuole targeting(49–54). In turn, Nd9p is needed for the discharge ofParamecium trichocysts and was suggested to be involvedin their fusion with the plasma membrane (25). Yeastcells deleted for Vac8 do not inherit vacuoles from themother to the daughter cell and manifest a mild tohighly fragmented vacuole morphology (55). Although it istempting to speculate on a similar function of ARO, forinstance for rhoptry membrane fusion, complementationassays using PfARO on a vac8 deficient background doesnot rescue the fragmented vacuolar morphology and doesnot complement Vac8 function in an in vitro vacuolefusion assay (Figure S7). Gene disruption experiments

are consequently required for deciphering the precisefunction of ARO in apicomplexan parasites.

Material and Methods

Cell culture and transfection of Plasmodium

falciparumP. falciparum (3D7) was cultured in human O+ erythrocytes according tostandard procedures using complete RPMI medium (54). For transfection,ring-stage parasites (10%) were electroporated with 100 μg of plasmidDNA resuspended in cytomix as previously described (56). Transfectantswere selected using 10 nM WR99210 (57).

Nucleic acids and constructsPFD0720w was previously cloned as a GFP-fusion and localized in P.-falciparum (22). For the generation of a GST-fusion protein, full lengthPFD0720w was reamplified and cloned using the EcoRI and XhoI sitesof pGEX-4T-1 vector (GE Healthcare). PCR amplifications were performedusing Phusion DNA polymerase (New England Biolabs). Mutations ofPFD0720w and the synthetic chimera were produced by PCR using therespective oligonucleotides summarized in the Table S1. All constructswere digested with KpnI and AvrII and cloned into a derivate of pARL-1a(58) containing GFP (37) that ensures expression of C-terminally GFP inlate stages (59).

PFD0720w orthologs were identified using the blast search toolon NCBI and the BLAST tool in PlasmoDB and ToxoDB. Proteinsequences of all orthologs in apicomplexan parasites were alignedusing Praline multiple sequence alignment (www.ibi.vu.nl/programs/pralinewww).

Genes encoding proteins that are predicted to be myristoylated andpalmitoylated were identified using the protein motif pattern tool availablein PlasmoDB searching for the pattern M1GX0-17C in the wholeP. falciparum genome. Each sequence was run into the myristoylationand palmitoylation prediction softwares: Myristoylator (60), NMT (61) andCSS-Palm (62).

The 20 amino acid N-termini of PF08_0062, PFL1119c, PF14_0578,PF10_0107 PFI0675w, PFB0815w, Mal13P1.51, Mal13P1.310, PFI1500w,Mal8P1.109, Mal13P1.44, PFL1090w, PF11_0307, PF14_0354,PF08_0064, PFI1005w and Mal7P1.300 were generated using overlappingoligonucleotides, summarized in Table S1. Subsequently, the KpnI/AvrIIgene fragments were cloned into the same pARL1a-vector used forall other transfection experiments. All constructs were sequenced foraccuracy.

Recombinant protein expression and antiseraMouse antiserum was raised against PFD0720w-GST fusion protein. Thefusion protein was expressed in Escherichia coli BL21-RIL and induced with1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The GST fusion proteinwas purified by affinity on a high-affinity GST resin (GenScript) accordingto the manufacturer instructions. Purity was assessed on SDS-PAGE, andconcentration was calculated using its absorbance at 280 nm. Mice wereimmunized according to good laboratory practice using TiterMax® GoldAdjuvant (Sigma).

Western blot analysisP. falciparum proteins were extracted using 0.03% saponin (Sigma), andpellets were resuspended in adequate amount of PBS and 5× sodiumdodecyl sulphate (SDS) loading dye. Proteins were separated on 10%SDS–PAGE and transferred to nitrocellulose membranes (Schleicher &Schuell). Monoclonal mouse anti-GFP (Roche) was diluted 1:1000, rabbit

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anti-RALP1-C (28) and mouse anti-PFD0720w were diluted 1:500 in 5%(w/v) skim milk. The secondary antibody was horseradish peroxidase(HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) (1:3000, JacksonIR) and HRP conjugated goat anti-mouse IgG (1:3000, Jackson IR). Theimmunoblots were developed by chemiluminescence using AmershamECL (GE Healthcare).

