From the
Institute of Parasitology, University of Zürich, CH-8057 Zürich, Switzerland, the Electron Microscopy Unit, Institutes of Veterinary Anatomy and Virology, University of Zürich, CH-8057 Zürich, Switzerland, and the ¶Institute of Parasitology, University of Bern, CH-3012 Bern, Switzerland
Received for publication, February 27, 2003 , and in revised form, April 23, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asexually dividing, motile trophozoite forms of Giardia colonize the upper intestine of vertebrate hosts, and are shed as environmentally resistant, infectious cysts. The intracellular organization of the binucleate trophozoite is unusually simple, also in comparison with other phylogenetically basal groups such as the trichomonads (e.g. Trichomonas vaginalis (9)) and the kinetoplastids (e.g. Trypanosoma cruzi (10)). A compartment with functional and structural characteristics of the endoplasmic reticulum (ER)1 has been identified and characterized as an endomembraneous network, which extends throughout the cell body. Immunoelectron microscopy studies demonstrated the localization of giardial BiP (11), three protein-disulfide isomerase paralogues (12), as well as trophozoite and cyst surface antigens in the Giardia ER (13). Other morphologically recognizable membrane compartments are the peripheral vesicles (PVs, approximately 150200 nm in size), which are thought to perform both lysosomal and endosomal activities (14). PVs underlie the plasma membrane of the cell body except at the flagella or where it covers the cytoskeleton structures of the ventral disk (14). A third intracellular compartment, encystation-specific vesicles (ESVs) (15), arises only during the highly orchestrated process of encystation. Formation and maturation of the large ESVs (up to 1 µm diameter) is functionally linked to the regulated expression of exported cargo (i.e. cyst wall proteins), and there is increasing evidence that ESVs may be the cisternae of a unique Golgi equivalent in Giardia (1618). We recently demonstrated that sorting of exported proteins to the regulated secretory pathway via ESVs or a second constitutively active export pathway to the plasma membrane occurred at or immediately after ER exit (18). This also argued for the absence of a conventional Golgi apparatus in all developmental stages. Thus, current data suggests the presence of a primordial secretory system in Giardia, but very little is known about its molecular characteristics. Here we report a broad approach to analyze the functional anatomy of this unusual and dynamic membrane system and to investigate its putatively ancestral nature. Specifically, we addressed the question whether the cellular machinery for the generation of the Golgi-like ESV compartments indeed arises de novo during encystation, or whether a functional, or even a morphologically identifiable Golgi equivalent is present in trophozoites that could be used as a template on which encysting cells build ESVs. We established a molecular framework for secretory transport in this ancient eukaryote using a comparative genomics approach and provide evidence for the presence of membrane compartments with Golgi characteristics in trophozoites as well. Despite significant morphological changes, there is little evidence for regulation on a molecular level, indicating that generation of ESVs as a stabilized Golgi equivalent in Giardia is achieved in large part though re-organization of constitutive elements of this secretory system.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of Giardia Genes and Sequence ConfirmationEach putative Giardia orthologue was identified individually using the online tBLASTN2 tool to search Giardia HTGS sequences (20). The genome coverage as of April 2002 was 7.3-fold with an estimated 98.3% of the sequence determined.3 Contigs of assembled single-pass reads were manually scanned for the AT-rich region immediately preceding a translation start codon and the consensus Giardia polyadenylation signal AGTPurAAPyr at, or shortly after the stop codon. Identities of putative ORFs were confirmed using the BLASTP tool, and e-values and GenBankTM accession numbers of best hits were determined (see also Table I). Conserved domains were used to further confirm tentative identifications with the Pfam tool4 using default parameters. Predictions of transmembrane domains and prenylation motifs were performed using PSORTII.5
|
Semi-quantitative RT-PCRTotal RNA from 5 x 106 trophozoites or encysting cells (3, 7, 15, and 24 h after induction of encystation) was prepared using the Stratagene total RNA kit (Stratagene, La Jolla, CA) following digestion with 30 units of DNase for 15 min at 37 °C. For first strand cDNA synthesis, 3.5 µg of total RNA were reverse-transcribed with 100 ng of primer k-anchor and 50 units of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) at 45 °C in 25 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, and 500 µM dNTP (Clontech, Palo Alto, CA). The reaction was incubated at 45 °C for 50 min and heat inactivated at 70 °C followed by digestion with 10 units of RNase H for 20 min at 37 °C. Single-stranded cDNA was purified with the Concert DNA purification spin cartridge system (Invitrogen). Single-stranded cDNA corresponding to 70 ng of total RNA was used as template for semi-quantitative PCR analysis with 8 pmol each of primer k-adaptor and a gene-specific primer. A list of all primers used for RT-PCR and semi-quantitative PCR is available as Supplementary Material. Primer sequences were chosen to amplify fragments between 100 and 500 bp, containing the end of the gene ORF and the complete 3'-untranslated region. Reaction conditions were 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 200 µM each dNTP, and 1 unit of recombinant Taq polymerase (Sigma). Thermal cycling conditions were as follows: "hot start cycle," 94 °C for 5 min, 80 °C for 2 min (addition of Taq polymerase), 64 °C for 30 s, 72 °C for 30 s, followed by 2232 cycles (depending on the copy number of a specific cDNA) at 94 °C for 30 s, 64 °C for 30 s, and 72 °C for 30 s. PCR products were separated on a 1.5% agarose gel containing 0.01% SYBR Green I (Molecular Probes, Eugene, OR). Data collection was performed on a fluorimager (Alpha Innotech Corp., San Leandro, CA) using ChemiImager 5500 software. For all the cDNA products, the log-linear range was between 20 and 30 PCR cycles. Negative controls included: 1) omission of cDNA template in the PCR reaction and 2) omission of enzyme in the RT reaction.
