©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Developmental Induction of Golgi Structure and Function in the Primitive Eukaryote Giardia lamblia(*)

(Received for publication, October 3, 1994; and in revised form, November 10, 1994)

Hugo D. Luján (1)(§) Alex Marotta (2) Michael R. Mowatt (1) Noah Sciaky (2) Jennifer Lippincott-Schwartz (2) Theodore E. Nash (1)

From the  (1)Laboratory of Parasitic Diseases, NIAID and the (2)Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A fundamental characteristic of eukaryotic cells is the presence of membrane-bound compartments and membrane transport pathways in which the Golgi complex plays a central role in the selective processing, sorting, and secretion of proteins. The parasitic protozoan Giardia lamblia belongs to the earliest identified lineage among eukaryotes and therefore offers unique insight into the progression from primitive to more complex eukaryotic cells. Here, we report that Giardia trophozoites undergo a developmental induction of Golgi enzyme activities, which correlates with the appearance of a morphologically identifiable Golgi complex, as they differentiate to cysts. Prior to this induction, no morphologically or biochemically identifiable Golgi complex exists within nonencysting cells. Remarkably, protein secretion in both nonencysting and encysting trophozoites is inhibited by brefeldin A, and brefeldin A-sensitive membrane association of ADP-ribosylation factor and beta-COP is observed. These results suggest that the secretory machinery of Giardia resembles that of higher eukaryotes despite the absence of a Golgi complex in nonencysting trophozoites. These findings have implications both for defining the minimal machinery for protein secretion in eukaryotes and for examining the biogenesis of Golgi structure and function.


INTRODUCTION

Giardia lamblia is a flagellated protozoan that inhabits the upper small intestine of its vertebrate host and is a major cause of enteric disease worldwide. Infection is initiated by ingestion of cysts, followed by excystation and colonization of the small intestine by the trophozoites. In the intestine, some trophozoites are induced to encyst, and mature cysts are excreted in the feces(1, 2) . The induction of cyst wall proteins and their secretion play direct roles in the transformation of the trophozoites into cysts, underlying the ability of Giardia to survive relatively harsh conditions and to infect other hosts(1, 2) . Understanding the nature and regulation of protein transport in Giardia, therefore, offers therapeutic potential in the treatment of this parasite. It is also likely to provide insight into the minimal requirements for secretory transport, since Giardia represents one of the most ancient lineages among eukaryotes(3) . Its ribosomal RNA shares more sequence homology with prokaryotes than does the corresponding RNA of any other eukaryote(3) . Giardia trophozoites contain two nuclei and lack many of the subcellular organelles characteristic of higher eukaryotes, including mitochondria and peroxisomes(4, 5, 6) . In addition, they have been reported to lack a Golgi complex(4, 5, 6) , although a membranous Golgi-like structure has been observed during encystation in vitro(7) .

The Golgi complex plays a central role in the transport, processing, and sorting of proteins and usually consists of a series of flattened stacks of cisternae that are enriched in glycoprotein processing enzymes(8, 9, 10, 11, 12) . Traffic of proteins from the ER (^1)into the Golgi complex and between Golgi cisternae occurs within membrane-bound intermediates whose formation is controlled by cytosolic protein complexes which undergo regulated association/dissociation from membranes(11, 13) . The small GTP-binding protein ARF (14) and a set of proteins called coatomer (15, 16) are constituents of these complexes(17, 18) . ARF is required for coatomer binding to Golgi membranes(19, 20, 21, 22) . Coatomer and ARF exist free in the cytosol, but assembly is initiated when a GDP molecule present in the cytosolic ARF is exchanged for GTP, allowing ARF to bind to Golgi membranes(13, 19, 20, 21, 22) . Brefeldin A (BFA), a fungal metabolite that rapidly and reversibly inhibits transport of secretory proteins in many mammalian cells, acts by blocking the exchange of guanine nucleotides in ARF, thereby preventing assembly of ARF and coatomer onto membranes(23, 24) . This, in turn, blocks secretion and causes the Golgi complex to disassemble and redistribute into the ER(23, 24) .

