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(Received for publication, October 3,
1994; and in revised form, November 10, 1994) From the
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 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 ( 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. (
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) .
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:
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).
Figure 3:
BFA treatment results in a redistribution
of Golgi markers in encysting trophozoites. Giardia homogenates prepared from encysting or nonencysting trophozoites,
treated (
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 (
Figure 6:
ARF and
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 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 In summary, our results
suggest that the minimal machinery for protein secretion in primitive
eukaryotic cells like Giardia utilizes membrane association of
ARF and
Volume 270,
Number 9,
Issue of March 3, 1995 pp. 4612-4618
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
)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) .
)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.
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
10
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 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 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 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 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 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 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-
H]galactose or N-acetyl-[6-H]galactosylamine (DuPont
NEN) as substrates, respectively. Protein concentration was determined
according to Lowry et al.(46) .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--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 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.
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.
) none; 
, 12.5 µg/ml;
, 25 µg/ml; 
, 50 µg/ml; 
, 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.
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).
) or not treated () 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 µg of protein
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(36) and by
a nonglycosylated VSP ()were studied.
, ) and encysting (, )
metabolically radiolabeled trophozoites were incubated in the presence
(, ) or in the absence (, ) 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
To test whether secretory machinery involving ARF and
coatomer functions in Giardia, we used antibodies to ARF and
-COP in Response to
BFA-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 -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).
-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, -COP localize to
small perinuclear vesicles, which greatly diminish after BFA
treatment.
-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 -COP was morphologically distinct from
that of NBD-ceramide in encysting trophozoites. ARF and -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
-COP function), this could explain the differential distribution
of -COP and NBD labeling in these cells. Previous work in
mammalian cells has demonstrated that -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 -COP and ARF or to the compartment enriched in NBD-ceramide. -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.-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.
)-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl).))
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 H. G. Morrison, A. G. McArthur, F. D. Gillin, S. B. Aley, R. D. Adam, G. J. Olsen, A. A. Best, W. Z. Cande, F. Chen, M. J. Cipriano, et al. Genomic Minimalism in the Early Diverging Intestinal Parasite Giardia lamblia Science, September 28, 2007; 317(5846): 1921 - 1926. [Abstract] [Full Text] [PDF]
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N. Gottig, E. V. Elias, R. Quiroga, M. J. Nores, A. J. Solari, M. C. Touz, and H. D. Lujan Active and Passive Mechanisms Drive Secretory Granule Biogenesis during Differentiation of the Intestinal Parasite Giardia lamblia J. Biol. Chem., June 30, 2006; 281(26): 18156 - 18166. [Abstract] [Full Text] [PDF]
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S. Stefanic, D. Palm, S. G. Svard, and A. B. Hehl Organelle Proteomics Reveals Cargo Maturation Mechanisms Associated with Golgi-like Encystation Vesicles in the Early-diverged Protozoan Giardia lamblia J. Biol. Chem., March 17, 2006; 281(11): 7595 - 7604. [Abstract] [Full Text] [PDF]
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M. Marti, A. Regos, Y. Li, E. M. Schraner, P. Wild, N. Muller, L. G. Knopf, and A. B. Hehl An Ancestral Secretory Apparatus in the Protozoan Parasite Giardia intestinalis J. Biol. Chem., June 27, 2003; 278(27): 24837 - 24848. [Abstract] [Full Text] [PDF]
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M. C. Touz, H. D. Lujan, S. F. Hayes, and T. E. Nash Sorting of Encystation-specific Cysteine Protease to Lysosome-like Peripheral Vacuoles in Giardia lamblia Requires a Conserved Tyrosine-based Motif J. Biol. Chem., February 14, 2003; 278(8): 6420 - 6426. [Abstract] [Full Text] [PDF]
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J. G. Ellis IV, M. Davila, and R. Chakrabarti Potential Involvement of Extracellular Signal-regulated Kinase 1 and 2 in Encystation of a Primitive Eukaryote, Giardia lamblia. STAGE-SPECIFIC ACTIVATION AND INTRACELLULAR LOCALIZATION J. Biol. Chem., January 10, 2003; 278(3): 1936 - 1945. [Abstract] [Full Text] [PDF]
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M. C. Touz, N. Gottig, T. E. Nash, and H. D. Lujan Identification and Characterization of a Novel Secretory Granule Calcium-binding Protein from the Early Branching Eukaryote Giardia lamblia J. Biol. Chem., December 20, 2002; 277(52): 50557 - 50563. [Abstract] [Full Text] [PDF]
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N. Iwabe and T. Miyata Kinesin-Related Genes from Diplomonad, Sponge, Amphioxus, and Cyclostomes: Divergence Pattern of Kinesin Family and Evolution of Giardial Membrane-Bounded Organella Mol. Biol. Evol., September 1, 2002; 19(9): 1524 - 1533. [Abstract] [Full Text] [PDF]
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J. B. Dacks and W. F. Doolittle Novel syntaxin gene sequences from Giardia, Trypanosoma and algae: implications for the ancient evolution of the eukaryotic endomembrane system J. Cell Sci., April 15, 2002; 115(8): 1635 - 1642. [Abstract] [Full Text] [PDF]
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M. C. Touz, M. J. Nores, I. Slavin, C. Carmona, J. T. Conrad, M. R. Mowatt, T. E. Nash, C. E. Coronel, and H. D. Lujan The Activity of a Developmentally Regulated Cysteine Proteinase Is Required for Cyst Wall Formation in the Primitive Eukaryote Giardia lamblia J. Biol. Chem., March 1, 2002; 277(10): 8474 - 8481. [Abstract] [Full Text] [PDF]
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A. B. Hehl, M. Marti, and P. Köhler Stage-Specific Expression and Targeting of Cyst Wall Protein-Green Fluorescent Protein Chimeras in Giardia Mol. Biol. Cell, May 1, 2000; 11(5): 1789 - 1800. [Abstract] [Full Text]
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S. E. Francis, R. Banerjee, and D. E. Goldberg Biosynthesis and Maturation of the Malaria Aspartic Hemoglobinases Plasmepsins I and II J. Biol. Chem., June 6, 1997; 272(23): 14961 - 14968. [Abstract] [Full Text] [PDF]
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B. Soltys, M Falah, and R. Gupta Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy and antibody to Bip J. Cell Sci., January 7, 1996; 109(7): 1909 - 1917. [Abstract] [PDF]
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H. D. Luján, M. R. Mowatt, J. T. Conrad, B. Bowers, and T. E. Nash Identification of a Novel Giardia lamblia Cyst Wall Protein with Leucine-rich Repeats J. Biol. Chem., December 8, 1995; 270(49): 29307 - 29313. [Abstract] [Full Text] [PDF]
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