1

The Longin Domain Regulates the Steady-state Dynamics of Sec22 in 1

P. falciparum 2

3

Lawrence Ayong1, Avanthi Raghavan1, Timothy G. Schneider3, Theodore F. Taraschi3, 4

David A. Fidock2, and Debopam Chakrabarti1 5

6

Department of Molecular Biology & Microbiology, Burnett School of Biomedical 7

Sciences, University of Central Florida, Orlando, Florida 328261, 8

Departments of Microbiology and of Medicine, Columbia University, New York 100322 9

Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 10

Philadelphia, Pennsylvania 191073 11

12

13

Running title: Sec22 trafficking in P. falciparum 14

15

16

To whom correspondence should be addressed: Debopam Chakrabarti, 12722 17

Research Parkway, University of Central Florida, Orlando, Fl 32826-3227, Fax: 407-18

384-2062, E-mail: dchak@mail.ucf.edu 19

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00092-09 EC Accepts, published online ahead of print on 17 July 2009

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Abstract1

The specificity of vesicle-mediated transport is largely regulated by the 2

membrane-specific distribution of SNARE proteins. However, the signals and 3

machineries involved in SNARE protein targeting to their respective intracellular 4

locations are not fully understood. We have identified a Sec22 ortholog in 5

Plasmodium falciparum (PfSec22) that contains an atypical insertion of the 6

Plasmodium export element within the N-terminal longin domain. This Sec22 7

protein partially associates with membrane structures in the parasitized-8

erythrocytes when expressed under control of the endogenous promoter 9

element. Our studies indicate that the atypical longin domain encodes signals 10

that are required for both ER/Golgi recycling of PfSec22, and partial export 11

beyond the ER/Golgi interface. ER exit of PfSec22 is regulated by motifs within 12

the α3 segment of the longin domain, whereas the recycling and export signals 13

require residues within the N-terminal hydrophobic segment. Our data suggest 14

that the longin domain of PfSec22 exhibits major differences when compared 15

with the yeast and mammalian orthologs, perhaps indicative of a novel 16

mechanism for Sec22 trafficking in malaria parasites. 17

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1

Introduction 2

Plasmodium falciparum exhibits a complex network of endomembrane organelles 3

that are unique to this obligate intracellular parasite of human erythrocytes. 4

These include parasite-induced tubules and vesicles in the infected host cell, and 5

specialized secretory structures collectively known as the apical complex. The 6

asexual blood stages of the parasite develop within a parasitophorous vacuole 7

(PV), and thus are separated from the external milieu by three lipid bilayers: the 8

parasite plasma membrane (PPM), the parasitophorous vacuole membrane 9

(PVM), and the erythrocyte plasma membrane (EPM). To survive inside these 10

terminally differentiated human erythrocytes, P. falciparum remodels the host cell 11

compartment by exporting numerous proteins into the erythrocyte cytoplasm [1-12

5]. The mechanisms by which both soluble and membrane-bound proteins are 13

transported, first into the PV lumen, followed by translocation across the PVM 14

and transport within the erythrocyte cytosol are not fully understood [6]. A 15

majority of the exported proteins contain bipartite signals that comprise a 16

‘recessed’ N-terminal signal sequence, and a Plasmodium export 17

element/vacuolar translocation sequence (PEXEL/VTS) that is characterized by 18

the consensus sequence Rx(L,I)x(D,E,Q). These signals are predicted to 19

facilitate transport of proteins into the PV (using their recessed or N-terminal 20

signal sequence) and translocation across the PVM (using their PEXEL/VTS 21

motif) [7-10]. However, a subset of the exported proteins lack either one or both 22

signal elements and may require novel targeting motifs for transport beyond the 23

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PPM [11, 12]. A majority of the proteins enter the parasite secretory system via 1

the ER, where they are incorporated into ER-derived vesicles, and then 2

transported through the ‘unstacked’ Golgi bodies to their final destinations [13-3

16]. Membrane-bound vesicular elements have been detected in the infected 4

host cell cytosol, suggesting the existence of an extraparasitic vesicle-mediated 5

transport process in malaria parasites [17-19]. How vesicle targeting is achieved 6

in P. falciparum parasites remains elusive. 7

Vesicle targeting and fusion in eukaryotic cells involves proteins of the 8

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) 9

family [20-23]. SNAREs are ‘tail-anchored’ proteins that function by forming 10

complexes that bridge vesicle and target membranes during fusion [24-26]. 11

Distinct sets of SNARE proteins localize to different intracellular transport 12

pathways using processes that are not well understood. Increasing evidence 13

suggests that the N-terminal regions of SNARE proteins encode signals required 14

for their subcellular localization [27-29]. These N-terminal regions include the 15

three-helical Habc bundles of syntaxin SNAREs, and the ‘profilin-like’ folds of 16

long VAMPs (vesicle associated membrane proteins) also known as longin 17

domains [25, 30-33]. The Sec22 gene products in mammals and yeast are longin 18

domain-containing SNAREs that cycle between the ER and Golgi compartments 19

[29, 34-36]. We have identified a Sec22 ortholog in P. falciparum (PfSec22) that 20

contains a PEXEL/VTS sequence insertion between the α2 and α3 segments of 21

the longin domain preceded by a stretch of hydrophobic residues that spans a 22

region between the β5 and the α2 segments [37]. In this study, we examined the 23

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distribution of PfSec22 in P. falciparum-infected erythrocytes, and investigated 1

the role of the atypical longin domain in its steady-state localization. Our data 2

show that the P. falciparum ortholog of Sec22 partially associates with non-3

canonical destinations (TVN and intraerythrocytic vesicles) in the infected 4

erythrocytes and that the N-terminal longin domain exhibits a dual function, 5

mediating ER to Golgi trafficking as well as retrieval from the Golgi. 6

7

Materials and methods 8

9

Plasmid constructs and transfection. Transfection plasmids were 10

designed to express wild type or mutant green fluorescent protein (GFP)-tagged 11

Sec22 proteins under the control of its upstream homologous promoter 12

sequence. To achieve this, forward (5’-CCGGGGCCCTTTCCCTTCCCCGAAA-13

3’) and reverse (5’-CGGCCTAGGTGTTCTTTTTTTATTATTTTCTTCT-3’) primers 14

were designed to amplify a 995 bp segment of the 1781 bp intergenic region (5’ 15