Expression and Purification of Bacterial Fusion ProteinsThe nucleotide sequences encoding amino acids 10170 of Gi'COP, 8189 of GiSar1p, 430705 of GiCLH, 25176 of GiRab11, 97270 of GiSyn1, as well as 8201 and 344714 of GiDLP and 3111 of GiYip were PCR amplified from genomic DNA and subcloned into the polylinker region of the pMal-2Cx expression vector (New England Biolabs), downstream of the maltose-binding protein (MBP) gene, giving rise to fusion genes MBP-Gi
'COP, MBP-GiSar1p, MBP-GiCLH, MBP-GiRab11, MBP-GiSyn1, MBP-GiDLPn, and MBP-GiDLPc (corresponding to N- and C-terminal fragments of GiDLP, respectively), and MBP-GiYip. Bacterial overexpression of fusion proteins was induced by adding 0.5 mM isopropyl-
-D-thiogalactopyranoside for2hat37 °C, and fusion proteins were affinity purified from bacterial cold shock lysates on amylose resin according to manufacturers protocols and lyophilized as previously described (21). Primers used for the amplification of marker gene fragments are available as Supplementary Material.
Peptide SynthesisSynthesis of a polypeptide for antibody production was performed by Eurogentec (Seraing, Belgium). A peptide corresponding to Glu-613 through Lys-630 of the GiDLP protein, NH2C-ESVPEKIKAQGPLSEAEK-COOH, was synthesized and coupled to keyhole limpet hemocyanin.
Production of Polyclonal AntibodiesBALB/c mice were immunized intraperitoneally on days 0, 15, and 30 with 50 µg of fusion protein or 10 µg of keyhole limpet hemocyanin-coupled peptide resuspended in 100 µl of phosphate-buffered saline (PBS) and emulsified with an equal volume of RIBI adjuvant (Corixa, Hamilton MT). Blood was collected prior to initial immunization and after each boost from the tail vein, the serum fraction was assayed for specific antibody content.
SDS-PAGE and ImmunoblottingCells were harvested as described above, cell pellets were washed once in ice-cold PBS and counted. SDS sample buffer was added to obtain a uniform concentration of 5 x 105 cells per sample, and samples were immediately boiled for 3 min. 10% Polyacrylamide gels were run under reducing conditions with 7.75 mg/ml dithiothreitol in samples, and proteins were transferred to a nitrocellulose membrane according to standard methods. Antisera were diluted as specified below in PBS, 0.05% Tween 20, and 5% nonfat milk powder. CWP2 was detected with mouse mAb 7D2 (22) and diluted 1:20,000. Mouse pAbs raised against Giardia marker proteins GiSar1p, Gi'COP, GiCLH, GiSyn1, and GiYip1p were diluted 1:1000, and to GiCLH and GiDLP 1:5000. Primary antibodies were detected with a peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit antibody (both Sigma), respectively, and visualized using the ECL system (PerkinElmer Life Sciences).
Immunofluorescence MicroscopyAll manipulations were carried out at 4 °C. Trophozoites and encysting cells were harvested as described above, washed twice in ice-cold PBS, and fixed with 3% paraformaldehyde for 30 min at room temperature, followed by a 5-min incubation with 100 mM glycine in PBS. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min and blocked >1 h in 2% bovine serum albumin in PBS. Fixed and permeabilized cells were incubated with primary antibodies diluted in 2% bovine serum albumin, 0.1% Triton X-100% in PBS for 1 h. Mouse polyclonal antibodies have been diluted 1:200 (except GiDLPn and GiCLH, these pAbs were diluted 1:1000), and Texas Red-conjugated mouse mAb A300-TR (Waterborne, New Orleans, LA), an anti-CWP antibody, 1:30. After washing with ice-cold PBS, cells were incubated for 1 h with FITC-conjugated sheep anti-mouse antibody (Sigma). Fluorescence microscopy was performed on a Leica DM-IRBE microscope using a x100 HCX PL Fluotar lens (Leica Microsystems GmbH, Wetzlar, Germany) and digital images were recorded using a cooled CCD camera (Diagnostic Instruments Inc.) and processed with the Metaview software package (Visitron Systems GmbH, Puchheim, Germany).
Electron MicroscopyTrophozoites or encysting cells were cultivated as described above. After collection, cells were washed twice in ice-cold PBS and transferred into 6-well plates containing 1012 sapphire glasses per well in pre-warmed PBS (37 °C). To promote attachment to these 30-µm thick carbon-coated sapphire disks, cells were then incubated at room temperature for 5 min. Sapphire disks covered with a monolayer of attached parasites were subsequently plunged into a mixture of liquid propane/ethane (8/2) cooled by liquid nitrogen using a custom device. The ultra-rapidly frozen samples were substituted at 90 °C in acetone containing 0.5% osmium tetroxide and 0.25% glutaraldehyde (23) overnight. The temperature was then continuously (5 °C/h) raised to 0 °C, and the samples embedded in Epon at 4 °C. After polymerization at 60 °C for 2 days, ultrathin sections were cut parallel to the sapphire surface, stained with uranyl acetate and lead citrate, and examined in a CM 12 electron microscope (Philips, Netherlands) equipped with a slow scan CCD camera (Gatan, Pleasanton, CA) at an acceleration voltage of 100 kV. Recorded pictures have been processed with the Digital micrograph 3.34 software (Gatan).
