From the Department of Internal Medicine, Section of Infectious Diseases, Yale University School of Medicine, New Haven, Connecticut 06520-8022
Received for publication, September 12, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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Toxoplasma gondii dense
granules are morphologically similar to dense matrix granules in
specialized secretory cells, yet are secreted in a constitutive,
calcium-independent fashion. We previously demonstrated that secretion
of dense granule proteins in permeabilized parasites was augmented by
the non-hydrolyzable GTP analogue guanosine
5'-3-O-(thio)triphosphate (GTP Remarkable progress has been made recently in understanding the
protein machinery responsible for secretory events in mammalian and
yeast cells (1). The components involved in both constitutive secretion
as well as regulated release in secretory cells are broadly conserved
evolutionarily (2). Although this suggests that the same should be true
in protozoan parasites, the presence of unusual secretory organelles in
these organisms confounds this simple hypothesis. By the same argument,
parasites provide a unique means to explore selected issues in
regulation of secretion from preformed organelles (reviewed in Ref.
3).
We have used this logic to explore the organization of the secretory
pathway in the protozoan parasite Toxoplasma gondii. This
parasite is an obligate intracellular pathogen that resides in the host
cell within a specialized compartment, the parasitophorous vacuole
(PV),1 which is separated
from the host cell cytoplasm by the parasitophorous vacuole membrane
(PVM). The success of infection depends on the ability of the parasite
to modify the PV and the PVM by secreting proteins to the
extra-parasite environment (reviewed in Ref. 4). Thus, secretion is a
critical feature of parasite survival.
T. gondii has a well-developed secretory system that
includes endoplasmic reticulum, Golgi, and three morphologically
distinct secretory organelles: micronemes, rhoptries, and dense
granules (DG), which discharge sequentially during and after cell
invasion (reviewed in Ref. 5). Micronemes and rhoptries are
morphologically unique organelles, which discharge only at the time of
cell invasion, and are therefore strictly regulated. Dense granules are
morphologically similar to dense matrix granules in specialized
secretory cells. Although a burst of dense granule exocytosis is
described immediately following cell invasion by T. gondii,
potentially consistent with regulated release (6, 7), substantial dense
granule release occurs throughout the intracellular residence of the
organism, consistent with a constitutive process. We have previously
argued that soluble proteins are delivered quantitatively and by bulk flow to dense granules (8), rather than to more conventional constitutive secretory vesicles, containing surface proteins of the
parasite such as SAG1, making the dense granule pathway highly unique.
Because constitutively secreted dense granule proteins are packaged in
a distinctive organelle, T. gondii provides a more tractable
system for studying control of vesicle docking and fusion at the plasma
membrane than is the case in many other cells.
For this reason, we previously developed a permeabilized cell secretion
assay, using the pore forming protein streptolysin O (9), to explore
the machinery mediating the unusual exocytic process for dense
granules. Release was not only constitutive, but also
calcium-independent (9, 10), an important distinction when compared to
release of dense matrix granules in mammalian cells. Addition to
permeabilized parasites of hamster NSF and bovine To pursue this last point, we have now characterized the role of the
GTP binding protein ADP-ribosylation factor-1 (ARF1) in dense granule
release. ARF proteins, which are broadly conserved, were first
characterized by their ability to activate the cholera toxin-mediated
ADP-ribosylation of the Our interests for the current work relate to the role of ARF1 in
secretion from the trans-Golgi network. In mammalian cells, ARF1
stimulates release of nascent secretory vesicles from the trans-Golgi
network (19), acting through phospholipase D (20). A role of ARF1 in
regulated exocytosis in mammalian cells has also been identified (21,
22). In this situation, ARF1 enhances the regulated secretion of
preformed granules, release of which is induced physiologically by a
rise in intracellular calcium following secretagogue addition. Although
no evidence has previously existed to support a role for ARF1 in the
docking and fusion of constitutively secreted vesicles, we provide such
evidence in the current paper. The finding that ARF1 can facilitate
docking and fusion of constitutively secreted dense granules was made possible by the unusual features of the T. gondii secretory system.
Cloning of ARF1
An expressed sequence tag (EST) clone encoding a fragment of an
ARF1 homologue (W66111/gi:374337) was identified from the T. gondii dbEST sequence data base. The ARF1 open reading frame in
this sequence appeared to be interrupted by an unknown fragment. To
subclone a full-length ARF1 cDNA, T. gondii RNA was
reverse-transcribed with Superscript II Reverse Transcriptase (Life
Technologies, Inc., Gaithersburg, MD) for use as the PCR template. A
3'-RACE PCR strategy was employed in which the 5'-end (sense strand)
oligonucleotide primers TARFUT (5'-AAA CAC GCG TCC TCT CTC TGC AAG
CGA-3') and TARF1 (5'-GGG CAC CAT GGG TTT GAG CGT CAG C-3') were used
in sequential PCR reactions with the 3'-(antisense)
oligo-dT17 polyadenylation site anchor primer. In the first
round, TARFUT and oligo-dT17 primers were used for cDNA
amplification with Taq DNA polymerase (Roche Molecular
Biochemicals, Branchburg, NJ). The products were purified on QIAquick
PCR columns (Qiagen, Valencia, CA) and used as template in a second
round PCR reaction with primers TARF1 and oligo-dT17. The
resulting products were again purified on QIAquick columns and
subcloned into pGEM-T (Promega, Madison, WI). To identify 3'-RACE PCR
product clones from the resulting colonies, an internal ARF1 probe was
generated by PCR from T. gondii cDNA using the known EST
sequence with primers TARF1 and TARF2 (5'-GCG ATC TCT GTC GTT GCT
GT-3'). The single PCR product was agarose gel-purified and labeled by
random oligonucleotide priming with [32P]dCTP and the
Rediprime II kit (Amersham Pharmacia Biotech, Piscataway, NJ). Colony
hybridization with this probe identified several clones which contained
~0.8-kbp inserts, and sequencing analysis confirmed the presence of a
full ARF1 homologue open reading frame (ORF) and 3'-untranslated
region. For expression in the parasite, an HA epitope tag with a
BglII cloning site was engineered for targeting of the
3'-antisense end of the ARF1 ORF in primer ARFHAR (5'-ACT AGA TCT AAG
CGT AGT CTG GGA CGT CGT ATG GGT AAT CGA TGT TTT TCT GCG CAA G-3'). The
TgARF1 RACE clone ORF was PCR-amplified with TARF1, encoding an
NcoI site, and ARFHAR, and subcloned into the NcoI-BglII cloning site of pNTPRab11, utilizing
the T. gondii NTPase3 promoter cassette for
expression.2 The TgARF1
cDNA sequence has been deposited in GenBankTM (accession number
AF227524).
Mutagenesis of TgARF1
Site-directed mutagenesis of TgARF1-HA was performed using a
two-independent PCR amplification approach followed by triple ligation.
For TgARFQ71L, the 237-bp PCR product from primers TARF1 and ARFQ71L
(5'-GAA GTA GTG GCG CCA CAG AGG ACG AAT CTT GTC CAG TCC ACC
G-3') was digested with NcoI and NarI. A
333-bp TgARF1 fragment was prepared by digestion with NarI
and BglII, and the two products were subcloned in vector
pNTPRab11 following removal of the Rab11 open reading frame with
digestion by NcoI and BglII. A plasmid encoding
the untagged TgARF1T31N mutation was obtained from Kristen Hager and
David Roos (University of Pennsylvania) and used for amplification with
TARF1 and ARFHAR, digested with NcoI and BglII,
and subcloned into pHXNTPRab11 as above. TgARF1 lacking the 17 N-terminal residues (TgARF1d17-HA) (19, 23, 24) was constructed in the
same vector. Plasmid sequences were verified by dideoxynucleotide
sequencing at the WM Keck Sequencing Center, Yale University School of Medicine.