UTR) between PFC0890w and PFC0886w by PCR using P. falciparum 3D7 16

genomic DNA. The amplified sequence was ligated into the pGEMT-Easy vector 17

(Promega) and subsequently subcloned into the PspoM I/Avr II sites of the 18

previously generated construct pDC2-cam-GFP-PfSec22 [37, 38]. This replaced 19

the cam promoter with the 0.995kb 5’-UTR sequence of PfSec22 to obtain the 20

construct pDC2-0.995. The resulting construct expressed full-length PfSec22 21

proteins with GFP fused to its N-terminus (GFP-PfSec22). DNA encoding 22

PfSec22 mutant proteins were also generated by PCR, and subcloned into the 23

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Bgl II and Xho I sites of the pDC-0.995vector. The primer sets 5’-1

GGAAGATCTATGTGCGATGTAGTATTACTT-3’ and 5’-2

CCGCTCGAGTTATTTTAGATTTAATACCCTTGA-3’ or 5’-3

GGATCCAGCTTTTTTATTTTTAAACGAT-3’ and 5’-4

CCGCTCGAGTTAAAAATAATTTTTAAAAATTATAATT-3’ were used to amplify 5

PfSec22 gene fragments to construct the GFP-PfSec22∆198-221 and the GFP-6

PfSec22∆1-78 fusion genes, respectively. DNA encoding amino acids 1-58 of the 7

full length Sec22 protein was also amplified using primers 5’-8

GGAAGATCTATGTGCGATGTAGTATTACTT-3’ and 5’-9

GGATCCAAAATGGTAATTAAAATTGTTAG-3’ and subsequently ligated via the 10

BamHI site to the C-terminal fragment of PfSec22 (GFP-PfSec22∆1-78) cloned 11

in a pGEMT-Easy vector. The ligated fragments were then subcloned into pDC2-12

0.995 vector to obtain the GFP-PfSec22∆58-78 mutant construct. To generate 13

plasmids with GFP appended to the C-terminus of PfSec22 (PfSec22-GFP), the 14

full-length sequence was amplified using primers 5’-15

CCTAGGATGTGCGATGTAGTATTACTT-3’ and 5’-16

AGATCTAAAATAATTTTTAAAAATTATAATTAAT-3’ and subsequently cloned 17

into the AvrII/BglII sites of pDC2-0.995 vector. The GFP-PfSec22∆198-18

221.PEXEL(R>A) construct was generated from the GFP-PfSec22∆198-221 19

mutant plasmid by site-directed mutagenesis of the PEXEL motif-specific 20

arginine, replacing it with an alanine codon. All constructs were confirmed by 21

sequencing prior to transfection of P. falciparum 3D7 parasites. Ring stage 22

parasites were transfected by electroporation (Biorad Gene pulser II at 0.31kV, 23

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950µF, maximum capacitance) using 100µg of purified plasmid (Qiagen 1

Maxiprep kit). Positive selection for transfected parasites was achieved using 2.5 2

nM WR99210 as previously described [37, 39]. 3

4

Anti-peptide antibodies and immunoblot analyses. The decapeptide 5

‘YKDPRSNIAI’, corresponding to residues 131-140 of PfSec22, was synthesized 6

(Genscript) and conjugated to Keyhole Limpet Hemacyanin (KLH) following the 7

manufacturer’s instructions (Pierce). Antisera were raised in rabbit against the 8

conjugated peptide (Harlan Bioproducts for Science, Inc) and affinity purified by 9

column chromatography using peptide-conjugated agarose beads (Pierce). The 10

reactivity of these antibodies against the endogenous and/or GFP-tagged 11

PfSec22 proteins was analyzed by standard Western blot techniques using 12

Supersignal West Femto detection kit (Pierce). 13

14

Immunofluorescence and confocal microscopy. Both live and fixed 15

cells were examined using a laser scanning confocal microscope (LSM 510, Carl 16

Zeiss). The excitation/emission spectra settings were 488/505 nm for GFP, 17

543/555 for Alexa Fluor-555 conjugated antibodies, or 543/594 nm for Alexa 18

Fluor-594 conjugates. To image live parasites, the cells were mounted under a 19

cover slip in 50% glycerol and observed within 15 minutes of removal from 20

cultures. Immunofluorescence assays were performed in suspension as 21

previously described by Tonkin et al [40]. Rabbit anti-PfErd2 (MRA-1) and rat 22

anti-PfBip (MRA-19) antibodies were obtained from the Malaria Research and 23

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Reference Resource Center (MR4) and were each used at a dilution of 1 in 1

1,000. The purified rabbit anti-PfSec22 antibodies were also used at a 1 in 500 2

dilution. Secondary antibodies consisted of goat anti-rabbit Alexa Fluor-488, goat 3

anti-rabbit Alexa-Fluor-555, or goat anti-rat Alexa Fluor-594 (Molecular Probes), 4

each used at a dilution of 1 in 1,000. 5

6

Brefeldin A treatment. To investigate the effects of Brefeldin A (BFA) on 7

the steady-state location of PfSec22 and its mutant proteins, GFP-expressing 8

cells were cultured in the presence of this agent at 5 µg/ml for 3 hours [41] and 9

then examined live as described above. Control experiments consisted of the 10

same culture samples prepared in equivalent amounts (0.5% methanol in RPMI 11

medium) of the drug solvent. 12

13

Subcellular fractionation analyses 14

i) Freeze/thaw fractionation of soluble and membrane-associated proteins. 15

P. falciparum 3D7 parasites were purified by saponin treatment and resuspended 16

in a TBS buffer (10 mM Tris-HCL, pH 7.4 + 150 mM NaCl), containing a cocktail 17

of protease inhibitors (Roche). The cells were subjected to five cycles of freezing 18

and thawing in liquid nitrogen followed by a brief sonication for 15 seconds to 19

release both the cytosolic and the lumenal proteins. The disrupted cells were 20

clarified by centrifugation at 100,000 x g for 1 h at 4ºC to separate the soluble 21

proteins from the membrane-associated fractions. The pellet sample was then 22

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normalized with the TBS buffer to the volume of the supernatant, and equivalent 1

