From the Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022
Received for publication, September 13, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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Toxoplasma gondii relies on
protein secretion from specialized organelles for invasion of host
cells and establishment of a parasitophorous vacuole. We identify
T. gondii Rab6 as a regulator of protein
transport between post-Golgi dense granule organelles and the Golgi.
Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated
transport vesicles, and microdomains of dense granule and endosomal
membranes. Overexpression of wild-type Rab6 or GTP-activated Rab6(Q70L)
rerouted soluble dense granule secretory proteins to the Golgi and
endoplasmic reticulum and augmented the effect of brefeldin A on
Golgi resorption to the endoplasmic reticulum. Parasites
expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N))
mutant accumulated dense granule proteins in the Golgi and
associated transport vesicles and displayed reduced secretion of
GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi
membranes colocalized with centrin during mitosis, and parasite clones
expressing Rab6 mutants displayed a partial shift in cytokinesis from
endodyogeny (formation of two daughter cells) to endopolygeny (multiple
daughter cells). We propose that Toxoplasma Rab6 regulates
retrograde transport from post-Golgi secretory granules to the parasite Golgi.
The Golgi complex coordinates secretory protein maturation and
sorting and is a central intermediary of bidirectional transport between exocytic and endocytic pathways. In most protozoa, the early
secretory pathway is well conserved in that of other eukaryotes, performing the critical function of biosynthetic transport to organelles, the cell surface, and the extracellular environment. Members of Apicomplexa, a diverse phylum of obligate
intracellular parasites, are distinct from other eukaryotes in
harboring three unique polarized secretory organelles, termed
micronemes, rhoptries, and dense granules. As exemplified in
Toxoplasma gondii, sequential secretion from these
organelles is essential for host cell invasion and the concomitant
formation of an intracellular parasitophorous vacuole
(PV)1 enveloping the parasite
(1). Micronemes and rhoptries are apically tethered and secreted during
attachment and penetration of a host cell. Dense granules in
Toxoplasma secrete aggregates of soluble and transmembrane
proteins constitutively at a basal level and are stimulated for
enhanced release following parasite invasion, suggesting a regulated,
but Ca2+-independent, component (2).
Immunohistochemical analysis indicates that the sorting of dense
granule proteins from those targeted to micronemes and rhoptries occurs
at the late Golgi cisternae or trans-Golgi network (TGN). Although rhoptries originate from a precursor organelle formed by the
Golgi during parasite division, the process regulating biogenesis of
Toxoplasma dense granules is completely unknown. Two
hypotheses are proposed to account for the biogenesis of regulated secretory vesicles in specialized mammalian cells (reviewed in Ref. 3).
In the selective aggregation model, secretory proteins aggregate in the
TGN and are sorted for entry into secretory granules by receptors in
the TGN membrane. Proteins that fail to aggregate may exit the TGN
independently in constitutive secretory vesicles. In the
sorting-by-retention model, regulated proteins are retained and sorted
from constitutive secretory cargo after formation of immature secretory
granules. In this model, immature secretory granules budding from the
TGN contain the bulk of biosynthetic cargo and undergo maturation by
selective budding of constitutive secretory vesicles containing
non-aggregate proteins. Toxoplasma dense granule protein
aggregates may dissociate and insert post-translationally into target
membranes only after secretion into the PV (4-6). Understanding the
biogenesis of Toxoplasma dense granules and the mechanisms
involved in regulating dense granule protein transport will provide
fundamental insight into the minimal requirements for sorting
between regulated and constitutive secretory pathways in eukaryotes.
Molecular evidence and genomic sequencing efforts indicate that the
molecular machinery regulating vesicular transport in Apicomplexa is
partially conserved in other eukaryotes, particularly early in the
secretory pathway. Morphologically, these protozoa possess an
endoplasmic reticulum (ER) that is contiguous with the nuclear envelope
and a Golgi apparatus that ranges from single dispersed cisternae in
malarial parasites (genus Plasmodium) to a stacked apically
oriented Golgi in Toxoplasma. Components of the vesicle
budding, transport, and fusion machinery have been cloned in
Toxoplasma, including N-ethylmaleimide-sensitive
fusion protein, multiple Rab proteins (7), ARF1 (8), and
subunits of the coatomer (9) and adapter complexes (10).
Evolutionarily conserved sorting motifs function in the transport of
proteins to rhoptries and micronemes (10, 11). In contrast, targeting motifs have not been found in dense granule proteins, and heterologous soluble proteins expressed with an N-terminal signal sequence are
constitutively secreted into the PV through dense granules (12).
To further define regulation of dense granule secretion, we sought to
identify molecular effectors of dense granule protein transport. In
conjunction with effector proteins, monomeric GTPases of the Rab family
localize to distinct intracellular compartments and confer one level of
specificity on vesicle transport and fusion by mediating tethering or
docking of opposing membranes prior to pairing of SNAREs. The small
GTPase Rab6 appears to function in intra-Golgi transport (13, 14) and
COPI/ARF1-independent retrograde transport from the Golgi to the ER in
mammalian cells (15, 16). The yeast homolog of Rab6, Ypt6p, was
initially reported to function in early Golgi transport (17, 18).
Current evidence now indicates that Ypt6p aids recycling of
endosome-derived vesicles with late Golgi membranes and is dependent
upon a guanine nucleotide exchange factor complex, SNAREs, and the VPS
(vacuolar protein sorting) complex
(19-21).
In this report, we show that Toxoplasma Rab6 regulates
protein transport at a step between dense granules and the late Golgi cisternae and that overstimulation or inhibition of Rab6 function partially blocks constitutive secretion of proteins by
disregulating their transport to dense granules. The effect of
Rab6 overstimulation appears to enhance the brefeldin A-induced
resorption of constitutively secreted cargo and Golgi cisternae to the
ER. Furthermore, expression of either GTP-activated or
dominant-interfering Rab6 mutants altered parasite cytokinesis, leading
to a partial switch in daughter parasite budding to endopolygeny. These
results suggest that Toxoplasma Rab6 mediates a retrograde
pathway from post-Golgi secretory organelles to the late Golgi and may
provide a link between Rab6 function and coupled mitosis and cytokinesis.
T. gondii Growth and Isolation--
T. gondii RH
strain cells were maintained in vitro by infection of Vero
cell monolayers as previously described (22).
Hypoxanthine-xanthine-guanine phosphoribosyltransferase
(HXGPRT)-deficient T. gondii cells were obtained through the
National Institutes of Health AIDS Research Reference Reagent Program
(23).
T. gondii Nucleic Acid Isolation, cDNA Synthesis, and PCR
Amplifications--
T. gondii RNA was extracted with TRIzol
(Invitrogen). cDNA synthesis was performed with Superscript II
reverse transcriptase (Invitrogen) and primer
oligo(dT17)XX. PCR amplification of cDNA was
performed with Taq DNA polymerase (Roche Molecular
Biochemicals) using degenerate oligonucleotides and targeting domains
conserved either among Rab proteins in general (primer URABF, encoding
the conserved PM3-binding site QLWDTAGQE) or Rab6 proteins in
particular (primers AVVYDIT and AQEYNT). PCR cycling conditions were as
follows: 94 °C for 45 s, 52.5 °C for 1 min, and 72 °C for
1 min for 35 rounds of amplification. Amplification products from the
URABF/oligo(dT17)XX primer set were used as template for
nested 3'-RACE/PCR with the AIVVYDIT/oligo(dT17)XX primer
set. Template for 5'-RACE/PCR was generated by reverse transcription
with Rab6R and dCTP tailing with terminal deoxynucleotide transferase
(New England Biolabs Inc.) and used for PCR with Rab6R and the RACE
anchor primer (Invitrogen). Nested 5'-RACE/PCR was performed with Rab6I
and the anchor primer. Nested PCR products were cloned into pGEM-T
(Promega) and screened by colony hybridization. Sequence analysis
of independent RACE clones confirmed the 5'- and 3'-ends of
T. gondii rab6 cDNA,
including an overlapping region of 133 bp. The full-length open reading frame (ORF) of T. gondii rab6 was amplified from
cDNA using the Rab6F/Rab6R primer set, overlapping the initiation
and stop codons, respectively. Sequences of independent clones
confirmed the identity of the full-length 597-bp rab6 ORF to
the 5'- and 3'-RACE clones, and Rab6 homology was confirmed by TBLASTN
analysis. The T. gondii rab6 cDNA sequence
has been deposited in the GenBankTM/EBI Data Bank
(accession number AF228419). The sequences of the oligonucleotide
primers used in the isolation of rab6 cDNA are as
follows (where positions of deoxynucleotide base degeneracy are in
parentheses and I is inosine): oligo(dT17)XX,
5'-T17(G/A/C)(G/A/T/C)-3'; URABF, 5'-CA(G/A) AT(A/T/C) TGG
GA(T/C) AC(G/A/T/C) GCI GG(G/A/T/C) CA(G/A) GA-3'; AIVVYDIT, 5'-GCI
AT(A/T/C) GTI GT(G/A/T/C) TA(T/C) GA(T/C) AT(A/T/C) AC-3'; AQEYNT,
5'-AAC ATI GT(G/A) TG(G/A) TA(T/C) TC(T/C) TG(G/A/T/C) GC-3'; Rab6R,
5'-AAC AGA TCT TTC AGC AAG AGC AGG ATG-3'; Rab6I, 5'-GAT CCA CTT GGT
TGT ATT GAG-3'; Rab6HANsi, 5'-GTC AAC ATG CAT TAC CCA TAC GAC GTC CCA
GAC TAC GCG GAG GCG ACA GTT GCG-3'; and R6rPacI, 5'-TCT TTA ATT AAC AAG
AGC AGG ATG AAG TC-3'.