Sucrose Density Gradient Centrifugation and Subcellular FractionationAll manipulations were carried out at 4 °C. Cells were grown at 37 °C in triple surface flasks (Nunc, Roskilde, Denmark) to a density of approximately 1 x 109 cells/flask, harvested as described above, and washed once in ice-cold PBS. For quantitative assays (densitometric quantification, biochemical assays), cell numbers of different populations (trophozoites and encysting cells) were normalized after determining the absolute cell number with a Neubauer chamber. The adjusted cell pellet was then resuspended in 4 ml of ice-cold PBS containing a 2x protease inhibitor mixture (1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM EDTA, 2 µM E-64, 2 µM leupeptin, and 300 nM aprotinin; Calbiochem, San Diego, CA) and 1 mM phenylmethylsulfonyl fluoride. This suspension was transferred into a 15-ml Falcon tube (BD Biosciences, Franklin Lakes, NJ) and processed on a fixed stand with a sonifier (Branson, Danbury, CT) using a total of 5 pulses of 30 s each (setting 2, 10% pulse intensity). Microscopic examination was used to confirm that trophozoites and encysting cells (but not cysts) were completely disintegrated. After adding sucrose to a final concentration of 250 mM, the suspension was centrifuged for 10 min at 1000 x g and the supernatant harvested. 1.8 ml of this postnuclear supernatant were layered onto a discontinuous sucrose gradient in 12-ml polyallomer tubes (Beckman-Spinco) made from four 0.5-ml layers of 60, 55, 50, and 45% sucrose and five 1.5-ml layers of 40, 35, 30, 25, and 20% sucrose, which resulted in a total volume of 11.3 ml including the loaded cell suspension sample. This discontinuous sucrose gradient was centrifuged at 100,000 x g for 18 h at 4 °C. The gradient was eluted from the bottom into 18 fractions of 600 µl each, and stored as 300-µl aliquots at 20 °C for further analysis or directly processed for biochemical assays (see below). Protein content in each fraction was measured with the BCA protein assay kit (Pierce). For SDS-PAGE, aliquots were further diluted with PBS to 1 ml, and proteins were precipitated by addition of 250 µl of concentrated trichloroacetic acid. Precipitated proteins were collected by centrifugation at 10,000 x g for 10 min, washed once with acetone, and air dried for 1 h at room temperature. Dried and washed protein pellets were redissolved in 100 µl of SDS-PAGE sample buffer containing 7.75 mg/ml dithiothreitol, boiled for 3 min, loaded on a 10% SDS-PAGE gel, and processed for Western analysis as described above. The Western data have been analyzed densitometrically using ChemiImager 5500 software (Alpha Innotech, San Leandro, CA) and are indicated relative (in %) to the maximum value (100%). Protein content (indicated in µg/ml) was determined for each fraction in both developmental stages, and CWP2 was used as an internal marker for ESV localization. In addition, the activity of the usually Golgi-specific enzyme GlcNAc-transferase was measured in both developmental stages (see below).
Glycosyltransferase AssayN-Acetylglucosamine transferase activity assay was performed essentially as described by Vischer and Hughes (24). Briefly, 300-µl aliquots of each subcellular fraction were diluted with an equal volume of assay buffer to a final concentration of 50 mM sodium phosphate, pH 6.9, 5 mM MgCl2, 5 mM MnCl2, 10 mM KCl, 0.1% Triton X-100, and 5 mM pyrophosphate. To saturate non-catalytic binding of substrate, samples were preincubated with 1 mM cold (unlabeled) UDP-N-acetylglucosamine and 2 mg of ovalbumin as a donor for 2 h at the restrictive temperature of 4 °C. The reaction was started by addition of labeled UDP-N-acetyl-[3H]glucosamine (1.67 kBq per fraction) and shifting the temperature to 37 °C. After 1.5 h of incubation, the reaction was terminated with 1 ml of ice-cold 0.5 M HCl containing 1% phosphotungstic acid. The precipitates were collected by centrifugation and the pellet carefully resuspended in 1 ml of H2O to completely resolve co-precipitated sugars, centrifuged, and incubated with 1 ml of ice-cold 95% ethanol for 5 min at 4 °C. Finally, precipitates were neutralized with 500 µl of 0.5 M NaOH overnight at room temperature and counted in 10 ml of scintillation mixture (PerkinElmer Life Sciences) on a liquid scintillation counter (PerkinElmer Life Sciences).
