characterization of snares determines the absence of a typical

Characterization of SNAREs Determines the Absence of aTypical Golgi Apparatus in the Ancient EukaryoteGiardia lamblia*□S

Received for publication, August 22, 2008, and in revised form, October 15, 2008 Published, JBC Papers in Press, October 16, 2008, DOI 10.1074/jbc.M806545200

Eliana V. Elias‡, Rodrigo Quiroga‡, Natalia Gottig‡, Hideki Nakanishi§, Theodore E. Nash¶, Aaron Neiman§,and Hugo D. Lujan‡1

From the ‡Laboratory of Biochemistry and Molecular Biology, School of Medicine, Catholic University of Cordoba/National Councilfor Science and Technology, Cordoba CP X5004ASK, Argentina, the §Department of Biochemistry and Cell Biology, Stony BrookUniversity, Stony Brook, New York 11794, and the ¶Laboratory of Parasitic Diseases, National Institutes of Health, NIAID,Bethesda, Maryland 20892

Giardia is a eukaryotic protozoal parasite with unusual char-acteristics, such as the absence of a morphologically evidentGolgi apparatus. Although both constitutive and regulatedpathways for protein secretion are evident in Giardia, little isknown about themechanisms involved in vesicular docking andfusion. In higher eukaryotes, soluble N-ethylmaleimide-sensi-tive factor attachment protein receptors (SNAREs) of the vesi-cle-associated membrane protein and syntaxin families playessential roles in these processes. In this work we identified andcharacterized genes for 17 SNAREs in Giardia to define theminimal set of subcellular organelles present during growth andencystation, in particular the presence or not of a Golgi appara-tus. Expression and localization of all Giardia SNAREs demon-strate their presence in distinct subcellular compartments,whichmay represent the extent of the endomembrane system ineukaryotes. Remarkably, Giardia SNAREs, homologous toGolgi SNAREs fromother organisms, do not allow the detectionof a typical Golgi apparatus in either proliferating or differenti-ating trophozoites. However, some features of theGolgi, such asthe packaging and sorting function, seem to be performedby theendoplasmic reticulum and/or the nuclear envelope. Moreover,depletion of individual genes demonstrated that severalSNAREs are essential for viability, whereas others are dispensa-ble. Thus, Giardia requires a smaller number of SNAREs com-paredwith other eukaryotes to accomplish all of the vesicle traf-ficking events that are critical for the growth and differentiationof this important human pathogen.

Giardia, which is considered one of the most ancienteukaryotes, is a flagellated, binucleated protozoan that parasit-izes the upper small intestine of an extensive variety of verte-brate hosts (1). Human infections are caused by Giardia lam-blia, the most commonly reported intestinal parasite in theworld. Giardia has a simple life cycle alternating between dis-ease-causing trophozoites and environmentally resistant cysts,which are responsible for transmission of the parasite amongsusceptible hosts (2).The typical Golgi apparatus of most eukaryotic organisms

consists of a number of flattened cisternae arranged in a stack,which functions as the protein delivery center of the cell andperforms multiple modifications of lipids and proteins (3).G. lamblia trophozoites possess an interesting secretory sys-tem in which a morphologically identifiable Golgi apparatusseems to be absent, although the packaging and sorting func-tions of this organelle are obvious in this organism (4, 5). Forexample, transport to the plasma membrane (PM)2 and releaseinto the culture medium of variant-specific surface proteins(VSPs) (6, 7), as well as trafficking of both membrane and solu-ble enzymes to peripheral vacuoles (PVs), which are thought toperform both lysosomal and endosomal activities (4, 8), are evi-dence for constitutive protein transport. Regulated secretionhas been reported to take place only during trophozoite differ-entiation into cysts (9). Encystation can be reproduced in thelaboratory by replacing the culturemediumwith an encystationmedium containing high concentrations of bile and a pHresembling that of the small intestine (10). By using this in vitrosystem, it was found that cyst formation comprises differentsteps that include the expression of encystation-specific genes,such as those necessary for the synthesis and processing of cystwall components (11–13) as well as the biogenesis of electron-dense secretory granules (encystation-specific secretory vesi-cles (ESVs)) that transport cyst wall material to the cell periph-ery (11, 12). Before exocytosis, the ESVs interact with the PVs, a

* This work was supported, in whole or in part, by the National Institutes ofHealth NIAID Intramural Research Program. This work was also supportedby the Agencia Nacional para la Promocion de la Ciencia y la Tecnología(FONCYT), the Consejo Nacional de Investigaciones Científicas y Tecnicas(CONICET), the Howard Hughes Medical Institute (HHMI), and the Univer-sidad Catolica de Cordoba. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S10 and Tables S1–S5.

1 An International Research Scholar of the HHMI and a member of the Scien-tist’s Career of CONICET. To whom correspondence should be addressed:Laboratorio de Bioquimica y Biologia Molecular, Facultad de Medicina,Universidad Catolica de Cordoba, Jacinto Rios 571, CP X5004ASK, Cordoba,Argentina. Tel.: 54-351-4860708. E-mail: [email protected].

2 The abbreviations used are: PM, plasma membrane; ER, endoplasmic retic-ulum; ESV, encystation-specific secretory vesicle; PV, peripheral vacuole;HA, hemagglutinin; FITC, fluorescein isothiocyanate; IFA, immunofluores-cence assay; mAb, monoclonal antibody; CWP, cyst wall protein; VSP, vari-ant-specific surface protein; SNARE, soluble N-ethylmaleimide-sensitivefactor attachment protein receptor; gSNARE, Giardia SNARE; VAMP, vesi-cle-associated membrane proteins; ORF, open reading frame; HMW, highmolecular weight; TGN, trans-Golgi network.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 51, pp. 35996 –36010, December 19, 2008Printed in the U.S.A.

35996 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 51 • DECEMBER 19, 2008

by guest on February 18, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/cgi/content/full/M806545200/DC1

http://www.jbc.org/

step required for the processing of cyst wall proteins (CWPs)before extracellular cyst wall assembly (4, 9).During the last decade, many proteins involved in Giardia

secretory pathways have been identified and characterized:endoplasmic reticulum (ER) chaperones such as BiP/GRP78(14) andprotein-disulfide isomerase (15), cystwall proteins 1–3(CWPs) (11, 12, 16), encystation-specific cysteine protease (17),granule-specific protein (17), and giardins (18), among others(5). Recently, the completion of theGiardia genome project (1)has allowed the identification of a number of moleculesinvolved in protein trafficking (i.e. the vesicular coat compo-nents COPI, COPII, clathrin, and adaptor protein complexesplus Rabs, dynamin, and members of the SNARE proteins);however, most have not been fully characterized (12, 19–21).Unfortunately, similar to other anaerobic organisms, chimerasusing fluorescent proteins cannot be used to study the dynam-ics of the secretory pathway ofGiardia (22), and therefore littleis known about the processes of vesicle transport and mem-brane fusion in this parasite.Intracellular membrane fusion is a complex and multistage

process essential for cell growth, proliferation, and differentia-tion. Both homotypic and heterotypic fusion of intracellularmembranes along the secretory and endocytic pathways aremediated by a family of proteins called SNAREs (solubleN-eth-ylmaleimide-sensitive factor attachment protein receptors)(23). These proteins mediate vesicle fusion in essentially all or-ganisms from yeast to human (24). SNAREs share �-helicalcoiled-coil domains, called SNARE motifs, that probablyevolved from a common ancestor and are composed of a hydro-phobic heptad repeat (25) interrupted at a central interface(termed the zero layer) by a conserved arginine or glutaminepolar residue (26). Depending on whether arginine or gluta-mine is present at this position, the SNAREs are referred to asR- or Q-SNAREs (27). In addition, they are categorized as v- ort-SNAREs, depending on their localization to either the trans-port vesicle (v) or the target (t) membrane (23).Originally, it was assumed that there was a strict separation

between SNAREs on the “donor” compartment and the “accep-tor” compartment. However, this terminology is not useful indescribing homotypic fusion events and certain SNARE func-tions in several transport steps with varying partners (28).Functional analysis of these proteins in higher eukaryotes hasled to the “SNARE hypothesis,” which states that interactionsbetween R-SNAREs and Q-SNAREs mediate vesicle fusionwith target membranes (29). Specific R-SNAREs will oligomer-ize productively only with a limited number of Q-SNAREs (28).Such restricted interaction of SNARE proteins is thought toform the basis for specificity of vesicle targeting.SNARE-mediated fusion typically results from the formation

of a complex that comprises a supercoil of four �-helical coils,consisting of one R-SNARE and three Q-SNARE motifs(26). In this context, Q-SNAREs can be subdivided into Qa-SNAREs (syntaxins), Qb-SNAREs (25-kDa synaptosomal-associated protein (SNAP-25) N-terminal SNARE motif),and Qc-SNAREs (SNAP-25 C-terminal SNARE motif) (30).The R-SNAREs also can be subdivided into short VAMPs (ves-icle-associated membrane proteins), or “brevins” (e.g. synapto-brevin), and long VAMPs, or “longins,” on the basis of whether

they contain a short and variable domain or a conserved longindomain of 120–140 amino acids at theirN termini (31). SNAREcomplexes may be assembled from three or four proteins(SNAP-25 homologs typically contribute two helices), and thecomplete complex is a four-helix bundle composed of one helixfrom each sequence family, Qa, Qb, Qc, and R (24).Like Q-SNAREs, R-SNAREs can include transmembrane

domains or can be covalently anchored to a lipid at conservedcysteine residues (31). Indeed, a phylogenetic analysis ofSNARE sequences from Saccharomyces cerevisiae, Arabidopsisthaliana, some protozoa, and mammals shows that these fourSNARE subfamilies are highly conserved and diverged early ineukaryotic evolution (21).Because Giardia is an early divergent eukaryote (1, 2), the

study of the organization of membrane compartments andmembrane fusion processes in this organismmay allow a betterunderstanding of the evolution of transport pathways fromprimitive to higher eukaryotes. In addition, because of theirparasitic life style, Giardia is an interesting system in which toanalyze how microorganisms could have lost complexorganelles and, therefore, to illuminate the minimal set oforganelles needed for a given cellular function. For these rea-sons, the aim of the present work was to identify and character-ize the complete set of SNARE proteins inGiardia to define theminimal set of subcellular compartments present duringgrowth and encystation, in particular the existence or not of atypical Golgi apparatus. Our results suggest that gSNAREs arefunctional and distinctively localized to known subcellularcompartments, suggesting a role in regulating membrane traf-ficking to and from these organelles. Surprisingly, a comparisonof Giardia SNAREs homologous to Golgi SNAREs from otherorganisms does not allow the detection of a typical Golgi appa-ratus in either proliferating or differentiating trophozoites.