Colony and Northern and Southern Blot Hybridization
Analyses--
Northern blot analysis was performed after
electrophoresis of 20 µg of total RNA on denaturing
formaldehyde-agarose gels (1.2% Sea Kem GTG agarose (FMC Corp.), 20 mM HEPES, pH 7.8, 0.2 mM EDTA, and 6%
formaldehyde) in HEPES/Formalin running buffer (20 mM
HEPES, pH 7.8, 0.2 mM EDTA, and 0.1% formaldehyde). RNA
samples were transferred to Zetaprobe membranes (Bio-Rad) in sodium
phosphate transfer buffer (25 mM
Na2HPO4/NaH2PO4), pH
6.5.
Southern and colony blot hybridization analyses were performed as
described (24). A rab6 cDNA fragment generated by PCR primers AIVVYDIT and AQEYNT was agarose gel-purified and labeled by
random oligonucleotide priming with [ Phylogenetic Analysis--
Sequences homologous to Rab6 were
aligned using the Genetics Computer Group Wisconsin package.
Phylogenetic reconstruction was performed using the PAUP program
(phylogenetic analysis underln]using parsimony). Bootstrap analysis was carried out with 1000 replicates. The consensus tree derived from bootstrap analysis was
topologically identical to the single best parsimony tree. Graphical
output from the tree file was provided using TreeView (R. Page,
University of Glasgow, Glasgow, Scotland).
Construction of Expression Vectors--
A cytosolic T. gondii HA-Rab6 expression vector was developed by amplifying the
rab6 ORF using the Rab6HANsi/R6rPacI primer set. The product
was subcloned into the NsiI-PacI sites of pSAG, a
SAG1 expression vector (25), modified by removal of the SAG1 signal
sequence for cytosolic protein expression, creating vector pSAGRab6HA.
HA-Rab6(Q70L) and HA-Rab6(N124I) were generated by amplification of the
rab6 ORF with mutagenic primers and cloned by triple
ligation into the NsiI-PacI sites of pSAG.
Expression vectors encoding HA-Rab6(T25N) were created by site-directed
mutagenesis of the corresponding HA-Rab6 vectors with complementary
mutagenic primers by amplification using Pfu Turbo
polymerase (Stratagene).
For bacterial expression, a 1.7-kilobase pair
NcoI-BamHI fragment was excised from pNTPRab6 and
subcloned into NcoI-BamHI-digested pET15b.
HA-Rab6(Q70L) and HA-Rab6(N124I) were subcloned into the same sites
after amplification of pSAGRab6 with appropriate mutagenic primers. The
rab6 ORF in all vectors was fully sequenced for confirmation of the site-directed mutations and exclusion of additional mutations. All restriction enzymes were purchased from New England Biolabs Inc.
Dual Secretory Reporter Constructs--
Plasmid vector
pminCATBAP was modified for coexpression with HA-Rab6 or HA-Rab6 point
mutants. This vector contains the heterologous secretory reporter
Escherichia coli alkaline phosphatase (BAP), expressed from the T. gondii
nucleoside-triphosphate hydrolase-3 promoter and fused to the
nucleoside-triphosphate hydrolase-3 signal sequence (12). The
rab6 ORFs were excised as
HindIII-BamHI fragments from pSAGRab6HA and
subcloned by replacement of the dihydrofolate reductase-chloramphenicol
acetyltransferase fusion cassette in pminCATBAP, generating the
pHXBAPRab6HA series plasmids.
Expression Analysis in T. gondii and Selection of Stable
Lines--
Parasites were transfected by electroporation according to
published protocols (22). For selection of stable lines,
hypoxanthine-xanthine-guanine phosphoribosyltransferase-deficient
parasites were transfected, passaged twice in human foreskin fibroblast
cultures under selection with 25 µM mycophenolic acid and
50 µM xanthine, and cloned by limiting dilution on
96-well plates (22).
Immunoblot Analysis of Endogenous and Recombinant T. gondii
Rab6--
Bacterial expression plasmids pET15HARab6,
pET15HARab6(Q70L), and pET15HARab6(N124I) were transformed into
E. coli BL21(DE3) pLysS (Novagen). Crude extracts from
isopropyl- GTP Overlay Assay of Recombinant T. gondii
Rab6--
Nitrocellulose transfer blots containing crude
recombinant T. gondii HA-Rab6, HA-Rab6(Q70L), and
HA-Rab6(N124I) proteins were analyzed for GTP-binding activity as
previously described (26), except that phosphate buffer was substituted
for Tris-HCl in the binding buffer (50 mM
Na2HPO4/NaH2PO4, pH
7.0, 5 mM MgCl2, 1 mM EGTA, and
0.3% Tween 20). Blots were preincubated in binding buffer supplemented
with 1 mM ATP for 30 min. [ Immunofluorescence Microscope Analysis--
Human foreskin
fibroblast cell monolayers grown on coverslips were infected with
freshly transfected (106 cells/coverslip) or untransfected
(105 cells/coverslip) T. gondii cells
for 16 h at 37 °C. Coverslips were prepared for
immunofluorescence assay as previously described (12). Primary
antibodies included mouse anti-HA-11 (Covance), rabbit anti-BAP
(5 Prime Electron Microscopy--
For immunohistochemistry, infected Vero
cell monolayers were fixed with 8% paraformaldehyde in 0.25 M HEPES, pH 7.4. Sections (95 nm) were obtained from the
Yale Center for Cell Imaging. Immunolabeling was performed with mouse
anti-HA mAb (1:100; Babco), followed by rabbit anti-mouse antibody
(1:50; Cappel or ICN) and protein A-gold (1:70; J. Slot, University of Utrecht, Holland). The sections were
contrasted with neutral uranyl acetate (2%), infiltrated with methyl
cellulose (1.8%) and uranyl acetate (0.5%), and examined with a
Philips 410 transmission electron microscope. For transmission electron
microscopy, infected Vero cell monolayers were fixed in 8%
paraformaldehyde and 100 mM cacodylate buffer for 2 h.
Blocks were prepared for Epon embedding by the Yale Center for Cell
Imaging. Ultrathin 60-80-nm sections were collected on grids,
contrasted with lead citrate and uranyl acetate, and examined as
described above.