Several ARF genes have been identified in mammalian cells(25) , and it is likely that they encode proteins with different functions and intracellular location(26, 27, 28, 29, 30, 31) . A gene encoding ARF was also found in Giardia trophozoites (32) and gARF partially complemented ARF function in yeast(33) . However, the function and localization of ARF in Giardia are unknown.

In Giardia, both constitutive and regulated protein secretion pathways (12) appear to exist. Evidence for continuous protein secretion is the transport to the plasma membrane and the release into the culture medium of variant-specific surface proteins, VSP(34) . Regulated protein secretion is exemplified by the formation of the cyst wall during encystation, which is characterized by the appearance in the trophozoite cytoplasm of dense encystation-specific vesicles that transport cyst wall materials. The vesicle contents are released by exocytosis to form the cyst wall(7) . The molecular mechanisms of these phenomena are, however, poorly understood.

To determine whether the secretory apparatus of Giardia shares any of the features of the secretory machinery of higher eukaryotes, we applied biochemical and morphological techniques to study protein secretion in Giardia using markers for Golgi membranes, coatomer, ARF and two Giardia specific secretory proteins, VSP 1267 (35) and a cyst wall protein(36) , CWP. (^2)BFA affected the transport and secretion of both newly synthesized VSP in nonencysting and encysting trophozoites and CWP during encystation, indicating an ARF/coatomer-mediated mechanism of transport. Golgi-resident enzymes were induced and a Golgi complex became evident only during encystation. These results indicate that Golgi structure is not required for the transport of a simple protein such as VSP, but Golgi apparatus assembly is required for the production of complex glycoproteins during parasite differentiation into cysts.


EXPERIMENTAL PROCEDURES

Organisms and Encystation in Vitro

G. lamblia trophozoites (WB isolate, 1267 clone) (35) were cultured axenically in TYI-S-33 medium supplemented with 10% adult bovine serum and bovine bile(37) . Cultures were grown to confluence in 8-ml glass tubes. The medium and nonadherent trophozoites were discarded and replaced with the same volume of fresh medium. The tubes were chilled, inverted 10 times, and the trophozoites counted with a Coulter model ZB1 electronic counter (Coulter Electronic, Hialeah, FL). Trophozoites were induced to encyst as reported(38) .

BFA Treatment of the Cells

To investigate the effect of BFA (Epicentre Biotechnologies, Madison, WI; stock solution 10 mg/ml of dimethyl sulfoxide) on trophozoite growth and viability, 1 times 10^5 cells/ml of TYI-S-33 medium were allowed to attach to the glass wall of tubes for 30 min. The media was then discarded and fresh medium containing different concentrations of BFA or solvent added. The morphology of the trophozoites and the number of viable and attached cells were determined at different time periods(39) .

Labeling of Cells with NBD-ceramide

Nonencysting and encysting cells were washed twice with culture medium, loaded onto glass slides, incubated for 15 min at 37 °C in a humidified chamber in a CO(2) incubator, treated for 30 min with 50 µg/ml BFA or diluent alone, fixed with 1% (v/v) glutaraldehyde in 250 mM sucrose, 2 mM MgCl(2)/2 mM EGTA, 10 mM sodium cacodylate, pH 7.4, washed twice with PBS, and incubated with a complex of N-(-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-erythrosphingosine (NBD-ceramide, Molecular Probes) formed with defatted bovine serum albumin (40, 41) for 15 min. Excess lipid was removed by washes with complete medium, and the samples were observed on an optical tower microscope (Jona Instruments, Co), equipped with a cooled CCD camera (Photometrics) and Macintosh interface. Fluorescence plus differential interference contrast images were analyzed using BDS image software.

Measurement of Protein Transport and Secretion

Trophozoites were incubated in labeling medium (PBS, pH 7.4, 20 µM of bathocuproine sulfonate, trace metals (Mediatech)/vitamin solution (Sigma)/amino acid mixture (Sigma), 0.5% bovine serum) containing 50 µCi of L-[S]cysteine (Amersham Corp.)/ml, for 15 min at 37 °C(42) . After removal of the radioactive medium, organisms were washed twice with PBS, 10 mML-cysteine at 37 °C, and trophozoites were then resuspended in labeling medium without serum containing L-cysteine (6 mM) and BFA (50 µg/ml) for different time periods. Cells were separated by centrifugation for 60 min at 100,000 times g. Proteins were precipitated by addition of trichloroacetic acid (20% final concentration) for 1 h at 4 °C. Trichloroacetic acid-insoluble associated radioactivity in the cell pellet and the supernatant was determined in a Beckman LS 9000 liquid scintillation system.