Overexpression and Purification of Recombinant TgARF1-HA
Protein
The TgARF1 coding sequence with a C-terminal epitope tag (HA)
was subcloned into pET-24d for overexpression in BL21(DE3) E. coli cells, which do not express TgARF1 was inserted into the pET vector between the restriction sites
NcoI (at the initiating Met of ARF) and BamHI.
Expression of the protein was induced with
isopropylthio- SDS-PAGE and Immunoblotting
The expression of recombinant protein TgARF1-HA was monitored
using SDS-PAGE, and immunoblots were performed as previously described
(8). Immunoblots were developed using an anti-HA monoclonal antibody
(1:1000) or a goat anti-human ARF1 (1:500), followed by goat anti-mouse
or rabbit anti-goat IgG-horseradish peroxidase conjugation (1:2000) and
analysis using the ECL detection system (Amersham Pharmacia Biotech, UK).
Parasites
T. gondii tachyzoites were maintained by serial
passage in monolayers of either African Green Monkey (Vero) cells or
human foreskin fibroblasts (HFF) grown in modified Eagle's minimal
medium or Parasite Permeabilization with SLO and Cytosol Depletion
Permeabilization of extracellular parasites with SLO was
performed using a protocol described earlier (9). Assessment of permeabilization was done by staining SLO permeabilized parasites with
4 µg/ml propidium iodide for 5 min at room temperature. The percentage of positive nuclear staining was quantitated by fluorescence microscopy.
Immunofluorescence Assay (IFA)
Use of HA Epitope Tag to Localize the Transgenes in Transiently
Transfected Parasites--
The detailed technique is described in
Karsten et al. (8). Briefly, confluent HFF cell monolayers
(12-mm coverslips) were infected with transiently transfected
parasites. Transient transfection was performed by electroporation.
After 16-24 h, cells were fixed and permeabilized with 3%
paraformaldehyde in phosphate-buffered saline and 0.1% Triton X-100
and incubated with anti-HA monoclonal antibody (Babco, Richmond, CA)
(1:200), followed by FITC or tetramethyl rhodamine-conjugated goat
anti-mouse IgG (1:500). Coverslips were mounted in Mowiol and
observed with an epifluorescence microscope. Images were captured with
a charge-coupled device camera.
Localization of Stably Expressed BAP-LDLR--
Confluent
monolayers of HFF cell were infected with a Toxoplasma
stable line expressing BAP-LDLR (26). Cells were fixed and
permeabilized as described earlier after 16-24 h of infection. As
previously described (8), a purified rabbit anti-BAP polyclonal was
used as the first antibody (1:1000) and a goat anti-rabbit rhodamine-conjugated was used as secondary antibody (1:500).
Anti-TgARF1 Antibody and Localization of Endogenous
TgARF1--
Purified recombinant ARF1-HA was sent to Cocalico
Biologicals, Inc. for production of a polyclonal rabbit antiserum.
Localization of endogenous TgARF1 in T. gondii was done by
IFA. HFF cells were infected with tachyzoites (RH) for 16-24 h. After
fixation and permeabilization as described above, cells were stained
with the anti-TgARF1-HA antibody (1:100) followed by FITC-conjugated
goat anti-rabbit antibody (1:500).
Secretion Assay and Enzymatic Assay for BLA
Secretion assays were performed as described in a previous study
(9). In brief, extracellular parasites (5 × 107
parasites/ml) expressing the soluble secretory reporter BLA were incubated with various reagents in modified potassium acetate buffer
(115 mM potassium acetate, 2.5 mM
MgCl2, 10 mM glucose, 25 mM HEPES,
pH 7.2) at 37 °C for 30 min. After the incubation, parasites were
centrifuged at 760 × g for 10 min at 4 °C.
Supernatant was further centrifuged at 7000 × g for 10 min at 4 °C.
Determination of BLA in parasite supernatants was done by the method
described earlier (9) with minor modifications. 40 µl of parasite
supernatant (5 × 107 parasites/ml) were added to each
well of 96-well plate. 160 µl of nitrocefin mix (0.2 mM
nitrocefin, 0.25 mg/ml bovine serum albumin, 50 mM
potassium phosphate, pH 7.0) was added to develop the reaction. Samples
were incubated for 20 min at 25 °C. Plates were read at 492 nm in an
enzyme-linked immunosorbent assay reader.
Electron Microscopy
Immunocytochemistry and Electron Microscopy--
Infected Vero
cell monolayers were washed twice with phosphate-buffered saline and
fixed with 8% paraformaldehyde in 0.25 M Hepes, pH 7.4, for 2 days at 4 °C. Sections were obtained in the Yale Center for
Cell Imaging using a Leica Ultracut microtome with fetal calf serum
cryoattachment at Tannic Acid Fixation--
Extracellular parasites were
permeabilized with SLO as described previously (9). GTP GTP Binding Assay
Binding of [35S]GTP Augmented Dense Granule Protein Release with GTP
Because this did not address the influence of GTP binding proteins on
DG protein release, we next asked whether there were any qualitative or
quantitative differences in the morphology of DG exocytosis, in the
presence or absence of GTP
Altogether, these results suggest that a T. gondii GTP
binding protein enhances exocytosis of preformed dense granules in a
calcium-independent fashion. We therefore turned our attention to the
role of the T. gondii GTP binding protein, ARF1, in this process.
Cloning and Sequence Analysis of ARF1--
The
Toxoplasma data base of expressed sequence tags (EST)
revealed a cDNA sequence (W66111) with partial homology to human ARF1. Using a PCR RACE approach, we amplified from parasite cDNA the full-length T. gondii ARF1 homologue. Sequence analysis
of cDNA clones revealed an open reading frame encoding a
polypeptide of 183 amino acids, with a predicted molecular mass
of 21 kDa and an isoelectric point of 6.8. The protein sequence
contains prototypical GTP and Mg2+ binding sites and switch
regions (Fig. 3) characteristic of
RAS-related proteins, and an additional C-terminal signature sequence
for the ADP-ribosylation factor family. BLASTN and BLASTP analyses of
the sequence data bases through NCBI revealed strongest homology to
ARF1 proteins of plants, animals, yeast, and protozoa, and we named the
T. gondii homologue TgARF1 accordingly. The highly conserved
switch regions of the ARF1 subfamily serve in part as effector binding
sites for phospholipase D. Although the T. gondii sequence
varies little in these conserved domains from those in the well
characterized mammalian and yeast sequences, a Ser-83 residue adjacent
to the switch 2 domain is at a site conferring differential stimulation
of phospholipase D1 between yeast and mammalian ARF1 (30).
TgARF1-HA Localizes to the Golgi/TGN by Immunofluorescence and
Immunoelectron Microscopy--
We examined the localization of TgARF1
in T. gondii. A C-terminal HA epitope tag was added, and the
TgARF1-HA construct was transiently expressed in T. gondii.