Metabolic Labeling and Quantitative
Immunoprecipitation--
Parasite cultures were washed three times
with cysteine/methionine-free minimal essential medium
(Invitrogen) and metabolically labeled in 1 ml of minimal essential
medium containing 5% fetal bovine serum and 100 µCi of
[35S]Met/Cys (Promix, Amersham). For analysis of
BAP and GRA2 secretion, parasites were labeled for 30 min. For
pulse-chase analysis of GRA4, parasites were labeled for 10 min and
chased after three washes for 0, 15, and 30 min. Parasites were freed
from host cells by syringe passage in 1 ml of phosphate-buffered saline
containing proteinase inhibitors (2) and centrifuged at 1400 × g for 10 min at 4 °C. Cell pellets were lysed in
radioimmune precipitation assay buffer (24) containing Complete
protease inhibitor mixture (Roche Molecular Biochemicals). Supernatants
following the 1400 × g spin (containing T. gondii soluble secreted proteins of the vacuolar space) were used
for immunoprecipitation after addition of Nonidet P-40 to 1% as
previously described (28) with antibody to BAP, GRA4 (mAb T8-4B9), or
GRA2 (mAb T4-1F5) as described above. Gels were dried and exposed to
Biomax film (Kodak). Images were scanned and quantitated using Scion
Image software.
Identification and Phylogeny of the T. gondii Rab6
Homolog--
Because Rab6 functions in Golgi transport in yeast and
mammalian cells, we sought to identify this protein in
Toxoplasma as a potential regulatory target for secretory
protein trafficking. Combinatorial RACE/PCR amplification was used
to clone a T. gondii rab6 cDNA. In genomic
digests, a rab6 cDNA probe strongly hybridized to a
single T. gondii restriction fragment in each case (Fig. 1A), indicating that
rab6 is a single-copy gene in T. gondii. Northern
blot analysis indicated a major RNA transcript size of ~1.3 kb, along
with a larger, possibly unprocessed transcript (Fig. 1B). In
neither case did the rab6 probe hybridize to nucleic acid
controls derived from host cell cultures, confirming that Rab6 is of
parasite origin.
The Toxoplasma rab6 cDNA encodes a 209-amino
acid protein (Fig. 1D) with a predicted molecular mass of
23.7 kDa. Of the available sequences, the Toxoplasma Rab6
protein is most homologous to Rab6 of the Apicomplexa malarial
parasites Plasmodium falciparum and Plasmodium
berghei (76-77% similarity and ~72% identity using BESTFIT
analysis). The phylogenetic relationship of T. gondii Rab6
to other Rab6 homologs was examined by alignment (Fig. 1D) and parsimony analysis using the PAUP program (Fig. 1C). The
Rab family domains are well conserved, including the nucleotide-binding pocket domains and switch regions, which confer
nucleotide-dependent conformational changes and interaction
with Rab effectors (Fig. 1D). Distinct from
Saccharomyces and Plasmodium,
Toxoplasma Rab6 encodes a conservative Ser85
residue within a motif essential for interaction with a
cytoskeleton-associated Rab6 effector, Rabkinesin-6 (29). The
hypervariable C-terminal region and extreme N terminus of Rab6,
important in conferring functional specificity and intracellular
distribution between Rab proteins, are divergent among Rab6 homologs. A
single most parsimonious phylogenetic tree, well supported by bootstrap
analysis, was generated with representatives of animal, plant, and
fungal Rab6 homologs (Fig. 1C).
Recombinant T. gondii HA-Rab6 and HA-Rab6(Q70L), but Not
HA-Rab6(N124I), Are Competent GTP-binding Proteins--
To initiate
a functional analysis of T. gondii Rab6, we developed
expression plasmids encoding Toxoplasma Rab6 and Rab6
GTP-binding domain mutants predicted to be altered in their GTP-binding
affinity. An N-terminal 9-HA epitope tag was fused to the
Toxoplasma rab6 ORF or to point mutants
Rab6(Q70L) and Rab6(N124I). Rab6(Q70L) is predicted to be deficient in
GTPase activity, whereas Rab6(N124I) is predicted to lack affinity for
guanine nucleotide, based upon homology to known Ras-related protein
mutations (30, 31). Soluble bacterial lysates were probed by
immunoblotting with antiserum to a synthetic peptide (T. gondii Rab6-(192-206)) (Fig.
2B, lanes 2-4) or
with antibody to the HA epitope (data not shown); in each case,
proteins with an apparent molecular mass of 30 kDa were recognized.
GTP-binding activity was assessed for HA-Rab6, HA-Rab6(Q70L), and
HA-Rab6(N124I). Both HA-Rab6 (Fig. 2C, lane 2)
and HA-Rab6(Q70L) (lane 3) bound [ Toxoplasma Rab6 Localizes to the Golgi in Both Parasite and CHO
Cells--
In transiently or stably transfected Toxoplasma
tachyzoites, both HA-Rab6 (Fig.
3A) and HA-Rab6(Q70L) (Fig.
3C) proteins localized, as determined by indirect
immunofluorescence assay, predominantly to a discrete irregular
structure apical to the parasite nucleus. HA-Rab6 signal was detected
in lower amounts throughout the parasite cytoplasm and in one or more
dense granules (Fig. 3, A and C). We reasoned
that Rab6 motifs necessary for Golgi localization would be functionally
conserved among eukaryotes, given the general ability to functionally
complement subsets of Rab proteins between yeast and mammals. To
compare the distribution of Toxoplasma Rab6 within a higher
organism, T. gondii Rab6 was transiently expressed in CHO-K1
cells and localized with endogenous markers, including the
Golgi-associated tethering proteins giantin and GRASP65, the ER protein
calnexin, and mannose 6-phosphate receptor protein, a marker
principally of late endosomes. In addition to labeling a cytosolic
pool, T. gondii HA-Rab6 localized in a juxtanuclear pattern
typical of the Golgi (Fig. 3, E and H) and
colocalized in this region with giantin (Fig. 3, E-G).
Likewise, HA-Rab6 signal overlapped GRASP65 on the Golgi (Fig. 3,
H-J). In contrast, HA-Rab6 exhibited minimal colocalization
with the mannose 6-phosphate receptor and calnexin (data not
shown).
To more precisely define Rab6 localization in Toxoplasma,
HA-Rab6(Q70L) parasite clones were subjected to immunoelectron
microscopy (Fig. 4, A-E).
Activated Rab6 was most heavily concentrated on late
Toxoplasma Golgi cisternae (Fig. 4, A and
F). Specifically, Rab6 was distributed on the apical
(trans) cytosolic face of the late Golgi cisternae and the
TGN, on the cisternal rims of the trans- and medial-Golgi
cisternae, and on peripheral vesicles closely associated with the
cisternal rims. Labeling of the cis-Golgi, the intermediate
compartment between the Golgi and the nuclear envelope, and the
transitional ER of the nuclear envelope was minimal. Rab6 also strongly
labeled microdomains on membranes of a subset (~42%,
n = 38) of dense granule sections (Fig. 4, B, C, and E, arrows). Rab6
was sporadically associated with microdomains on membranes of large
electron lucent vesicles, putatively of endosomal function, but was not
associated with membranes of mature rhoptries, micronemes, apicoplasts,
or mitochondria (Fig. 4, A and E). This
distribution implicates activated Rab6 in a transport process to the
late Golgi in Toxoplasma, possibly involving dense granules.
Localization of Toxoplasma Rab6 in both the parasite (Fig.
5) and transiently transfected CHO cells
(data not shown) was also sensitive to the fungal metabolite brefeldin
A (BFA), an inhibitor of the ARF1 GTPase exchange protein. Following
addition of 5 µg/ml BFA, transiently expressed HA-Rab6 (Fig.
5C) collapsed to the perinuclear envelope, along with a
reticular and cytosolic labeling pattern throughout the parasite (Fig.
5, F and I). Activated HA-Rab6(Q70L) was likewise
dispersed by BFA treatment to a prominent perinuclear and reticular
pattern, suggesting a complete collapse of the parasite Golgi into the
ER, similar to tubulovesicular retrograde Golgi-derived elements in
mammalian cells. In total, these results indicate that
Toxoplasma Rab6 contains evolutionarily conserved
Golgi-targeting signals, despite significant C-terminal divergence of
Rab6 proteins.
Transiently Overexpressed Toxoplasma Rab6 and Rab6 Mutants Block
Transport of Dense Granule Proteins--
To examine the role of
Toxoplasma Rab6 in transport and secretion of dense granule
proteins, HA-Rab6, HA-Rab6(Q70L), HA-Rab6(N124I), or GDP-restricted
HA-Rab6(T25N) were coexpressed along with the secretory reporter
protein BAP. This protein, fused to the nucleoside-triphosphate hydrolase-3 signal sequence, behaves as a soluble secreted dense granule protein in Toxoplasma tachyzoites (12). Secretory
BAP localized to dense granules and the PV upon secretion (Fig. 5, A and D). In the presence of transiently
overexpressed HA-Rab6 (Fig. 5B) or BFA (Fig. 5, D
and G), BAP secretion into the PV and distribution in dense
granules were partially inhibited, with signal accumulating in the
Golgi and the ER-associated perinuclear envelope. Rab6 overexpression
augmented the BFA-induced retrograde appearance of BAP in the parasite
ER within the first 15 min of treatment (Fig. 5J).