volumes were analyzed by western blotting. 2

ii) Alkaline extraction of peripheral from integral membrane proteins. 3

Membrane pellets were prepared as described above and resuspended in 3 4

volumes of 0.1 M Na2CO3, pH 11. The suspension was mixed by rotation at 4oC 5

for 30 min, followed by centrifugation as above to separate the peripheral 6

membrane proteins in the supernatant from the integral membrane protein in the 7

pellet. The resulting pellet was normalized as described above, and equal 8

volumes analyzed by western blotting. 9

iii) Triton X-114 phase separation of hydrophobic from hydrophilic 10

membrane proteins. Integral membrane fractions were prepared by alkaline 11

extraction as described above and solubilized using the Membrane Protein 12

Extraction Reagent kit (Pierce). The solubilized fraction was clarified by 13

centrifugation at 10,000 x g for 3 minutes at 4oC, and then clouded in a 37ºC 14

water bath for 20 minutes. This was followed by centrifugation at 10,000 x g for 2 15

min at room temperature to separate the hydrophilic phase (top layer) from the 16

hydrophobic protein phase (bottom layer). Equal volumes of the phase-separated 17

samples were further diluted in Laemmli sample buffer and analyzed by Western 18

blotting. 19

iv) Sucrose density gradient fractionation of subcellular compartments. 20

250-300 mg of parasite pellets were resuspended in ice-cold TBS buffer, 21

containing protease inhibitor cocktail (Roche) in the presence of 2 mM EDTA or 5 22

mM MgCl2. Cells were disrupted by 5 freeze-thaw cycles and then cleared at 23

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10,000 x g for 5 minutes at 4oC. The suspension was then adjusted to 47% final 1

sucrose concentration, and deposited (in a final volume of 1.5 ml) at the bottom 2

of a discontinuous sucrose gradient comprising 1 ml of 40% sucrose, 1 ml of 3

35% sucrose, 0.75 ml of 25% sucrose, and 0.75 ml of 20% sucrose. The gradient 4

was centrifuged at 100,000 x g for 16 hours at 4oC in an Beckman SW50.1 rotor, 5

and fractionated into 0.5 ml aliquots by piercing the bottom of the tube using a 6

fraction recovery system (Beckman). Equal volumes of each fraction were 7

analyzed by Western blotting using anti-PfSec22, anti-PfErd2, or anti-PfBip 8

antibodies, diluted 1 in 1000. 9

10

Cryo-immunoelectron microscopy 11

P. falciparum-infected erythrocytes were enriched by Percoll density 12

centrifugation and fixed for 30 minutes at room temperature in 4% 13

paraformaldehyde/0.1% glutaraldehyde (EMS) in 0.1M phosphate buffer, pH 7.2 14

(EMS). IRBC were washed three times with phosphate buffer and pelleted into 15

10% gelatin in phosphate buffer and infused with 2.3 M sucrose in phosphate 16

buffer. The cryo-protected pellets were frozen in liquid nitrogen and cryo-17

transferred to a Leica EMFCS chamber. Ultrathin cryosections were obtained at -18

120oC on a Leica Ultracut UCT using a Drukker ultramicrotome diamond knife. 19

The resulting ribbons of frozen sections were collected onto carbon and formvar 20

substrates mounted on 300 mesh nickel grids. The grids were floated on drops of 21

BD JL8 anti-GFP (diluted 1:100 in 0.05M phosphate buffer containing 1%BSA) at 22

5oC overnight and then incubated at room temperature for 1 hour with a 1:50 23

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dilution of 12 nm colloidal gold-conjugated affinipure goat anti-mouse IgG/IgM 1

(Jackson ImmunoResearch Laboratories) in 0.05M phosphate buffer containing 2

1% BSA. The grids were fixed with 1% gluteraldehyde and then embedded in 3

1% methylcellulose and 2.5% uranyl acetate prior to examination in a FEI 4

Technai 12 TEM. The images were captured using a AMT XR111 digital camera. 5

6

RESULTS 7

8

Steady-state locations of endogenous PfSec22 protein. Sequence analysis of 9

the Sec22 gene product in P. falciparum revealed a significant divergence from 10

orthologs in yeast and mammals (overall identities of 26.2% and 30.8%, 11

respectively) [42]. The N-terminal longin domain of PfSec22 contains a bipartite 12

signal sequence typical of proteins that are exported into the host cell 13

compartment of P. falciparum-infected erythrocytes. These features include a 14

PEXEL-like motif (105RSIIE109), located at an unusual loop-like region between 15

the α2 and α3 segments of the longin domain, and an N-terminal hydrophobic 16

segment that is predicted (with a prediction probability ratio of 0.8192, 17

http://bioserve.latrobe.edu.au/cgi-bin/pfsigseq.py) to function as a ‘recessed’ 18

signal sequence capable of targeting the protein for export beyond the PPM (Fig. 19

1A and B). We have previously shown, however, that expression of a GFP-20

tagged version of PfSec22 under control of a heterologous cam promoter results 21

in partial association of the protein with the parasite ER and Golgi, but not with 22

the host cell compartment [37]. To investigate whether over-expression of the 23

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protein in transgenic parasites had any influence in its steady-state dynamics, we 1

generated anti-peptide antibodies that were used to detect the endogenous 2

proteins by immunofluorescence microscopy and by Western blot analyses of 3

cellular fractions. As shown in Fig. 2A, the affinity purified antibodies specifically 4

recognized a 26-kDa protein in cell-free extracts from ring, trophozoite, and 5

schizont stage parasites, corresponding to the predicted molecular mass of the 6

full-length PfSec22 protein. Expression of PfSec22 protein in all the asexual life-7

cycle stages is consistent with the gene transcription profile (shown in 8

www.PlasmoDB.org). By immunofluorescence analyses, we observed that the 9

majority of the PfSec22 signal localized to membrane-like compartments inside 10

the parasite cytoplasm. Consistently, albeit in a small proportion of cells (5.2% of 11