Nucleotide Sequence Accession NumbersThe sequence data used for (i) phylogenetic inferences of translated ORFs and/or (ii) for recombinant expression and antibody production have been confirmed by microsequencing (Microsynth GmbH, Balgach, Switzerland) and are available from EMBL/GenBankTM/DDBJ under the following accession numbers: putative adaptor protein complex large chain subunit BetaB AF503489
[GenBank]
; putative adaptor protein complex large chain subunit BetaA AF503488
[GenBank]
; G. intestinalis -adaptin gene AF486294
[GenBank]
; G. intestinalis
-adaptin gene AF486293
[GenBank]
; putative adaptor protein complex medium subunit (MuA) AY078979
[GenBank]
; putative adaptor protein complex medium subunit (MuB) AY078978
[GenBank]
; putative adaptor protein complex small chain subunit (SigmaA) AY078976
[GenBank]
; putative adaptor protein complex small chain subunit (SigmaB) AY078977
[GenBank]
; putative coatomer protein complex I subunit gene AF456417
[GenBank]
; G. intestinalis t-SNARE-like protein (SYN1) AF456415
[GenBank]
; G. intestinalis strain WBC6 RabA (RabA) (GiRabA) AF481768
[GenBank]
; G. intestinalis strain WBC6 RabB (GiRabB) (RabB) AF481767
[GenBank]
; G. intestinalis strain WBC6 RabF (RabF) (GiRabF) AF481766
[GenBank]
; G. intestinalis strain WBC6 RabD (RabD) (GiRabD) AF481765
[GenBank]
; G. intestinalis dynamin-like protein (GiDLP) gene, with complete cds AF456416
[GenBank]
; G. intestinalis Yip1 homologue (GiYip) AY219652.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The Molecular Basis for a Generic Eukaryotic Secretory System in GiardiaIn a survey of the Giardia Genome Data base, we addressed the question whether this early diverged eukaryote harbors a classical secretory system despite its completely divergent morphology. Because most of the genes coding for key proteins involved in vesicular coating, budding, and fusion are members of gene families (2628), the membrane compartment organization and complexity of a cell can be predicted by genomic approaches (26). We sought to define the molecular basis for Giardia secretory transport by a systematic genomic search for members of these gene families in the Giardia HTGS data base. These included sequences coding for subunits of the COPI, COPII, and adaptor protein (AP) coat complexes, the SNARE family of tethering factors and their corresponding adaptors, which belong to the Sec1 family, the Rab GTPases and, finally, the large vesicle tethering complexes Sec34/35 and VPS52/53/54 and the exocyst complex, which are organized in three gene families.
By pairwise reciprocal BLAST searching of the Giardia Genome Data base for subunits we identified genes coding for five of seven postulated Giardia COPI subunits -,
-,
'-,
-, and
-COP (Table I). A Giardia
-COP subunit has not been identified yet, and
-subunits are not sufficiently conserved to be identified with BLAST searches. We also identified all COPII subunits, i.e. the complete ORFs of two Giardia Sec24 paralogues, Gisec24a and GiSec24b, as well as the Giardia orthologues of Sec23, Sec13, and Sec31 (Table I). Two complete sets of heterotetrameric Giardia AP subunits (Table I) were also found using subunits from AP-1 to AP-4 from Homo sapiens and Arabidopsis thaliana, or AP-1 to AP-3 from S. cerevisiae as query sequences. A re-Blast of the Giardia HTGS data base with these eight putative AP subunits as query sequences did not reveal additional hits, making the existence of a third AP complex in Giardia highly unlikely. Consequently, Giardia is the first organism examined to date with only two putative AP complexes. Furthermore, we identified an orthologue of the clathrin heavy chain in the genome (Table I), termed GiCLH. The putative ORF encodes a 1871-amino acid protein with three (S. cerevisiae: seven) C-terminal clathrin repeats and one N-terminal propeller, according to a Pfam analysis. Not surprisingly, identification of a clathrin light chain in the Giardia genome was not successful, presumably because clathrin light chain orthologues share exceedingly low sequence homology.
A Giardia ARF gene homologue has been cloned and sequenced previously (29) and was used to rescue a lethal yeast mutant (30). In this study we identified ORFs predicting three additional members of the ARF family in Giardia (Table I). Phylogenetic analysis (data not shown) suggests that one protein belongs to the same subgroup as ARF (ARF2), whereas the two other proteins comprise the Giardia ARL subgroup (ARL1 and ARL2). We also found a predicted related Giardia Sar1p protein to be highly homologous to the corresponding A. thaliana orthologue (Table I).
Specific factors on acceptor membranes and transport vesicles confer specificity to fusion events and are therefore critical for maintaining organelle integrity. These include the membrane-associated SNARE family of proteins and the small Rab GTPases, which act as molecular switches.
SNAREs comprise a family of related integral membrane proteins that mediate a variety of membrane docking and fusion reactions in eukaryotic cells. The family of SNARE proteins can be divided into Q-SNAREs with a conserved central glutamine residue (syntaxin and SNAP subgroups) and R-SNAREs with a conserved central arginine residue (vamp/synaptobrevin subgroup), whereby three Q-SNAREs and one R-SNARE are required for the action of a functional tetrameric helical bundle. In our attempts to identify Giardia orthologues of members of the two SNARE subclasses we validated initial BLAST results using the Pfam tool to confirm the presence of conserved structural syntaxin (Syn) or synaptobrevin (Vamp) domains. In a first round of data base searches we uncovered four putative Giardia SNAREs (Table I): two Q-SNAREs (GiSyn1 and GiSyn4) and two R-SNAREs (GiVamp1 and GiVamp2). Using these Giardia SNARE orthologues as query sequences, we found an additional syntaxin-like Q-SNARE with closest homology to GiSyn1 (termed GiSyn2, Table I). The presence of predicted C-terminal transmembrane domains in GiSyn1, GiSyn2, GiSyn4, and GiVamp2 and a C-terminal prenylation site in GiVamp1 further supported the correct identification of these SNARE orthologues. The cytosolic Sec1 proteins interact directly with members of the syntaxin subfamily of SNAREs. A previous phylogenetic analysis of members of the Sec1 family of proteins (31) revealed that of the four Sec1 members in yeast, Sec1p, Sly1p, and VPS45p belong to one subgroup, and VPS33p constitutes a second subgroup. Additional gene duplications in Caenohrabditis elegans and human have only been observed with Sec1p. Giardia seems to contain only two Sec1 paralogues, a prototypic GiSec1p and second Sec1 paralogue most closely related to the yeast VPS33 protein (GiVPS33p, Table I).