The role of activated and inhibitory Rab6 mutants in secretory
transport was assessed by transient overexpression in transfected parasites. GTP-activated mutant HA-Rab6(Q70L) (Fig.
6A) induced a similar but more
pronounced accumulation of BAP in the Golgi and the ER-associated
perinuclear envelope (Fig. 6B) in comparison with
overexpressed wild-type Rab6. HA-Rab6(N124I) was cytosolic in
distribution (Fig. 6D), whereas HA-Rab6(T25N) was partially Golgi-localized (Fig. 6G). Each of these mutants induced
dispersal of BAP within the parasite in a small vesicular and reticular pattern independent of dense granules (Fig. 6, E and
H), with BAP accumulating at or near the late Golgi in the
case of HA-Rab6(T25N) (Fig. 6H), although secreted BAP was
still detectable in parasite vacuoles (Fig.
7A). Transient overexpression
of HA-Rab6(N124I) was toxic because parasite vacuoles often contained
rounded degenerative bodies of atypical parasite morphology (data not
shown).
Parasites expressing Rab6 mutants were further analyzed for transport
of endogenous dense granule proteins GRA1-4. In the case of
HA-Rab6(T25N), GRA1 accumulated in the same Golgi-proximal anterior
compartment (Fig. 7B, arrows), colocalizing with
BAP (Fig. 7C). Although BAP was dispersed from dense
granules, these remained weakly positive for GRA1; both secreted
proteins were still apparent in the PV (Fig. 7, B and
C, near the asterisks). Identical results were
obtained when parasites were labeled with endogenous GRA2-4,
suggesting an attenuated effect on transport of dense granule protein
aggregates in comparison with soluble secretory BAP. In contrast,
transport of glycosylphosphatidylinositol-anchored proteins to
the cell surface and microneme or rhoptry proteins from the ER and
Golgi was uninhibited in the presence of the Rab6 mutants (data not
shown). Altogether, these results indicate that alteration of Rab6
function by overexpression leads to partial disruption in the transport
of constitutively secreted proteins between dense granules and the Golgi.
Dense Granule Secretion Is Differentially Inhibited in Transient
and Stable Rab6 Mutants--
To determine whether bulk secretion into
the PV was altered by Rab6 levels, secretion of BAP was quantitatively
assessed in intracellular parasites by metabolic pulse labeling. BAP
secreted from parasites transiently overexpressing HA-Rab6 or
HA-Rab6(Q70L) was ~50-60% of that secreted from parasites
expressing BAP alone, whereas expression of HA-Rab6(N124I) reduced BAP
secretion by ~10% (Fig.
8A), despite the loss in
labeling of dense granules. This result suggests that constitutive
secretion may not require transport through dense granules, although
this is the default route in wild-type parasites (2, 12). We then
examined the effect of low level expression of Rab6 mutants in
tachyzoites by isolating stable parasite clones coexpressing
HA-Rab6(Q70L) or Rab6(N124I) along with BAP. These clones retained
endogenous Rab6 along with a single integrated copy of the mutant
proteins expressed from the low level tachyzoite
Sag1 promoter (data not shown). In contrast to
parasites transiently overexpressing HA-Rab6(Q70L), BAP secretion was
essentially unaltered in HA-Rab6(Q70L) clones, whereas HA-Rab6(N124I)
parasite clones exhibited a similar attenuation upon secretion of BAP
compared with those transiently expressing the mutant
(n = four independent experiments) (Fig.
8B).
To confirm these results for endogenous dense granule proteins,
we examined the secretion of GRA4 by quantitative immunoprecipitation in parasite clones expressing HA-Rab6(Q70L) or HA-Rab6(T25N). Once
again, the activated Rab6 mutant had little effect on dense granule
secretion compared with untransfected parasites, whereas stable
expression of the dominant inhibitory mutant was attenuated upon
secretion of GRA4 (Fig. 8C). To address the possibility that protein modification in the parasite Golgi could be altered in the
presence of the Rab6 mutants due to retrograde imbalance of glycosylation enzymes, the O-linked glycosylation of GRA2
was examined by metabolic pulse-chase. Within minutes of synthesis, GRA2 is modified by O-linked glycosylation from an ~26-kDa
precursor to an ~28-kDa mature protein (27, 32). At 15 min of chase, a kinetic lag was observed in the mobility of GRA2 in parasites stably
expressing HA-Rab6(T25N), whereas GRA2 mobility in parasites expressing
HA-Rab6(Q70L) was unaltered compared with wild-type parasites (Fig.
8D). By 30 min of chase, nearly 100% of GRA2 was modified
in all parasite clones (Fig. 8D). In summary, elevated overexpression of wild-type and GTP-activated Rab6 blocks
constitutive protein secretion, whereas GDP-bound and nucleotide-free
Rab6 mutants appear to attenuate dense granule protein transport and glycosylation. Consequently, Rab6 appears to regulate a retrograde pathway involving transport of dense granule proteins to the Golgi.
Rab6 Marks Golgi Fission, and Rab6 Mutants Alter Cytokinesis
in Toxoplasma Tachyzoites--
Upon both plaque assay and uracil
incorporation analysis, growth or invasiveness of activated and
inhibitory Rab6 clones was reduced in comparison with wild-type
parasites (Fig. 9). Although Rab6 mutants
did not alter transport of rhoptry proteins to the post-Golgi
organelle, HA-Rab6(Q70L) and HA-Rab6(N124I) parasites were enriched in
tubulovesicular precursor rhoptry organelles (data not shown). Because
rhoptry biogenesis is itself developmentally regulated, we examined the
parasite clones for developmental alterations. Daughter
Toxoplasma merozoites form in pairs within the maternal parasite by a process of internal budding termed endodyogeny. This
process is initiated through a pair of cytoplasmic centrosomes, which
form the intranuclear spindle poles and behave as a
microtubule-organizing center (33). During mitosis, the Golgi stacks
partition in two near the centrosomes. This was easily visualized in
parasites expressing Golgi-localized HA-Rab6(Q70L). The medial fission
of the Golgi stacks occurred in close juxtaposition with the duplicated centrosomes and appeared to be complete prior to nuclear division (Fig.
10, K-N). During
cytokinesis, activated Rab6 and centrin were perfectly colocalized
(Fig. 10M, arrow with asterisk), suggesting a
tethering of the late Golgi with the centrosomes.
Concurrently with Golgi partitioning, a scaffold for
daughter cell assembly is formed from subpellicular microtubules and an
associated network of flattened membranes, the IMC. During cytokinesis,
the IMC migrates posteriorly toward and around each nuclear pole,
partitioning the replicated nucleus into the two forming daughters.
Strikingly, some HA-Rab6(Q70L) and HA-Rab6(N124I) parasite vacuoles
exhibited unusual patterns of division, forming multiple daughters
(Fig. 10A), indicative of replication by endopolygeny rather
than endodyogeny. The IMC of each daughter enveloped a complete set of
organelles, including Golgi, apicoplasts, and pre-rhoptries, indicating
that appropriate organellar replication and segregation were occurring
concurrent with nuclear division. The frequency of endopolygeny was
quantitated using antibody to IMC1, an antigen of the IMC in both adult
and forming daughter parasites, thus serving as a hallmark of daughter
cell formation by microscopy (34). In nearly all untransfected
tachyzoites, parasites budded by endodyogeny, with two forming
daughters distinguishable (Fig. 10B), although on rare
occasions (~2% of all vacuoles containing dividing parasites) (Fig.
10F), multiple daughter parasites could be found in a single
parent. However, in HA-Rab6(Q70L) (data not shown) and HA-Rab6(N124I)
(Fig. 10, E-J, arrows) clones, vacuoles harboring parasites replicating by endopolygeny were 5 and 10 times
more common, respectively (Fig. 10F). These results suggest that altered Rab6 function enhances uncoupling of cytokinesis and
karyokinesis in developing parasites.