Giardia Fractionation, Biochemical Assays, SDS-Polyacrylamide Gel Electrophoresis, and Western Blotting

Approximately 5 times 10 nonencysting trophozoites or trophozoites cultured in encystation medium for 24 h, treated or not treated with 50 µg of BFA/ml for 30 min, were washed and resuspended in 1.5 ml of 250 mM sucrose containing 1-chloro-3-tosylamido-7-amino-2-heptanone (5 mM), PMSF (5 mM), and leupeptin (20 µg/ml), homogenized by sonication three times (30 s, 20 A, in a Tekmar Sonic Disruptor, at 4 °C), and centrifuged at 1,000 times g for 10 min to remove unbroken cells and nuclei. The supernatant (750 µl) was then layered onto a discontinuous sucrose gradient formed by layering 750 µl of 60, 55, 50, 45, 40, 35, 30, and 25% (w/w) sucrose into an SW 40 polyallomer centrifuge tube. The gradient was centrifuged 18 h at 100,000 times g and fractionated from the bottom using a peristaltic pump into 17 fractions (400 µl each). Malate dehydrogenase and acid phosphatase were determined according to Lindmark(43, 44) ; alkaline phosphatase was assayed using kits from Sigma. GalT and GalNAcT activities were measured as described (45) using ovalbumin and UDP-D-[6-^3H]galactose or N-acetyl-[6-^3H]galactosylamine (DuPont NEN) as substrates, respectively. Protein concentration was determined according to Lowry et al.(46) .

Proteins in the fractions were precipitated with 20% trichloroacetic acid, washed once with acetone at -20 °C, and dried after collection by centrifugation. SDS-polyacrylamide gel electrophoresis in 4-20% gradient gels and electrophoretic transfer of proteins to nitrocellulose were performed as reported(47, 48) . CWP was visualized by reacting with the monoclonal antibody 5-3C at 1/400 dilution, for 2 h(36) .

Antisera Preparation

Purified rgARF (32) was used to elicit polyclonal antibodies in rabbits. Affinity-purified anti-rgARF antibodies were obtained by chromatography on protein A-agarose (Bio-Rad). To isolate rgARF-specific IgG, purified rgARF was coupled to CNBr-Sepharose (Pharmacia Biotech Inc.) and the IgG fraction (200 mg of protein at 10 mg/ml in PBS) passed over the ARF-Sepharose column (0.8 mg rgARF/ml, bed volume 5 ml) at 0.1 ml/min. The column was then washed with 100 ml of PBS, and the rgARF-specific IgG eluted, buffer exchanged, concentrated to 0.32 mg/ml, and used at 1/100 dilution. Rabbit anti-beta-COP serum (49) were used at 1/100 dilution.

Immunofluorescence Microscopy

Trophozoites grown in either normal (37) or encystation media (38) were detached from tubes by chilling and allowed to reattach to glass slides precoated with poly-L-lysine, at 37 °C, for 30 min, in a CO(2) incubator. The adherent trophozoites were rinsed twice with PBS, cells treated with BFA or solvent alone at different time points, as described above, and then fixed and permeabilized for 5 min with methanol:acetone (1:1) at -20 °C. Cells were then blocked for 30 min with 5% normal goat serum (Vector Laboratories) in PBS (PBS/goat) and incubated for 1 h with the first antibody in PBS/goat. After rising three times (15 min total), fluorescein-conjugated goat anti-Ig (Organon-Teknika-Cappel) diluted in PBS/goat was added followed by three PBS washes. The specimens were mounted in Vectashield (Vector Laboratories) and viewed as described above.