EXPERIMENTAL PROCEDURES

Construction of Expression Vectors—For expression andlocalization of SNARE proteins in Giardia, the entire openreading frame of each SNARE gene was amplified by PCR withthe following sense oligonucleotides containing BamHI or BglIsites and antisense oligonucleotides containing a NotI site:gQa1_f, 5�-GCGGATCCGAGAACATGTATGACGAC-3�;gQa1_r, 5�-ATA AGA ATG CGG CCG CTT ACT TAA AGATTGTGCTC-3�; gQa2_f, 5�-GCGGATCCACAGTGACCATAG ATC TAA C-3�; gQa2_r, 5�-ATA AGA ATG CGG CCGCCT ATA TAA GTC GCA GAA A-3�; gQa3_f, 5�-GAA GATCTACCGATTTTGATGCACCA-3�; gQa3_r, 5�-ATAAGAATGCGGCCGCCTACTTTTTCACGGCACG-3�; gQa4_f,5�-GCG GAT CCT CTC TAG ATT TTA CCG GCA TC-3�;gQa4_r, 5�-ATA AGA ATG CGG CCG CTC AGT TGA TATAAT CTA G-3�; gQa5_f, 5�-CGC GGA TCC GAG TTC ACGTGCCTGTTTGG-3�; gQa5_r, 5�-ATAAGAATGCGGCCGCCT AGA TCC GCC TTA TTG CCC-3�; gQb1_f, 5�-ATAAGAATGCGGCCGCCCCAAGCCGAGGATGAAGA-3�;gQb1_r, 5-ATA AGA ATG CGG CCG CCT AGC GTT TTAAAA GGC CCA CAA T-3�; gQb2_f, 5�-CGC GGA TCC TCCTAT TCT TCG CGC CCT C-3�; gQb2_r, 5�-ATA AGA ATGCGG CCG CTC AAA GTA GCT TCA TAA GTA AAT ATACTAATGAG-3�; gQb3_f, 5�-CGCGGATCCAATGCTCAG

SNAREs Define Vesicular Compartments in Giardia

DECEMBER 19, 2008 • VOLUME 283 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 35997


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

CTG ATT CGG G-3�; gQb3_r, 5�-ATA AGA ATG CGG CCGCTTAAGGAAGAAACGGGGGA-3�; gQb4_f,5�-CGCGGATCC GAA GAG ATA GAA TGT TCA CTC ATG G-3�;gQb4_r, 5�-ATA AGA ATG CGG CCG CTC AAT ATC TGATCTCTGAGGGC-3�; gQb5_f, 5�-CGCGGATCCTCTGAGGAG TAT GAC ATC CAA AAG-3�; gQb5_r, 5�-ATA AGAATG CGG CCG CTC ACT TCT TGG TGG CCT GTG-3�;gQc1_f 5�-CGC GGA TCC TCT ATA GTA CCC GTG CTGGTG-3�; gQc1_r, 5�-ATA AGA ATG CGG CCG CTC AACCAG TGA AGT TTA TAG CAA T-3�; gQc2_f, 5�-CGC GGATCC TAC AGT TTC ATT GAG ATC GAC TAT AAG-3�;gQc2_r, 5�-ATA AGA ATG CGG CCG CAA AGG GCG CACGAC CAG-3�; gQc3_f, 5�-CGC GGA TCC GAC GAT TCTGGTAACGATATTGG-3�; gQc3_r, 5�-ATAAGAATGCGGCCG CTT AAG GAC ACT GCC ACG TGA-3�; gQc4_f,5�-CGC GGA TCC GAA GTA AAC GGG CCG AGA AA-3�;gQc4_r, 5�-ATA AGA ATG CGG CCG CCT AAT GGA GAACCA TGT ACA GCA AG-3�; gR1_f, 5�-GCG GAT CCC TTCCTG TCG ATA CCA AAA GG-3�; gR1_r, 5�-ATA AGA ATGCGG CCG CTT ACC TGA AGA AAA TCT TTA CC-3�;gR2_f, 5�-GCG GAT CCG GGG CAT TTT TAA AAA TGAC-3�; gR2_r, 5�-ATA AGA ATG CGG CCG CTC ATA GAACAGCACATAGTC-3�; gR3_f, 5�-GCGGATCCAGTGCCGGGAACATTTATG-3�; gR3_r, 5�-ATAAGAATGCGGCCGCTT AAG AAA ACT TGA TCG C-3�. Restriction sites areunderlined. The products were purified, restricted, and clonedinto the vector pTubHApac N terminus (4). This plasmidallows the constitutive and stable expression of genes in Giar-dia trophozoites by the presence of the�-tubulin promoter andselection with puromycin. The sequences of all constructs wereverified by sequencing using dye terminator cycle sequencing(Beckman Coulter).gSNARE Antisense Silencing—For gSNARE functional analy-

sis, we used the following sense oligonucleotides containing aNotI site and antisense oligonucleotides containing BamHI orBg1I sites to amplify by PCR-specific regions of each gSNAREgene: gQa1A_f, 5�-ATAAGAATGCGGCCGCGCGGATCCGAG AAC ATG TAT GAC GAC-3�; gQa1A_r, 5�-GCG GATCCG TCT GCC CTT TAT CTC CGC GAC CTT AC-3�;gQa2A_f, 5�-ATAAGAATGCGGCCGCACGGGCGGAGTTGA GTT CAG AGG AGA-3�; gQa2A_r, 5�-GCG GAT CCGATA AGA ATG CGG CCG CCT ATA TAA GTC GCA GAAA-3�; gQa3A_f, 5�-ATAAGAATGCGGCCGCGAAGATCTACC GAT TTT GAT GCA CCA-3�; gQa3_r, 5�-GAA GATCTA CTT TGC CTC CGC GTC CTC TTT-3�; gQa4A_f,5�-ATA AGA ATG CGG CCG CGT CCG GCG CAC AGAACA AAC AGC-3�; gQa4A_r, 5�-GCG GAT CCT TAA GAATGCGGCCGCTCAGTTGATATAATCTAG-3�; gQa5A_f,5�-ATA AGA ATG CGG CCG CGC TTC GCT TCC GTCCGTT-3�; gQa5A_r, 5�-CGCGGATCCGACCGCTCAGCAGTT CGC-3�; gQb1A_f, 5�-ATA AGA ATG CGG CCG CCCCAA GCC GAG GAT GAA GA-3�; gQb1A_r, 5�-CGC GGATCC GGG CGC CGG ACT AAA CC-3�; gQb2A_f, 5�-ATAAGA ATG CGG CCG CTA TGC CCG CCC CCA ACT T-3�;gQb2A_r, 5�-CGCGGATCCCCAGCGCCTTGATATCATCTT CA-3�; gQb3A_f, 5�-ATA AGA ATG CGG CCG CGCGAAGTT TGTGGCAGA TG; gQb3A_r, 5�-CGCGGA TCCACG GGG GAT AAA GGT GGA T-3�; gQb4A_f, 5�-ATA

AGAATGCGGCCGCGCGCCGTGCAGACGCTAAAA-3�; gQb4A_r, 5�-CGC GGA TCC GAG CCG GCT CTG GATTGTTGTG-3�; gQb5A_f, 5�-ATAAGAATGCGGCCGCTCCCG AGA GGC TGC GAT GA-3�; gQb5A_r, 5�-CGC GGATCC TCA CTT CTT GGT GGC CTG TGC T-3�; gQc1A_f5�-ATA AGA ATG CGG CCG CGG CAA TGT GCA ATAAAT ATT TG-3�; gQc1A_r, 5�-CGC GGA TCC GTT CGCGCT CAC ATT TTC TT-3�; gQc2A_f, 5�-ATA AGA ATGCGG CCG CAA AGG GCG CAC GAC CAG-3�; gQc2A_r,5�-CGC GGA TCC TTA TAG CCA TAT GAG GCA AACG-3�; gQc3A_f, 5�-ATAAGAATGCGGCCGCACCGAGATCTCAGGGGCACTT-3�; gQc3A_r, 5�-CGCGGATCCGCCCCC TCA TCC AGC TTC TC-3�; gQc4A_f, 5�-ATA AGAATGCGGCCGCGAAGTAAACGGGCCGAGAAAGC-3�;gQc4A_r, 5�-CGCGGATCCCGCTGCTGGCATCACCGAGTA G-3�; gR1A_f, 5�-ATA AGA ATG CGG CCG CGC GGATCCCTTCCTGTCTGATACCAAAA-3�; gR1A_r, 5�-GCGGAT CCG CAC CCC GCA TAT GAG CAC CGA GAT-3�;gR2A_f, 5�-ATA AGA ATG CGG CCG CGC GGA TCC GGGGCATTTTTAAAAATGAC-3�; gR2A_r, 5�-GCGGATCCGGGT AAT GGT TTG CGA AGC GGA TGG C-3�; gR3A_f,5�-ATA AGA ATG CGG CCG CGC GGA TCC AGT GCCGGG AAC ATT TAT G-3�; gR3A_r, 5�-GCG GAT CCG GGCTGA CAA AAG TGC GCG GGA AAG T-3�. Restriction sitesare underlined.PCR products were purified, cut, and cloned into the vector

PtubHApac N terminus (4). In this way, each SNARE gene wasinserted inside the PtubHApac N terminus inversely, thus gen-erating each SNARE antisense vector. Sequences were con-firmed by dye terminator cycle sequencing (Beckman Coulter).Transfection of G. lamblia trophozoites was performed byelectroporation as described previously (32), and cells carryingthe antisense construct were selected using puromycin (33).G. lamblia Cultivation and Transfection—Trophozoites of

the isolate cloneWB/1267 (34) were cultured as described pre-viously (35). Encystation of the trophozoite monolayer wasaccomplished by the method described by Boucher and Gillin(36). Trophozoites were transfected with the constructs byelectroporation and selected with puromycin as described pre-viously (37). After transfection, clones with low expression ofgSNARE proteins were selected by immunoblotting and IFA.Immunofluorescence Analysis of Giardia Trophozoites—

Cells cultured in either growth or encystation medium wereharvested and processed as described previously (12). The cellswere fixedwith 4%paraformaldehyde and permeabilized for 1 hat room temperature in phosphate-buffered saline, 0.1% TritonX-100, 10% goat serum. The cells then were incubated with theantibodies diluted in phosphate-buffered saline, 0.1% TritonX-100, 3% goat serum. For indirect staining, slides were incu-bated with the specific mAbs (final dilution 1:200) or anti-hem-agglutinin (HA) mAb (Sigma-Aldrich, final dilution 1:1000) for1 h at 37 °C and then with anti-mouse secondary antibodylabeled with FITC (final dilution 1:250) for 1 h at 37 °C. Fordirect double staining, anti-HA mAb FITC (Sigma-Aldrich,final dilution 1:500) was used to detect the transgenic proteins;9C9, 5B11, 5C1, and 7D2 mAbs were labeled directly withTexas Red (Zenon One, Molecular Probes, Inc., Eugene, OR)for the detection of BiP, PVs, VSP1267, and the endogenous