The highly polarized secretory pathway of T. gondii,
complete with a single stack of apically oriented Golgi cisternae,
provides a simple model for protein transport in eukaryotes. We have
identified the T. gondii homolog of the highly conserved
GTPase Rab6 as the first marker of the parasite Golgi and as a
potential mediator of retrograde transport from dense granule secretory
organelles. Whereas Rab6 paralogs function in retrograde transport from
post-Golgi-, intra-Golgi-, and Golgi-to-ER transport pathways in
mammalian cells, and Ypt6p functions in recycling of Golgi proteins
from the endosomal pre-vacuole in yeast, Toxoplasma Rab6
appears to regulate in part post-Golgi transport of constitutively
secreted proteins to the parasite Golgi. Our results further suggest
that Rab6 function may be required for normal cytokinesis or that
parasite cell division may be sensitive to perturbation of the
Rab6-mediated retrograde pathway.
We propose that the role of Rab6 in Toxoplasma dense granule
protein transport is retrograde from a post-Golgi compartment, based
upon several observations. Generally, activated forms of Rab proteins
concentrate on the target membrane for vesicle transport. Both
wild-type and GTP-activated Rab6 localize to the late Golgi cisternae
and inhibit constitutive secretion of dense granule proteins only when
highly overexpressed, resulting in Golgi and ER retention of the dense
granule proteins. In this instance, Rab6 augments the effect of BFA on
retrograde transport to the ER, resulting in resorption of the Golgi
cisternae and associated cargo. BFA inhibits GTPase exchange proteins
mediating ARF1 recruitment that is essential for COPI-regulated vesicle
budding (35). Toxoplasma ARF1 mutants induce a similar block
in the early transport of dense granule proteins (8), indicating that
both COPI-mediated and Rab6-mediated pathways function in dense granule
protein transport.
In contrast to activated Rab6, dominant inhibitors Rab6(N124I) and
Rab6(T25N) alter post-Golgi transport of dense granule proteins,
accumulating these proteins in the Golgi and altering glycosylation
rates, indicative of an imbalance in Golgi modification enzymes.
However, the viability of the mutants and the relatively modest effects
on constitutive secretion in stable clones indicate that Rab6 function
is dispensable for secretion in Toxoplasma, in common with
Ypt6p in yeast. Nonetheless, the specific association of Rab6 with
dense granule membranes and small vesicles near the trans-face of the Golgi complex, in addition to the
entrapment of dense granule proteins in the Golgi induced by
dominant-negative mutants, implicates a transport pathway linking the
two organelles. This pathway may serve a recycling function from mature
dense granules or alternatively play a direct role in dense
granule biogenesis or maturation through protein sorting.
A novel Rab6-mediated Golgi-to-ER retrograde transport in mammalian
cells was initially proposed based upon the BFA-like phenotypes of
activated Rab6 on Golgi enzymes (14). This pathway was further defined
by following transport of Shiga and Shiga-like toxins to the ER (15)
and the finding that fluorescent protein-Rab6 transits along
microtubule tracks between the Golgi and ER sites at the mammalian cell
periphery (16). It is thought that this retrograde pathway may regulate
recycling of proteins lacking ER retrieval motifs, recycling of lipids,
and the slow continuous cycling of resident Golgi proteins. A
post-Golgi function for mammalian Rab6 in recycling between early or
recycling endosomes and the TGN has now distinguished the highly
homologous Rab6A' isoform from Rab6A, which is implicated in
Golgi-to-ER transport (36). Evidence for Rab6 association with
post-Golgi secretory vesicles is less clear, although Rab6 has been
postulated to play a role in transport of rhodopsin on post-Golgi
vesicles in retinal rod photoreceptor cells (37) and to function at an
early step in the biogenesis of small synaptic vesicles of embryonic
neurons (38) and was found to be associated with secretory granules in
rat atrial myocytes (39).
The role of the yeast homolog of Rab6, Ypt6p, is increasingly well
understood. Yeast late Golgi membrane proteins cycle through a
pre-vacuolar endocytic compartment and are retrieved to the Golgi.
Ypt6p mutants disrupt the cycling of the endoprotease Kex2p and
Vps10p, the sorting receptor for the vacuolar hydrolase
carboxypeptidase Y, which is consequently partially missorted to the
plasma membrane (40). Recent identification of effector and binding
proteins acting on Ypt6p has elucidated a mechanistic role for
GTP-activated Ypt6p, the trimeric VPS52-VPS53-VPS54 complex, and the
SNARE Tlg1p in sorting and docking of transport vesicles to the late
Golgi. The Ypt6p GTP exchange factor has been identified as a stable heteromeric complex of Ric1p-Rgp1p peripherally associated with Golgi
membranes (20). After GDP/GTP exchange, activated Ypt6p recruits the
VPS52-VPS53-VPS54 complex to the Golgi membrane, which in turn binds
the endosomal/late Golgi SNARE Tlg1p on endosome-derived vesicles and
allows fusion upon SNARE pairing (19).
It is possible that an intermediate endosomal compartment may exist
between dense granules and the Golgi. Toxoplasma Rab5A localizes to a tubulovesicular compartment closely opposed to, but
distinct from, Golgi cisternae, and Rab5 mutants alter cholesterol ester-rich lipid body formation (41). The yeast Rab5 homolog, Ypt51p, controls transport of Golgi-derived vesicles to the
yeast pre-vacuole and vacuole, opposite in nature to Ypt6p-mediated transport (42). Toxoplasma Rab5 and Rab6 may act as opposing partners in similar pathways. For now, identification of endosomal and
recycling compartments in Toxoplasma awaits characterization with biochemical and molecular markers in addition to Rab5.
A novel role for Rab6 function was suggested by the appearance of Rab6
mutant parasites altered in cytokinesis. Paired internal budding
(endodyogeny) is the predominant mechanism of cytokinesis in T. gondii. Although karyokinesis and cytokinesis can be uncoupled in
Toxoplasma tachyzoites, as demonstrated through the use of microtubule inhibitors (33), organellar and nuclear divisions are
coupled through association with centrioles. Endopolygeny, a derivative
of endodyogeny, occurs as a consequence of multiple nuclear and
organellar divisions paired with new apical complex and IMC
formation anywhere in the cytoplasm of the parent cell. This differs
from schizogony, a common form of asexual division in Apicomplexa in
which multiple daughter nuclei are ordered along the maternal cell
periphery before cytokinesis initiates. Using the IMC as a hallmark of
nascent daughter formation, HA-Rab6(N124I) and HA-Rab6(Q70L) parasite
clones partially shifted to endopolygeny. Furthermore, activated Rab6
on Golgi membranes colocalized with centrin specifically during
cytokinesis, suggesting a role for Rab6 in coordinating cytokinesis
with the nuclear cycle.
Recently, effectors of Rab6 have been identified that may function
directly in cytokinesis. A Rab6-binding kinesin, alternately named
Rabkinesin-6, RB6K, and Rab6-KIFL, was first identified through a Rab6
protein two-hybrid screen (43). This motor protein interacts with the
effector domain of GTP-activated Rab6 and binds to microtubules
both in vitro and in vivo. Rab6 may regulate the association and dissociation of Rabkinesin-6 with the microtubule cytoskeleton and, as a complex, control movement of Golgi membranes and
transport vesicles along microtubules (43). However, Rab6-KIFL is also
proposed to function in cell division during cytokinesis (44, 45).
Rab6-KIFL was shown to localize to the mid-zone of the mitotic spindle
and to the cleavage furrow and midbody during late stages of mitosis,
and overexpression of the protein induced a defect in cleavage furrow
formation, blocking cytokinesis. A second effector, GAPCenA, has been
identified by a yeast two-hybrid screen and serves as a Rab6-specific
GTPase-activating protein, with the dual capacity to transiently
interact with centrosomes (46). GAPCenA bears homology to spindle
checkpoint proteins in yeast, can form complexes with
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP and the
Rediprime II kit (Amersham Biosciences) to a specific activity of
>108 cpm/µg. Hybridizations were performed in high
stringency buffer.