Staining with an anti-HA antibody revealed that TgARF1-HA localized
predominantly to a region anterior to the nucleus, consistent with the
Golgi/TGN of the parasite (Fig.
4A). No staining was apparent
on dense granules. This localization pattern was confirmed with rabbit
antiserum to TgARF1, raised against the recombinant protein expressed
in E. coli (Fig. 4D). Brefeldin A, which disrupts
the T. gondii Golgi (31), induced a redistribution of TgARF1
to the nuclear envelope and ER (not shown). These results are all
concordant with the known localization of ARF1 to the Golgi complex in
mammalian cells.
This result was confirmed by two separate approaches. First, thin
section cryoimmunoelectron microscopy was done using the rabbit
anti-TgARF1 antiserum. As shown in Fig.
5, TgARF1 localized to the Golgi stacks.
No TgARF1 staining of dense granules was observed. Second, a stable
line of T. gondii expressing bacterial alkaline phosphatase
fused with the LDL receptor (BAP-LDLR) was transiently transfected with
TgARF1-HA. As previously assessed by immunoelectron microscopy,
BAP-LDLR localizes to the Golgi/TGN of the parasite (26). Transiently
transfected TgARF1-HA co-localized with BAP-LDLR in the Golgi region
(Fig. 6A), confirming
localization of TgARF1-HA to the Golgi/TGN of the parasite.
Expression and Localization of TgARF1 Mutants--
We generated
two mutants of TgARF1, which are impaired in the GTP binding or
hydrolysis cycle. By analogy to the Ras-Q61L mutation, which inhibits
GTP hydrolysis but not binding, a Q71L mutation was introduced in
T. gondii TgARF1-HA (25) and transiently expressed in the
organism. The Q71L mutant localized to the Golgi as well as to
dispersed punctate structures (Fig. 4B) in comparison to the
discrete Golgi/TGN structure observed with wild type TgARF1-HA. Thus,
the Q71L mutant appears to behave as in mammalian cells, where this
mutant has distinct effects on the structure and/or function of ER,
Golgi apparatus, and endocytic pathway (32). We also generated a T31N
mutant of T. gondii TgARF1, a mutant predicted to be
defective in GTP binding (19). Following transient transfection of the
epitope-tagged protein, the T31N mutant was diffusely distributed
throughout the parasite cytosol (Fig. 4C), likely due the
inability to bind GTP. Together, these results demonstrate that the
localization of TgARF1 within the parasite is coupled to the GTP cycle,
as has been described for ARF1 in other systems (reviewed in Ref.
33).
Effect of TgARF1 Mutants on Organelle Structure--
The effect of
overexpression of TgARF1Q71L-HA on the localization of BAP-LDLR was
assessed. TgARF1Q71L-HA was transiently transfected into the BAP-LDLR
stable line. In contrast to wild-type TgARF1-HA, this mutant altered
the localization of BAP-LDLR, consistent with partial disruption of the
Golgi/TGN (Fig. 6B), as shown above. Mechanistically, this
alteration in BAP-LDLR localization may be due to both an enhancement
of retrograde transport and to augmented anterograde flow from the TGN.
A similar partial dispersion of the BAP-LDLR signal was seen with the a
T31N mutant (not shown).
Effects of TgARF1 Mutants on Localization of Dense Granule
Proteins--
We assessed the effects of TgARF1 mutants on transport
of proteins to dense granules. Native TgARF1, and TgARF1 mutants
ARF1T31N and ARF1Q71L, were transiently overexpressed in a parasite
clone stably expressing the soluble dense granule secretory reporter BAP (8) or in wild type parasites. As shown in Fig.
7, in parasites transfected with
TgARF1T31N, discrete labeling of BAP (Fig. 7B) or GRA3 (Fig.
7K) in dense granules was reduced and replaced by a
reticular endoplasmic reticulum-like staining pattern. Similarly, in
parasites transfected with the TgARF1Q71L mutant, labeling of dense
granules for BAP (Fig. 7E) or for GRA3 (Fig. 7N)
was partially replaced by perinuclear and posterior reticular staining. Less GRA3 was detected at the parasitophorous vacuole membrane, with
both the TgARF1T31N (Fig. 7K) and TgARF1Q71L (Fig.
7N) mutants. Staining for GRA3 in the wild type TgARF-1
transfectants was variably altered, with mild effects in most cells
(Fig. 7H). These results suggest that one predominant effect
of both Q71L and T31N mutants is to block delivery of soluble proteins
to dense granules and to induce accumulation of the secretory proteins
in the ER. This observation precluded an assessment of ARF1 effects on
post-Golgi release of dense granule proteins.
Expression and Purification of Recombinant TgARF1 Protein--
We
therefore opted to analyze the effects of TgARF1 on dense granule
secretion in the permeabilized cell assay, using purified TgARF1-HA
protein. TgARF1-HA was expressed in E. coli using the pET
system. Induction with 1 mM IPTG of BL21(DE3) cells
carrying the TgARF1-HA/pET-24d construct resulted in a
time-dependent increase in the accumulation of TgARF1-HA
protein, which continued for at least 120 min (not shown). The induced
protein migrated at ~21 kDa on SDS-PAGE.
Previous work overexpressing human and bovine ARF1 in E. coli has shown that recombinant ARF1 proteins are easily
solubilized from the bacterial pellet with TX-100 and lysozyme
treatment or using a French press cell. Our results confirmed these
facts. Recombinant TgARF1-HA was solubilized from IPTG-induced
bacterial cell pellets (Fig. 8,
lane 2). Different protocols were attempted to purify
recombinant TgARF1-HA. First, the protocol described for human and
bovine ARF1 (25) was used. This protocol consists of a DEAE-Sephacel
column as a first step, where typically almost all of the bacterial
proteins are absorbed, while ARF1 is not retained. In our experiments,
recombinant TgARF1-HA was adsorbed to the DEAE matrix along with the
bacterial proteins (not shown), necessitating further purification on
an AcA 54 Ultrogel column (not shown). Second, we used an
immunoaffinity column, containing monoclonal anti-HA antibodies
cross-linked to a Sepharose matrix (Babco, Richmond, CA). Even though
the antibody recognized TgARF1-HA in immunoblots, the recombinant
TgARF1-HA was not absorbed to the column under the various conditions
tested (not shown). Finally, purification of the soluble TgARF1-HA was
done using a Cibacron G3 column, equilibrated with buffer A. Cibracon
G3 is a triazine dye column, used as an affinity resin for binding
nucleotide-dependent enzymes. TgARF1-HA remained adsorbed
to this column, and was not present in the column flow (Fig. 8,
lane 3), even after a 1 M NaCl wash (not shown).
TgARF1 was eluted with 1.2 M NaCl (Fig. 8, lane
4). The second purification step consisted of a gel filtration column, AcA 54 Ultrogel, equilibrated in buffer B (10 mM
potassium phosphate, pH 7.4, 1 mM EDTA, 100 mM
NaCl, 1 mM DTT). The column was developed in the same
buffer at a flow rate of 19 ml/h, and 2.5-ml fractions were collected.
Pooled fractions containing purified TgARF1-HA from the Ultrogel column
are shown in Fig. 8, lane 5. This band was determined to be
epitope-tagged TgARF1-HA by specific recognition (not shown) with the
monoclonal anti-HA antibody, the correct molecular size, and the
absence of the same band in control cells, carrying the pET24-d plasmid
without the coding sequence for TgARF1 (Fig. 8, lane 1).