Live microscopy and immunofluorescence assaysImages of unfixed GFP-expressing parasites were captured using a ZeissAxioskop 2plus microscope with a Hamamatsu Digital camera (ModelC4742-95, Zeiss axiovision) with 1 μg/mL DAPI (Roche) for nuclei stain.Immunofluorescence microscopy was performed on ice-cold methanol-fixed P. falciparum parasites (alternatively, parasites were fixed with amixture of 4% formaldehyde and 0.0075% glutaraldehyde).

Parasites were permeabilized with 0.2% Triton X100 and blocked with2% bovine serum albumin (BSA). First, antibodies were incubated for 1 hin the following dilutions: anti-GFP (Roche, 1:500), anti-PfGAP45 (1:5000),anti-RALP1-C [1:500 (28)] and anti-PFD0720w (1:500). Subsequently, cellswere washed three times with PBS and incubated for an hour with Alexa-Fluor 594 goat anti-rabbit IgG or Alexa-Fluor 488 goat anti-mouse IgGantibodies (1:2000, Molecular Probes) and 1 μg/mL DAPI (Roche).

After removal of unbound antibodies by three times washing with PBS,methanol fixed slides or paraformaldehyde fixed coverslips were mountedwith Fluoromount G (Sigma) with coverslips or slides, respectively, andkept at 4◦C until evaluation.

Proteinase K protection assaySaponin extracted P. falciparum parasites were resuspended in 1.5 mLof cold SoTE (0.6 M sorbitol, 20 mM Tris–HCl pH 7.5 and 2 mM EDTA)and aliquoted in three tubes (0.5 mL each). Cold SoTE was added to tube1 and kept as control. Parasites on tubes 2 and 3 were permeabilizedadding 0.5 mL of cold 0.02% digitonin (Sigma) in SoTE. Samples werecarefully mixed by inversion and incubated on ice for 10 min. This wasfollowed by a 10 min centrifugation at 800 × g at 4◦C. Supernatants werediscarded and 0.5 mL cold SoTE was added on tubes 1 and 2. About0.5 mL of cold 0.1 mg/mL Proteinase K (Sigma) in SoTE was added ontube 3. All tubes were gently mixed by inversion and incubated on icefor 30 min. Proteinase K was inactivated adding cold trichloroacetic acid(TCA) to a final concentration of 10% and further incubating on ice for 30min. All samples were centrifuged at full speed for 20 min, washed oncewith acetone, dried briefly, resuspended in TE buffer and prepared forSDS–PAGE. SDS–PAGE was performed as previously described runningsamples 1–3 side to side and testing with different antibodies: anti-GFP(1:1000), anti-GAPDH (63) (1:1000) and anti-RhopH3 (64) (1:1000).

S-acyl biotin switch assaySaponin lysed parasite pellets were subjected to biotin switch protocol aspreviously described (29). Parasites were washed in ice-cold lysis buffer(50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.4), resuspended in lysisbuffer containing 1.7% TX100, 10 mM N-ethylmaleimide (NEM) (Pierce)and protease inhibitor cocktail (Roche), and incubated at 4◦C for 1 h withrotation. Parasite lysates were then chloroform/methanol precipitated(65), and the pellet was resuspended in solubilization buffer (4% SDS,50 mM Tris, 5 mM EDTA, pH 7.4) containing 10 mM NEM. Solubilizedprotein was diluted with lysis buffer containing 1 mM NEM, 0.2% TX100and protease inhibitors, and incubated overnight at 4◦C with rotation.After chloroform/methanol precipitation, proteins were resuspended insolubilization buffer and divided into two equal sets; first set was dilutedwith control buffer (50 mM Tris, 1 mM biotin-BMCC, 0.2% TX100, pH 7.4)and the second with hydroxylamine buffer (0.7 M Hydroxylamine, 1 mM

biotin-BMCC, 0.2% TX100, pH 7.4), and incubated at room temperature for2 h. After chloroform/methanol precipitation the pellet was resuspendedin solubilization buffer, diluted in low-biotin-BMCC buffer (50 mM Tris,150 mM NaCl, 5 mM EDTA, 0.2 mM biotin-BMCC, 0.2% TX100, pH 7.4)

and incubated at room temperature for 2 h with rotation. The samplewas then precipitated again by chloroform/methanol precipitations andresuspended in solubilization buffer. The samples were diluted to 0.1%SDS by addition of lysis buffer with 0.2% TX100 and protease inhibitors,and incubated for 30 min at room temperature with rotation. The sampleswere centrifuged at 20,000 × g to remove any insoluble material,high-capacity NeutrAvidin-agarose (Pierce) was added, and incubated atroom temperature for 2 h with rotation. Beads were washed four timeswith lysis buffer with 0.1% SDS and 0.2% TX100. Beads were eluted with4× SDS sample buffer containing β-mercaptoethanol and boiled at 80◦C.Sample was run on SDS–PAGE and blotted on WB for analysis. Controland hydroxylamine sample was run side by side.

