From the Section of Infectious Diseases, Department
of Internal Medicine, and the ¶ Department of Cell Biology, Yale
University School of Medicine, New Haven, Connecticut 06520-8022
Received for publication, August 13, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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We have previously demonstrated that
Toxoplasma gondii has a tyrosine-based sorting system,
which mediates protein targeting to the lysosome-like rhoptry secretory
organelle. We now show that rhoptry protein targeting is also dependent
on a dileucine motif and occurs from a post-Golgi endocytic organelle
to mature rhoptries in an adaptin-dependent fashion. The
T. gondii AP-1 adaptin complex is implicated in this
transport because the µ1 chain of T. gondii AP-1
(a) was localized to multivesicular endosomes and the
limiting and luminal membranes of the rhoptries; (b) bound to endocytic tyrosine motifs in rhoptry proteins, but not in proteins from dense granule secretory organelles; (c) when mutated
in predicted tyrosine-binding motifs, led to accumulation of the
rhoptry protein ROP2 in a post-Golgi multivesicular compartment; and
(d) when depleted via antisense mRNA, resulted in
accumulation of multivesicular endosomes and immature rhoptries. These
are the first results to implicate AP-1 in transport from a post-Golgi
compartment to a mature secretory organelle and substantially expand
the role for AP-1 in anterograde protein transport.
Obligate intracellular protozoa of the phylum Apicomplexa are
highly sophisticated secretory cells. Although the host cell type and
the nature of the intracellular vacuole differ when
Plasmodium, Toxoplasma,
Cryptosporidium, Eimeria, Theileria,
and Babesia are compared, these organisms share common
secretory organelles: micronemes, rhoptries, and dense granules.
Differential secretion from these organelles directs invasion of host
cells, formation of an intracellular parasitophorous vacuole,
and subsequent modification of the vacuole for replication (reviewed in
Ref. 1).
Most unique morphologically and biochemically are the rhoptries. These
are flask-shaped secretory organelles that are assembled and positioned
at the apical pole of the invasive forms of Apicomplexa (reviewed in
Refs. 2-4). At the onset of host cell invasion, prepackaged proteins
and membranous materials from rhoptries are secreted through an apical
opening. Rhoptry constituents are likely involved in a plethora of
essential functions. These include directing host cell attachment and
invasion (3), expansion and maintenance of the parasitophorous vacuole
membrane surrounding the intracellular parasite (5), and attachment of
host mitochondria and the endoplasmic reticulum to the parasitophorous
vacuole membrane (6). Furthermore, rhoptry proteins are targets of the
normal immune response; antibodies to rhoptry proteins block invasion;
and immunization with selected rhoptry proteins provides partial
protection against parasite challenge (2). Hence, the unique rhoptry
organelle can serve as a potential target for blocking parasite
invasion and growth.
The biogenesis and phylogenetic relationship of rhoptries
to secretory organelles in other cells are not well understood. Rhoptries and pre-rhoptries are described as the only acidified organelles in the parasite (7) and are packaged with specialized hydrolases (8) and cholesterol and membranes for secretion (9, 10),
characteristics of a secretory lysosome or lysosome-like organelle (11,
12). We have recently demonstrated that an endocytic tyrosine-based
motif (YXX Another implication of these results is that
heterotetrameric adaptin complexes (reviewed in Refs. 9, 14, and 15)
are potentially involved in protein sorting to rhoptries. In other eukaryotes, tyrosine-based (YXX On both a functional and genomic basis, it is increasingly clear that
the Toxoplasma gondii secretory pathway can be considered a
stripped-down version of the more complicated machinery present in
higher eukaryotes (24, 25).2
Coupled with the presence of multiple distinct and easily recognized secretory organelles, the parasite permits insight into processes not
readily discerned in other systems. We now show that rhoptry proteins
are transported to mature rhoptries from a post-Golgi endocytic
compartment in an AP-1-dependent fashion. Other adaptins and adaptors, including AP-3, GGA proteins, stonins, and
Cloning of the T. gondii µ1 Chain (Tgµ1) Gene--
A
cDNA clone (zz38e01) with strong homology to the mouse
µ1A chain was located in the T. gondii expressed sequence
tag data base (27).3
The clone was found to contain the 117 C-terminal codons of a T. gondii µ1 homolog, which we designated Tgµ1. The sequence of the clone was extended by PCR from parasite cDNA using degenerate primers to the conserved sequence YELLDE (amino acids 113-118 of the
mouse µ1A sequence), and full-length Tgµ1 was finally obtained by
5'-rapid amplification of cDNA ends from T. gondii
cDNA. The Tgµ1 cDNA sequence has been deposited in the
GenBankTM/EBI Data Bank under accession number AY117037.
T. gondii and human foreskin fibroblast DNAs were isolated
as previously described (13).