We tested the ability of purified recombinant TgARF1-HA to bind
GTP Recombinant TgARF1 Stimulates BLA Release from Permeabilized
Parasites--
Finally, we examined the influence of recombinant
TgARF1-HA on the release of the secretory reporter BLA in permeabilized T. gondii. Extracellular tachyzoites were permeabilized with
1 unit/ml streptolysin O, cytosol was depleted, and BLA release was
measured, as described previously (9) and under "Materials and
Methods." Three separate experiments are illustrated in Fig. 10, A, B, and
C. Slightly different experimental variables were tested in
the three experiments. ARS alone but not GTP Our data suggest that T. gondii TgARF1 stimulates
post-Golgi secretion from preformed DG. This represents a specialized
function for TgARF1 in this apicomplexan parasite. A related function
has been described for ARF1 in selected secretory cells, in which ARF1
mediates the release of preformed granules in the presence of GTP In mast cells, ARF1-mediated enhancement of granule secretion is
thought to occur via activation of phospholipase D. PLD hydrolyses phosphatidylcholine to generate PA and choline. The conversion of
phosphatidylcholine to PA alters the lipid bilayer properties, replacing a non-fusogenic phospholipid with a fusogenic one (34), potentially stimulating preformed granules to fuse and release their
contents to the extracellular environment. In addition, PA generated by
PLD regulates a phosphatidylinositol 5-kinase, resulting in
enhanced synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). Elevated levels of PIP2 stimulate mast
cell exocytosis. In contrast, in both chromaffin and PC12 cells, ARF1
stimulates granule release in a process not dependent on PLD
activation. It is of note that ethanol, which diverts PA to PE and
blocks PIP2 generation in mammalian cells, also inhibits
dense granule secretion in T. gondii (35). This result
suggests that TgARF1 augments DG release in a PLD-dependent
fashion. Successful purification of functionally active TgARF1d17-HA
capable of binding GTP There are several non-mutually exclusive alternatives to the scenario
presented above. It is possible that TgARF1 is involved in a triggered
component of dense granule release. In this scenario, the secretion of
preformed dense granules measured in the permeabilized cell assay in
the absence of GTP TgARF1 was not detected on the dense granule membrane, either by
immunofluorescence or immunoelectron microscopy. This does not obviate
a role for TgARF1 in mediating release of preformed dense granules,
especially if a TgARF1-regulated phospholipase D activity localizes to
the cortical cisternae or plasma membrane (37). Furthermore,
translocation of ARF1 to secretory granule membranes of rat parotid
acinar cells, specifically in the presence of GTP Our data suggest that TgARF1 is also involved in maintaining the
structure of the T. gondii Golgi and TGN. In mammalian
cells, ARF1 supports the GTP-dependent association of COPI
with Golgi membranes (39-41). Localization of T. gondii
TgARF1 to the Golgi/TGN is consistent with such a function in
vivo in the parasite. Although it is apparent that transfected
TgARF1 affects delivery of secretory reporters to dense granules (Fig.
7), likely by effects on the Golgi, this is not likely to be
the case in the permeabilized cell assay.
The functions for ARF proteins in the secretory and endocytic pathways
of mammalian cells are now quite broad. Although it is assumed that
TgARF1 will have most analogous functions in T. gondii, this
is yet to be formally established. The parasite has a Golgi-ER
retrieval system that is reminiscent of the process in higher
eukaryotes (42) and has components of a COP1 coat (31), confirming the
expectation from the morphologic consequences of Brefeldin A treatment.
T. gondii expresses the AP-1 adaptor complex, and sorts
proteins in an AP-1-dependent fashion, suggesting that
TgARF1 will also participate in this step
(26).4
AP-3-dependent sorting is also ARF1-dependent
(43, 44), and circumstantial evidence (26) argues in favor of an
AP-3-dependent sorting pathway in Toxoplasma. We
expect that the unusual features of the T. gondii secretory
and endocytic pathways may allow further insights into ARF1 functions,
as these pathways are explored in the parasite.
S) (Chaturvedi, S., Qi,
H., Coleman, D. L., Hanson, P., Rodriguez, A., and Joiner, K. A. (1998) J. Biol. Chem. 274, 2424-2431). As now
demonstrated by pharmacological and electron microscopic approaches,
GTP
S enhanced release of dense granule proteins in the
permeabilized cell system. To investigate the role of ADP-ribosylation
factor 1 (ARF1) in this process, a cDNA encoding T. gondii ARF1 (TgARF1) was isolated. Endogenous and transgenic
TgARF1 localized to the Golgi of T. gondii, but not to
dense granules. An epitope-tagged mutant of TgARF1 predicted to be
impaired in GTP hydrolysis (Q71L) partially dispersed the Golgi signal,
with localization to scattered vesicles, whereas a mutant impaired in
nucleotide binding (T31N) was cytosolic in location. Both mutants
caused partial dispersion of a Golgi/trans-Golgi network marker.
TgARF1 mutants inhibited delivery of the secretory reporter,
Escherichia coli alkaline phosphatase, to dense granules,
precluding an in vivo assessment of the role of TgARF1 in
release of intact dense granules. To circumvent this limitation,
recombinant TgARF1 was purified using two separate approaches, and used
in the permeabilized cell assay. TgARF1 protein purified on a Cibacron
G3 column and able to bind GTP stimulated dense granule secretion in
the permeabilized cell secretion assay. These results are the first to
show that ARF1 can augment release of constitutively secreted vesicles
at the target membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-SNAP increased
the secretion of the stably transfected dense granule secretory
reporter
-lactamase (BLA). In contrast, bovine Rab GDP
dissociation inhibitor blocked BLA secretion, suggesting altogether
that the NSF/SNAP/SNARE/Rab machinery participates in dense granule
release. The non-hydrolyzable GTP analogue GTP
S significantly
enhanced BLA secretion in the presence of an ATP regenerating system
(ARS), indicating that one or more GTP binding proteins were implicated
in dense granules exocytosis.