308 trophozoite-infected erythrocytes), we observed a specific staining of 12

isolated vesicular structures in the host cell cytoplasm using the anti-PfSec22 13

antibodies (Fig. 2B). When compared with the ER marker PfBip, PfSec22 was 14

observed to often be associated with the ER (Fig. 3A), consistent with a potential 15

role in ER-to-Golgi transport in the malaria parasite. A similar co-16

immunofluorescence assay on the association of PfSec22 with the Golgi was 17

precluded because the available anti-PfErd2 and anti-PfSec22 antibodies 18

originated from the same animal species. 19

To establish that PfSec22 is membrane-anchored in the parasite, we 20

investigated its membrane properties by i) freeze-thaw fractionation of 21

membrane-associated proteins from soluble fractions, ii) alkaline extraction of 22

peripheral membrane proteins from integral transmembrane proteins, and iii) 23

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Triton X-114 solubilization of integral membrane proteins followed by 1

immunodetection. The ER-restricted lumenal chaperone PfBip was used as a 2

marker for the soluble non-membrane-associated proteins, whereas the 3

presence of the KDEL receptor PfErd2 in the fractions marked the membrane-4

associated proteins. As shown in Fig. 3B, the endogenous PfSec22 protein 5

partitioned alongside PfErd2 in the membrane fractions and was efficiently 6

solubilized into the hydrophilic phase after detergent extraction using Triton X-7

114. PfErd2 was only partly solubilized by the detergent, consistent with its 8

topological function as a multi-pass membrane protein containing up to seven 9

transmembrane domains [41]. These results suggest that PfSec22 is an integral 10

membrane protein, consistent with a requirement in vesicle trafficking. 11

12

Localization of PfSec22 in transgenic parasites. To better understand the 13

intracellular dynamics of this unusual Sec22 gene product, we generated a 14

transgenic cell line expressing the GFP-tagged proteins under control of the 15

endogenous PfSec22 promoter sequence. Additionally, to ensure that a suitable 16

tagging approach was being employed, we first investigated the effect of the GFP 17

tag. Transgenic parasites were developed that expressed the PfSec22 protein 18

with GFP appended either to its C-terminus (PfSec22-GFP) or to the N-terminus 19

(GFP-PfSec22). We confirmed the expression of each fusion protein by 20

immunoblot analyses using either anti-PfSec22 or anti-GFP antibodies (Fig. 4A). 21

As indicated in Fig. 4A, two protein bands corresponding to the expected sizes of 22

the GFP-tagged proteins (~54 kDa) and the endogenous protein (26 kDa) were 23

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detected in each transgenic cell lysate using the anti-PfSec22 antibodies. In 1

contrast, only the 54-kDa bands were detected in these extracts using the anti-2

GFP antibodies. These data established that full-length proteins were expressed 3

in the respective cell lines, and that PfSec22 is not processed at the N-terminus 4

as has been reported for most PEXEL-containing proteins [43]. 5

Live cell imaging of GFP fluorescence revealed significant differences in 6

the distribution of GFP-PfSec22 and PfSec22-GFP (Fig. 4B and 4C, 7

respectively). In transgenic parasites expressing the C-terminally tagged protein, 8

the GFP fluorescence was observed predominantly in the parasite cytosol (Fig. 9

4B). In some trophozoite-infected cells, representing 9.3% of 302 trophozoite-10

infected erythrocytes, the GFP fluorescence was also observed inside the 11

erythrocyte cytosol (Fig. 4B, second row). In comparison, the N-terminally tagged 12

protein (GFP-PfSec22) localized predominantly to membranous structures, 13

located inside the parasite cytoplasm (Fig. 4C). Similar to the untagged PfSec22 14

protein, the GFP-PfSec22 protein was also detected inside the host cell 15

compartment in a small proportion (approximately 5%) of trophozoite-infected 16

cells. There, it associated with various tubovesicular extensions (Fig. 4C, second 17

row), and also with mobile vesicular structures (data not shown). The cellular 18

locations of GFP-PfSec22 were further investigated at the ultrastructural level 19

using monoclonal anti-GFP antibodies, and by immunofluorescence analyses 20

(Fig. 5 and Fig. 6, respectively). Immunogold labeling of ultrathin cryosections 21

confirmed that GFP-PfSec22 localized predominantly to the parasite ER (Fig. 22

5A). Membrane vesicles were also labeled in the parasite cytoplasm (Fig. 5A, 23

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black arrowheads) and in the erythrocyte cytosol of some trophozoite-infected 1

cells (Fig. 5B and C, white arrowheads). Together, these findings suggest that 2

PfSec22 might function as a v-SNARE in the malaria parasite. Further attempts 3

to fractionate the exported proteins by limited permeabilization of the host cell 4

membrane were unsuccessful, presumably due to its membrane-localization 5

and/or low levels in the erythrocyte cytosol. 6

To validate the use of GFP-tagged proteins in our study, we compared the 7

GFP and PfSec22 signals by immunofluorescence analysis using the anti-8

PfSec22 antibodies. As shown in Fig. 6A, top row, the anti-PfSec22 signal in the 9

GFP-PfSec22 expressing parasites was limited to the GFP fluorescence 10

structures, suggesting that the untagged wild-type protein also localized to the 11

same cytoplasmic compartments as was the GFP-tagged chimera. GFP-PfSec22 12

also co-localized significantly with PfBip (Fig. 6A, middle row) and PfErd2 (Fig. 13

6A, bottom row), suggesting that PfSec22 is predominantly a v-SNARE of the 14

parasite ER/Golgi pathway. As an alternative approach to confirm the association 15

of GFP-PfSec22 with ER and Golgi, we investigated its co-fractionation with i) the 16

untagged PfSec22 protein, ii) the ER marker PfBip, and iii) the Golgi marker 17

PfErd2, by sucrose density gradient ultracentrifugation and immublot analyses 18

(Fig. 6B). To overcome common difficulties in resolving the parasite ER from 19

other membrane-bound compartments, the co-fractionation experiments were 20

done in the presence of the chelating agent EDTA, or in the presence of the 21

rough ER stabilizing agent MgCl2 [44, 45]. By this approach, a redistribution of 22

the PfSec22 proteins alongside the ER marker PfBip, from a low-density fraction 23

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(in the presence of EDTA) to a high-density fraction (in the presence of MgCl2), 1

suggested its association with the rough ER. As shown in Fig. 6B, the untagged 2

(PfSec22) and the tagged (GFP-PfSec22) proteins similarly redistributed 3

alongside PfBip to the high sucrose density fractions following treatment with 4

MgCl2. However, only a limited amount of these PfSec22 proteins shifted to the 5

high-density fractions when compared with PfBip, which was completely 6

redistributed to the high-density fractions in presence of the Mg++ cations. These 7

results also suggest that PfSec22 associates with non-ER structures presumably 8

including transport vesicles as revealed by our electron microscopy studies (Fig. 9