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

CWP2, respectively. Controls included the omission of primaryantibodies and staining of cells transfected with the empty vec-tor. Confocal images were collected using a Zeiss LSM5 Pascallaser-scanning confocal microscope equipped with an argon/helium/neon laser and an�100 1.4NA oil immersion objective(Zeiss Plan-Apochromat). Single confocal sections (0.3 �m)were taken parallel to the coverslip (xy sections). Images wereacquired and processed with LSM software; blind two-dimen-sional deconvolution was performed with AutoDeblur &Auto-Visualize Gold.Nucleic Acid Analysis—Genomic DNA used for the PCRs

was obtained as described (7). To determine whether the genescoding for gSNARE proteins were transcribed, we isolated totalRNA from Giardia non-encysting trophozoites using TRIzol�reagent (Invitrogen) according to the manufacturer’s protocol.Samples then were treated with RQ1 RNase-free DNase (Pro-mega) to remove contaminating DNA. After one phenol-chlo-roform extraction, RNAwas precipitated, and 5 �g was used tosynthesize first-strand cDNA with SuperScriptTM III reversetranscriptase (Invitrogen) andPolyTprimer following theman-ufacturer’s instructions. One-tenth of the final material wasused as a template for separate quantitative real-time PCR reac-tions with QuantitectTM SYBR� Green PCR (Qiagen) using theChromo 4 real-time PCR detector. The same primers used ingSNARE antisense silencing were used to amplify fragments100–500 pb in length. A negative control contained all reagentsexcept the cDNA (RNA was included instead). One positivecontrol was also used, the constitutively expressed gene gdh(GenBankTM accession number M84604) (58). We usedMicrosoft Excel� to open the exported Ct (threshold cycle) file.Reverse transcription PCR products were analyzed by electro-phoresis on a 0.8% agarose gel in Tris acetate-EDTA buffer.Analysis of the DNA sequence was performed with the com-puter program DNAStar (Lasergene).Bioinformatic Analysis—The latest version of the Giardia

genomewas downloaded. AnORF from the giardia13 assembly(ORF 10803) was included in the data set, as this ORF had beenamplified by PCR in our laboratory and proved to be a SNAREprotein through bioinformatics, although it was discontinuedin the giardia14 assembly of the giardial genome. HMMsearchand HMMPfam were performed with the HMMER program(38). SMART searches were performed with the SMARTWebserver (39). HHPred (40) was performed with varying options,against either the Protein Data Bank or COG/KOG data bases.Secondary structure scoring was set to “predicted” versus“pre-dicted only.” Searches that resulted in a top hit with a p valueabove 1e�4were run through theHHSenser program (41). Pho-bius 5.1was used to predict transmembrane domains and signalpeptides (42).Protein sequenceswere alignedwithMUSCLE (43) andman-

ually curedwithGenedoc, taking into account secondary struc-ture information. Accession numbers for proteins used inalignment were: AAA53519.1 (Syntaxin 1A), NP_001007026.1(GOSR1), EAL24137.1 (Bet1), and CAG33270.1 (YKT6). Thepresence of Habc domains, characteristic of the syntaxin pro-tein group, was determined using PCOILS (44), PSIPRED (45),and HHPfam.

Western Blot Analysis—Protein extraction was performed byresuspending the final membrane pellet in SDS sample buffer(62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 5% 2-mer-captoethanol, and 0.01% bromphenol blue). In all experiments,bromphenol blue was omitted initially to allow absorbancemeasurements of themembrane extracts at 280 nm; it then wasadded later to verify that the amount of protein extracted didnot vary substantially between individual samples. Sampleswere incubated at different temperatures (at 37, 42, and 80 °Cfor 5 min to detect SDS-resistant SNARE complexes and at100 °C for 15 min to disrupt SDS-resistant SNARE complexes)and subjected to SDS-PAGE. Ten �g of total protein fromtransfected trophozoites grown in normal and encystationmedium was loaded per lane. Electrophoretic transfer of pro-teins to nitrocellulose was performed as reported (7, 11, 12).Filters were blocked with 3% defatted milk, 0.1% Tween, Tris-buffered saline and then incubated with anti-HA or 3B5 mAbs(1:1000 dilution). Following incubation with alkaline phospha-tase-conjugated goat anti-mouse IgGs at a 1:2000 dilution,Giardia proteins were visualized by color development with analkaline phosphatase color development reagent (Bio-Rad).

RESULTS

G. lamblia SNARE Homologs

Through runs of HMMsearch, HMMPfam (38), SMART(39), and HHPred (40) on the giardia13 genomic assembly, weidentified 17 Giardia proteins as putative SNAREs (Table 1).The HMM profiles, recently published by Kloepper et al. (21)for several species, allowed us to classify these proteins moreefficiently. RunningHMMsearchwith theHMMfiles describedin Kloepper et al. (21) against the giardia14 assembly proteindata base resulted in very fewQb andQc proteins with E valuessmaller than the standard 0.1 E value cut-off line. Taking intoconsideration that Giardia is such a divergent eukaryote,whether because of primitiveness, rapid evolution, or a combi-nation of both (5), wemodified our protocol to increase the sensi-tivity of the search. All sequenceswith E values lower than 10 (285protein sequences) were run through the HMMPfam programagainst a custom data base containing the complete Pfam-A databaseand theHMMs(supplementalTableS1) (fromKloepper etal.(21)).All protein sequences inwhich the tophitwas anon-SNAREHMMwere removed from the data set.To make a prediction about the possible function of the dif-

ferent SNAREs, we performed bioinformatic analyses expect-ing that functional conservation should be reflected bysequence conservation. Because the signals that drive the local-ization of SNAREs are not well understood, we searched forhomologs of the entire sequence of each molecule and theSNARE domain only. FASTA searches against the Swiss-ProtandUniprotKB data bases provided homologs for eachGiardiaSNARE protein (supplemental Table S2). However, low E val-ues prompted us to use amore sensitive searchingmethod; thusHHPred andHHSenser were used to search the COG andKOGdata bases (44) for the SNARE subfamily that is more closelyhomologous to each Giardia SNARE (supplemental Table S3).Using HMMsearch and HMMpfam, we were able to classify

putative SNAREs into four different groups (Qa,Qb,Qc, and R)




ww

.jbc.org/D

ownloaded from




http://www.jbc.org/

in accordance with genome-wide classification performed withother organisms. Five SNAREs corresponded to theQa-SNAREsubfamily (gQa-SNAREs 1–5), five to theQb-SNARE subfamily(gQb-SNAREs 1–5), four to the Qc-SNARE subfamily (gQc-SNAREs 1–4), and three to the R-SNARE subfamily (gR-SNAREs 1–3) (Table 1). This classification was supported byperforming local and global alignment HHPred searchesagainst the Protein Data Bank data base, confirming that thetop hit for each gSNARE corresponded to the Qa, Qb, Qc, or RSNAREs for the 2 nps Protein Data Bank entry (46) (supple-mental Tables S4 and S5).

Giardia SNARE Characteristics

The putative gQ-SNAREs display all of the structural char-acteristics defined for this family of proteins. They have thetypical size (104–345 amino acids), a single transmembranedomain near the C terminus (with a predicted type II integralmembrane protein orientation), and a coiled-coil SNAREdomain preceding the transmembrane domain (Table 1). Inaddition, using PCOILS (45), PSIPRED (47), and HHPfam, wecould identify the presence of an N-terminal helical bundle(called Habc) (48) in gQa1, gQa2, and gQa3 (supplemental Fig.S1), characteristic of the syntaxin family, that can fold back intoa closed conformation to interact with the SNARE motif (49).Secondary structure predictions for Qb-, Qc-, and R-SNAREsidentified a similar series of helixes in gQb3, gQb4, gQb5, gQc1,and gQc3 (supplemental Figs. S2–S4). These could representdivergent Habc domains, as HMMPfam and HMMsearch werenot able to detect homology to the Habc domain. These find-ings are consistent with the presence of Habc domains inmem-bers of the Qb and Qc families of Leishmania major and inhuman Syntaxin 6 (belonging to the Qc group) and Vti1(belonging to the Qb group) (50).Animals, plants, and fungi express SNAP-25 family proteins,

which contain two SNARE motifs belonging to the Qb and Qcgroup. In addition, few Qbc-SNAREs were reported to be pres-ent in protists like Plasmodium falciparum and Dictyosteliumdiscoideum (21). Giardia, however, similar to other protozoasuch as L.major andTrypanosoma brucei (51), does not possess

anymember of this SNARE family. All of the SNARE sequencesidentified contain a single, C-terminal SNARE motif.As shown in Table 1, four gQ-SNAREs sequences belong to

the specific syntaxin paralog family called gQa1–4, and thesewere characterized previous to this work (19, 32, 52, 53). Wefound that mAb 3B5 (raised against Giardia encysting tropho-zoite microsomes) recognizes gQa1 (supplemental Fig. S5).ThismAbwas useful in comparing the level of expression of thewild type gQa1 with the transgenic gQa1-HA expressed underthe control of the �-tubulin promoter (supplemental Fig. S5).We also identified gQa2 by searching an incomplete version ofthe Giardia genome data base and subsequently performedsequencing of the entire gene from different cDNA andgenomic DNA libraries (see additional methods and informa-tion in the supplemental data for Fig. S5). Giardia Qa3 andgQa4 were characterized similarly by other groups (52, 53).The three gR-SNAREs correspond to the family of VAMP

proteins and we called them gR1–3 (Table 1). Similar to find-ings in L. major, gR1–3 are longer than the classical shortVAMPs (brevins) so they could be classified as long VAMPs(longins) (31). These three long gVAMPs contain the R-SNAREcoiled-coil region. Giardia R1 and gR3 have the characteristictransmembrane domain near the C terminus; gR3 has an addi-tional predicted transmembrane domain near the N terminus(amino acids 71–90). Giardia R2 has an isoprenylation site(CAAX box at amino acid 218) instead of the transmembranedomain, which mediates membrane attachment (Table 1).