-subunit of the heterotrimeric G proteins
(11). ARFS exist in two stages, bound to guanidine nucleotides: the
GDP-bound "off" form, which is cytosolic, and the GTP-bound active
form, which interacts with Golgi membranes and phospholipid vesicles. A
major function of ARF1 is to drive, in a GTP-dependent
cycle, the assembly of sets of cytosolic coat proteins onto Golgi
membranes facilitating vesicle budding and conferring the timing for
the release of coat proteins to the cytosol (12, 13). ARF1 was
initially described to recruit the COPI coat involved in traffic
between the ER and Golgi. More recent data demonstrate that ARF1 plays
a role in generating clathrin-coated vesicles, by first recruiting
adaptor complexes on vesicles at the trans-Golgi network (TGN) and
immature secretory vesicles (14-17). Additional effects of ARF1 on the
cytoskeleton are also described (18).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-lactamase (BLA). This
precludes contamination of purified TgARF1-HA with BLA, which is
essential for subsequent assays monitoring BLA release from stably
transfected T. gondii. Even highly purified preparations of
human ARF1 and ARF1 mutants (obtained from D. Shields, New York, NY)
generated in standard vectors have sufficient residual BLA
contamination to invalidate secretion
assays.3
-galactoside (IPTG, 1 mM) for 4 h at
37 °C and bacterial cells were collected by centrifugation. TgARF1-HA was solubilized from the bacterial pellet using a French press at 8,000 p.s.i. in buffer A (50 mM Tris, 2 mM EDTA, 1 mM DDT and 1 mM
phenylmethylsulfonyl fluoride). The lysate was clarified by
centrifugation at 18,000 × g. Two different protocols
were used to purify recombinant TgARF1. First, the protocol described for human and bovine ARF1 (25) was used, consisting of a DEAE-Sephacel column as a first step. The second purification step consisted of a gel
filtration column, AcA 54 Ultrogel, equilibrated in buffer B (10 mM potassium phosphate, pH 7.4, 1 mM EDTA, 100 mM NaCl, 1 mM DTT). The column was developed in
the same buffer at a flow rate of 19 ml/h; 2.5-ml fractions were
collected. Pooled fractions containing ARF1-HA from the Ultrogel column
were concentrated using a Centricon-3 concentrator (Amicon, Beverly,
MA). Samples were kept at
80 °C. Second, a purification protocol
for soluble TgARF1-HA was developed using as a first step a dye column,
Cibacron G3, equilibrated in buffer A. Subsequent wash with 1 M NaCl was performed, and the elution of the active protein
was performed with 1.2 M NaCl. Fractions containing
TgARF1-HA were pooled and concentrated using Centricon-3 concentrators
(Amicon). The second step was a gel filtration column, AcA 54 Ultrogel,
as described above. Purified recombinant TgARF1-HA was frozen
immediately at
80 °C.
-minimal essential medium, respectively,
supplemented with 7.5% fetal bovine serum. The RH strain and a stable
transgenic clone of the RH strain, expressing the soluble foreign
secretion reporter E. coli
-lactamase (BLA) were
described previously (8). The stable transgenic clone of the RH strain,
expressing bacterial alkaline phosphatase fused with the LDL receptor
(BAP-LDLR) is as previously described (26). For experiments with
extracellular parasites, infected cells were scraped, and parasites
were isolated by two passages through a 27-gauge needle.
108 °C. Immunolabeling was performed with rabbit
anti-TgARF1 antisera (1:50) followed by protein A-gold (1:70, from the
laboratory of J. Slot, Utrecht, Holland). The sections were contrasted
with neutral uranyl acetate (2%), infiltrated with methyl cellulose
(1.8%) and uranyl acetate (0.5%), air-dried, and examined with a
Philips 410 transmission electron microscope.
S and tannic
acid arrest of secretion is similar to the methodology described in
Newman et al. (29). For 8 min at 37 °C, permeabilized
parasites were incubated with or without 100 µM GTP
S,
2% tannic acid, and an ATP-regenerating system containing 2 mM ATP. Between washes, samples were fixed sequentially
with 2% glutaraldehyde and 1% osmium tretroxide in 100 mM
sodium cacodylate buffer. Fixed samples were processed for Epon
embedding and sectioned for transmission electron microscopy using
standard protocols.
S to TgARF1-HA was
determined essentially as described previously (11). Purified
recombinant TgARF1-HA or TgARF1 mutants were incubated at 30 °C with
1 µM [35S]GTP
S (2.8 × 106 cpm) in 100 µl of 2 mM Tris-HCl (pH 8),
50 mM potassium acetate, 2.5 mM magnesium
acetate, 1 mM EDTA, and 1 mM DTT. After 2 h at 30 °C, samples were filtered through nitrocellulose filters.
The filters were washed three times with the same buffer. Radioactivity retained by the nitrocellulose filters was quantified with a liquid scintillation analyzer (Packard 2500 TR).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S Reflects
Increased Dense Granule Docking and Fusion--
We have previously
argued that soluble proteins are delivered to and retained
quantitatively in T. gondii dense granules, via a bulk flow
pathway (8). Because this result was not expected, we considered at the
time the alternative that a portion of dense granule proteins was
released via a budding process from forming dense granules, analogous
to the situation in pancreatic
-cells (27). Failure to block dense
granule protein release using Brefeldin A was interpreted at the time
to provide evidence that all release was from preformed dense granules.
However, it has recently been demonstrated that Brefeldin A can augment
release of endosomes containing soluble proteins previously routed
through immature secretory granules (28). To determine if this process
was operative in T. gondii, parasites were treated with
wortmannin, which blocks trafficking of lysosomal proenzymes to
endosomes, by inhibiting AP-1/clathrin-coated vesicles formation
from immature secretory granules (28). No inhibition of either BLA or
GRA3 secretion was observed in intact parasites (Table
I). This result supports the original
argument that soluble proteins in T. gondii are delivered and retained quantitatively within dense granules.
Wortmannin does not alter release of T. gondii dense granule proteins
S, using the permeabilized cell system
(9). We took advantage of a previously described approach, involving
fixation with tannic acid (29), to allow the capture of multiple DG
docking/fusion events. As shown in Figs.
1 (A and B) and
2, DG docking and fusion events were
readily detected and quantitated (see morphologic definitions for
docking and fusion in the legend of Fig. 2) in the presence of tannic
acid, ARS, and GTP
S. In untreated parasites, the total percentage of
dense granules that were docked or fused was <2%. In the presence of
SLO and tannic acid alone, 29% of all dense granules
(n = 119 analyzed) were docked and 4% were fusing.
With SLO, tannic acid, ARS, and GTP
S, 24% of dense granules
(n = 101 analyzed) were docked and 16% were fusing. Of
note, no budding of coated vesicles from DG, nor dense granule protein
coats, were noted in either the presence (Figs. 1 and 2) or absence
(not shown) of GTP
S. Formation of coated vesicles at the anterior
nuclear envelope, and at other locations within cell, was readily
visualized in the presence of GTP
S (Fig. 1C). The later
events in dense granule fusion and luminal content release were readily
captured using tannic acid fixation (29) (Fig. 2, D-F).
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Fig. 1.
GTP S augments the
formation of coated vesicles and the exocytosis of dense granules.
Extracellular parasites were permeabilized with SLO, incubated with
GTP
S, ARS, and tannic acid, then prepared for transmission electron
microscopy. Although the cytoplasm appears depleted (A,
B) in comparison to control non-permeabilized cells (not
shown), membranes of the nucleus (N), perinuclear
endoplasmic reticulum, and rhoptries (R) are undamaged.
Treatment with GTP
S promotes a significant increase in the budding
of coated vesicles (small arrow) from the ER and plasma
membrane (PM) as illustrated in C (a higher
magnification of the bar region in A). Dense
granules (DG) arrested by tannic acid in their docking and
fusion to the cell surface are readily observed (arrowhead
in A, B). CC, cortical cisternae.
Magnifications: A, × 6,500; B, × 4,000;
C, × 20,000.
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Fig. 2.
Steps in dense granule exocytosis. Dense
granules in extracellular parasites treated as described in the legend
to Fig. 1 were classified in three stages. Preformed dense granules
(A) were not associated with the cortical cisternae and had
an intact delimiting membrane (arrowhead). Docking dense
granules (B and C) were in contact with the
cortical cisternae, often via membranous threads (unlabeled
arrow in B), with no or minimal discontinuity in the
cortical cisternae. An electron dense band was commonly observed at the
junction of the docked DG and the cortical cisternae (arrow
in C). Fusing dense granules (D,
E) were associated with a discontinuity in the
cortical cisternae. Contents from the dense granule lumen are secreted
into the extracellular space, occasionally with evagination of the
plasma membrane (E), and loss of clear definition of the
delimiting membrane surrounding the dense granule. The secreted
contents often have a membranous appearance (F).