RESULTS

Brefeldin A Inhibits Giardia Growth

To study the secretory system of this primitive eukaryote, we first investigated whether BFA, a drug that blocks protein transport through the Golgi complex in animal cells(23) , affects Giardia trophozoites. BFA was added to the culture medium at different concentrations and the viability of the trophozoites measured at different time points. BFA concentrations of 100 µg/ml or higher were rapidly toxic to the parasites (Fig. 1). At 75 µg/ml trophozoite morphology changed from healthy half-pear shaped to rounded forms indicative of cell disfunction. Between 25 and 50 µg/ml, growth ceased, but cells were viable at 48 h. BFA concentrations lower than 25 µg/ml affected neither viability nor growth (Fig. 1). Thus, we used BFA at 50 µg/ml for subsequent experiments.


Figure 1: Effect of Brefeldin A on the growth response of G. lamblia. Trophozoites were cultured as described under ``Experimental Procedures,'' washed twice with PBS at 37 °C, and then incubated in complete TYI-S-33 medium containing BFA: circle) none; bullet, 12.5 µg/ml; down triangle, 25 µg/ml; , 50 µg/ml; box, 75 µg/ml; , 100 µg/ml. The growth is expressed cells/ml. Each point represents a mean of three independent experiments ± S.D.



A NBD-ceramide-labeled, BFA-sensitive Structure Appears during Giardia Encystation

To examine whether a secretory compartment equivalent to the Golgi complex of higher eukaryotes is present in either nonencysting or encysting trophozoites, we labeled cells with the fluorescent lipid analogue NBD-ceramide, which preferentially integrates into the Golgi complex of mammalian cells(40, 41) . Incubation of nonencysting trophozoites with NBD-ceramide revealed no specific intracellular labeling (Fig. 2A). In contrast, encysting trophozoites showed prominent intracellular labeling of discrete perinuclear structures (Fig. 2B). Interestingly, these were usually present over one of the two nuclei of Giardia (Fig. 2, E and F). As shown in Fig. 2C, BFA treatment caused the NBD-labeled perinuclear structures to disappear, redistributing the fluorescent label onto structures dispersed throughout the cytoplasm, as well as onto nuclear envelopes.


Figure 2: The Golgi apparatus is present only in encysting trophozoites. Nonencysting (A) and encysting (B) trophozoites were labeled with the fluorescent lipid analogue NBD-ceramide. Prominent perinuclear structures resembling a Golgi complex were observed only in encysting trophozoites (B) and redistributed into the cytoplasm and nuclear envelopes after BFA treatment (C). In a representative encysting trophozoite (D-F), the perinuclear localization of the NBD-ceramide labeled structure (E) is shown by superimposition (F) to the differential interference contrast-visualized cell (D).



Effects of BFA on the Distribution of Golgi Enzymes in Giardia

The specific activity of several enzymes was determined in subcellular fractions of Giardia homogenates. Fig. 3shows that similar enzyme distribution patterns for alkaline phosphatase, acid phosphatase, and malate dehydrogenase were observed in both encysting and nonencysting trophozoites. In contrast, the activities of the Golgi specific enzymes GalT and GalNAcT (50) were readily detected in encysting trophozoites and sedimented with densities between 1.1 and 1.15 g/ml. In nonencysting trophozoites, by contrast, no detectable Golgi enzyme activities were present. When the cells were treated with BFA for 30 min and subsequently fractionated, the activities of both transferases shifted to less dense fractions (Fig. 3). Controls treated with solvent showed no difference in the activity or localization of any marker (results not shown).


Figure 3: BFA treatment results in a redistribution of Golgi markers in encysting trophozoites. Giardia homogenates prepared from encysting or nonencysting trophozoites, treated (bullet) or not treated (circle) with BFA, were subjected to isopycnic gradient centrifugation and the resulting 17 fractions analyzed for malate dehydrogenase (cytosol), alkaline phosphatase (plasma membrane), acid phosphatase (lysosome-like organelles), galactosyltransferase (Golgi), and N-acetylgalactosamine transferase (Golgi) activities. Specific activity of acid phosphatase, alkaline phosphatase, and malate dehydrogenase are expressed as micromoles of product formed times µg of protein times min. Values represent the average of two independent experiments performed in duplicate.