Rab GTPases are molecular switches cycling between an active, membrane-associated, GTP-bound and an inactive, cytosolic, GDP-bound state. Because different Rabs preferentially localize to distinct vesicles and organelles in their activated state, they are assumed to be critical for the specificity of membrane fusion. Our genomic survey revealed eight genes coding for Rab proteins in Giardia (Table I). These include orthologues of Rab1 (GiRab1) and Rab11 (GiRab11), with high homology to the corresponding proteins in S. cerevisiae, C. elegans, and H. sapiens, and two orthologues of Rab2 (GiRab2a and GiRab2b). In various organisms, Rab1/2 and Rab11 have well characterized functions in anterograde transport from ER to Golgi and in cycling of vesicles from endosomes to the TGN or exit from the TGN, respectively. In addition, we identified four highly diverged Giardia Rab homologues, GiRabA/B/D/F, whose identities could not be assigned with confidence based on sequence homology alone. However, all four of these highly diverged Giardia GTPases show highest similarity to the five functional Rab domains (RabF15 as defined by Ref. 32) rather than to the corresponding domains of the Ras/Rho/Arf groups within the superfamily of Ras proteins.
Three recently identified multisubunit protein complexes are involved in vesicle tethering at distinct trafficking steps in yeast and human cells. The COG complex (33) (formerly termed Sec34/35 complex (34)) is involved in intra-Golgi recycling and recycling from endosomes back to the Golgi (35). Four of the eight COG subunits are structurally and phylogenetically related with each other and share sequence homology with four subunits of the exocyst and two of the GARP complex. The exocyst (36) is a complex associated with the TGN in yeast and mammalian cells and is involved in trafficking to the plasma membrane, mostly at sites of polarized secretion. The GARP complex (formerly termed VPS52/53/54 (37)) is required for retrograde transport from endosomes to the TGN. Surprisingly, our survey indicated that Giardia lacks each of these complexes. We were unable to identify any subunits of these quatrefoil complexes involved in vesicle tethering during trafficking to and from the TGN. The conspicuous absence of these factors adds molecular support to the hypothesis that a conventional Golgi is not present in this protozoan.
The Secretory Machinery Components Are Not Stage Specifically Expressed in GiardiaEncystation causes significant morphological changes of the endomembrane system of Giardia, notably the appearance of discrete ER exit sites followed by the apparent neogenesis of large ESVs. If ESVs indeed corresponded to a Golgi, and are generated de novo, this developmental induction of a compartment structure should be reflected in changes of steady-state mRNA levels of the broad panel of markers and complexes detailed in Table I. To address this question and to identify markers potentially associated with the developmentally regulated genesis of ESV compartments, we performed a semiquantitative RT-PCR analysis and compared mRNA levels in trophozoites with those in parasites in an early phase of encystation in vitro (7 h postinduction). Message levels of only one representative subunit gene of each COPI, COPII, and putative API/II complex were measured. As a positive control for encystation, we determined induction of endogenous CWP1 transcripts. mRNA levels of the constitutively expressed protein phosphatase 2 (PP2) (16) were determined as a control that equal amounts of cDNA were being compared. Cycle numbers for PCR amplification were adapted to individual cDNA levels to allow densitometric quantification of each pair of SYBR green-stained PCR products (trophozoites/cells 7 h postinduction of encystation) within the linear range of the Fluoroimager detection filter. Semi-quantitative RT-PCR of 24 different mRNAs and densitometric analysis revealed no or only minor changes in transcript levels during stage conversion (Figs. 2, A and B). In comparison with the mRNA coding for the CWP1 protein (90-fold induction), mRNA levels of the analyzed markers were only very moderately elevated in encysting cells, if at all. The levels of six of these weakly induced mRNAs (GiSec24a, GiRab2a, GiRabA, GiRab11, GiSar1, and GiVPS33) were determined more precisely at five time points during the encystation process (trophozoites, 3/7/15/24 h postinduction of encystation) by Light Cycler PCRTM (Fig. 2C), using PP2 mRNA as the constitutive standard. GiRabF was not analyzed further despite some indications of up-regulation in encysting cells because of the extremely low levels of steady-state mRNA. This approach confirmed the modest induction levels of certain mRNAs (GiSec24a, GiRabA, and GiRab2a), and demonstrated that none of the secretory components investigated in this study were induced only during encystation.
|
Generation of Polyclonal Antibodies Against Seven "Sentinel" Proteins and Analysis of Compartment Dynamics during EncystationUsing comparative genomics, we showed that Giardia holds the basic machinery constituting a eukaryotic secretory system, and that the major components of this machinery were not stage-regulated. To analyze stage-specific dynamics of the Giardia secretory system in more detail, we generated a novel set of antibodies against selected proteins predicted to be associated with different components and compartments of this apparatus. These pAbs were used as tools in a systematic investigation of the membrane compartments anatomy in Giardia and specifically, to address the question whether some of these sentinel proteins were involved in the generation and/or maintenance of ESVs. As a key criterion for the latter, we addressed the question whether some of these marker proteins re-localized to ESVs from cytoplasmic pools or other membrane compartments during encystation. The markers used in this investigation were: GiSar1p, Gi'COP, GiCLH, GiSyn1, and GiRab11 (see also Table I). Two additional and separately identified proteins were included: (i) a Giardia orthologue of the yeast dynamin-like protein VPS1p (38) termed GiDLP; (ii) GiYip, a homologue of the recently identified protein Yip1p in yeast, which interacts with the transport GTPase Ypt1p at the Golgi (39). Mouse antisera against fusion proteins or the GiDLP peptide were analyzed for specificity by Western blot using total protein from vegetatively dividing trophozoites. All antibodies detected a major band that corresponded to the molecular mass of the predicted proteins (Fig. 3A).
