(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 -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.
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 ()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. ()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.
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: ) 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.
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 () 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.
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 (,
) 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.
Figure 6:
ARF and -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.
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 -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.
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 -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 -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.