Giardia SNAREs Possess Atypical Amino Acid Residues at the“0” Layer

It has been suggested that R-SNAREs provide an arginineresidue (R) to the conserved ionic layer, whereas Q-SNAREprovide complementary glutamines (Q) for the formation ofthe ternary complexes (26). As shown in Fig. 1, many putativeGiardia SNAREs do not possess either residue at the zero layer.Present at this position were atypical amino acid residues thatinclude aspartic acid (D), asparagine (N), serine (S), glycine (G),and isoleucine (I). Recently, atypical zero layer amino acidshave been reported for other organisms, such as serine for the

TABLE 1Sequence characteristics of gSNARE proteinsGiardia SNARE proteins were classified into fourmain groups: Qa, Qb, Qc, and R. TheORF, as listed in the Giardia genome project data base, is indicated for each SNARE.Sizes of the gSNARE proteins and positions of SNARE motifs and transmembrane (TM) domains are indicated. Phobius 5.1 was used to locate SNARE motifs andtransmembrane domains. gR2 has an isoprenylation site CAAX box at amino acid 218 instead of the transmembrane domain.

Gene product Function ORF GL50803_ Length SNARE domain TM domain Molecular mass Referencesaa aa aa kDa

Qa-SNARE 1 Qa1 7,309 293 207–259 270–292 32.51 17, 19, 53Qa-SNARE 2 Qa2 3,869 271 185–237 250–268 30.81 17, 19, 52, 53Qa-SNARE 3 Qa3 96,994 307 217–269 284–305 34.79 52, 53Qa-SNARE 4 Qa4 11,220 345 264–316 326–344 38.62 53Qa-SNARE 5 Qa5 10,803 268 185–237 248–265 30.46 This studyQb-SNARE 1 Qb1 16,054 212 127–179 188–210 23.57 This studyQb-SNARE 2 Qb2 17,464 204 124–176 182–201 22.45 This studyQb-SNARE 3 Qb3 5,161 215 122–174 184–204 24.54 This studyQb-SNARE 4 Qb4 5,785 226 125–173 186–206 24.74 This studyQb-SNARE 5 Qb5 10,315 222 135–183 191–214 25.10 This studyQc-SNARE 1 Qc1 5,927 337 249–297 312–332 38.47 This studyQc-SNARE 2 Qc2 7,590 257 168–227 239–256 27.93 This studyQc-SNARE 3 Qc3 10,013 220 128–176 189–207 24.46 This studyQc-SNARE 4 Qc4 19,509 104 34–83 87–103 11.80 This studyR-SNARE 1 R1 9,489 238 156–208 217–237 26.14 This studyR-SNARE 2 R2 7,306 221 161–213 CAAX 24.02 This studyR-SNARE 3 R3 14,469 239 152–204 70–90 215–235 26.34 This study




ww

.jbc.org/D

ownloaded from







http://www.jbc.org/

yeast Bet1p, aspartate for the human Vti1 and Slt1, and aspar-agine and isoleucine amongmembers of the syntaxin subfamilyof P. falciparum and in some synaptobrevins of Plasmodiumtetraurelia (51). The presence of a glycine residue in place ofglutamine at the zero layer position of gQc3 is a novel findingamong the SNARE superfamily.

Subcellular Distribution of gSNARE Proteins in G. lamblia

Defining the subcellular localization of SNARE proteins isessential to understand their function, especially in an orga-nism with an atypical secretory apparatus such as Giardia. Totest whether the bioinformatic data correctly predict the com-partment or pathway of a given SNARE, the 17 coding genes forputative gSNAREs were amplified by PCR from G. lambliagenomic DNA, cloned into an HA fusion vector, and trans-fected into G. lamblia trophozoites (37). It was shown previ-ously that small tags such as the HA epitope do not influencethe behavior ofGiardia proteins and do not affect their subcel-lular localization (4, 20). The HA epitope was inserted at the Nterminus of these constructs because SNARE proteins do nothave an N-terminal signal peptide (31) and also because it hasbeen shown that this procedure accurately reflects the localiza-tion of SNARE proteins in other eukaryotic cells (54). Therewas a possibility of mistargeting to improper locations becauseof overexpression, and thus care was taken to select cell clones

expressingmoderate or low levels oftagged proteins for localizationexperiments. We colocalized thefluorescence patterns obtained foreach gSNARE with cellular markersto tentatively identify the labeledcompartments. In addition, allSNAREs were analyzed under twodevelopmental conditions, non-en-cysting as well as encysting cells.The latter displays a unique secre-tory compartment, secretory gran-ules called ESVs, which seem todevelop directly from the ER, asshown recently (55).Giardia Qa-SNAREs—Immuno-

fluorescence assays in non-encyst-ing Giardia trophozoites showedthat HA-gQa1 localized at the PVs,as determined by their colocaliza-tion with the mAb 5B11, which spe-cifically labels PVs by recognizinglysosomal cathepsin B3 (Fig. 2A).The same pattern was observedusing either the anti-HAmAbor thegQa1-specific mAb 3B5, showingthat the HA tag does not affect thelocalization of this protein (supple-mental Fig. S5). When these cellswere induced to encyst, HA-gQa1still localized to the PVs (Fig. 2B). Inaddition, using the anti-HA mAband anti-cyst wall protein 2 (CWP2)

mAb, partial colocalization between HA-gQa1 and someencystation-specific ESVs that transportCWPswas observed inmany encysting cells (Fig. 2B). This result indicates a closeinteraction between PVs and ESVs, necessary for the release ofsecretory granule content before extracellular cyst wall forma-tion, as suggested previously (17).In non-encysting cells, HA-gQa2 localized to the ER sur-

rounding both nuclei of the parasite (Fig. 2A),as determined byits colocalization with the ER-resident chaperone BiP (14).During encystation, HA-gQa2 showed the same ER pattern aswell as colocalization with ESVs (Fig. 2B).In non-encysting trophozoites, HA-gQa3 appeared on

broadly distributed punctate structures distributed throughoutthe cell and in the cell cortex. Moreover, partial localization atthe ER also was observed, particularly in the perinuclear region.On the other hand, the staining at the cell cortex colocalizedwith mAb 5B11, suggesting that HA-gQa3 also localizes to thePVs (Supplemental Fig. S6). In encysting trophozoites, gQa3showed the same pattern, with less staining surrounding thenuclei, and the vesicles were in the proximity of, but distinctfrom the ESVs (Fig. 2B).In non-encysting and encysting cells, HA-gQa4 localized in a

punctate pattern at the ER and surrounded both nuclear enve-

3 H. D. Lujan, unpublished results.

FIGURE 1. Multiple sequence alignment of Giardia Qa-SNAREs, Qb-SNAREs, Qc-SNAREs, and R-SNAREs.Each alignment also includes the SNARE protein from the 2 nps Protein Data Bank entry used for classifying theSNARE proteins (Syntaxin 13, Vti1a, Syntaxin 6, and VAMP 4), and a model SNARE for each of the classifications(Syntaxin 1A, GOSR1, Bet1, and YKT6). A higher degree of conservation in the residues predicted to interactwith other SNAREs in the formation of the SNARE complex is observed. Only layers �7 to �8 are depicted,because this region is more highly conserved and is also the region used to generate the HMMs (21).




ww

.jbc.org/D

ownloaded from



http://www.jbc.org/

lopes (Fig. 2). During encystation, HA-gQa4 displayed this pat-tern aswell as colocalizationwith ESVs (Fig. 2B). In non-encyst-ing trophozoites, HA-gQa5 was found not only at the PMcolocalized with the integral membrane protein VSP1267, asdetermined using the specific mAb 5C1 against this protein(56) (Fig. 2A), but in addition, it colocalized with PVs (supple-mental Fig. S6), which indicates an interaction between PVsand PM, showing that the PV is a dynamic endosomal compart-ment. This interaction was confirmed by colocalizing the PVs(using mAb 5B11) with the PM (using mAb 5C1) in non-trans-fected trophozoites (supplemental Fig. S6). During encystationof these cells, we observed that the localization of HA-gQa5remained at the PM/PVs interphase.Thus, the gQa-SNAREs display distinct localizations: gQa1

to the PVs, gQa2 and gQa4 to the ER, gQa5 to the PM/PVs, andgQa2, gQa4, and gQa3 to membranes that are either subdo-mains of the ER or are closely associated with it. In addition,gQa4 appears to localize to ESVs during the process ofencystation.Giardia Qb-SNAREs—Immunofluorescence assays of trans-

fected trophozoites reflected that both HA-gQb1 andHA-gQb2 localized in a similar vesicular pattern but one thathad not been described before in non-encysting and encystingcells, e.g. lack of colocalization to the ER, PVs, or ESVs (Fig. 3).However, as some ER proteins display a dot-like staining pat-

tern, it is possible that HA-gQb1and HA-gQb2 localize to some por-tions of the ER or to ER exit sites (5,19).In non-encysting trophozoites,

HA-gQb3 was found in a number ofvesicles with a distribution similarto “mitosomes” (57), as a vesiculartube between the two Giardianuclei and in close proximity to thebasal bodies, and in vesicles atthe lateral and posterior parts of thecell. These vesicles were clearly dis-tinct from the ER and the PVslocated underneath the PM andclearly similar to the mitosomesdescribed in Fig. 2 of Dolezal et al.(57). During encystation, the num-ber of vesicles decreased and thepattern changed; lateral vesicles dis-appeared and a prominent vesicleappeared in the apical, posteriorpart of the cell, which conceivably isa result of the fusion of vesicles (Fig.3B). However, the tubular vesiclefound between the nuclei had thesame localization pattern describedby Dolezal et al. (57).