Magnification, × 30,000. These events were assessed quantitatively,
and the results are provided in the text.
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Fig. 3.
ClustalW alignment of phylogenetically
divergent ARF1 homologues with TgARF1. Highly conserved residues
are boxed in black. The conserved myristoylation
site at Gly2 (asterisk), the phosphate/magnesium
(PM) loops and GTP binding (G) domains and switch
regions (SW) thought to interact with phospholipase D are
all indicated above the corresponding sequence. Organism sources and
GenBankTM data base accession numbers for sequences are:
Arabidopsis thaliana (M95166), Oryza sativa
(rice) (AF012896), Zea mays (X80042), Chlamydomonas
reinhardtii (U27120), human (M84326), Drosophila
melanogaster (P35676), Crypotococcus neoformans
(L25115), Saccharomyces cerevisiae ARF1 (J03276), S. cerevisiae ARF2 (M35158), Schizosaccharomyces pombe
(L09551), Histoplasma (Ajellomyces) capsulatus (L25117),
T. gondii (AF227524), Plasmodium falciparum
(U57370), and Giardia lamblia (M86513).
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Fig. 4.
Localization of wild type TgARF1 and TgARF1
mutants by IFA. Tachyzoites (RH) were transiently
transfected with either wild type TgARF1 or TgARF1 mutants, both of
which were tagged at the C terminus with an HA epitope
(A-C). Monolayers of HFF cells were infected for 18 h
and fixed with paraformaldehyde and permeabilized with Triton X-100.
IFA images were obtained staining with an HA monoclonal antibody,
followed by FITC-goat anti-mouse. Wild type TgARF1 and mutants were
expressed under the control of T. gondii NTPase promoter.
ARF-1-HA localizes to the Golgi/TGN region (A). The Q71L-HA
mutant is more dispersed in the parasite, with small punctate
structures in addition to Golgi region staining (B). The
T31N mutant is diffusely distributed throughout the parasite cytosol
(C). Immunofluorescence localization of endogenous ARF-1,
detected with rabbit anti-TgARF1 (D). Corresponding phase
contrast images are shown.
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Fig. 5.
TgARF1 localizes to the Golgi by thin section
cryoimmunoelectron microscopy. Polyclonal antibody to recombinant
TgARF1 stains the Golgi stacks. No labeling of dense granules is
observed.
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Fig. 6.
The TgARF1 Q71L mutant disperses the
Golgi/TGN marker BAP-LDLR. A T. gondii stable
line expressing bacterial alkaline phosphatase fused with the
LDL receptor (BAP-LDLR) was transiently transfected with TgARF1-HA
(A) or TgARF1 (Q71L) (B). Monolayers of HFF cells
were infected for 18 h, fixed, and permeabilized with PFA/Triton
X-100. IFA images were obtained co-staining with the HA monoclonal and
rabbit anti-BAP antibodies, followed by FITC-goat anti-mouse and
rhodamine-goat anti-rabbit. Phase-contrast image of a parasite vacuole
and corresponding fluorescence images are shown. A,
TgARF1-HA co-localizes with BAP-LDLR at the Golgi/TGN of intracellular
parasites by IFA. B, TgARF1 Q71L-HA disrupts the
localization of BAP-LDLR.
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Fig. 7.
Transiently overexpressed ARF1 mutants block
transport and secretion of dense granule proteins. Parasites
stably expressing the soluble dense granule secretory reporter BAP
(A-F) or wild type parasites (G-O) were
transiently transfected with TgARF1T31N (A-C;
J-L), TgARF1Q71L (D-F;
M-O), or wild type TgARF1-HA (G-I). The apical
ends of parasite vacuoles expressing the ARF1 proteins as detected with
antibody to the HA epitope tag are denoted in each image
(asterisks in A, D, G,
J, M). TgARF1Q71L is predominately localized to
the Golgi and perinuclear envelope (arrows in D,
M) whereas TgARF1T31N localization is predominately
cytosolic with minor Golgi association (arrows in
A, J). Transiently expressed TgARF1-HA is
concentrated in the Golgi and cytosol (G). BAP and GRA3
proteins label dense granules in untransfected parasites (small
arrows in B, E, H, K).
Transport of BAP (B, E) or GRA3 (H,
K, N) to dense granules is partially blocked, and
the dense granule proteins accumulate in an reticular ER-like pattern
throughout the ARF1T31N-transfected parasites (arrows in
B, K) and ARF1Q71L parasites (arrows
in E, N). Additionally, less secreted GRA3 signal
is detected at the parasitophorous vacuolar membrane in parasites
expressing ARF1T31N and ARF1Q71L than in untransfected parasites
(long arrows in K, N). Transiently
overexpressed ARF1HA induces only partial accumulation of GRA3 in the
Golgi region (H).
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Fig. 8.
Purification of TgARF1 recombinant protein
(TgARF1-HA) on Cibacron G3. TgARF1-HA was subcloned for expression
into pET-24d. Analysis by SDS-PAGE of recombinant TgARF1-HA expression
is shown (Coomassie Blue staining). Lane 1, extract from
BL21(DE3) cells transfected with vector without the insert. Lane
2, soluble fraction from cells transfected with vector containing
T. gondii TgARF1-HA after a 2 h induction with 1 mM IPTG. Lane 3, pooled fractions from 1m NaCl
elution from Cibacron G3, Lane 4, pooled fractions after 1.2 M NaCl elution from Cibacron G3. Lane 5, pooled
Ultrogel AcA 54 fractions containing purified TgARF1-HA. The arrow
illustrates the position of the band recognized by SDS/PAGE immunoblot
(anti-HA monoclonal antibody).
S. TgARF1-HA purified on the Cibacron G3 column bound 35[S]GTP
S (Fig. 9,
lanes 1 and 2). In contrast, TgARF1-HA purified by the standard protocol using DEAE chromatography did not bind 35[S]GTP
S (Fig. 9, lane 3). Despite
multiple attempts, it was not possible to purify either the
TgARF1Q71L-HA, TgARF1T31N-HA, or TgARF1d17-HA mutants by Cibacron G3
chromatography, nor on other dye resin columns. Although all of these
proteins were successfully purified by the standard DEAE method (not
shown), the purified proteins lacked GTP binding activity (Fig. 9,
lanes 4 and 5), likely explaining the failure of
the proteins to bind to dye resin columns. Control samples (containing
no protein or bovine serum albumin) did not bind the nucleotide (Fig.
9, lane 6).
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Fig. 9.
Binding of
[35S]GTP S by purified,
recombinant TgARF1 and TgARF1 mutants. Shown in the molar ratio of
[35S]GTP
S bound to protein. The protocol for
[35S]GTP
S binding is described under "Materials and
Methods." Lanes 1 and 2, TgARF1-HA (10 pmol) purified on Cibacron G3. Bar 1, storage at 4 °C;
bar 2, stored at
80 °C. Bars 3-5, proteins
purified on DEAE and stored at
80 °C; bar 3, TgARF1-HA
(26 pmol); bar 4, TgARF1d17-HA (59 pmol); bar 5,
TgARF1Q71L-HA (31 pmol); bar 6, buffer. All purified protein
preparations migrated at 21 kDa by SDS-PAGE.