|
The seven pAbs were used to localize the corresponding marker proteins within the Giardia cytoplasm and/or endomembrane system by immunofluorescence assay (IFA), and to determine its dynamics during encystation and ESV formation. A mAb against a cyst wall protein was used as an internal marker to label cyst wall material in ESVs. Specific pAbs against GiSar1p-labeled nuclear envelope membranes and the clamp-shaped ER compartment in both developmental stages (Fig. 4A) but not ESVs. Co-localization of CWP and GiSar1p was occasionally detected in areas where ER exit sites had been found previously (18). GiSyn1, a Giardia syntaxin homologue, did not re-localize to ESV membranes at all, but remained associated with internal and peripheral membrane structures in both developmental stages (Fig. 4D). In contrast, the putative Giardia COPI coat complex, represented by the Gi'COP subunit, partially redistributed to ESVs during stage conversion (Fig. 4B). Interestingly, the antibody labeled as yet uncharacterized structures in trophozoites as well. Because COPI is associated exclusively with Golgi membranes in all eukaryotes, this was the first direct evidence for the existence of a putative Golgi-like organelle in Giardia trophozoites. Similarly, GiRab11 exhibited ESV labeling in encysting cells in addition to the peripheral distribution observed in trophozoites (Fig. 4E). GiYip was mostly confined to punctate structures along the clamp-shaped reticular ER in trophozoites and encysting cells, reminiscent of ER exit sites (18), and partially localized to ESV membranes (Fig. 4F). GiCLH was detected exclusively in close association with PVs in trophozoites at the periphery of the cell body, and re-localized to ESVs in late encysting cells only (18). GiDLP showed a similar distribution with two major differences: (i) in trophozoites the marker was distinctly associated with PVs but also other internal structures, and (ii) in contrast to GiCLH, antibodies against GiDLP already labeled early, immature ESVs and the marker remained associated with these compartments until secretion of the cyst wall material. The results showed that the seven markers investigated here were expressed in both trophozoites and encysting cells and thus provided independent confirmation of the semi-quantitative RT-PCR experiment. Moreover, we showed that this set of antibodies including the anti-CWP mAb can be used to distinguish the three known membrane compartments ER, PVs, and ESVs. Finally, the IFA data showed that the markers, which partially (Gi
'COP, GiYip, and GiRab11) or mostly (GiCLH and GiDLP) redistributed to ESVs during encystation, localized to distinct compartments in trophozoites. This supported the notion that the vesicular transport system in Giardia is constitutive and argued against the hypothesis that ESVs are de novo generated compartments.
|
Trophozoites Contain a Compartment with Golgi CharacteristicsClear identification of endomembrane compartments using IFA experiments remained difficult because of the very limited information available on the structure of the Giardia endomembrane system. We used subcellular fractionation techniques in conjunction with this novel set of antibodies as a complementary approach to improve compartment characterization, and to investigate the stage-specific dynamics of the secretory apparatus. In particular, we were interested if marker proteins that were seen to associate with ESVs by IFA were recruited from cytoplasmic pools or compartments with completely different characteristics in trophozoites, as predicted if ESVs were indeed a novel Golgi-like compartment. Using the antibodies described above we first demonstrated that we could indeed reproducibly generate and separate distinct microsome populations from trophozoites and encysting cells with a dynamic range between sucrose densities of 20 and 55%. Sucrose gradients were eluted into 18 fractions of equal volume after centrifugation and proteins were separated on SDS-PAGE and blotted to nitrocellulose membranes. We showed that detection of marker proteins with specific antibodies resulted in distinct distribution profiles (Fig. 5), reflecting the association of the markers with microsomes of defined densities, or the presence of the marker as a soluble protein in the cytoplasm (e.g. the cytoplasmic pool of GiSar1p). Each profile was verified in at least three fractionation experiments; densitometric analysis of a representative experiment was used for graphical presentation of Western blot data in Fig. 5B. A representative Western blot reflecting the subcellular distribution of GiDLP in both developmental stages is shown in Fig. 5A. ESV-derived microsomes prepared from encysting cells were identified using a mAb against the cargo protein CWP1 (fractions 1012). The lack of soluble CWP in the top fractions (cytoplasm) demonstrated the faithful resealing of ESVs during the cell disruption process. The peak of the Golgi marker Gi'COP overlapped significantly (fractions 11 to 13) with those of the Golgi-associated GlcNAc-transferase enzyme activity and CWP at a sucrose concentration of 22 to 26%. Similar density values have also been reported in initial fractionation studies for the purification of ESVs (17) and of Golgi microsomes in mammalian systems (40). The five sentinel proteins (Gi
'COP, GiCLH, GiYip, GiDLP, and GiRab11), which localized to ESVs by IFA in encysting cells also showed a major peak at sucrose concentrations between 25 and 29%, whereas the GiSyn1 peak (69) did not overlap with CWP-containing fractions. Most importantly, Gi
'COP, GiYip, GiDLP, and GiRab11, as well as GlcNAc-transferase activity, were also associated with microsomes from trophozoites with identical densities. This showed for the first time that membrane-bound compartments with characteristics equivalent to those of ESVs were present in trophozoites. Consistent with IFA data, the trophozoite GiCLH peak did not overlap with the peak generated by the ESV-associated clathin. Three markers (GiCLH, GiDLP, and GiSyn1) showed a second peak at densities similar to those reported for lysosomes (4044%) or, alternatively, microsomes derived from rough ER (3854%) of higher eukaryotes (40). Considering the observed localizations in IFA experiments we favor the interpretation that these markers are associated with PVs, which apparently have similar densities as reported for lysosomes, but we cannot exclude association of GiDLP with ER-derived microsomes during encystation (fractions 13). Microsomes derived from the ER usually peak at two densities in fractionation studies of other eukaryotic cells: a rough ER fraction with a significantly higher density than Golgi microsomes, and a smooth ER fraction, which may overlap with the Golgi microsomes (40). The extensive Giardia ER is likely to be organized in distinct subdomains (including ER exit sites, which sometimes appear as punctate structures in IFA (18)), and may even be directly connected to PVs (41, 42). IFA data showed that GiSar1p evenly localized to the clamp-shaped ER/nuclear envelope membranes. This is in agreement with the GiSar1 peaks detected in fractions 3, 4, and 912 (811 in encysting cells), whereas the large peak in fractions 1418 represents the cytoplasmic pool. GiYip is a predicted integral membrane protein and showed a punctate distribution along the ER structure in IFA. Its distribution is correspondingly different to that of GiSar1p with only a minor overlap (fractions 10 and 11) and no cytoplasmic pool. Finally, a third peak in the subcellular distribution of GiCLH, GiDLP, and GiSyn1 between 30 and 36% sucrose (fractions 68) could not be assigned to a specific compartment with confidence. The variety of profiles obtained by different markers indicated a more complex anatomy of the Giardia endomembrane system than tEM studies would suggest. All markers except GiSyn1 and GiYip are peripheral membrane proteins, which cycle between a cytoplasmic and a compartment-bound state. It is therefore important to note that these proteins have sometimes large cytoplasmic pools, represented by a peak in fractions at the top of the gradient.