Solubility assaysP. falciparum proteins were extracted using 0.03% saponin (Sigma) (if nototherwise indicated, all steps were carried out on ice). After three timeswashing with PBS, complete protease inhibitor (Roche) was added andparasites were lysed in 100 μL of water and frozen at −80◦C. Preparationwas thawed and mechanically disrupted passing through a needle andfrozen again at −80◦C. Extraction was done sequentially: the lysate wasthawed and centrifuged at 14 000 rpm for 5 min. The supernatant wasremoved, centrifuged again for 5 min to remove residual insoluble materialand saved as the soluble protein fraction. The pellet after hypotonic lysiswas washed once with PBS and resuspended in 100 μL freshly prepared0.1 M Na2CO3 and kept on ice for 30 min to extract peripheral membraneproteins. After centrifugation at full speed for 5 min the supernatant waskept and centrifuged again. The pellet was washed once with PBS andextracted for 30 min with 100 μL 1% Triton X100 and centrifuged at fullspeed for 5 min to obtain the integral membrane protein fraction in thesupernatant. The final pellet was washed once with PBS and resuspendedon 100 μL PBS containing the insoluble fraction. Equal amounts of allsupernatants were analyzed by immunoblotting with adequate amountsof 5× SDS loading dye. Proteins were detected using anti-GFP antibodies,the cytosolic protein GAPDH was used as a control (63).

Acknowledgments

We would like to thank C. Daubenberger, P. Sharma and B. Cooke forproviding antibodies against GAPDH, GAP45, RhopH3, respectively. Weare grateful for WR99210 provided by Jacobus Pharmaceuticals and thesupport of the Hamilton Health Sciences McMaster Hospital TransfusionMedicine. We are in debt to Julian Rayner and Matthew Jones for invalu-able advices for establishing the biotin switch experiments in our laboratory.This study was supported by grants from the Canadian Institutes for HealthResearch (MOP#111196, T. G.), the Canadian Foundation for Innovation (T.G.), the Deutsche Forschungs-gemeinschaft (GI312, T. G.), the Universityof Osnabruck (GK UOsBio, A. T. and C. U.), the Hans-Muhlenhofffoundation (C. U.), the European Molecular Biology Laboratories (EMBOshort-term fellowship, A. C.), the University of Geneva (DSF, J. S. and S.B.) and the Vereinigung der Freunde des Tropeninstituts Hamburg (A. C.).

Supporting Information

Additional Supporting Information may be found in the online version ofthis article:

Figure S1: Alignment of PFD0720w apicomplexan orthologs (related

to Figures 1 and 2). A) Secundary structure prediction (www.compbio.dundee.ac.uk/www-jpred/) of PFD0720w indicates multiple helices asdepicted by arrows. Although only two armadillo domains are predicted byMotifScan (http://hits.isb-sib.ch/cgi-bin/PFSCAN), five armadillo domainsmight be present in PFD0720w given three additional sets of triple helices(orange, red and purple arrows). B) PFD0720w homologs were alignedusing ClustalW multiple sequence alignment. Armadillo repeats are inboxes following the same color format than (A). Asterisks (*) show fullyconserved residues, colons (:) show conserved residues of a group with

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strongly similar properties and periods (.) show conserved residues of agroup with weakly similar properties.

Figure S2: ARO in Toxoplasma gondii co-localize with the rhoptries

(related to Figures 1 and 4). A) Domain structure of T. gondii ARO(TgARO); light red: armadillo domains, black: TY1 epitope. B) Co-localizationstudies of TgARO-TY1 with antibodies against the TY1 epitope (green) andthe rhoptry bulb marker ROP-1 (red), (C) ROP-7 (red), (D) the rhoptry neckmarker RON4 (red) and (E) the microneme marker MIC3 (red). Nucleistained with DAPI (blue). F) Schematic representation and localizationof the minimal TgARO construct (20TgARO) fused to GFP (green) and(G) co-localized with the rhoptry marker ROP-7 (red). Nuclei stained withDAPI (blue). Generation of cell lines and assays described in Appendix S1,Supporting Information.