Brefeldin A Inhibits Protein Transport and Secretion in Both Encysting and Nonencysting Trophozoites

To determine whether BFA affects the transport of Giardia proteins, the secretory pathways followed by one CWP^2(36) and by a nonglycosylated VSP (^3)were studied.

In nonencysting trophozoites, no CWP could be detected by immunoblot analysis (Fig. 4A, top and bottom, lane 1). After 2 h of culture in encysting medium, however, CWP appeared as a single 26-kDa protein, which was later processed to both higher and lower molecular weight species (Fig. 4A, top panel). The intracellular pool of CWP localized primarily in large encystation specific secretory vesicles (Fig. 4B). BFA treatment did not inhibit the release of secretory vesicle contents, but prevented passage of newly synthesized CWP into secretory vesicles and caused CWP accumulation in nuclear envelopes (Fig. 4B, 15-90 min). Consistent with the failure of CWP to enter the secretory pathway in BFA-treated encysting trophozoites, no post-translational processing of CWP occurred in cells incubated in the presence of the drug (Fig. 4A, bottom panel).


Figure 4: BFA inhibits CWP transport during encystation. A, Western blot analysis of CWP in trophozoites cultured in encystation medium for different time periods, in the absence or in the presence of BFA. BFA inhibits the processing of CWP to higher and lower molecular weight products. B, immunofluorescence of CWP in encysting trophozoites. BFA treatment causes a progressive accumulation of CWP into the nuclear envelopes. The nuclei are localized by propidium iodide staining in the left column.



We also compared the regulated transport of CWP in encysting trophozoites with the constitutive transport and secretion of a VSP. VSP is synthesized and released to the medium (34) by both nonencysting and encysting trophozoites and in nonencysting trophozoites is the major protein species secreted (Fig. 5, top panel). BFA inhibited protein secretion, including VSP secretion (Fig. 5, bottom panel), in both stages of Giardia.


Figure 5: BFA inhibits VSP secretion in encysting and nonencysting trophozoites. Top, trichloroacetic acid-insoluble material released into culture supernatants by metabolically radiolabeled nonencysting trophozoites, in the presence or in the absence of BFA, were analyzed by electrophoresis and autoradiography. The arrow indicates the position of the 1267 VSP detected by Western blot in a duplicate gel using monoclonal antibody 5C1(34) . Bottom, nonencysting (circle, bullet) and encysting (box, ) metabolically radiolabeled trophozoites were incubated in the presence (bullet, ) or in the absence (circle, box) of BFA. Cells were removed by centrifugation and the radioactivity associated with the trichloroacetic acid-insoluble material released into the supernatants measured. Values represent a mean of three experiments performed in duplicate ± S.D.



Redistribution of ARF and beta-COP in Response to BFA

To test whether secretory machinery involving ARF and coatomer functions in Giardia, we used antibodies to ARF and beta-COP (a subunit of coatomer) to assay for the presence of these proteins in encysting and nonencysting trophozoites. Immunoblot analysis using a polyclonal antibody raised against rgARF revealed a bimodal distribution of ARF in both encysting and nonencysting trophozoites (Fig. 6, top). BFA treatment led to the dissociation of ARF from membranes and its accumulation in cytosolic fractions at the top of the gradient in both trophozoite stages. Furthermore, antibodies to beta-COP and ARF (not shown) labeled discrete vesicular structures surrounding the nuclei, which disappeared upon BFA treatment, in encysting and nonencysting cells (Fig. 6, bottom).


Figure 6: ARF and beta-COP redistribute after BFA treatment in both encysting and nonencysting trophozoites. Top, ARF detection in Giardia subcellular fractions obtained from trophozoites treated or not treated with BFA. The vesicular association of ARF is inhibited by BFA. Bottom, in both encysting and nonencysting trophozoites, beta-COP localize to small perinuclear vesicles, which greatly diminish after BFA treatment.