In non-encysting and encystingcells, HA-gQb4 was found not onlyat the PM colocalizing with mAbanti-VSP1267 (Fig. 3) but also colo-calizing with the PVs labeled with

mAb 5B11 (supplemental Fig. S6).HA-gQb5 localized at the ER in non-encysting cells as deter-

mined by its colocalization with BiP (Fig. 3A). When these cellswere induced to encyst, HA-gQb5 still showed a more promi-nent perinuclear distribution with less staining at the ER. Inter-estingly, colocalization with ESVs also was observed near thenuclei (Fig. 3B).Overall, the distribution of the gQb-SNAREs is somewhat

different than that for the gQa subfamily. Giardia Qb4 andgQb5 can be assigned with high confidence to the PVs and theER, respectively, but gQb1 and gQb2 are more broadly distrib-uted, perhaps in transport vesicles, ER patches, or ER exit sites.The gQb3 protein displayed a unique distribution that mightcorrespond to the mitosome, a recently discovered organellethat seems to be a remnant of mitochondria (57).Giardia Qc-SNAREs—Immunofluorescence assays in non-

encysting and encysting trophozoites showed that HA-gQc1and HA-gQc4 localized at the PVs as determined by their colo-calization with mAb 5B11 (Fig. 4). In addition, we observedcolocalization of these two SNAREs with the PM labeled withmAb 5C1 (supplemental Fig. S7). Surprisingly, in encysting tro-phozoites, HA-gQc4 colocalized with ESVs in proximity to thePVs/PM (Fig. 4B). In non-encysting and encysting trophozo-ites, HA-gQc2 localized at the PM (Fig. 4).Moreover, HA-gQc2colocalized with PVs (supplemental Fig. S7). HA-gQc3 local-

FIGURE 2. Localization of gQa-SNAREs (gSyntaxins) in non-encysting and encysting Giardia trophozo-ites. A, confocal microscopy of direct immunofluorescence assays of non-encysting transfected Giardia tro-phozoites shows the colocalization (yellow) of HA-gQa1 (green) with the PVs (in red) and of HA-gQa2 andHA-gQa4 (green) with BiP (red) at the ER. HA-gQa3 localizes at internal vesicles, nuclear envelope, and PVs/plasma membrane (green); and HA-gQa5 (green) colocalizes (yellow) with the PM (VSP1267, in red). HA-taggedgQ-SNARE proteins are stained with FITC-conjugated anti-HA mAb (left panels); the PVs and BiP are detectedwith Texas Red-labeled (red) mAbs 5B11 and 9C9, respectively. B, confocal microscopy of direct immunofluo-rescence assays showing the localization of HA-gQa SNAREs (green) in encysting cells. The ESVs are labeled withmAb 7D2 specific for CWP2 (red).




ww

.jbc.org/D

ownloaded from






http://www.jbc.org/

ized at the ER in non-encysting cellsas determined by its colocalizationwith BiP (Fig. 4A). In contrast, inencysting trophozoites, HA-gQc3showed a pattern of vesicles next tothe ESVs without colocalizing withthem.Therefore, most of the gQc-

SNAREs could be assigned to thePVs/PM compartment with theexception of gQc3 that seems tolocalize at the ER. Moreover, gQc4appears to localize to ESVs duringthe process of encystation.Giardia VAMPs—Immunofluo-

rescence assays of transfected tro-phozoites showed that HA-gR1localized at the ER in either non-en-cysting or encysting cells (Fig. 5).In addition, during encystation,HA-gR1 colocalized with ESVs (Fig.5). HA-gR2 was found mainly in aregion surrounding the nuclei of theparasite and in a diffuse intracellularcytoplasmic pattern, colocalizing insome regions with the ER in non-encysting trophozoites and encyst-ing trophozoites (Fig. 5A). However,in encysting trophozoites, HA-gR2colocalized partially with ESVs (Fig.5B). HA-gR3was found at the PM innon-encysting and encysting cells(Fig. 5).Therefore, the distribution of gR

SNAREs is similar to that of thegQa-SNAREs, with gR1 and gR2localized to the ER and surroundingboth nuclei of the parasite and gR3to the PM. Furthermore, gR1 andgR2 appear to localize to ESVs dur-ing encystation.

Expression of SNAREs in G. lamblia

To verify which of the genesencoding for the 17 gSNAREs weretranscribed, we isolated total RNAfromGiardia trophozoites and per-formed quantitative real-time PCRusing gene-specific primers. Asshown in Fig. 6, top, the expectedsize of cDNAs corresponding toeach gSNAREs was amplified,establishing the expression of all ofthese SNARE proteins in proliferat-ing G. lamblia trophozoites. By thisanalysis, we also demonstrated thatthe gQa5 (ORF: 10803), absent inthe latest Giardia DB release, giar-

FIGURE 3. Localization of gQb-SNAREs in non-encysting and encysting Giardia trophozoites. A, confocalmicroscopy of direct immunofluorescence assays of transfected non-encysting Giardia trophozoites shows thelocalization of HA-gQb1, HA-gQb2, and HA-Qb3 in small vesicles (green). HA-gQb4 colocalizes (yellow) with PVs (red),and HA-gQb5 colocalizes (yellow) with BiP at the ER and nuclear envelope (red). HA-tagged gQ-SNARE proteins arestained with FITC-conjugated anti-HA mAb (green), and the PVs and BiP are detected with Texas Red-labeled (red)mAbs 5B11 and 9C9, respectively. B, confocal microscopy of direct immunofluorescence assays showing the local-ization of HA-gQb-SNAREs (green) in encysting cells. The ESVs are labeled with mAb 7D2 specific for CWP2 (red).

FIGURE 4. Localization of gQc-SNAREs in non-encysting and encysting Giardia trophozoites. A, confocalmicroscopy of direct immunofluorescence assays of non-encysting transfected Giardia trophozoites shows thecolocalization (yellow) of HA-gQc1 and HA-gQc4 (green) with PVs (red) and HA-gQc2 (green) with PM (red). HA-gQc3colocalizes (Merge) with BiP at the ER (red) and localizes in small vesicles (green). HA-tagged gQc-SNARE proteins arestained with FITC-conjugated anti-HA mAb (green). The PVs and BiP are detected with Texas Red-labeled (red) mAbs5B11 and 9C9, respectively. B, confocal microscopy of direct immunofluorescence assays showing the localization ofHA-gQc-SNAREs (green) in encysting cells. The ESVs are labeled with mAb 7D2 (red).




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

dia14, is expressed in this parasite, suggesting not only theexistence of this protein in the genome but also that it isexpressed normally. As a positive control, the glutamate dehy-

drogenase (gdh) transcript wasamplified (58). When we comparedthe threshold cycle values (Ct) ofeach amplicon with gdh (Fig. 6, bot-tom), we observed that all of thetranscripts except gR3 have slightlylower expression levels than gdh.We also determined that theexpression levels of gQa2, gQa4,gQa5, and gQc1 were slightly lowerwhen compared with the otherSNARE genes.

Western Blot Assays of GiardiaPutative SNAREs Revealed SlowlyMigrating Bands That Might BeAttributed to SDS-resistantComplexes

SNARE heterocomplexes consistof a parallel four-helix bundle ofextraordinary stability derived fromone R-SNARE and three Q-SNAREmotifs (26). The four helices form acoiled-coil structure that in mostcases is SDS-resistant at tempera-tures of �80 °C but not at boilingtemperature (59). SDS-resistantSNARE complexes often exist invarious formswith different electro-phoretic motilities that give rise tomultiple anti-SNARE-immunore-active bands on Western blots (59).To verify whether SDS-resistantcomplexes exist inGiardia, proteinswere extracted in SDS-samplebuffer without boiling, separated bySDS-PAGE, blotted and immuno-stained with an anti-HAmAb.Withthis antibody, wewere able to detectone or more bands with molecularmasses between �60 to �200 kDa,consistent with complexes formedby the conserved coiled-coil domainof putative gSNAREs in both non-encysting and encysting trophozo-ites (Fig. 7). Fastermigrating speciesthat corresponded to the molecularweight of themonomeric formweredetected in boiled samples (supple-mental Fig. S8). It is known that highmolecular weight (HMW)bands arenot detected in samples that havebeen boiled prior to electrophoresis.However, inGiardia, when the sam-ples were boiled, we found some

peculiarities that had not been described in other eukaryotes(59). First, some of the putative gSNAREs (gQa1–4; gR1–3)presented almost the same bands obtained in unboiled samples,

FIGURE 5. Localization of gR-SNAREs (gVAMPs) in non-encysting and encysting Giardia trophozoites. A, con-focal microscopy of direct immunofluorescence assays of non-encysting transfected Giardia trophozoites shows thecolocalization (yellow) of HA-gR1 (green) with BiP at the ER (red). HA-gR2 (green) is localized in the cytoplasm andsurrounds both nuclei. HA-gR3 (green) colocalizes (yellow) with the surface protein VSP1267 at the plasma mem-brane (red). HA-tagged gR-SNARE proteins are stained with FITC-conjugated anti-HA mAb (green), and BiP andVSP1267 are detected with Texas Red-labeled mAbs 9C9 and 5C1 (red), respectively. B, confocal microscopy of directimmunofluorescence assays showing the localization of HA-gVAMPs (green) in encysting cells. The ESVs are labeledwith mAb 7D2 (red).

FIGURE 6. Expression analysis of gSNARE proteins by quantitative real-time PCR. After reverse transcription ofmRNA extracted from trophozoites cultured in growth medium, quantitative real-time PCR was performed for eachgSNARE in triplicate using the primers shown under “Experimental Procedures.” Top, the products obtained afterquantitative real-time PCR were loaded onto a gel to reveal the presence of one fragment. Bands of the expectedsizes (in bp) were amplified for: gQa1 (293); gQa2 (326); gQa3 (351); gQa4 (265); gQa5 (347); gQb1 (245); gQb2 (203);gQb3 (388); gQb4 (380); gQb5 (372); gQc1 (392); gQc2 (299); gQc3 (311); gQc4 (169); gR1 (192); gR2 (339); gR3 (413).These indicate the presence of RNA coding for all putative gSNAREs found in Giardia. The constitutively expressedgene gdh was used as a positive control. PCR reaction without input cDNA but with RNA instead served as a negativecontrol. Bottom, Ct profiles of gSNARE proteins. Data were shown as the mean � S.E. (vertical bar) of triplicateexperiments.




ww

.jbc.org/D

ownloaded from


http://www.jbc.org/

suggesting that the majority of HMW bands are not heat-sen-sitive (supplemental Fig. S8). Second, other putative gSNAREs,such as Qa5, Qb4, Qc1, Qc2, and Qc4, showed not only the

HMWbands but also a derivative of the lower band that repre-sents themonomeric form (supplemental Fig. S8). On the otherhand, gQb1–3 and gQc3 presented only the lower band, with

FIGURE 7. Evidence of SDS-resistant SNARE complexes of gSNAREs in non-encysting and encysting Giardia trophozoites. Western blots of HA-taggedgSNAREs are shown. Trophozoites transfected with gSNARE proteins were cultured in either growth (N) or encystation (E) medium for 24 h. Analysis of unboiledtotal proteins using anti-HA mAb shows the expression of the HA-tagged proteins and the formation of putative gSNARE complexes. A, gQa-SNARE proteins.B, gQb-SNARE proteins. C, gQc-SNARE proteins. D, gR-SNARE proteins. The expected sizes of the fusion proteins (in kDa) are as shown in Table 1.




ww

.jbc.org/D

ownloaded from



http://www.jbc.org/

more intensity, which corresponded to themolecular weight ofthe monomeric protein (supplemental Fig. S8). In highereukaryotes, it is known that a property described for nativeSNARE complexes and for complexes formed from recombi-nant SNARE proteins is the temperature dependence of theirSDS resistance (59). To test whether this was also true for SDS-resistant complexes in Giardia, we investigated the tempera-ture sensitivity of the higher bands found with some putativeSNAREs such gQc3, gQa5, and gQb4. When Giardia proteinswhere incubated in SDS-containing sample buffer for 5 min atdifferent temperatures, the amount of HMW bands decreasedwith increasing temperatures for gQc3 but not for gQa5 andgQb4 (supplemental Fig. S8). Moreover, some occasional addi-tional bands lower than the monomeric molecular weight ofeach gSNARE appeared, which likely were degradation prod-ucts. In most cases we were unable to observe behavior ofgSNAREs under SDS-PAGE similar to that reported in highereukaryotes, and we conjectured that it might be due to the par-ticular amino acids present at the zero layers of SNAREproteins.In addition, when we used the gQa1-specific mAb 3B5

instead of the anti-HA mAb, the monomeric form of �33 kDaas well as a �115-kDa band, which may represent an SDS-resistant complex, were found (supplemental Fig. S5). Thus, itis possible that the failure of the anti-HA antibody to detect the115-kDa highmolecularmass gQa1 complexes (andmost likelymore complexes formed by the other gSNAREs) is attributableto a hidden HA epitope. In any case, the structural basis forthese multiple forms of SNARE complexes remains unknown.Accordingly, the question of whether only one or several, all, ornone of the SDS-resistant complexes represent fusion-compe-tent SNARE structures remains unanswered.