Figure S3: TgARO is attached to the cytosolic face of the rhoptry

membrane (related to Figure 3 and Figure S1). A) expression of TgARO-Ty1 on transgenic parasites and not in RH wild type. B) Topology ofTgARO-TY1 using Proteinase K (PK) protection assay on TgARO-TY1permeabilized with digitonin (D). TgARO-TY1 was detected with anti-TY1antibodies. Antibodies against a luminal rhoptry protein (anti-ROP1) andagainst the cytosolic protein profilin (anti-PRF) were used as controls. Firstlane D− PK− not treated control. Second lane: D+ PK− permeabilizationcontrol. And third lane: D+ PK+ permeabilized and digested with proteinaseK. Assay described in Supplementary material and methods.

Figure S4: Positional effect of arginine 9 and role of the individual

cysteines 5 and 6 in rhoptry membrane attachment (related to Figure

6 and Discussion.) A) Substitution of G8/R9 with R8/G9 (20AROGR9RG)do alter rhoptry localization of the fusion protein to a plasma membraneassociation. B) The replacement of C5 with an alanin (20AROC5A) hadno effect on rhoptry membrane localization of the fusion protein. C)Substitution of the C6 with an alanin (20AROC6A) targets the protein tothe parasite plasma membrane. Enlargement of selected areas are markedwith white squares and referred as Zoom. Scale bar, 1 μm.

Figure S5: Contribution of K16 for rhoptry membrane specificity and

expandable negative charge E19 (related to Figure 7). A) Substitutionof positively charged K16 by glutamic acid (20AROK16E) leads to a mixedphenotype with some cells that show mainly apical (33%), some cells showapical and plasma membrane (47%) and some cells show predominatelyplasma membrane (20%) targeting of the fusion protein with an extendedcytosolic pool (A1–2). The mixed phenotypes were quantified based on30 cells and categorized. Example for each category (A3) and percentageof cells resembling this phenotype (A4) are given. B) The replacement ofK16 with another positively charged residue (20AROK16R) had no effecton rhoptry membrane localization of the fusion protein. C) Substitutionof the negative charged E19 with the positive charged residue lysine(20AROE19K) does not interfere with rhoptry membrane targeting (A1–2).Enlargement of selected areas are marked with white square and referredas Zoom. Scale bar, 1 μm.

Figure S6: A K14/K16 minimal construct and positional effect of

positive charges (related to Figure 7). A) The replacement of all butK14 and K16 within the amino acid 11–20 alanine (20ARO11 – 20AK14,16)leads to a mixed phenotype with some cells that show mainly apical (7%),some apical and plasma membrane (78%) and some predominately plasmamembrane (15%) targeting of the fusion protein with an extended cytosolicpool (B1–2). This mutant cannot fully resemble the wild type targetingsequence and results in a mixed phenotypes quantified in C4. B) The shiftof the positive charges from K14/16 to K12/15 by the substitution of aminoacid 12–18 with an amino acid stretch of an unrelated protein (PF14_0578,20ARO12 –18 0578 ) does not impair rhoptry membrane localization (C1–2).Enlargement of selected areas are marked with white square and referredas Zoom. Scale bar, 1 μm.

Figure S7: Vac8 complementation in yeast (related to Discussion).

A) Complementation analysis by microscopy. Localization of GFP-taggedPfAROVac8N fusion protein in yeast cells. Wild-type Vac8 and PfAROVac8Nand PfKaryoAVac8N were C-terminally GFP-tagged and expressed in a vac8deletion strain. FM4-64 staining was used to monitor vacuole morphology

by fluorescence microscopy. Bar, 10 μm. B) Number of vacuoles per cellfor all constructs. C) In vitro vacuole fusion assay. Vacuoles purified fromtester strains BJ3505 (pep4) and DKY6281 (pho8) were incubated in thefusion reaction buffer containing ATP-regenerating system for 90 min at26◦C and then developed. Fusion activity was measured as described inAppendix S1, Supporting Information.

Table S1: Primers used in this study. Restriction endonuclease sites areunderlined.

Table S2: Palmitoyl acyl transferases in P. falciparum (related toDiscussion). Late transcribed DHHC palmitoyl acyl transferases (PAT) wereretrieved from PlasmoDB. PATs with a transcriptional maximum at 42 ± 4h with fourfold induction are in black, PATs with < fourfold upregulation ingrey.

Table S3: Plasmids and yeast strains used in this study.

Appendix S1: Materials and methods.

Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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