5). Neither EDTA nor MgCl2 affected the buoyant density of the Golgi in our 10

gradients as evident from the distribution of PfErd2. Taken together, our data 11

indicate that PfSec22 is predominantly a v-SNARE of the parasite ER/Golgi 12

interface and that this SNARE protein might also play a limited role in the host 13

cell remodeling process, specifically in late trophozoite-infected cells. 14

15

Trafficking determinants for PfSec22. In an attempt to decipher the signals 16

that determine the steady-state localization of PfSec22 in the parasite, we 17

investigated the role of the atypical longin domain, the two hydrophobic 18

segments, and of the PEXEL/VTS-like sequence. Transgenic parasites were 19

generated that expressed various N-terminally tagged PfSec22 mutants. As 20

shown in Figure 7A, truncation of the longin domain at position 78 (GFP-21

PfSec22∆1-78 mutant) dramatically shifted its steady-state accumulation from 22

the ER to the Golgi, based on its co-localization with PfErd2 (Fig. 7A, bottom 23

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row). These findings suggest that GFP-PfSec22∆1-78 presumably has a 1

preference for the parasite Golgi and that residues 1-78 might encode signals 2

required for ER retrieval of PfSec22. Interestingly, deletion of the entire longin 3

domain at position 124 (GFP-PfSec22∆1-124) resulted in an ER localization of 4

the protein (Fig. 7B and co-localization data not shown), suggesting that this 5

mutant was not exported beyond the ER compartment. In contrast, a minimized 6

deletion of the N-terminal hydrophobic segment (GFP-PfSec22∆58-78 mutant) 7

resulted in Golgi localization (Fig. 7C). Similar to the GFP-PfSec22∆1-78 mutant, 8

this GFP-PfSec22∆58-78 mutant co-localized significantly with the Golgi marker 9

PfErd2 but not with the ER marker PfBip (Fig. 7C, middle and bottom rows, 10

respectively). Golgi targeting of this N-terminal hydrophobic domain mutant was 11

sensitive to brefeldin A (BFA) treatment (Fig. 8A), suggesting its association with 12

ER/Golgi transport vesicles. As shown in Fig. 8A, bottom row, BFA induced a 13

partial redistribution of both the GFP-PfSec22∆58-78 mutant and PfErd2 to the 14

ER, a phenomenon typical of most Golgi-localized proteins [41, 46]. We 15

confirmed the differential localization of GFP-PfSec22∆58-78 to the Golgi by 16

discontinuous sucrose density ultracentrifugation in the presence of EDTA or 17

MgCl2 (Fig. 8B). Compared to the endogenous wild-type protein, which 18

redistributed alongside PfBip to the high-density fractions in the presence of 19

MgCl2, the buoyant density of the GFP-PfSec22∆58-78 associated 20

compartments did not change in the presence of EDTA or MgCl2 (Fig. 8B). A 21

small proportion of the mutant protein remained in the low-density zone, 22

consistent with an association with transport vesicles and/or other lipid-rich 23

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compartments. Together, our data suggest that the ER exit of PfSec22 is 1

independent of residues 1 to 78 of the longin domain, and that the N-terminal 2

hydrophobic region (amino acid 58-78) is required for the retrograde transport of 3

PfSec22 back to the ER. 4

To establish the role of the C-terminal hydrophobic segment, we 5

generated a transgenic cell line expressing the deletion mutant GFP-6

PfSec22∆198-221. Deletion of this hydrophobic segment (amino acids 198 to 7

221) resulted in a cytosolic protein that, occasionally, was also released into the 8

erythrocyte cytosol in a pattern similar to the C-terminally tagged full-length 9

protein (compare Fig. 9A and Fig. 4B). These results support a role of the C-10

terminal hydrophobic segment in membrane anchorage, and suggest that the 11

occasional export of PfSec22 into the infected erythrocyte may involve processes 12

independent of its membrane insertion. Such processes, however, are not likely 13

to involve a canonical PEXEL/VTS signal, given that PfSec22 does not exhibit N-14

terminal cleavage at this motif. Furthermore, mutation of the critical arginine 15

residue using our C-terminal hydrophobic domain mutant failed to ablate the 16

protein export (Fig. 9B). 17

18

DISCUSSION 19

20

The N-terminal longin domains of SNARE proteins have been proposed to 21

mediate the differential localization of these proteins to intracellular 22

compartments, and in some cases, like for ykt6 gene products, to regulate the 23

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SNARE activity [28, 36, 47-50]. The longin domain of PfSec22 exhibits significant 1

differences, compared to yeast and human orthologs, which might influence its 2

cellular locations and trafficking mechanisms. These features include a 3

decapeptide sequence insertion within an unusual loop-like region of the longin 4

domain (Fig. 1A), and an N-terminal hydrophobic segment predicted to function 5

as a recessed signal sequence. We have examined the subcellular distribution of 6

PfSec22, and have investigated the role of the atypical longin domain and of the 7

C-terminal hydrophobic segment in its steady-state localization. Consistent with 8

the subcellular locations of Sec22 proteins in model organisms [34, 51-54], 9

PfSec22 localized predominantly to the ER-Golgi interface in P. falciparum. 10

However, we consistently detected the GFP-tagged and the untagged proteins in 11

tubovesicular structures, and in single vesicular compartments, inside the 12

infected host cell (Fig. 2B, Fig. 4B, Fig. 6A). These findings are consistent with 13

the findings of Trelka et al. [18] in which single vesicles, or vesicular aggregates 14

of variable sizes, were observed in trophozoite-infected erythrocytes. Until now, 15

the significance of these less frequently occurring vesicular structures in the host 16

cell compartment has not been determined. In the previous report, treatment of 17

the parasites with aluminum tetrafluoride, an activator of GTP-binding proteins, 18

resulted in the accumulation of electron-dense vesicles inside the parasitized 19

erythrocyte cytosol. A similar aluminum fluoride treatment of the GFP-PfSec22 20

expressing parasites in our study, however, did not accumulate the GFP 21

fluorescence in the host cell compartment (data not shown), suggesting that the 22

PfSec22-associated structures might represent a distinct sub-population of the 23

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extraparasitic vesicles. It was not clear in our studies whether export of the 1