DISCUSSION

A stack of flattened cisternae is the main feature of the Golgi apparatus in plant and mammalian cells. Nevertheless, neither the structure nor the distribution of the Golgi complex has been defined in Saccharomyces cerevisiae(51) , many fungi (52) and protozoa (6) . The protozoan G. lamblia has been reported to lack Golgi apparatus(1, 2, 4, 5, 6, 7) ; however, protein sorting to different cellular compartments is evident in this organism(5, 7, 53) , indicating that Giardia is capable of vectorial protein transport. The apparent absence of a Golgi structure in Giardia and other lower eukaryotes leads to the question of what type of machinery is used for protein transport, sorting, and secretion in these organisms.

In this study, we have identified several novel properties of the secretory pathway of Giardia. The most striking characteristic was the developmental induction of Golgi enzyme activities and Golgi structure upon encystation of trophozoites. Prior to this induction, no morphologically or biochemically identifiable Golgi complex existed within these cells. Specifically, we observed no subcellular staining with NBD-ceramide, a fluorescent lipid analogue specific for Golgi membranes(41) , and no Golgi enzyme activities (50) (including GalT and GalNAcT) in nonencysting trophozoites. In contrast, in trophozoites undergoing encystation (when a filamentous, N-acetylgalactosamine-rich cyst wall is being produced) (54, 55, 56) , we identified a discrete perinuclear structure labeled by NBD-ceramide and the presence of Golgi transferase activities in subcellular fractions. Importantly, the distribution of NBD-ceramide labeling within encysting cells and Golgi transferase activity in membrane fractions prepared from these cells was altered by treatment with BFA, a drug which has profound effects on mammalian Golgi structure and function(23) . Since induction of Golgi enzyme activities and NBD-ceramide staining occurred rapidly, within 4 h of placing nonencysting cells into encysting medium, specific signals appear to be involved in Golgi biogenesis within Giardia. These signals presumably coordinate the biosynthesis of Golgi enzymes and carbohydrate-containing secretory substrates for efficient cyst wall production in encysting trophozoites.

The interaction of the cytoplasmic domain of Golgi-resident enzymes with an intercisternal matrix, in addition to other protein-protein interactions such as oligomerization, has been proposed as a mechanism for maintaining the structural integrity of the Golgi complex in higher eukaryotes(57) . A bilayer-mediated mechanism of retention in the Golgi might also maintain the identity and morphology of this organelle(58, 59) . Several groups have shown that the length of the hydrophobic transmembrane domain of Golgi-resident enzymes is both necessary and sufficient for retention in the Golgi apparatus(50, 60) . Brestscher and Munro (58) suggested that the lipid content and thickness of Golgi membranes might favor the retention of particular enzymes in specific Golgi cisternae. Additionally, although the mechanism of NBD-ceramide accumulation in the Golgi apparatus is still unknown, it most likely occurs through physical interaction with specific lipids present in the membranes of this organelle(41, 61) . Taken together, these observations indicate an intimate relationship among composition, structure, and function of the Golgi apparatus. Since one of the principal functions of the Golgi apparatus is the biosynthesis of complex carbohydrate portions of lipids and proteins(10) , cells lacking such enzymes, or expressing them at very low level, may also lack a characteristic Golgi structure.

Golgi function is likely to be crucial for the biogenesis of the Giardia cyst wall during encystation, when this carbohydrate-rich structure must rapidly be synthesized. Nevertheless, BFA inhibited protein secretion in both nonencysting and encysting trophozoites. This suggests that despite the absence of Golgi structure in nonencysting trophozoites, the intracellular pathway followed by proteins within these cells resembles that of higher eukaryotes.