S alone augmented BLA
release slightly, as previously reported (9). Treatment with 100 µg/ml TgARF1-HA (purified on Cibacron G3 and stored at
80 °C)
resulted in 1.4-fold (Fig. 10A, lanes 2 and
4), 1.8-fold (B, lanes 2 and
4), or 2.0-fold (C, lanes 3 and
4) enhancement of release of the secretory reporter BLA from
permeabilized parasites in the presence (but not the absence) of ARS.
Addition of GTP
S to ARS and TgARF1-HA did not substantially augment
BLA release (Fig. 10A, lanes 4 and 5).
Addition of TgARF-1 to GTP
S-treated parasites, in the presence of
ARS, did not substantially augment BLA release in comparison to GTP
S
plus ARS alone (Fig. 10B, lanes 4 and
5). Treatment of non-permeabilized cells with recombinant TgARF1-HA did not result in stimulation of secretion, either in the
absence or presence of ARS and GTP
S (Fig. 10A,
lanes 6 and 7). TgARF1-HA purified by DEAE
chromatography, and lacking GTP binding activity, did not stimulate BLA
release from permeabilized parasites (not shown). These results
illustrate that TgARF-1 augments release of dense granule proteins, and
that the magnitude of the effect is similar to that of GTP
S alone or
in combination with GTP
S.
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Fig. 10.
Purified TgARF1-HA stimulates BLA release in
permeabilized parasites. Methodology and results are described in
the text. The experiment was repeated on three occasions, shown in
A, B, and C. The
manipulations/additions for each experiment are indicated in the tables
below the graphs. The -fold increase in BLA release with TgARF1-HA
addition to SLO-permeabilized parasites (+ARS) varied from
1.4 (A) to 1.8 (B) to 2.0 (C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S
(21, 22). Nonetheless, a fundamental difference between our system and
release observed in mast cells, chromaffin cells, or PC12 cells is that
DG release in the parasite is a constitutive process, which is not
triggered by calcium. Hence, our results have direct relevance to
constitutive secretion. Moreover, we have been able to identify an
effect of TgARF1 at the final step of constitutive vesicle release
because of the unique features of the T. gondii system.
S would have allowed a direct test of this
hypothesis (19, 23, 24) in the permeabilized cell system, but this was
not possible (Fig. 9).
S would correspond to the constitutive component
of DG release and would not be dependent on ARF1 activity. The putative
burst of DG release following invasion would be an ARF1-dependent process, analogous to the situation already
described in selected secretory cells (21, 22). Nonetheless, the
relationship of T. gondii dense granules to dense core
secretory granules in mammalian cells is not clear, and there are many
unusual features of the T. gondii system. The process is
calcium-independent, and no physiological trigger has been identified
for dense granule release (9, 10). No immature secretory granule
precursors (36) are visible in T. gondii. Soluble proteins
are routed by the bulk flow pathway quantitatively to dense granules
(8). The presence of putative membranous material within the dense granule matrix (Fig. 2) is also unique. Altogether, these features suggest that T. gondii dense granules, despite their
morphologic appearance, are evolutionarily distinct from dense core
granules in mammalian secretory cells. As another alternative, ARF1 may augment release of a sub-population of dense granules, although no
convincing evidence yet exists for dense granule heterogeneity. TgARF1
may also enhance formation of nascent secretory vesicles at the
trans-Golgi network (19, 20), in addition to or even instead of
augmenting release of intact DG. Finally, different pathways may be
triggered by GTP
S and by TgARF1 in the permeabilized cell system. In
particular, the effects on GTP
S on release of intact dense granules
could be mediated either via a Rab protein or another member of the ARF
family, such as ARF6 (34). Although all of these possibilities exist,
and several processes may operate concurrently, the morphology,
kinetics, and pharmacological inhibition profile suggest at a minimum
that TgARF1 is enhancing constitutive release of preformed dense granules.
S, has been
previously demonstrated (38), and such a process may also occur in
T. gondii.
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ACKNOWLEDGEMENTS |
---|
We thank Marc Pypaert and Kim Murphy in the Yale Center for Cell Imaging for assistance with electron microscopy. We also thank K. Hager and D. Roos (University of Pennsylvania) for preparing the TgARF1T31N mutation. We appreciate the helpful suggestions of D. Shields (Bronx, NY) and the generous gift of recombinant purified human ARF1 and ARF1 mutants.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grant ROI-AI30060 from the National Institutes of Health (NIH) and a Scholar Award in Molecular Parasitology from the Burroughs Wellcome Fund (to K. A. J.), by NIH Training Grants T32-AI 07404-09 (to A. L.) and T32 AI07404 (to H. M. N.), by National Research Service Awards 1F32-AI09938-01 (to T. S.) and 1F32-AI10044-01A1 (to H. M. N.), and by a South African Foundation for Research Development postdoctoral fellowship (to H. C. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF227524.
To whom correspondence should be addressed: Dept. of Internal
Medicine, Section of Infectious Diseases, Yale University School of
Medicine, LCI 808, 333 Cedar St., New Haven, CT 06520-8022. Tel.:
203-785-4140; Fax: 203-785-3864; E-mail: keith.joiner@yale.edu.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M008352200
2 T. Stedman and K. A. Joiner, unpublished.
3 A. Liendo and K. A. Joiner, unpublished observations.
4 H. M. Ngô, M. Yang, M. Pypaert, H. Hoppe, and K. A. Joiner, submitted.
5 H. M. Ngô et al., manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PV, parasitophorous
vacuole;
PVM, parasitophorous vacuole membrane;
ARF, ADP-ribosylation
factor;
ARS, ATP-regenerating system;
BAP, bacterial alkaline
phosphatase;
BAP-LDLR, fusion between BAP and the low density
lipoprotein receptor;
BLA, -lactamase;
HFF, human foreskin
fibroblasts;
IFA, immunofluorescence assay;
PFA, paraformaldehyde;
SLO, streptolysin O;
TgARF, T. gondii ADP-ribosylation factor;
GTP
S, guanosine 5'-3-O-(thio)triphosphate;
DG, dense
granules;
RACE, rapid amplification of cDNA ends;
TGN, trans-Golgi
network;
EST, expressed sequence tag;
PCR, polymerase chain reaction;
kbp, kilobase pair(s);
ORF, open reading frame;
HA, hemagglutinin;
bp, base pair(s);
IPTG, isopropyl-1-thio-
-D-galactopyranoside;
DTT, dithiothreitol;
PAGE, polyacrylamide gel electrophoresis;
LDL, low
density lipoprotein;
FITC, fluorescein isothiocyanate;
AP-1 and -3, adaptor protein complex 1 and 3;
ER, endoplasmic reticulum;
PLD, phospholipase D;
PIP2, phosphatidylinositol
4,5-bisphosphate;
NSF, N-ethylmaleimide-sensitive fusion
protein;
SNAP, soluble NSF attachment protein;
SNARE, SNAP receptor;
PA, phosphatidic acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Mellman, I., and Warren, G. (2000) Cell 100, 99-112[Medline] [Order article via Infotrieve] |
2. | Ferro-Novick, S., and Jahn, R. (1994) Nature 370, 191-193[CrossRef][Medline] [Order article via Infotrieve] |
3. | Ngô, H., Hoppe, H. C., and Joiner, K. A. (2000) Trends Cell Biol. 10, 67-72[CrossRef][Medline] [Order article via Infotrieve] |
4. | Coppens, I., and Joiner, K. A. (2001) Expert Rev. Mol. Med., http://www- ermm.cbcu.cam.ac.uk./01 0022 77h.htm |
5. | Stedman, T., and Joiner, K. A. (1999) in Advances in Cell and Molecular Biology of Membranes and Organelles (Gordon, S., ed) , pp. 233-261, JAI Press, Greenwich, CT |
6. | Carruthers, V. B., and Sibley, L. D. (1997) Eur. J. Cell Biol. 73, 114-123[Medline] [Order article via Infotrieve] |
7. | Dubremetz, J. F., Achbarou, A., Bermudes, D., and Joiner, K. A. (1993) Parasitol. Res. 79, 402-408[Medline] [Order article via Infotrieve] |
8. |
Karsten, V.,
Qi, H.,
Beckers, C. J. M.,
Dubremetz, J. F.,
Webster, P.,
and Joiner, K. A.