-D-thiogalactopyranoside-induced cultures and
extracts from T. gondii and Vero cells were subjected to
12% SDS-PAGE and prepared for immunoblotting or GTP binding by
electrophoretic transfer to nitrocellulose membranes. Immunoblot analysis was performed with the ECL detection system (Amersham Biosciences) using, as primary antibody, rabbit serum (Zymed
Laboratories, Inc.) immunized with a synthetic peptide encompassing
T. gondii Rab6-(192-206) and displaying strong
enzyme-linked immunosorbent assay activity. Membranes were exposed on
X-Omat AR autoradiography film (Eastman Kodak Co.). Scanned
images were semiquantitatively analyzed with Scion Image software.
-32P]GTP was
added to a concentration of 1 µCi/ml and incubated for 2 h, and
the blots were washed with several changes of binding buffer for 2 h. Gels were dried and exposed at
70 °C on X-Omat AR film.
3 Prime, Inc.), anti-IMC1 monoclonal antibody (mAb) 45.5 (gift of Gary Ward, University of Vermont, Burlington, VT), and
anti-GRA1 mAb T5-2B4 and anti-GRA2 mAb T4-1F5 (27). Secondary
antibodies included fluorescein isothiocyanate-conjugated sheep
anti-mouse IgG (Calbiochem), rhodamine-conjugated goat anti-rabbit IgG
(Calbiochem), and Alexa-conjugated equivalents (Molecular Probes,
Inc.). Epifluorescence and phase-contrast microscope images were
captured on a Photometrics SenSys CCD camera and processed using Image
Pro Plus software (Media Cybernetics).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Molecular analysis of T. gondii rab6. A, Southern
analysis of 10 µg of gDNA derived from T. gondii and host Vero cells. Lane M, molecular size
standards in kilobase pairs (kbp); lane 1,
T. gondii HindIII digest; lane 2, Vero
HindIII digest; lane 3, T. gondii NcoI
digest; lane 4, Vero NcoI digest; lane
5, T. gondii HindIII and NcoI digest;
lane 6, Vero HindIII and NcoI digest.
B, left panel, ethidium bromide-stained gel of
total T. gondii (T) and Vero (V)
cellular RNAs. Lane M, RNA ladder. Right panel,
Northern blot analysis results indicating a mature T. gondii
Rab6 mRNA of ~1.3 kb. C, phylogenetic analysis of Rab6
protein homologs. Parsimony analysis using the PAUP program was
performed on the alignment in D corresponding to amino acids
6-185 of T. gondii Rab6 (180/209 amino acids), excluding
the divergent amino and carboxyl termini. A single most parsimonious
tree (shown) was found. Bootstrap analysis was then run using 1000 replicates of the alignment, and a consensus tree with identical
topology was produced. Values at tree nodes represent bootstrap
confidence values supporting nodal placement. D, sequence
alignment of Rab6 proteins. Highly conserved residues are
boxed in black. Functionally conserved domains
include the phosphate/magnesium loops (PM), GTP-binding
domains (G), switch regions, effector loops (L),
and junctions between helices and loops ( 2-L5 and
3-L7) (30, 31,
47). GenBankTM/EBI and Swiss-Prot sequence accession
numbers that were used in the alignment and analysis are as follows:
human Rab6A (M28212), human Rab6B (Q9NRW1), Nicotiana
tabacum (L29273), Plasmodium falciparum (X92977),
Drosophila melanogaster (D84314), Plasmodium
berghei (AF093827), Arabidopsis thaliana (L26984),
Saccharomyces cerevisiae (S.c.) Ypt6p (X59598),
Schizosaccharomyces pombe Ryh1 (X52475),
Caenorhabditis elegans (P34213), and T. gondii (AF228419).
-32P]GTP
nearly equally, but HA-Rab6(N124I) (lane 4) did not bind detectable amounts of GTP, confirming the anticipated GTP-binding characteristics of the mutants.
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Fig. 2.
Western and GTP blot assays of recombinant
Toxoplasma Rab6 and Rab6 mutant proteins.
A, protein gel of bacterially expressed Rab6 proteins
induced by isopropyl- -D-thiogalactopyranoside.
Lane M, molecular mass standards; lane 1, pET15b
expression control; lane 2, HA-Rab6; lane 3,
HA-Rab6(Q70L); lane 4, HA-Rab6(N124I). Induced protein at
~30 kDa is visible in Rab6 expression lysates. B, Western
blot analysis of gel in A probed with rabbit
anti-T. gondii Rab6-(192-206). The 30-kDa band is
present in Rab6 lysates (lanes 2-4), but not in
control pET15b lysate (lane 1). C,
[
-32P]GTP blot overlay of identical gel run in
parallel to that in A.
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Fig. 3.
Toxoplasma HA-Rab6 and
HA-Rab6(Q70L) localization in parasites and Golgi localization in CHO
cells. For orientation, the apical end of a single
tachyzoite in each transfected parasite vacuole is labeled with an
asterisk. Transiently expressed HA-Rab6 (A) and
HA-Rab6(Q70L) (C) were localized to the single T. gondii Golgi stack (arrows), cytoplasm, and dense
granules (arrows with asterisks). The corresponding
phase-contrast images for A and C are shown in
B and D. Transiently expressed T. gondii Rab6 partially colocalized with giantin (E-G)
and GRASP65 (H-J) in CHO cells. T. gondii
HA-Rab6 transiently expressed in CHO cells was localized to a
juxtanuclear compartment (E and H). Also shown is
the cisternal labeling of the Golgi with giantin (F) and
with GRASP65 (I). Merged images show colocalization of
Toxoplasma Rab6 (red) with giantin
(green) (G) or with GRASP65 (green)
(J) along Golgi cisternae.
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Fig. 4.
Immunolocalization of Rab6 in T. gondii HA-Rab6(Q70L) clones. Immunogold labeling
was enriched at the lateral ends of the trans-most Golgi
(Go) cisternae (A), on microdomains of dense
granule (DG) membranes (B, C, and
E), on lucent vacuole membranes (E), and
associated with cytoplasmic centrosomes (Ce) (D).
No labeling was observed in rhoptries (Rh), micronemes
(Mi), apicoplasts (Ap), and the nucleus
(Nu). Control cryosections of wild-type parasites with
equivalent antibody dilutions were completely free of immunogold label
(data not shown). Also shown is the frequency distribution of
HA-Rab6(Q70L) antigen (Ag) on cryosections (F).
19 random cryosections encompassing 34 parasites were quantitated for
total gold distribution. DG, dense granules;
cytopl+sv, labeling of the cytoplasm and small vesicle
(these were co-quantitated because vesicle membranes were frequently
difficult to differentiate); ER+tER, labeling of the ER and
perinuclear envelope; ERGIC, intermediate compartment
between the Golgi and nuclear envelope; endosome, electron
lucent vacuoles; mic, micronemes; rhop,
rhoptries; mito, mitochondrion; apicopl,
apicoplast; IMC/PM, inner membrane complex and
plasma membrane.
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Fig. 5.
Overexpressed Rab6 augments the brefeldin
A-induced retrograde transport of dense granule proteins.
A, in untreated parasites, BAP was secreted through
dense granules and accumulated in the PV. In D-I, treatment
with BFA induced intracellular retention, ultimately accumulating BAP
in the perinuclear ER. In parasites transiently overexpressing HA-Rab6
(C, arrows), BAP secretion through dense granules
was partially inhibited, accumulating in the Golgi and perinuclear ER
(B, arrows and asterisks). Both BAP (E
and H) and transiently expressed HA-Rab6 (F and
I) were partially collapsed to the perinuclear ER following
treatment with 5 µg/ml BFA for 15 or 60 min. In J, shown
is the quantitation of the appearance of BAP in the parasite ER
following BFA (5 or 20 µg/ml) addition. Perinuclear and reticular
labeling was more pronounced and rapidly induced in cells
overexpressing HA-Rab6.
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Fig. 6.
Transient overexpression of Rab6 mutants in
T. gondii inhibits trafficking of BAP to
dense granules and secretion into the PV. In parasites
overexpressing GTP-activated HA-Rab6(Q70L) (A), BAP
accumulated in the Golgi, perinuclear envelope, and ER (B,
arrow). In parasites transiently overexpressing
HA-Rab6(N124I), which is predominately cytosolic (D), BAP
accumulated in a dispersed vesicular pattern throughout the parasite
(E). In parasites transiently overexpressing HA-Rab6(T25N)
(G), BAP was dispersed in vesicles and accumulated at or
juxtaposed to the Golgi (H, arrow). Corresponding
phase-contrast images are shown in C, F, and
I.