Down-regulation of Some gSNAREs Affects Cell Viability

To determine the functionality of individual gSNAREs inregard to cell viability and their participation in the secretorypathway,Giardia cloneWB/1267 was transfected with vectors

carrying fragments of 150–400 bp of the antisense sequence ofeach gSNARE. Cells were selected with puromycin (33) andanalyzed by quantitative real-time PCR to determine the levelof silencing of the corresponding gSNARE gene (reduction ofRNA expression varied from 70 to 60%; see supplemental Fig.S9). No viable clones were obtained for gQa1, gQa2, gQa3,gQa4, gQb3, gR1, gR2, and gR3, suggesting that these SNAREsare essential. However, for gQa5, gQb1, gQb2, gQb4, gQb5, andgQc1–4 we obtained viable clones in which, when cultured ingrowth medium, neither cell viability nor proliferation wasaffected when compared with either untransfected trophozo-ites or trophozoites transfected with an HA-tagged senseSNAREs. After 48 h in encystation medium, trophozoitesexpressing antisense SNAREs formed cysts at normal levels(results not shown).

Golgi SNARE Homologs in Giardia Fail to Establish thePresence of a Typical Golgi Apparatus

As we have shown, gQa2, gQb5, gR1, and gR2, which donot localize to a Golgi-like structure, have roles in vesicletransport during the differentiation of Giardia. However,homologs to these SNAREs were predicted by sequencecomparisons to the Golgi and trans-Golgi network (TGN;see Table 2). HHPred analysis of gQa2 indicated thatgSNAREs are highly similar to the Syntaxin 16 subfamily (seesupplemental Table S4). Syntaxin 16 is a ubiquitous proteinin eukaryotic cells that localizes at the Golgi apparatus andTGN and is involved in protein transport in this organelle(60). Moreover, the closest subfamily for gQb5 is Vti1, whichcomprises SEC20, Vti1a, and Vti1b (see supplemental TableS4). Vti1A is localized predominantly to the Golgi and theTGN area in higher eukaryotes.Giardia SNARE-R1 was found at the ER/nuclear envelope in

non-encysting cells. Bioinformatics analysis predicted that thisprotein localizes at the Golgi/ER-Golgi and is homologous tothe Sec22 subfamily of SNAREs (see supplemental Table S4).

TABLE 2Summary of Giardia SNARE localizationSubcellular localization of Giardia SNAREs determined by IFA was compared with the predicted localization determined by FASTA, HMMPfam, and HHPRED. Todetermine the closest orthologous gene for each Giardia SNARE protein, FASTA searches were performed with standard parameters on the UniProtKB/Swiss-Prot database. The homology of the SNARE domain to the HMMs defined in Kloepper et al. (21) allowed us to predict the localization for each SNARE protein using HMMPfam.By using HHPRED and HHSENSER, the subcellular localization of the closest homologous KOG entry for eachGiardia SNARE was analyzed. Subcellular localization wasexperimentally determined by IFA using organelle markers for each compartment. Presumed intracellular sites are as follows: NE (nuclear envelope), ER, GA (Golgiapparatus), TGN, PVs, VC (vacuolar compartment), ESVs, PM (plasma membrane).

gSNARE Accession GL50803_ FASTA HMM HHPRED IFA localization innon-encysting cells

IFA localization inencysting cells

gQa1 7,309 EC PM PM PVs PVs/ESVsgQa2 3,869 TGN TGN GA/EC NE/ER NE/ER/ESVsgQa3 96,994 PM PM PM NE/small vesicles/PVs/PM small vesicles/PVs/PMgQa4 11,220 ER ER ER NE/ER/vesicles NE/ER/ESVsgQa5 10,803 VC PM EC PVs/PM PVs/PM/ESVsgQb1 16,054 EC TGN ER/EC Vesicles VesiclesgQb2 17,464 GA GA GA Vesicles VesiclesgQb3 5,161 ER ER ER/EC Mitosomes MitosomesgQb4 5,785 No hit EC ER/EC PVs/PM PVs/PMgQb5 10,315 EC TGN ER/EC NE/ER NE/ER/ESVsgQc1 5,927 PM EC EC PVs/PM PVs/PMgQc2 7,590 VC ER EC PVs/PM PVs/PMgQc3 10,013 PM EC PM ER/vesicles VesiclesgQc4 19,509 GA/ER-GA EC EC PVs/PM PVs/PM/ESVsgR1 9,489 ER GA ER NE/ER NE/ER/ESVsgR2 7,306 GA/ER-GA GA GA/ER-GA NE/ER cytoplasm NE/ER/cytoplasm ESVsgR3 14,469 TGN/EC EC EC PM PM




ww

.jbc.org/D

ownloaded from








http://www.jbc.org/

Sec22b functions in both anterograde (61) and retrograde (62)trafficking between the ER and the Golgi.The expression of Giardia SNARE-R2 was highly concen-

trated, surrounding both nuclei with a diffuse intracellularcytoplasmic pattern, colocalizing in some areas with the ER,and consistent with ER localization in non-encysting trophozo-ites. Giardia SNARE-R2 was predicted to localize to the Golgiand is highly similar to Ykt6 (see supplemental Table S3). Ykt6is localized to both the cytoplasm andmembranes. In mamma-lian cells, the majority of the membrane-bound pool of Ykt6 isassociated with the Golgi; however a broader perinuclear dis-tribution also has been observed (63). Consistentwith its exten-sive cellular distribution, Ykt6 has been implicated in multipletransport steps in the secretory pathway, including ER-to-Golgitransport as part of a SNARE complex composed of GosR1,GosR2, and Stx5 and in early/recycling endosome-to-TGNtransport as a part of a SNARE complex composed of Bet1L,GosR1, and Stx5 (63, 64). The localization of gR2 at the ER andthe role of its close homologs suggest that it may be involved inER homotypic and heterotypic fusion events. Furthermore,other Golgi SNARE homologs such as gQb1 and gQb2 showeda pattern of vesicles scattered throughout the cytoplasm; thesevesicles did not colocalize with the ER, ESV, or PVs.Giardia Qb1 was predicted to localize to TGN/endosomes;

its closest homolog is Vti1 from S. cerevisiae, which is known tobe involved in endosomal or vacuolar trafficking steps (see sup-plemental Table S3). Vti1p functionally interacts with Pep12pin trafficking from the Golgi to the pre-vacuolar compartmentand with Sed5p in retrograde trafficking to the cis-Golgi (65).GiardiaQb2was predicted to localize to theGolgi. Its closest

homolog is theGolgi SNARE 28 subfamily (Gos28p), of which amember is Gos1p of S. cerevisiae (see supplemental Table S3),which directly interacts with Sed5p, is localized primarily to theGolgi complex, and likely functions in ER-Golgi and/or intra-Golgi transport (66). In addition, the closest homolog to Giar-dia gQa3 is the yeast Sed5p, a molecule that participates in thetransport between the ER and the Golgi complex and also isfound in vesicular structures throughout the cytoplasm (67),suggesting that gQa3 could be involved in the same traffickingpathway of gQb1 and gQb2.

DISCUSSION

The protein trafficking pathway in eukaryotes takes a com-mon route from the ER through the Golgi apparatus to theTGN, where exit and sorting take place (68). Giardia tropho-zoites possess an endomembranous organization that includestwo nuclei, ER, PVs, constitutive transport vesicles (5), and theso-calledmitosomes (57).Giardia lacks other organelles typicalof higher eukaryotes such as classical mitochondria, peroxi-somes, and compartments highly involved in intracellular pro-tein trafficking and secretion, such as secretory granules andthe localized stack of cisternae that is the hallmark of the Golgiapparatus (2). In an early report, we showed that when the pro-teins that will form the cyst wall (CWPs) concentrate within denovo generated-specific secretory granules (or ESVs), a perinu-clear structure labeled with the Golgi-specific marker NBD-ceramide could be observed (7). However, in a later study wedemonstrated that ESVs develop directly from the ER (55), rais-

ing doubts about the nature of that NBD-ceramide-labeledstructure. Because secretory granules form at the TGN inhigher eukaryotes, it is still an open question as to whether aGolgi complex is present in this ancient eukaryote.In the present work, we first identified and characterized the

complete set of SNAREs in Giardia to define the extent of theendomembranous system, including the presence or not of atypical Golgi. From the 17 gSNAREs proteins that we charac-terized, five represent Qa-SNAREs, five Qb-SNAREs, four Qc-SNAREs, and three R-SNAREs. Compared with other unicellu-lar eukaryotes such as P. falciparum (18 SNAREs), L. major (27SNAREs), and Entamoeba histolytica (31 SNAREs) and orga-nisms suchDrosophilamelanogaster (20 SNAREs), S. cerevisiae(24 SNAREs), Homo sapiens (36 SNAREs), and A. thaliana (54SNAREs), Giardia has a relatively small number of encodedSNAREs. This is consistent with the very elementary repertoireof membrane-bound organelles that can be identified in Giar-dia (Fig. 8). Our bioinformatic analysis complements thatrecently reported by Kloepper et al. (21), as we were able toidentify two additional SNAREs and demonstrate that the pre-dicted subcellular localization of many of them conflicts withour experimental results (see below). Additionally, we haveshown here that one particular gSNARE is expressed and func-tional inGiardia, although it is not present in the latest versionof the Giardia genome data base.