(1998)
J. Cell Biol.
141,
1323-1333 |
9. |
Chaturvedi, S.,
Qi, H.,
Coleman, D. L.,
Hanson, P.,
Rodriguez, A.,
and Joiner, K. A.
(1998)
J. Biol. Chem.
274,
2424-2431 |
10. | Carruthers, V. B., and Sibley, L. D. (1999) Mol. Microbiol. 31, 421-428[CrossRef][Medline] [Order article via Infotrieve] |
11. | Kahn, R., and Gilman, A. (1986) J. Biol. Chem. 261, 1-6 |
12. |
Kahn, R.,
Kern, F.,
Clark, J.,
Gelmann, E.,
and Rulka, C.
(1991)
J. Biol. Chem.
266,
2606-2614 |
13. | Donaldson, J. G., and Klausner, R. D. (1994) Curr. Opin. Cell Biol. 6, 527-532[Medline] [Order article via Infotrieve] |
14. | Dittie, A. S., Hajibagheri, N., and Tooze, S. A. (1996) J. Cell Biol. 132, 523-536[Abstract] |
15. | Stamnes, M. A., and Rothman, J. E. (1993) Cell 73, 999-1005[Medline] [Order article via Infotrieve] |
16. |
Austin, C.,
Hinners, I.,
and Tooze, S. A.
(2000)
J. Biol. Chem.
275,
21862-21869 |
17. |
Le Borgne, R.,
Griffiths, G.,
and Hoflack, B.
(1996)
J. Biol. Chem.
271,
2162-2170 |
18. |
Fucini, R.,
Navarrete, A.,
Vadakkan, C.,
Lacomis, L.,
Erdjument-Bromage, H.,
Tempst, P.,
and Stamnes, M.
(2000)
J. Biol. Chem.
275,
18824-18829 |
19. |
Chen, Y.,
and Shields, D.
(1996)
J. Biol. Chem.
271,
5297-5300 |
20. |
Chen, Y. G.,
Siddhanta, A.,
Austin, C. D.,
Hammond, S. M.,
Sung, T. C.,
Frohman, M. A.,
Morris, A. J.,
and Shields, D.
(1997)
J. Cell Biol.
138,
495-504 |
21. | Glenn, D., Thomas, G., O'Sullivan, A., and Burgoyne, R. (1998) J. Neurochem. 71, 2023-2033[Medline] [Order article via Infotrieve] |
22. | Fensome, A., Cunningham, E., Prosser, S., Khoon Tan, S., Swigart, P., Thomas, G., Hsuan, J., and Cockcroft, S. (1996) Curr. Biol. 6, 730-738[Medline] [Order article via Infotrieve] |
23. | Jones, D., Bax, B., Fensome, A., and Cockcroft, S. (1999) Biochem. J. 341, 185-192[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Kahn, R.,
Randazzo, P.,
Serafini, T.,
Weiss, O.,
Rulka, C.,
Clark, J.,
Amherdt, M.,
Roller, P.,
Orci, L.,
and Rothman, J.
(1992)
J. Biol. Chem.
267,
13039-13046 |
25. | Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J., and Rothman, J. (1993) J. Cell Biol. 123, 1365-1371[Abstract] |
26. | Hoppe, H. C., Ngô, H. M., Yang, M., and Joiner, K. A. (2000) Nat. Cell Biol. 2, 449-456[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Kuliawat, R.,
Klumperman, J.,
Ludwig, T.,
and Arvan, P.
(1997)
J. Cell Biol.
137,
595-608 |
28. |
Turner, M.,
and Arvan, P.
(2000)
J. Biol. Chem.
275,
14025-14030 |
29. | Newman, T. M., Tian, M., and Gomperts, B. D. (1996) Eur. J. Cell Biol. 70, 209-220[Medline] [Order article via Infotrieve] |
30. |
Sung, T. C.,
Altshuller, Y. M.,
Morris, A. J.,
and Frohman, M. A.
(1999)
J. Biol. Chem.
274,
494-502 |
31. |
Hager, K. M.,
Striepen, B.,
Tilney, L. G.,
and Roos, D. S.
(1999)
J. Cell Sci.
112,
2631-2638 |
32. | Zhang, C. J., Rosenwald, A. G., Willingham, M. C., Skuntz, S., Clark, J., and Kahn, R. A. (1994) J. Cell Biol. 124, 289-300[Abstract] |
33. | Roth, M. (1999) Cell 97, 149-152[Medline] [Order article via Infotrieve] |
34. |
Caumont, A. S.,
Galas, M. C.,
Vitale, N.,
Aunis, D.,
and Bader, M. F.
(1998)
J. Biol. Chem.
273,
1373-1379 |
35. | Carruthers, V., Moreno, S., and Sibley, L. D. (1999) Biochem. J. 342, 379-386[CrossRef][Medline] [Order article via Infotrieve] |
36. | Tooze, S., Flatmark, T., Tooze, J., and Huttner, W. B. (1991) J. Cell Biol. 115, 1491-1503[Abstract] |
37. | Whatmore, J., Morgan, C. P., Cunningham, E., Collison, K. S., Willison, K. R., and Cockcroft, S. (1996) Biochem. J. 320, 785-794[Medline] [Order article via Infotrieve] |
38. | Dohke, Y., Hara-Yokoyama, M., Fujita-Yoshigaki, J., Kahn, R. A., Kanaho, Y., Hashimoto, S., Sugiya, H., and Furuyama, S. (1998) Arch Biochem. Biophys. 357, 147-154[CrossRef][Medline] [Order article via Infotrieve] |
39. | Serafini, T., Orci, L., Amherdt, M., Brunner, M., Kahn, R. A., and Rothman, J. E. (1991) Cell 67, 239-253[Medline] [Order article via Infotrieve] |
40. | Donaldson, J., Cassel, D., Kahn, R., and Klausner, R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6408-6412[Abstract] |
41. |
Palmer, D. J.,
Helms, J. B.,
Beckers, C. J.,
Orci, L.,
and Rothman, J. E.
(1993)
J. Biol. Chem.
268,
12083-12089 |
42. | Hoppe, H. C., and Joiner, K. A. (2000) Cell. Microbiol. 2, 569-578[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Ooi, C. E.,
Dell'Angelica, E. C.,
and Bonifacino, J. S.
(1998)
J. Cell Biol.
142,
391-402 |
44. | Faundez, V., Horng, J. T., and Kelly, R. B. (1998) Cell 93, 423-432[Medline] [Order article via Infotrieve] |