|
A synthesis of the combined IFA and subcellular fractionation data indicating the localization(s) of marker proteins is presented in Table II. Note that IFA images of encysting cells are representative but, as opposed to the subcellular fractionation data, cannot reflect the variations among the population. The subcellular fractionation data show a virtual lack of major shifts in peak localization and relative size between preparation from trophozoites and encysting cells (Fig. 6, Table II). The few exceptions (i.e. GiDLP and GiSar1p) concern peaks in fractions denser than ESVs and will have to be investigated further. The remarkably similar profiles obtained in both developmental stages again supported the existence of compartments in trophozoites that are biochemically related to ESVs. Together with the fact that mRNA expression levels of the major secretory pathway components including several Golgi marker proteins were not stage-specifically regulated, the data presented here argued against a previously postulated hypothesis that Golgi structure and function are induced only during encystation in Giardia (17) and are not present in proliferating trophozoites.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The absence of large Golgi stacks, or even Golgi "ministacks" as observed in Pichia pastoris (45) is evidence for an unconventional membrane transport system, but not proof that a functional Golgi does not exist in Giardia. Although morphological evidence could not be found, we demonstrated that on a molecular genetic level Giardia holds the basic modules constituting a classical eukaryotic secretory apparatus. Identification of a molecular framework for secretory transport in Giardia allowed to significantly extend insights from a recent study on eukaryotic membrane compartment organization based on genome sequence data from S. cervisiae, C. elegans, Drosophila melanogaster, and H. sapiens (26) (Table III). Our data implies an increase in complexity from the ancestral Giardia "core system" to its yeast counterpart similar to that from single cells (S. cerevisiae) to multicellular organisms (C. elegans). For example, we identified only a limited number of factors involved in ordered fusion of membrane vesicles in Giardia: three Q-SNAREs and two R-SNAREs (S. cerevisiae: 23, C. elegans: 26), and eight paralogues of the Rab protein family (S. cerevisiae: 11, C. elegans: 29). Interestingly, preliminary phylogenetic analysis of AP subunits (data not shown) supported the presence of only two putative AP complexes in Giardia, AP-I and AP-II. According to the current model on the evolution of AP complexes by a series of coordinated gene duplications (27), the two prototypic Giardia AP complexes predicted the point of separation of Giardia (i.e. the diplomonads as a whole) after the first coordinated round of gene duplications resulting in an AP-3 and an AP1/2/4 ancestor (Fig. 7). Based on the Giardia orthologues and functional categories identified in this study, one can specify the secretory apparatus of the last common ancestor of diplomonads and the eukaryotic lineage. Such a hypothetical ancestral secretory apparatus would consist of four vesicle coat complexes (two AP-clathrin complexes, COPI and COPII), six or seven Rab proteins (assuming group-specific gene duplications of the closely related GiRab2a/b), two SNARE complexes, and two corresponding SNARE adaptors of the Sec1 family.