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Fig. 7.
Rab6(T25N) alters transport of endogenous
dense granule proteins and BAP. A, BAP in
parasites coexpressing Rab6(T25N) was diffuse and accumulated near the
Golgi (punctate label centralized in each tachyzoite). B,
GRA1 partially accumulated near the Golgi with reduced dense granule
labeling. C, merged images show colocalization
(yellow) of BAP (red channel) and GRA1
(green channel) in the region of the Golgi and after
secretion into the PV (near the asterisk). D,
shown is a phase-contrast image. Identical distributions were evident
for dense granule proteins GRA2-4 in parasites transfected with
Rab6(T25N) or Rab6(N124I).
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Fig. 8.
Rab6 inhibits dense granule protein secretion
in T. gondii transiently
overexpressing Rab6 GTP-binding domain mutants. A,
inhibition of BAP secretion in parasites transiently overexpressing
HA-Rab6 or HA-Rab6 mutants. Shown are the results from quantitative
immunoprecipitation of intracellular and secreted BAP protein in
parasites transiently expressing BAP alone or coexpressing BAP and
HA-Rab6 or HA-Rab6 mutants and expressed as fractions secreted over 60 min. B, BAP secretion in Toxoplasma HA-Rab6(Q70L)
and HA-Rab6(N124I) stable clones. Shown are the results from
quantitative immunoprecipitation of intracellular and secreted BAP in
parasites stably expressing BAP alone or coexpressing BAP and
HA-Rab6(Q70L) or HA-Rab6(N124I) and expressed as fractions secreted
over 60 min. The results shown are representative of four independent
experiments with two clonal lines for each Rab6 mutant. C,
GRA4 secretion in Toxoplasma HA-Rab6(Q70L) and HA-Rab6(T25N)
stable clones. Upper panel, 40-kDa GRA4 immunoprecipitates
from intracellular and PV fractions as follows. Lanes 1 and
2, hypoxanthine-xanthine-guanine phosphoribosyltransferase
(HXGPRT)-knockout parental strain intracellular and secreted
fractions; lanes 3 and 4, Rab6(Q70L) clone
intracellular and secreted forms; lanes 5 and 6,
Rab6(T25N) clone intracellular and secreted fractions. Lower
panel, quantitation of GRA4 immunoprecipitates expressed as
fractions secreted over 60 min. D, glycosylation of GRA2 in
Toxoplasma wild-type (WT), HA-Rab6(Q70L), and
HA-Rab6(T25N) stable clones. Shown are the results from quantitative
immunoprecipitation of intracellular GRA2 expressed as fractions of a
28-kDa mature protein (double asterisks) to a total of a
26-kDa precursor (single asterisk) + a mature form following
a 12-min metabolic pulse with or without a chase of 15 and 30 min. Gel
results for Rab6(Q70L) (lanes 1-3; 0-, 15-, and 30-min
chases, respectively) and Rab6(T25N) (lanes 4-6; 0-, 15-, and 30-min chases, respectively) are shown at the top. P,
pellet; S, supernatant.
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Fig. 9.
Replication and survival competence of
parasite clones stably expressing HA-Rab6(Q70L) and
HA-Rab6(N124I). A, uracil incorporation assay
measuring DNA synthesis within Toxoplasma cells. After
infection for 24 h in host cells, intracellular parasites were
labeled with [3H]uracil for 1 h. B,
plaque assay determining survival rates after 9 days in culture.
Toxoplasma plaques were scored relative to 100 parasites
plated on human foreskin fibroblast (HFF) host cell
monolayers (n = three experiments). Relative to BAP
clones, HA-Rab6(Q70L) (42%) and HA-Rab6(N124I) (26%) parasites were
maturation-impaired. RH, wild type
Toxoplasma.
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Fig. 10.
Rab6 mutants are altered in cytokinesis, and
activated Rab6 colocalizes with centrin during cytokinesis.
A, transmission electron microscopy of a HA-Rab6(Q70L)
parasite clone in late endopolygeny, with three forming daughter
parasites displaying nuclei (N), apicoplasts (A),
and rhoptries (P), enveloped within an IMC.
B, immunofluorescence analysis of wild-type parasites
undergoing endodyogeny (arrows) with IMC1. In daughter
cells, IMC1 tracked the IMC plane as it migrated posteriorly toward the
nucleus (B). C, 4,6-diamidino-2-phenylindole
(DAPI) staining of DNA; D and E, merge
and phase-contrast images, respectively. F, rates of
endopolygeny in Toxoplasma hypoxanthine-xanthine-guanine
phosphoribosyltransferase (HXGPRT)-deficient, HA-Rab6(Q70L),
and HA-Rab6(N124I) clones. Occurrence of vacuoles with parasites
undergoing endopolygeny (more than two daughter parasites) is defined
by nascent IMC1 labeling and expressed as a fraction of all vacuoles
with daughter parasites. 1000 vacuoles with dividing parasites were
scored for each clone. G, HA-Rab6(N124I) parasites
undergoing endopolygeny (arrows) with anti-IMC1 antibody.
H, 4,6-diamidino-2-phenylindole staining of DNA;
I and J, merge and phase-contrast images,
respectively. In one cell, a daughter parasite is emerging from the
maternal body (G-I, asterisks). K-N,
Golgi membranes labeled with Rab6(Q70L) (K) undergoing
medial fission (lower parasite vacuole, horizontal and
vertical arrows) and during cytokinesis (arrows with
asterisks). Centrin (L) is juxtaposed with Golgi
membranes during mitosis (M), but only colocalized during
daughter cell budding (M, arrow with asterisk).
In the phase-contrast image (N), a forming daughter is
delimited by a phase-contrast lucent zone (arrow with
asterisk).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin, and
has a role in microtubule nucleation. Intriguingly, assembly of the
Toxoplasma conoid, an initial step in parasite cytokinesis,
involves
-tubulin. Although they remain to be identified, Rab6
effectors similar to Rabkinesin-6/Rab6-KIFL and GAPCenA may be
operative in Toxoplasma, and alterations in parasite
cytokinesis might be due to a dominant-interfering effect of Rab6
mutants on these effectors. In total, it appears that Rab6 or its
effectors may play bimodal roles, functioning not only in protein
transport, but also in cell division.
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ACKNOWLEDGEMENTS |
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We thank Marc Pypaert and Kim Murphy (Yale Center for Cell Imaging) for preparation of electron microscope samples.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant ROI AI30060 and a scholar award in molecular parasitology from the Burroughs Wellcome Fund (to K. A. J.) and by National Research Service Award 1F32 AI09938-01 (to T. T. S.).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/EBI Data Bank with accession number(s) AF228419.
To whom correspondence should be addressed: Dept. of Internal
Medicine, Yale University School of Medicine, 333 Cedar St., LCI 808, P. O. Box 208022, New Haven, CT 06520-8022. Tel.: 203-785-7573; Fax:
203-785-3864; E-mail: Stedman@biomed.med.yale.edu.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M209390200
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ABBREVIATIONS |
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The abbreviations used are: PV, parasitophorous vacuole; TGN, trans-Golgi network; ER, endoplasmic reticulum; ARF1, ADP-ribosylation factor-1; SNARE, soluble underln]N-ethylmaleimide-sensitive fusion protein attachment protein receptor; RACE, rapid amplification of cDNA ends; ORF, open reading frame; HA, hemagglutinin; BAP, bacterial alkaline phosphatase; IMC, inner membrane complex; mAb, monoclonal antibody; CHO, Chinese hamster ovary; BFA, brefeldin A.
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1. |
Joiner, K. A.,
and Roos, D. S.
(2002)
J. Cell Biol.
157,
557-563 |
2. |
Chaturvedi, S., Qi, H.,
Coleman, D.,
Rodriguez, A.,
Hanson, P. I.,
Striepen, B.,
Roos, D. S.,
and Joiner, K. A.