The GenBankTM/EBI accession numbers of adaptin sequences
used for the sequence analysis in Fig. 2 are as follows:
Arabidopsis thaliana µ1 (AF009631), µ2
(AC007354.2), and µ4 (AL035356.1); Saccharomyces
cerevisiae µ1 (NC_001148.1), µ2 (NC_001147.1), and µ3
(NC_001134.1); Caenorhabditis elegans µ1 (P35602)
and µ2 (B49837); Drosophila melanogaster µ1 (AJ006219.1)
and µ2 (AJ005962.1); ray Discopyge omnata µ2
(I50530); mouse µ1A (NMOO7456.1), µ1B (AF067146.1), and µ2
(I49327); rat µ2 (I49327), µ3A (L07074.1), and µ3B (L07073.1);
and human µ1B (NM005498.1), µ2 (D63475.1), µ3A (AF092092.1), and
µ4 (AF155158.1).
Mutation of the ROP2 Cytoplasmic Tail Dileucine Motif--
The
ROP2 cytoplasmic tail contains a dileucine motif at residues 529 and
530, 10 residues upstream of the YXX Production of Polyclonal Antibodies--
The Tgµ1 coding
region was subcloned into a pZT28a vector (Amersham Biosciences) to
produce a His-Tgµ1 recombinant protein. His-Tgµ1 was purified on a
column and sent to Cocalico Biologicals, Inc. for production of a
rabbit polyclonal antiserum. The antiserum (anti-Tgµ1) was
affinity-purified by adsorption and desorption to isolated
recombinant proteins on nitrocellulose membrane as described previously
(28). The affinity-purified antiserum recognized a polypeptide
migrating at ~47 kDa upon immunoblotting.
Parasite Growth and Transfection--
The growth conditions and
transient and stable transfection of the RH strain of T. gondii grown on monolayers of Vero cells and human foreskin
fibroblasts were as described previously (13).
Antisense RNA-targeted Depletion--
The complete coding region
of Tgµ1 was amplified by PCR and cloned in the antisense orientation
into the AvrII and EcoRI sites of the
ribozyme-histone cassette pNTPRZ (29). Transfection, selection, and
cloning of antisense clones were done as previously described (29).
Mutagenesis of Tgµ1--
An HA epitope tag was
introduced into Tgµ1 between codons 231 and 232 by PCR, yielding
Tgµ1-HA. (Addition of a c-Myc epitope tag at the N terminus of Tgµ1
resulted in a protein that was diffusely cytosolic and was presumed not
to be incorporated into an adaptin complex.) D176A and W415A mutations
were introduced in Tgµ1-HA by PCR, yielding Tgµ1(D176A)-HA and
Tgµ1(W415A)-HA. These mutations have previously been shown to alter
binding of rat µ2 to tyrosine-containing motifs without affecting
incorporation of the µ2 chain into the AP-2 complex (30). Both
Tgµ1-HA and Tgµ1(D176A)-HA were ligated into pNTP/Sec for transient
transfection and into pDHFR-TS/C3M2M3 for stable transfection as
described previously (13). Sequences of all PCR-amplified fragments
were verified by dideoxy sequencing at the W. M. Keck Sequencing
Center of the Yale University School of Medicine.
SDS-PAGE, Immunoblotting, and
Immunoprecipitation--
Immunoblotting was performed as previously
described (31) using anti-Tgµ1 antibody (1:100), anti-HA monoclonal
antibody (1:200), or anti-ROP2/3/4 monoclonal antibody T3-4A7 (1:250). Antibody binding was detected by incubation with protein
A-horseradish peroxidase (1:5000) and the ECL detection system
(Amersham Biosciences). Pulse-chase and immunoprecipitation followed
previously described protocols (32).
Immunofluorescence and Cryoimmunoelectron Microscopy Localization
of Tgµ1--
Immunofluorescence localization of endogenous Tgµ1
using affinity-purified anti-Tgµ1 antibody (1:20) followed a
published protocol (13, 31). Tgµ1-HA and Tgµ1(D176A)-HA were
localized using anti-HA monoclonal antibody (1:200).
For cryoimmunoelectron microscopy localization, monolayers of Vero
cells or human foreskin fibroblasts were infected with wild-type RH or
stably transfected parasite clones for 24 h, fixed in 8%
paraformaldehyde in 0.25 M HEPES (pH 7.4) for 48 h,
scraped off in phosphate-buffered saline, and pelleted in 10% fish
skin gelatin. The gelatin-embedded pellets were infiltrated overnight with 2.3 M sucrose and frozen in liquid nitrogen. Ultrathin
cryosections were prepared with a Leica Ultracut microtome with
cryoattachment and transferred to Formvar/carbon-coated specimen grids.