Sec22 gene product in the malaria parasite was as a direct consequence of the 2

atypical longin domain, or due to the unusual organization of the parasite 3

secretory system. 4

To gain insights into the PfSec22 targeting signals, as a model for SNARE 5

protein targeting in P. falciparum, we investigated the role of the atypical longin 6

domain. Our data suggest that this domain regulates the steady state localization 7

of PfSec22 to the parasite ER/Golgi interface, and its putative transport to the 8

erythrocyte cytoplasm. We found that ER exit of PfSec22 requires residues within 9

the α3 helix, but with no absolute requirement for the entire longin domain. These 10

findings are in contrast with studies in yeast, wherein truncation of the Sec22p 11

longin domain results in ER retention [29], thus suggesting a mechanistic 12

difference in the PfSec22 export process. It is well established in yeast and 13

mammals that the packaging of Sec22 into COPII vesicles involves interaction 14

between the α3 segment of the longin domain and a pre-formed Sec23/Sec24 15

complex [29, 36]. Two conserved residues of the α3 segment, I113 and D116 in 16

Sec22b (corresponding to I118 and D121 in PfSec22), are required for this 17

interaction and, therefore, for Sec22 exit from the ER [36]. Exposure of the α3 18

residues for binding to the Sec23/Sec24 complex is thought to be induced by a 19

conformational epitope that is formed by interaction of an ‘NIE’ motif of the 20

SNARE domain and the α1 and β3 segments [36]. Presumably, truncation of the 21

PfSec22 longin domain at position 78 was sufficient to trigger exit from the ER. 22

Alternatively, ER export of the Golgi-localized mutant proteins (GFP-PfSec22∆1-23

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78 and GFP-PfSec22∆58-78) might have involved interactions with other 1

endogenous proteins including the untagged PfSec22 protein. We interpreted 2

Golgi retention of the N-terminal hydrophobic domain mutants as a defect in the 3

recycling process of these proteins. In yeast, recycling of Sec22p requires 4

interactions with the COPI budding complex and with the SNARE proteins Ufe1 5

and Sec20 [34]. Expression of yeast Sec22p in sec20-1 or ufe1-1 mutant cells, or 6

in the COPI mutants sec21-1 and sec27-1, results in accumulation of the protein 7

in the Golgi, and a lack of ER staining by anti-Sec22 antibodies [34]. In support of 8

a Golgi retention of the PfSec22 mutant protein (GFP-PfSec22∆58-78), treatment 9

of parasites with BFA resulted in partial redistribution of the protein to the ER 10

(Fig. 8A), a phenomenon typical of most Golgi-localized proteins including the 11

cis-Golgi marker PfErd2 [41]. BFA is a GTPase inhibitor that disrupts vesicle 12

trafficking in both the retrograde and the anterograde pathway, resulting in 13

retention of Golgi resident proteins in the ER [46, 55-57]. 14

Taken together, our data indicate that the longin domain of PfSec22 15

exhibits both structural and functional differences when compared to yeast and 16

mammalian Sec22 proteins. The potential for these deviations is supported by 17

both the lack of a significant sequence identity between the longin domains, and 18

the unusual organization of the parasite ER and Golgi compartments. Our data 19

suggest that the N-terminal hydrophobic segment (amino acids 58 to 78) plays 20

an essential role in retrograde transport of this v-SNARE. To our knowledge, this 21

study provides the first experimental evidence for the role of the Sec22 longin 22

domain in transporter recycling, a process that is likely to involve interactions with 23

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other SNARE proteins and the COPI machinery. We have also shown in this 1

study that the C-terminal hydrophobic domain of PfSec22 is important for the 2

membrane association of the protein, and that the occasional export of PfSec22 3

into the host cell was independent of the PEXEL-like motif. In support of our 4

views, PfSec22 is not processed at this motif as has otherwise been reported for 5

canonical PEXEL motif-containing proteins [43]. Additionally, and in contrast with 6

point mutation studies using fusion proteins that are efficiently exported to the 7

erythrocyte cytosol in a PEXEL motif-dependent process [10], mutation of the 8

PEXEL-like arginine of PfSec22 did not inhibit export of the C-terminal 9

hydrophobic mutant (Fig. 9B). In light of the above results, and difficulties with 10

the occasional export of PfSec22, it will now be important to identify all PfSec22 11

interacting partners and characterize their subcellular locations. 12

13

FOOTNOTES 14

We thank Dr. Marcus Lee of Columbia University for helpful discussions and 15

critical reading of the manuscript. 16

17

Abbreviations: SNARE, Soluble N-ethylmaleimide sensitive factor attachment 18

protein receptor; PfErd2, P. falciparum homologue of endoplasmic reticulum 19

retention-deficient mutant 2; PfBip, P. falciparum binding protein. 20

21

Key Words: Plasmodium falciparum, vesicle targeting, SNARE, Sec22 22

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FIGURE LEGENDS 41

42

Figure 1. Sequence features of PfSec22 43

(A) Sequence alignment of PfSec22 with its homologues in humans and yeast. Identical amino 44

acids are highlighted in yellow while the PEXEL-like motif is shaded in red. The five beta strands 45

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(β1-5) and three alpha helix (α1-3) structures of the longin domain are indicated. Black boxes 1

indicate the predicted hydrophobic segments (N-terminal hydrophobic and C-terminal 2

hydrophobic segments). PfSec22 and human Sec22b are 30.9% identical; PfSec22 and yeast 3

Sec22p are 26.2% identical. In comparison, the human and yeast Sec22 polypeptides are 4

37.2% identical. (B) Ribbon structures of human Sec22b and PfSec22 longin domains showing 5

the location of the PEXEL-like motif (RSIIE). The PfSec22 structure was modeled via SWISS-6

MODEL 8.05 using the human Sec22b sequence as template. 7

8

Figure 2. Expression analyses of PfSec22 9

Antibodies were generated against a peptide sequence in the non-conserved region of PfSec22 10

and affinity purified as described in the ‘Materials and Methods’ section. (A) Immunoblot 11

analysis of parasite extracts showing expression of endogenous PfSec22 in ring (R), trophozoite 12

(T), and schizont (S) stage parasites. Absence of antibody reaction with the uninfected 13

erythrocyte lysate (UE) indicates high specificity of the antibodies to the parasite protein. (B) 14