|
|
Because the developmental induction of Golgi structure and function had been postulated previously, it was of interest to test whether the expression of any of the now uncovered structural components of the secretory machinery in Giardia was regulated or constitutively active. The lack of significant expression regulation of the genes investigated (Fig. 2) provided important evidence that the Giardia secretory system is not subject to stage-regulation during encystation on a molecular level, even though this was surprising considering the significant morphologic changes in membrane structures. In particular, the steady-state mRNA levels of Giardia Rab and SNARE family members were more stable than expected. In Trypanosoma brucei, for example, the Golgi-associated TbRab18 protein (46), or the endocytic TbRab11 (47) and coat components such as the clathrin heavy chain or AP1 (48), are developmentally regulated, in response to changes in Golgi functions or the different requirements for endocytotic traffic in bloodstream forms or procyclic stages, respectively. With respect to the idea that ESVs are Golgi cisternae, the transcriptional analyses suggested, that even though ESVs were novel structures by morphological criteria they may not be generated de novo, but correspond in their basic biochemical characteristics to hitherto unidentified Golgi-like compartments in trophozoites. Additional independent support for the latter came from results obtained with IFA and subcellular fractionation experiments. A set of seven novel antibodies against marker proteins was used as sentinels to investigate the dynamics of the Giardia endomembrane system during a developmental transition where major morphological changes could be observed accompanying the emergence of ESVs. In addition to the detection of major compartments, some unexpected and novel structures were recognizable whose exact nature will have to be determined in future investigations. Most significantly, the presence and association of all marker proteins with specific subcellular compartments in trophozoites indicated the existence of a constitutive transport system, and presumably some form of Golgi apparatus. The most interesting finding of the complementing cell fractionation experiments was the detection of a microsome population with the same density as ESVs rather than a "vesicle gap," as would have been predicted if there was no Golgi-like compartments in trophozoites. This was unexpected considering the previous absence of any morphological and biochemical evidence for a Golgi in trophozoites (17, 18), an idea that was also based on an earlier study where GlcNAc-transferase activity was detected only in encysting cells but not in trophozoites (17). Other data (49) and our own showed practically identical GlcNAc-transferase activity levels (peaks in fractions 1012) in trophozoites and encysting parasites (10 h postinduction), however. These results indicating a constitutive Golgi structure and function also agreed with reports that export of VSPs to the plasma membrane in trophozoites was sensitive to brefeldin A (17), suggesting coatomer-dependent transport processes to the cell surface in trophozoites. How does this fit with previous models (18), which feature ESVs as the only secretory compartments with Golgi characteristics? The unique nature of ESVs as transient Golgi-like compartments is because of their conspicuous morphology, their defined cargo, and their stability over a period of several hours. Functionally similar compartments in trophozoites (and presumably throughout the life cycle) evidently lack this stability and are bound to be difficult to identify because of this. Therefore, the existing models will have to be adapted to feature two Golgi-like systems, which exist simultaneously, at least in encysting cells. In addition to different degrees of stability, these parallel systems presumably have unique cargo specificities as already suggested in previous work (18). Because protein sorting appears to occur at a pre-Golgi level, the exported cargo itself may ultimately determine the functional identity of the compartment in which it will travel to its destination. These compartments will therefore display specific biochemical characteristics, e.g. making pre-Golgi vesicles containing CWP cargo selectively fusion competent, or mature ESVs receptive for the exocytosis signal. In trophozoites, the current data suggest that VSPs are exported through a direct pathway with fast kinetics to the plasma membrane. This pathway is brefeldin A-sensitive but VSPs, or similarly targeted reporters, are insubstantially modified by post-translational processes (50), if at all, in agreement with the hypothesis that their export involves passage through a constitutive short-lived Golgi-like organelle. Consequently, the simplest (and probably too simplistic) scenario would predict that the comparatively longlived ESVs could be generated from a constitutive apparatus by recruitment of only one or few factors, which mediate homotypic fusion of ER-derived transport intermediates and stabilize the nascent cisternae for the time until exocytosis, without the complex neogenesis of an entire secretory system. Considering the important role such putative factors play during stage conversion, their identification and characterization will be key to understanding the principles for generation and maintenance of Golgi-like cisternae in Giardia, and perhaps in general. This model is consistent with a specialized "pulsechase" version of the cisternal maturation model (51): (i) ESVs are generated from smaller pre-Golgi vesicles by homotypic fusion; (ii) ESVs mature by retrograde transport via COPI-coated vesicles; (iii) mature ESVs are analogous to single-cargo trans Golgi cisterna and associated with clathrin; (iv) ESVs disperse simultaneously into small secretory vesicles that fuse with the plasma membrane and release their contents.6
The results presented here indicate novel aspects concerning possible Golgi functions in the context of a primordial secretory system in this basal protozoan. The evidence we now find for such structures in trophozoites makes the Giardia model system more consistent, because the previously postulated de novo synthesis of Golgi cisternae had been difficult to explain. All the challenging questions remain, i.e. how are cargo proteins sorted during export, and to what extent do their targeting signals determine the nature and fate of transport vesicles. In addition, we will start looking for factors responsible for the selective stabilization of ESVs. These molecules will provide the answer to the question, how early eukaryotes maintained Golgi cisternae for extended periods of time and evolved mechanisms for maturation and controlled secretion of more extensively modified cargo.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Swiss National Science Foundation Grants 31-58912.99 and 31-100270/1, the Novartis Foundation, and the "Stiftung für wissenschaftliche Forschung an der Universität Zürich." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains tables.
** Present address: Division of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia.
Supported by a training grant from the China Scholarship Council. Present address: Dept. of Parasitology, Harbin Medical University, 150086, Harbin, Peoples Republic of China.
|| To whom correspondence should be addressed. Institute of Parasitology, University of Zürich, Winterthurerstrasse 266a, CH-8057 Zürich, Switzerland. Tel.: 41-1-635-8526; Fax: 41-1-635-8907; E-mail: ahehl{at}vetparas.unizh.ch.
1 The abbreviations used are: ER, endoplasmic reticulum; PV, peripheral vesicle; ESV, encystation-specific vesicles; RT, reverse transcriptase; ORF, open reading frame; MBP, maltose-binding protein; PBS, phosphate-buffered saline; mAb, monoclonal antibody; pAb, polyclonal antibody; FITC, fluorescein isothiocyanate; AP, adapter protein; TGN, trans Golgi network; PP2, protein phosphatase 2; ARF, ADP-ribosylation factor; IFA, immunofluorescence assay.
3 jbpc.mbl.edu/Giardia-HTML/summary.html.
4 www.sanger.ac.uk./cgi-bin/Pfam/nph-search.cgi.
6 M. Marti and A. B. Hehl, unpublished data.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|