We analyzed the expression patterns and subcellular local-ization of the entire SNARE repertoire of Giardia. Our resultsindicate that these proteins are functional and distinctivelylocalized to known subcellular compartments, suggesting a rolein regulating membrane trafficking to and from theseorganelles. Interestingly, by antisense knockdown assays wefound thatmost of the conservedGiardia SNAREs seem essen-tial, whereas others are dispensable, particularly those that dohave atypical amino acids in the zero layer. This suggests thatsome SNAREs have essential functions, whereas others mighthave redundant roles that can be compensated for by otherSNARE isoforms (69). In addition, because many of thegSNAREs are essential for the viability of the parasite, theymaypresent useful targets for the development of drugs against thisimportant human pathogen.Remarkably, gQb3 localizes to vesicles with a peculiar pat-

tern, similar to the distribution attributed previously to mito-somes (see Fig. 2 in Dolezal et al. (57)). During encystation, thenumber of these gQb3-containing vesicles decreased and thepattern changed. Mitosomes are small, double membrane-bound mitochondrial remnants that retain some functions inthe synthesis of iron-sulfur clusters (70). The possible presenceof one SNARE in a mitochondrial remnant is intriguing, as noSNARE molecules have been reported to mediate mitochon-drial fusion in more evolved cells except for a splice isoform ofVAMP1 that contains a mitochondrial targeting signal (71).Based on our bioinformatic and biological results, we reason

that gQa2, gQa3, gQa4, gQb5, gQc3, gR1, and gR2 are likely toplay roles in homotypic fusion events in the ER and in transportthrough the ER/nuclear envelope compartment (Fig. 8). Wefound that gQa2, gQa4, gQb5, and gR1 localized to the nuclearenvelopes and to the ER. Because the ER and nuclear envelopeare a continuous structure, most of these SNAREs appear to be




ww

.jbc.org/D

ownloaded from




http://www.jbc.org/

localized in both. During encystation, however, some of theseSNAREs colocalizedwith the ESVs in some particular spots of thenuclear envelope and ER. We suggested previously that a regionsurrounding both nuclei may be a compartment that represents aspecialized ER cisterna where the biogenesis of the ESVs takesplace (7). It is possible, therefore, that gQa2, gQa4, gQb5, and gR1have a specific role in ESV biogenesis during differentiation ofGiardia trophozoites, further supporting the idea that these spe-cific secretory granules form from the ER in this protozoan.Giardia possesses PVs located underneath the plasmamem-

brane that function as endosomes and lysosomes and are there-fore considered a primitive endosomal/lysosomal complex (8)(Fig. 8). The participation of PVs also has been described asinfluencing secretory granule discharge during cyst wall forma-tion (13), and PVs act as secretory organelles that release cystwall-disrupting enzymes during excystation (72). We wereunable to demarcate SNARE localization between PVs and theplasma membrane by immunofluorescence assays usingorganelle markers, probably because of the heavy trafficbetween them during exocytosis, endocytosis, and membrane

recycling. We found eight gSNAREs present in PVs/PM, threeQa-SNAREs, three Qc -SNAREs, one Qb-SNARE, and oneR-SNARE. Giardia SNAREs Qa1, gQa3, and gQa5 are pre-dicted to be located in the PM or endosomal compartments.The presence of three different gQa-SNAREs suggests that atleast three distinct SNARE complexes are present in this regionof the endomembrane system, perhaps one for exocytosis, onefor endocytosis, and one for PV-PV fusion. The closesthomologs for gQa1, gQa3, and gQa5 from other organisms areSyntaxin 12, Syntaxin 1A, and Pep12, respectively. Syntaxin 12and Pep12 function in endosomal transport, whereas Syntaxin1A is required for exocytosis. Based on these homologies, wesuggest that gQa3 is essential for Giardia exocytosis, whereasgQa1 and gQa5 are important for endosomal trafficking.Among the R-SNAREs, gR2 is most closely related in its

SNARE domain to Ykt6. Ykt6 is unique in associating withmembranes via a C-terminal isoprenylation and palmitoylationrather than through a transmembrane domain anchor. Strik-ingly, gR2 also contains a consensus sequence for isoprenylmodification at its C terminus as well as a second cysteine

FIGURE 8. Schematic summary of spatial and functional distribution of organelles determined by SNARE proteins in non-encysting and encystingGiardia trophozoites. Subcellular compartments are represented as follows: the nuclei (N), the nuclear envelope/endoplasmic reticulum compartment(NE/ER), the ER, punctate vesicles (V), mitosomes (M), ESVs, PVs, and plasma membrane (PM). A, In non-encysting trophozoites, the ER extends throughout thecell body and is continuous with the NE; punctate vesicles are distributed throughout the cell in an ER pattern; mitosomes are located between both Giardianuclei and in the lateral and posterior part of the cell; PVs localize at the cell cortex. B, In encysting trophozoites, the synthesis of Cyst Wall Proteins (CWPs) takesplace, which then are transported in ESVs that are part of the regulated transport at this stage of the parasite life cycle; during cyst wall formation, ESVs interactwith PVs; the pattern of mitosomes changed as a result of fusion of vesicles from the apical and posterior part of the cell. These are the minimal compartmentsfound in this primitive eukaryote in non-encysting A and encysting B trophozoites. Each Giardia SNARE is represented by one color and localizes to differentcompartments, some of them changing their localization during encystation. The trafficking pathways are indicated by arrows as follows: Green, anterogradepathway. Black, retrograde pathway. Pink, transport to the PVs. Orange, plasma membrane-endosomal recycling pathway.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

nearby that could be palmitoylated. Mutation of both of theputative modified cysteines causes a redistribution of the pro-tein from the membrane to the cytoplasm (supplemental Fig.S10). The conservation of lipidation of this SNARE suggeststhat a reversible membrane anchor may be important for theshuttling of the protein between different compartments.gQb1, gQb2, and gQa3, which are most closely related to

known Golgi SNAREs, were found in a punctate pattern thatdid not localize to any previously identified Giardia organelle,suggesting that these vesicles could be vestigial or remnantGolgi membranes. Organelles that resemble this pattern (e.g.punctate appearance scattered throughout the cytoplasm) arethe “atypical” Golgi apparatus of the fungi Saccharomyces,Arp-ergillus, and Pisolithus (73, 74). Nevertheless, there are cleardifferences between Giardia and those organisms regardingglycosylation (one of the classical known functions of the Golgiapparatus). It was recently shown that, except for the first twosteps that take place in the ER, complexN-glycosylation ismiss-ing inGiardia (75).Whenwe performed studies evaluating gly-cosyltransferase activities during encystation (the process dur-ing which a carbohydrate-rich cyst wall is generated), weutilized a Giardia protein extract in the reaction. The enzy-matic activities we measured at that time (7) may well corre-spond to the recently identified cyst wall synthase, a develop-mentally regulated enzyme that produces a cyst wallcarbohydrate polymer (76). Therefore, these activities, whichwe suggested were present in Golgi-like compartment, appar-ently were unrelated to typical Golgi glycosyltransferases.Unfortunately, the lack of specific Golgi markers in Giardiaprevented us from further characterization of these compart-ments. Additional evidence, however, plays against the possi-bility that vesicles containing gQb1, gQb2, or gQa3 are Golgiequivalents. For example, (a) whenwe expressed anHA-taggedversion of a heterologous glycosyltransferase or p115 in tropho-zoites, they localized in the perinuclear region of Giardia andnot in a punctate pattern dispersed throughout the cytoplasm(55); (b) a similar perinuclear pattern was observed by consti-tutive expression of the Giardia KDEL receptor (55); and (c)brefeldin A treatment of trophozoites expressing gQb1, gQb2,or gQa3 failed to disrupt these vesicular structures, whereas thesecretory pathway was certainly affected, as determined byrelocalization of gARF3 (the only gARF showing similar local-ization pattern) (not shown). Although those experiments donot provide a definitive answer whether the vesicles containingSNAREsQb1 andQb2 areGolgi structures, our results stronglysuggest that if Giardia has a Golgi, it is completely atypical.In summary, we have added new evidence regarding the lack of

a typicalGolgi apparatus in this ancient eukaryote.However, somefeatures of the Golgi, such us the packaging and sorting function,seem to be performed by the ER or the nuclear envelope, raisingthe question of whether the typical architecture of the Golgi inhigher eukaryotes is a cause or consequence of the presence ofglycosyltransferases needed for the biosynthesis of complex glyco-conjugates, molecules that are missing inGiardia (2, 75).

Acknowledgment—We thank T. H. Kloepper for providing the HHMdata base.

REFERENCES1. Morrison, H. G., McArthur, A. G., Gillin, F. D., Aley, S. B., Adam, R. D.,

Olsen, G. J., Best, A. A., Cande, W. Z., Chen, F., Cipriano, M. J., Davids,B. J., Dawson, S. C., Elmendorf, H. G., Hehl, A. B., Holder, M. E., Huse,S. M., Kim, U. U., Lasek-Nesselquist, E., Manning, G., Nigam, A., Nixon,J. E., Palm, D., Passamaneck, N. E., Prabhu, A., Reich, C. I., Reiner, D. S.,Samuelson, J., Svard, S. G., and Sogin,M. L. (2007) Science 317, 1921–1926

2. Adam, R. D. (2001) Clin. Microbiol. Rev. 14, 447–4753. Tamaki, H., and Yamashina, S. (2002) J. Histochem. Cytochem. 50,

1611–16234. Touz,M. C., Lujan, H. D., Hayes, S. F., andNash, T. E. (2003) J. Biol. Chem.

278, 6420–64265. Hehl, A. B., and Marti, M. (2004)Mol. Microbiol. 53, 19–286. Nash, T. E., and Mowatt, M. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,

5489–54937. Lujan, H. D., Marotta, A., Mowatt, M. R., Sciaky, N., Lippincott-Schwartz,

J., and Nash, T. E. (1995) J. Biol. Chem. 270, 4612–46188. Lanfredi-Rangel, A., Attias, M., de Carvalho, T. M., Kattenbach, W. M.,

and De Souza, W. (1998) J. Struct. Biol. 123, 225–2359. Lujan, H. D., Mowatt, M. R., and Nash, T. E. (1997) Microbiol. Mol. Biol.

Rev. 61, 294–30410. Lujan, H. D., Mowatt, M. R., and Nash, T. E. (1998) Parasitol. Today 14,

446–45011. Mowatt, M. R., Lujan, H. D., Cotten, D. B., Bowers, B., Yee, J., Nash, T. E.,

and Stibbs, H. H. (1995)Mol. Microbiol. 15, 955–96312. Lujan, H. D., Mowatt, M. R., Conrad, J. T., Bowers, B., and Nash, T. E.