Immunofluorescence microscopy of fixed cells using anti-PfSec22 antibodies and goat anti-15

rabbit Alexa Fluor-555 secondary antibodies. An intense ring of PfSec22 fluorescence is visible 16

in ring and trophozoite infected cells. Isolated foci of PfSec22 fluorescence (black arrow) are 17

also detected in the host cell compartment in trophozoite-infected cells suggesting export of 18

PfSec22 into the erythrocyte cytosol. Scale bar, 2µm 19

20

Figure 3. Co-immunolocalization and membrane-association of endogenous PfSec22 21

(A) The association of PfSec22 with the parasite ER was investigated by co-22

immunofluorescence assays using rabbit anti-PfSec22 and rat anti-PfBip antibodies as 23

indicated. Both proteins co-localized significantly within the parasite. (B) Membrane association 24

of PfSec22. Saponin-purified parasites were lysed by freeze–thaw/sonication, and then 25

centrifuged to separate the membrane-anchored (P1) from the soluble cytosolic and lumenal 26

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proteins (S1). Integral membrane proteins (P2) were separated from the peripheral membrane 1

proteins (S2) by alkaline extraction with 0.1 M Na2CO3 solution at pH 11. The solubility profiles 2

of the integral membrane proteins were further analyzed by Triton-X114 extraction to separate 3

the hydrophilic proteins in the aqueous (aq) phase from the hydrophobic proteins (hy) in the 4

detergent phase. Normalized volumes of the sample pairs were loaded into each well and 5

immunoblotted with antibodies against the PfSec22 protein, the soluble protein PfBip, or the 6

integral protein marker PfErd2. Thirty-five µg of the freeze-thaw lysate were loaded in the lane 7

denoted L. Scale bars, 2µm 8

9

Figure 4. Live cell imaging of GFP-tagged PfSec22 chimeras 10

(A) Immunoblot analyses using anti-PfSec22 antibodies (left blot) or monoclonal anti-GFP 11

antibodies (right blot) confirms expression of the N-terminal GFP-tagged PfSec22 (N-GFP) and 12

the C-terminal GFP-tagged PfSec22 (C-GFP) proteins (~54 kDa) in the respective transgenic 13

cell lines but not in untransfected parasites (3D7). The anti-PfSec22 antibodies also detected a 14

~26 kDa protein in whole cell extracts from all three cell lines that corresponds to the untagged 15

PfSec22 protein. (B) Live cell imaging of parasites expressing the C-terminal GFP-tagged 16

PfSec22 (PfSec22-GFP), showing diffuse localization of the protein throughout the parasite 17

cytoplasm and, occasionally, in the host cell compartment. (C) Confocal micrographs showing 18

localization of the N-terminal GFP-tagged protein (GFP-PfSec22) in early- (first row) and mid-19

trophozoite (second row) stage parasites. In addition to the ER-like profiles, GFP-PfSec22 20

associates with tubovesicular elements in the infected host cell. The micrographs represent 21

differential interference contrast (DIC), GFP fluorescence, and a merge between the two. Scale 22

bars, 2µm. 23

24

Figure 5. Immunoelectron micrographs depicting the association of GFP-PfSec22 with 25

transition vesicles and tubovesicular elements 26

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Ultrathin cryosections of the GFP-PfSec22 expressing cells were probed with monoclonal anti-1

GFP antibodies followed by immunogold detection using gold (12nm)-labeled anti-mouse 2

secondary antibodies. (A) Association of GFP-PfSec22 with ER-derived transition vesicles 3

(black arrowhead). (B) Association of GFP-PfSec22 with membrane-limited vesicles (white 4

arrowheads) in the host cell compartment. (C) Association of GFP-PfSec22 with the TVN-like 5

extension (dotted arrow) and membrane-bound vesicles (white arrowhead) in the infected 6

erythrocyte cytosol. N: nucleus, MC: Maurer’s cleft, RBC: red blood cell cytosol. Scale bars are 7

500 nm in (A), and 100 nm in (B) and (C). 8

9

Figure 6. Co-localization of GFP-PfSec22 with ER and cis-Golgi markers 10

(A) Immunoflorescence micrographs indicating co-localization of the GFP-PfSec22 and anti-11

PfSec22 signals in transgenic parasites (top row), and co-localization of GFP-PfSec22 with the 12

ER marker PfBip (middle row), and Golgi marker PfErd2 (bottom row). Scale bars, 2µm. (B) 13

Sucrose density gradient fractionation of GFP-PfSec22 and untagged PfSec22 in the presence 14

of 2 mM EDTA (left panel), or in the presence of 5 mM MgCl2 (right panel). Numbers 15

represent the fraction numbers, collected from bottom (fraction 1) to top (fraction 10) of 16

a discontinuous sucrose density gradient consisting of 1.5 ml of 47%, 1 ml of 40%, 1 ml 17

of 35%, 0.75 ml of 25%, and 0.75 ml of 20% sucrose. GFP-PfSec22 and the untagged 18

protein both partially redistributed to the high-density fractions (fractions 1-4) alongside 19

PfBip in the presence of MgCl2, suggestive of PfSec22 localization to the ER. 20

21

Figure 7. Role of the atypical longin domain in PfSec22 trafficking 22

Transgenic parasites were generated expressing PfSec22 proteins that lack amino acids 1 to 78 23

(GFP-PfSec22∆1-78)(A), or amino acids 1 to 124 (GFP-PfSec22∆1-124) corresponding to the 24

entire longin domain (B), or that lack residues 58 to 78 (GFP-PfSec22∆58-78) corresponding to 25

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the 21-amino acid N-terminal hydrophobic segment (C). Both the GFP-PfSec22∆1-78 and the 1

GFP-PfSec22∆58-78 mutants co-localize with PfErd2, but not with the ER marker PfBip, 2

suggesting Golgi retention of the N-terminal hydrophobic domain mutants. In contrast, the GFP-3

PfSec22∆1-124 mutant was retained in the ER (B). Scale bars, 2µm. 4

5

Figure 8. Effect of Brefeldin A and Sucrose density gradient fractionation of GFP-6

PfSec22∆∆∆∆58-78 7

(A) Treatment of the GFP-PfSec22∆58-78 expressing parasites with brefeldin A (BFA) resulted 8

in partial redistribution of the mutant protein to the ER (top row), similarly to the Golgi-localized 9

PfErd2 protein (bottom row). Treatment of the parasites with the drug solvent (0.5% methanol in 10