Dissection of the biochemical basis of BFA action revealed that this drug inhibits the membrane interaction of ARF, preventing the assembly of the coatomer onto Golgi membranes, an event required for ER-to-Golgi and intra-Golgi transport, as well as for maintenance of Golgi structure within mammalian cells(22, 62, 63) . In Giardia, subcellular localization of ARF and beta-COP established that both are associated with vesicular structures in encysting and nonencysting trophozoites and that association was sensitive to BFA. The localization of ARF and beta-COP was morphologically distinct from that of NBD-ceramide in encysting trophozoites. ARF and beta-COP were found primarily associated with small structures scattered around the nuclei. NBD-ceramide labeling, by contrast, was localized to a large juxtanuclear structure. The perinuclear location and overall appearance of the NBD-ceramide-labeled structures, and its sensitivity to BFA, resembled the Golgi complex in mammalian cells. This structure in Giardia, however, may represent a late Golgi compartment, as has been reported for NBD-ceramide labeling of the Golgi in mammalian cells(41) . If this compartment in Giardia is spatially segregated from early Golgi structures (where ARF and beta-COP function), this could explain the differential distribution of beta-COP and NBD labeling in these cells. Previous work in mammalian cells has demonstrated that beta-COP (and therefore ARF) is not restricted to central Golgi structures, but is also found in pre-Golgi membranes near the ER and in the nuclear envelope(64, 65) . Our analysis of subcellular fractions in encysting trophozoites showed a slightly different distribution of ARF and Golgi enzyme activities. We observed no difference in the distribution of ARF in the cellular fractions obtained from encysting and nonencysting cells, however. Future studies are needed to determine whether, upon encystation of Giardia trophozoites, Golgi enzymes localize to the compartment enriched in beta-COP and ARF or to the compartment enriched in NBD-ceramide.

These results and their implications lead to the intriguing question of how the Golgi complex in Giardia and other primitive eukaryotes should be defined. In cells with no morphologically evident Golgi apparatus, the minimal machinery for budding from the ER still involves ARF and beta-COP. In addition to their assumed membrane budding activity, coat proteins might posses other functions, such as sorting vesicles to different acceptor membranes(64) . It therefore may be useful to consider a broader definition of the Golgi complex involving one that includes the set of membranes and cytosolic factors involved in early transport, sorting, and processing events of molecules leaving the ER. Membranes comprising this set would exhibit diverse shapes and dynamics depending on the quantity and characteristics of the molecules being exported from the ER. Thus, upon induction of Golgi enzymes and substrates in Giardia, or after perturbation of late secretory transport steps as in budding yeast(51, 66) , Golgi structure and morphology might become evident, presumably due to the accumulation of substrates and Golgi enzymes within these membranes.

In summary, our results suggest that the minimal machinery for protein secretion in primitive eukaryotic cells like Giardia utilizes membrane association of ARF and beta-COP, but does not require a morphologically or biochemically identifiable Golgi complex to secrete simple, nonglycosylated proteins. Golgi biogenesis within these cells appears to coincide with the induction of Golgi-resident enzymes needed for the synthesis of carbohydrate-rich cyst wall components during encystation, indicating an intimate relationship between Golgi function and structure as discussed previously(9, 23, 57, 58, 59, 60, 65, 67) . This phenomenon was rapidly induced upon placing cells in encystation medium, suggesting that signal-dependent mechanisms regulate the biogenesis of this organelle. The results reported here are significant for our understanding of the evolution of the secretory pathways and make Giardia a unique model system for future studies examining Golgi biogenesis and function in eukaryotic cells.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Parasitic Diseases, NIAID, NIH, 9000 Rockville Pike, Bldg. 4, Rm. 126, Bethesda, MD 20892-0425. Tel.: 301-496-6920; Fax: 301-402-2689; hdl{at}4.niaid.nih.gov.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; ARF, ADP-ribosylation factor; gARF, Giardia ARF; rgARF, recombinant gARF; BFA, brefeldin A; VSP, variant-specific surface protein; CWP, cyst wall protein; PBS, phosphate-buffered saline; NBD, N-(-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl).

(^2)
Mowatt, H. D., Cotten, D. B., Bowers, B., Yee, J., Nash, T. E., Stibbs, H. H.,(1995) Mol. Microbiol., in press.

(^3)
H. D. Luján, M. R. Mowatt, J. J. Wu, Y. Lu, A. Lees, M. R. Chance, and T. E. Nash, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. J. Moss for the plasmid clone pGGARF, Dr. H. H. Stibbs for monoclonal antibody 5-3C, and Drs. J. G. Donaldson, J. S. Bonifacino, and R. D. Klausner for helpful discussions and critical reading of this manuscript.


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