(1995) J. Biol. Chem. 270, 29307–2931313. Jarroll, E. L., Macechko, P. T., Steimle, P. A., Bulik, D., Karr, C. D., van

Keulen, H., Paget, T. A., Gerwig, G., Kamerling, J., Vliegenthart, J., andErlandsen, S. (2001) J. Eukaryot. Microbiol. 48, 22–26

14. Lujan, H. D.,Mowatt, M. R., Conrad, J. T., andNash, T. E. (1996) Biol. Cell86, 11–18

15. Knodler, L. A., Noiva, R., Mehta, K., McCaffery, J. M., Aley, S. B., Svard,S. G., Nystul, T. G., Reiner, D. S., Silberman, J. D., and Gillin, F. D. (1999)J. Biol. Chem. 274, 29805–29811

16. Sun, C. H., McCaffery, J. M., Reiner, D. S., and Gillin, F. D. (2003) J. Biol.Chem. 278, 21701–21708

17. Touz, M. C., Gottig, N., Nash, T. E., and Lujan, H. D. (2002) J. Biol. Chem.277, 50557–50563

18. Weiland, M. E., Palm, J. E., Griffiths, W. J., McCaffery, J. M., and Svard,S. G. (2003) Int. J. Parasitol. 33, 1341–1351

19. Marti, M., Regos, A., Li, Y., Schraner, E. M., Wild, P., Muller, N., Knopf,L. G., and Hehl, A. B. (2003) J. Biol. Chem. 278, 24837–24848

20. Touz, M. C., Kulakova, L., and Nash, T. E. (2004) Mol. Biol. Cell 15,3053–3060

21. Kloepper, T. H., Kienle, C. N., and Fasshauer, D. (2007)Mol. Biol. Cell 18,3463–3471

22. Regoes, A., and Hehl, A. B. (2005) BioTechniques 39, 809–810, 81223. Ungar, D., and Hughson, F. M. (2003) Annu. Rev. Cell Dev. Biol. 19,

493–51724. Ferro-Novick, S., and Jahn, R. (1994) Nature 370, 191–19325. Weimbs, T., Mostov, K., Low, S. H., and Hofmann, K. (1998) Trends Cell

Biol. 8, 260–26226. Sutton, R. B., Fasshauer,D., Jahn, R., andBrunger, A. T. (1998)Nature395,

347–35327. Fasshauer, D., Eliason,W. K., Brunger, A. T., and Jahn, R. (1998) Biochem-

istry 37, 10354–1036228. Jahn, R., and Scheller, R. H. (2006) Nat Rev Mol. Cell. Biol. 7, 631–64329. Rothman, J. E., and Warren, G. (1994) Curr. Biol. 4, 220–23330. Bock, J. B., Matern, H. T., Peden, A. A., and Scheller, R. H. (2001) Nature

409, 839–84131. Filippini, F., Rossi, V., Galli, T., Budillon, A., D’Urso, M., and D’Esposito,

M. (2001) Trends Biochem. Sci. 26, 407–40932. Touz, M. C., Nores, M. J., Slavin, I., Carmona, C., Conrad, J. T., Mowatt,

M. R., Nash, T. E., Coronel, C. E., and Lujan, H. D. (2002) J. Biol. Chem.277, 8474–8481

33. Elmendorf, H. G., Singer, S.M., Pierce, J., Cowan, J., andNash, T. E. (2001)




ww

.jbc.org/D

ownloaded from


http://www.jbc.org/

Mol Biochem. Parasitol. 113, 157–16934. Nash, T. E., Aggarwal, A., Adam, R. D., Conrad, J. T., andMerritt, J. W., Jr.

(1988) J. Immunol. 141, 636–64135. Chaturvedi, S., Qi, H., Coleman, D., Rodriguez, A., Hanson, P. I., Striepen,

B., Roos, D. S., and Joiner, K. A. (1999) J. Biol. Chem. 274, 2424–243136. Boucher, S. E., and Gillin, F. D. (1990) Infect. Immun. 58, 3516–352237. Yee, J., and Nash, T. E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5615–561938. Eddy, S. R. (1998) Bioinformatics (Oxf.) 14, 355–36339. Letunic, I., Copley, R., and Pils, B., Pinkert, S., Schultz, J., and Bork, P.

(2006) Nucleic Acids Res. 34, D257–D26040. Soding J., Biegert, A., and Lupas, A. N. (2005) Nucleic Acids Res. 33,

W244–W24841. Soding, J., Remmert, M., and Biegert, A. (2006) Nucleic Acids Res. 34,

W137–W14242. Kall, L., Krogh, A., and Sonnhammer, E. L. (2005) Bioinformatics 21,

Suppl. 1, i251–i25743. Edgar, R. C. (2004) BMC Bioinformatics 5, 11344. Tatusov, R. L., Fedorova, N. D., Jackson, J. D., Jacobs, A. R., Kiryutin, B.,

Koonin, E. V., Krylov, D. M., Mazumder, R., Mekhedov, S. L., Nikolskaya,A.N., Rao, B. S., Smirnov, S., Sverdlov, A.V., Vasudevan, S.,Wolf, Y. I., Yin,J. J., and Natale, D. A. (2003) BMC Bioinformatics 4, 41

45. Lupas, A. (1996) Trends Biochem. Sci 21, 375–38246. Zwilling, D., Cypionka, A., Pohl, W. H., Fasshauer, D., Walla, P. J., Wahl,

M. C., and Jahn, J. (2007) EMBO J. 26, 9–1847. McGuffin, L. J., Bryson, K., and Jones, D. T. (2000) Bioinformatics 16,

404–40548. Fernandez, I., Ubach, J., Dulubova, I., Zhang, X., Sudhof, T. C., and Rizo, J.

(1998) Cell 94, 841–84949. Dulubova, I., Sugita, S., Hill, S., Hosaka, M., Fernandez, I., Sudhof, T. C.,

and Rizo, J. (1999) EMBO J. 18, 4372–438250. Besteiro, S., Coombs, G. H., andMottram, J. C. (2006)BMCGenomics 7, 25051. Ayong, L., Pagnotti, G., Tobon, A. B., and Chakrabarti, D. (2007) Mol.

Biochem. Parasitol. 152, 113–12252. Dacks, J. B., and Doolittle, W. F. (2002) J. Cell Sci. 115, 1635–164253. Dacks, J. B., and Doolittle, W. F. (2004) Mol. Biochem. Parasitol. 136,

123–13654. Hay, J. C., Hirling, H., and Scheller, R. H. (1996) J. Biol. Chem. 271,

5671–567955. Gottig, N., Elias, E. V., Quiroga, R., Nores, M. J., Solari, A. J., Touz, M. C.,

and Lujan, H. D. (2006) J. Biol. Chem. 281, 18156–18166

56. Mowatt, M. R., Nguyen, B. Y., Conrad, J. T., Adam, R. D., and Nash, T. E.(1994) Infect. Immun. 62, 1213–1218

57. Dolezal, P., Smid, O., Rada, P., Zubacova, Z., Bursac, D., Sutak, R., Nebe-sarova, J., Lithgow, T., and Tachezy, J. (2005) Proc. Natl. Acad. Sci. U. S. A.102, 10924–10929

58. Yee, J., and Dennis, P. P. (1992) J. Biol. Chem. 267, 7539–754459. Kubista, H., Edelbauer, H., and Boehm, S. (2004) J. Cell Sci. 117, 955–96660. Teng, F. Y., Wang, Y., and Tang, B. L. (2001) Genome Biol. 2,

REVIEWS301261. Liu, Y., and Barlowe, C. (2002)Mol. Biol. Cell 13, 3314–332462. Burri, L., Varlamov, O., Doege, C. A., Hofmann, K., Beilharz, T., Rothman,

J. E., Sollner, T. H., and Lithgow, T. (2003) Proc. Natl. Acad. Sci. U. S. A.100, 9873–9877

63. McNew, J. A., Sogaard,M., Lampen,N.M.,Machida, S., Ye, R. R., Lacomis,L., Tempst, P., Rothman, J. E., and Sollner, T. H. (1997) J. Biol. Chem. 272,17776–17783

64. Zhang, T., and Hong, W. (2001) J. Biol. Chem. 276, 27480–2748765. Murray, R. Z., Wylie, F. G., Khromykh, T., Hume, D. A., and Stow, J. L.

(2005) J. Biol. Chem. 280, 10478–1048366. McNew, J. A., Coe, J. G., Sogaard, M., Zemelman, B. V., Wimmer, C.,

Hong, W., and Sollner, T. H. (1998) FEBS Lett. 435, 89–9567. Hardwick, K. G., and Pelham, H. R. (1992) J. Cell Biol. 119, 513–52168. Griffiths, G., and Simons, K. (1986) Science 234, 438–44369. Kissmehl, R., Schilde, C., Wassmer, T., Danzer, C., Nuehse, K., Lutter, K.,

and Plattner, H. (2007) Traffic 8, 523–54270. Tovar, J., Leon-Avila, G., Sanchez, L. B., Sutak, R., Tachezy, J., van der

Giezen, M., Hernandez, M., Muller, M., and Lucocq, J. M. (2003) Nature426, 172–176

71. Isenmann, S., Khew-Goodall, Y., Gamble, J., Vadas, M., and Wattenberg,B. W. (1998)Mol. Biol. Cell 9, 1649–1660

72. Reiner, D. S., McCaffery, M., and Gillin, F. D. (1990) Eur. J. Cell Biol. 53,142–153

73. Kuratsu, M., Taura, A., Shoji, J. Y., Kikuchi, S., Arioka, M., and Kitamoto,K. (2007) Fungal Genet. Biol. 44, 1310–1323

74. Cole, L., Davies, D., Hyde, G. J., and Ashford, A. E. (2000) Fungal Genet.Biol. 29, 95–106

75. Samuelson, J., Banerjee, S., Magnelli, P., Cui, J., Kelleher, D. J., Gilmore, R.,and Robbins, P. W. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 1548–1553

76. Karr, C. D., and Jarroll, E. L. (2004)Microbiology 150, 1237–1243




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Aaron Neiman and Hugo D. LujanEliana V. Elias, Rodrigo Quiroga, Natalia Gottig, Hideki Nakanishi, Theodore E. Nash,

Giardia lambliaApparatus in the Ancient Eukaryote Characterization of SNAREs Determines the Absence of a Typical Golgi

doi: 10.1074/jbc.M806545200 originally published online October 16, 20082008, 283:35996-36010.J. Biol. Chem.

10.1074/jbc.M806545200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2008/11/07/M806545200.DC2

Supplemental material:


http://www.jbc.org/content/283/51/35996.full.html#ref-list-1

This article cites 75 references, 35 of which can be accessed free at by guest on February 18, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M806545200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;283/51/35996&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/283/51/35996

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=283/51/35996&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/283/51/35996

http://www.jbc.org/cgi/alerts/etoc



http://www.jbc.org/content/283/51/35996.full.html#ref-list-1

http://www.jbc.org/

characterization of snares determines the absence of a typical

Documents