RPMI medium) did not inhibit GFP-PfSec22∆58-78 transport to the Golgi (data not shown). 11

Scale bars, 2µm. (B) Sucrose density gradient fractionation and co-immunodetection of GFP-12

PfSec22∆58-78 with wild-type PfSec22 protein, PfBip, and PfErd2 antigens. Compared with the 13

untagged wild-type protein (PfSec22), which redistributed alongside PfBip in the presence of 14

MgCl2, the GFP-PfSec22∆58-78 mutant was consistently detected in the high-density fractions 15

in the presence of EDTA (left panel) or MgCl2 (right panel). Numbers represent fraction numbers 16

collected from the bottom (fraction 1) to the top (fraction 10) of a discontinuous sucrose density 17

gradient consisting of 1.5 ml of 47%, 1 ml of 40%, 1 ml of 35%, 0.75 ml of 25%, and 0.75 ml of 18

20% sucrose. A small amount of GFP-PfSec22∆58-78 was also detected in the low-density 19

fractions, suggesting an association with lipid-rich structures that might represent transport 20

vesicles. 21

22

Figure 9. Role of the C-terminal hydrophobic domain and PEXEL motif 23

(A) Deletion of the C-terminal hydrophobic domain (GFP-PfSec22∆198-221) resulted in a 24

diffuse localization within the parasite cytoplasm and in the infected host cell in a pattern similar 25

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31

to that of the C-terminally tagged full-length protein (see Fig. 4B). (B) Replacement of the 1

PEXEL arginine with alanine (i.e RSIIE to ASIIE) did not inhibit export of the soluble GFP-2

PfSec22∆198-221 protein to the erythrocyte cytoplasm. These results suggest that the C-3

terminal hydrophobic domain plays an essential role in membrane anchorage of PfSec22, and 4

that the export process of PfSec22 is independent of its membrane integration and the PEXEL-5

like motif. Scale bars, 2µm. 6

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BHuman Sec22b PfSec22

RSIIE

FIG. 1

M C D V V L L C R V S D G M T L V E T N S E T K S I S - - - - - H K L E L K K L C K K L Y S - F P N1 PfSec22

M V L L T M I A R V A D G L P L A A S M Q E D E Q S G R D L Q Q Y Q S Q A K Q L F R K L N E Q S P T1 human

- M I K S T L I Y R E D G L P L C T S V D N E N D P S - - L F E Q K Q K V K I V V S R L T P Q S A T1 Yeast

L S T V T S N N F N Y H F L I E N G I A Y I A V F P V T Y P K K L A F L F L N D I C K Q F N E E L M45 PfSec22

R C T L E A G A M T F H Y I I E Q G V C Y L V L C E A A F P K K L A F A Y L E D L H S E F D E Q H G51 human

E A T L E S G S F E I H Y L K K S M V Y Y F V I C E S G Y P R N L A F S Y L N D I A Q E F E H S F A48 Yeast

I Q Y G T H S I D Y R S I I E T I E K P Y S F I K F D R K I T K I K Q E Y K D P R S N I A I K K L N95 PfSec22

K K V P T - - - - - - - - - - - V S R P Y S F I E F D T F I Q K T K K L Y I D S R A R R N L G S I N101 human

N E Y P K P - - - - - - - - - - T V R P Y Q F V N F D N F L Q M T K K S Y S D K K V Q D N L D Q L N98 Yeast

E S L N E V S S I M K K N I D D I L M R G E N L E D V G R K A F N L K Y E S E K F K K V S R V L N L145 PfSec22

T E L Q D V Q R I M V A N I E E V L Q R G E A L S A L D S K A N N L S S L S K K Y R Q D A K Y L N -140 human

Q E L V G V K Q I M S K N I E D L L Y R G D S L D K M S D M S S S L K E T S K R Y R K S A Q K I N F138 Yeast

K Y A L Y Q Y G I L A S I V I F F F L I I I F K N Y F 195 PfSec22

- - M R S T Y A K L A A V A V F F I M L I V Y V R F WWL 189 human

D L L I S Q Y A P I V I V A F F F V F L F WWI F L K 188 Yeast

β1 β2

β3 β4 β5

α1

α2

α3

SNARE motif

A

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FIG. 2

A B

R T S

25

250

75

20

50

37

100

150

UE

Tro

ph

ozo

ite

Rin

gS

ch

izo

nt

DIC Anti-PfSec22 Merge

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B

FIG. 3

PfSec22

PfErd2

PfBip

P1 S1 P2 S2 aq hyL

Freeze-thaw Na2CO3 Triton-X114

Anti-PfSec22 Anti-PfBip MergeA DIC

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FIG. 4

C

A

B

25

75

20

50

37

100

150N-G

FP

C-G

FP

3D7

C-G

FP

3D7

N-G

FP

Anti-PfSec22 Anti-GFP

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FIG. 5

A

N

RBC

N

B

RBC

C

MC

RBC

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FIG. 6

Anti-PfBip

A

DIC GFP-PfSec22 Merge

Anti-PfSec22DIC GFP-PfSec22 Merge

DIC GFP-PfSec22 Anti-PfErd2 Merge

GFP-

PfSec22

PfSec22

PfBip

PfErd2

(+ MgCl2)

1 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 10

(+EDTA)B

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FIG. 7

CA

B

GFP-PfSec22∆∆∆∆1-78GFP-PfSec22∆∆∆∆58-78

GFP-PfSec22∆∆∆∆1-124

DIC ∆∆∆∆1-78 PfErd2 Merge DIC ∆∆∆∆58-78 PfErd2 Merge

DIC

DIC ∆∆∆∆1-78 Merge

DIC ∆∆∆∆1-124 Merge

DIC ∆∆∆∆58-78 Merge

∆∆∆∆58-78 PfBip Merge

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GFP-PfSec22∆58-78

PfSec22

PfBip

PfErd2

A

B

1 2 3 4 5 6 7 8 9 10

(+ MgCl2)

1 2 3 4 5 6 7 8 9 10

(+EDTA)

FIG. 8

GFP-PfSec22∆∆∆∆58-78 + BFA

DIC ∆∆∆∆58-78 PfErd2 Merge

DIC ∆∆∆∆58-78 Merge

IFA

Live

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FIG. 9

GFP-PfSec22∆∆∆∆198-221A

B GFP-PfSec22∆∆∆∆198-221.PEXEL(R>A)

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