(1999)
J. Biol. Chem.
274,
2424-2431 |
3. | Tooze, S. A. (1998) Biochim. Biophys. Acta 1404, 231-244[Medline] [Order article via Infotrieve] |
4. |
Lecordier, L.,
Mercier, C.,
Sibley, L. D.,
and Cesbron-Delauw, M. F.
(1999)
Mol. Biol. Cell
10,
1277-1287 |
5. | Labruyere, E., Lingnau, M., Mercier, C., and Sibley, L. D. (1999) Mol. Biochem. Parasitol. 102, 311-324[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Mercier, C.,
Cesbron-Delauw, M. F.,
and Sibley, L. D.
(1998)
J. Cell Sci.
111,
2171-2180 |
7. | Stedman, T. T., and Joiner, K. A. (1999) in Phagocytosis and Pathogens (Gordon, S., ed), Vol. 6 , pp. 233-261, JAI Press Inc., Greenwich, CT |
8. |
Liendo, A.,
Stedman, T. T.,
Ngo, H. M.,
Chaturvedi, S.,
Hoppe, H. C.,
and Joiner, K. A.
(2001)
J. Biol. Chem.
276,
18272-18281 |
9. |
Hager, K. M.,
Striepen, B.,
Tilney, L. G.,
and Roos, D. S.
(1999)
J. Cell Sci.
112,
2631-2638 |
10. | Hoppe, H. C., Ngo, H. M., Yang, M., and Joiner, K. A. (2000) Nat. Cell. Biol. 2, 449-456[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Di Cristina, M.,
Spaccapelo, R.,
Soldati, D.,
Bistoni, F.,
and Crisanti, A.
(2000)
Mol. Cell. Biol.
20,
7332-7341 |
12. |
Karsten, V., Qi, H.,
Beckers, C. J.,
Reddy, A.,
Dubremetz, J. F.,
Webster, P.,
and Joiner, K. A.
(1998)
J. Cell Biol.
141,
1323-1333 |
13. | Martinez, O., Schmidt, A., Salamero, J., Hoflack, B., Roa, M., and Goud, B. (1994) J. Cell Biol. 127, 1575-1588[Abstract] |
14. |
Martinez, O.,
Antony, C.,
Pehau-Arnaudet, G.,
Berger, E. G.,
Salamero, J.,
and Goud, B.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1828-1833 |
15. | Girod, A., Storrie, B., Simpson, J. C., Johannes, L., Goud, B., Roberts, L. M., Lord, J. M., Nilsson, T., and Pepperkok, R. (1999) Nat. Cell. Biol. 1, 423-430[CrossRef][Medline] [Order article via Infotrieve] |
16. |
White, J.,
Johannes, L.,
Mallard, F.,
Girod, A.,
Grill, S.,
Reinsch, S.,
Keller, P.,
Tzschaschel, B.,
Echard, A.,
Goud, B.,
and Stelzer, E. H.
(1999)
J. Cell Biol.
147,
743-760 |
17. |
Li, B.,
and Warner, J. R.
(1996)
J. Biol. Chem.
271,
16813-16819 |
18. | Li, B., and Warner, J. R. (1998) Yeast 14, 915-922[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Siniossoglou, S.,
and Pelham, H. R.
(2001)
EMBO J.
20,
5991-5998 |
20. |
Siniossoglou, S.,
Peak-Chew, S. Y.,
and Pelham, H. R.
(2000)
EMBO J.
19,
4885-4894 |
21. |
Bensen, E. S.,
Yeung, B. G.,
and Payne, G. S.
(2001)
Mol. Biol. Cell
12,
13-26 |
22. | Roos, D. S., Donald, R. G., Morrissette, N. S., and Moulton, A. L. (1994) Methods Cell Biol. 45, 27-63[Medline] [Order article via Infotrieve] |
23. |
Donald, R. G. K.,
Carter, D.,
Ullman, B.,
and Roos, D. S.
(1996)
J. Biol. Chem.
271,
14010-14019 |
24. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
25. | Soldati, D., and Boothroyd, J. C. (1993) Science 260, 349-352[Medline] [Order article via Infotrieve] |
26. | Lapetina, E. G., and Reep, B. R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2261-2265[Abstract] |
27. | Achbarou, A., Mercereau-Puijalon, O., Sadak, A., Fortier, B., Leriche, M. A., Camus, D., and Dubremetz, J. F. (1991) Parasitology 103, 321-329[Medline] [Order article via Infotrieve] |
28. | Karsten, V., Qi, H., Beckers, C. J., and Joiner, K. A. (1997) Methods Companion Methods Enzymol. 13, 103-111[CrossRef] |
29. |
Echard, A.,
Opdam, F. J.,
de Leeuw, H. J.,
Jollivet, F.,
Savelkoul, P.,
Hendriks, W.,
Voorberg, J.,
Goud, B.,
and Fransen, J. A.
(2000)
Mol. Biol. Cell
11,
3819-3833 |
30. | Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127[CrossRef][Medline] [Order article via Infotrieve] |
31. | Valencia, A., Chardin, P., Wittinghofer, A., and Sander, C. (1991) Biochemistry 30, 4637-4648[Medline] [Order article via Infotrieve] |
32. | Zinecker, C. F., Striepen, B., Tomavo, S., Dubremetz, J. F., and Schwarz, R. T. (1998) Mol. Biochem. Parasitol. 97, 241-246[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Shaw, M. K.,
Compton, H. L.,
Roos, D. S.,
and Tilney, L. G.
(2000)
J. Cell Sci.
113,
1241-1254 |
34. |
Hu, K.,
Mann, T.,
Striepen, B.,
Beckers, C. J.,
Roos, D. S.,
and Murray, J. M.
(2002)
Mol. Biol. Cell
13,
593-606 |
35. | Roth, M. G. (1999) Cell 97, 149-152[Medline] [Order article via Infotrieve] |
36. |
Mallard, F.,
Tang, B. L.,
Galli, T.,
Tenza, D.,
Saint-Pol, A.,
Yue, X.,
Antony, C.,
Hong, W.,
Goud, B.,
and Johannes, L.
(2002)
J. Cell Biol.
156,
653-664 |
37. |
Deretic, D.,
and Papermaster, D. S.
(1993)
J. Cell Sci.
106,
803-813 |
38. |
Tixier Vidal, A.,
Barret, A.,
Picart, R.,
Mayau, V.,
Vogt, D.,
Wiedenmann, B.,
and Goud, B.
(1993)
J. Cell Sci.
105,
935-947 |
39. |
Iida, H.,
Tanaka, S.,
and Shibata, Y.
(1997)
Am. J. Physiol.
272,
C1594-C1601 |
40. |
Tsukada, M.,
and Gallwitz, D.
(1996)
J. Cell Sci.
109,
2471-2481 |
41. | Robibaro, B., Stedman, T. T., Coppens, I., Ngo, H. M., Pypaert, M., Bivona, T., Nam, H. W., and Joiner, K. A. (2002) Cell Microbiol. 4, 139-152[CrossRef][Medline] [Order article via Infotrieve] |
42. | Gotte, M., Lazar, T., Yoo, J. S., Scheglmann, D., and Gallwitz, D. (2000) Subcell. Biochem. 34, 133-173[Medline] [Order article via Infotrieve] |
43. |
Echard, A.,
Jollivet, F.,
Martinez, O.,
Lacapere, J. J.,
Rousselet, A.,
Janoueix-Lerosey, I.,
and Goud, B.
(1998)
Science
279,
580-585 |
44. |
Hill, E.,
Clarke, M.,
and Barr, F. A.
(2000)
EMBO J.
19,
5711-5719 |
45. |
Fontijn, R. D.,
Goud, B.,
Echard, A.,
Jollivet, F.,
van Marle, J.,
Pannekoek, H.,
and Horrevoets, A. J.
(2001)
Mol. Cell. Biol.
21,
2944-2955 |
46. |
Cuif, M. H.,
Possmayer, F.,
Zander, H.,
Bordes, N.,
Jollivet, F.,
Couedel-Courteille, A.,
Janoueix-Lerosey, I.,
Langsley, G.,
Bornens, M.,
and Goud, B.
(1999)
EMBO J.
18,
1772-1782 |
47. | Stenmark, H., Valencia, A., Martinez, O., Ullrich, O., Goud, B., and Zerial, M. (1994) EMBO J. 13, 575-583[Abstract] |