Sections were incubated in phosphate-buffered saline containing
affinity-purified rabbit anti-Tgµ1 antibody (1:20) or mouse anti-HA
monoclonal antibody (1:100). Following phosphate-buffered saline
washes, the sections were incubated with 1% fish skin gelatin
containing 5-nm protein A-gold conjugate (1:70, from the laboratory of
J. Slot) for 1 h. Sections were subsequently washed in
phosphate-buffered saline, post-fixed in 1% glutaraldehyde, and
contrasted with 0.5% uranyl acetate in 2% methyl cellulose. Sections
were observed, and images were recorded with a Philips 410 transmission
electron microscope. Specificity of localization was determined by
stereological analysis of 35-50 random sections of whole cells. The
total number of gold particles and organelle surface areas were
estimated from positive prints of transmission electron microscope
negatives using a double-square test system (33).
Transmission Electron Microscopy Quantitative Analysis of
Organelles--
Monolayers of Vero cells or human foreskin fibroblasts
were infected with RH or stably transfected parasite clones for 24 h, fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate
(pH 7.4) for 30 min, washed, and post-fixed in 1% osmium tetroxide in
the same buffer. Fixed samples were processed for Epon embedding and
thin-sectioned for transmission electron microscopy using standard protocols.
To assess the morphological differences in organelle biogenesis in
wild-type RH-, Tgµ1-HA-, or Tgµ1(D176A)-HA-expressing parasites, 60 parasite sections from 20-30 random vacuoles were photographed at low
magnification (×10,400) to examine gross cell morphology and at high
magnification (×21,000) to analyze organelle structure. Total
organelle number and surface areas were calculated from prints of
transmission electron microscope negatives using a double-square overlay test system (33).
Yeast Two-hybrid Assay--
Yeast two-hybrid assays were
performed using the pGBT9 and pACT2 vectors
(Clontech) as previously described (13, 16). Cytoplasmic tails at the C termini of putative transmembrane proteins GRA4 (dense granule protein-4; 52 amino acids),
GRA7 (41 amino acids), ROP2 (75 amino acids), and ROP4 (85 amino
acids), all containing YXX Mutation of Dileucine Residues in Rop2 Abolishes Targeting to
Mature Rhoptries--
Members of the Rop2 gene
family contain dileucine residues upstream of an endocytic
YXX Cloning of Tgµ1 of the AP-1 Adaptin Complex--
Based on
a cDNA sequence (clone zz38e01) from the T. gondii data
base of expressed sequence tags, a full-length medium chain (µ) of a
clathrin adaptin was amplified from parasite cDNA. Sequence analysis of the cDNA clones predicted an open reading frame
encoding a polypeptide of 430 amino acids with a predicted molecular
mass of 47.6 kDa. Alignment and dendrogram analysis of all four known classes of µ chains indicated that this T. gondii
homolog separates into the µ1 cluster and shares the highest
similarities (75-79%) and identities (58-62%) with the
adaptin µ1 chain (Fig. 2) (data not shown). The sequence also contains the signature residues (173VFLD176, Lys203,
Val399, Leu402, and
414PWVR417) that are known to mediate binding
to tyrosine and hydrophobic residues within the YXX
Southern blotting of parasite and host genomic DNAs (data not shown)
indicated that the Tgµ1 gene is parasite-specific and single-copy, as
also suggested by the recently available T. gondii genome
sequence.2 Northern blotting of host and parasite total
RNAs (data not shown) suggested that the Tgµ1 gene is translated as a
precursor transcript and processed to a mature transcript, consistent
with the presence of an intron.
Endogenous Tgµ1 and Tgµ1-HA Localize to the
TGN--
In mammalian cells, AP-1 is an essential regulator of
membrane trafficking by mediating budding of clathrin-coated vesicles from the TGN, immature secretory granules, and endosomes. To define the
specific roles of Tgµ1 in the multiple secretory and endocytic pathways in T. gondii, we first examined the localization of
endogenous Tgµ1 using affinity-purified anti-Tgµ1 polyclonal
antibody. As shown by immunofluorescence microscopy, endogenous Tgµ1
localized in RH parasites predominantly to a region anterior to the
nucleus (Fig. 3), consistent with the
Golgi/TGN of the parasite, although additional punctate and cytosolic
staining was observed. Brefeldin A, which disrupts the T. gondii Golgi cisternae, but not the Golgi scaffold (25, 37), only
partially dispersed the Golgi/TGN staining of Tgµ1, suggesting that
the protein is partially localized to a brefeldin A-resistant
compartment(s) in addition to the Golgi.
We then stably expressed wild-type Tgµ1 tagged with the
HA epitope (Tgµ1-HA). Putative Golgi/TGN localization and partial redistribution by brefeldin A (Fig. 3) were observed for the transgenic Tgµ1-HA reporter. This result suggests that the transgenic
epitope-tagged Tgµ1 is incorporated into an AP-1 complex, which is
appropriately localized in the parasite. Tgµ1-HA partially
co-localized with the transgenic Golgi/TGN marker bacterial
alkaline phosphatase-low density lipoprotein receptor (13) as well as
with endogenous T. gondii proteins (dynamin and
ADP-ribosylation factor-1) (38) with a predominant Golgi localization
(Fig. 3B). Partial co-localization with the rhoptry
compartment was also detected (Fig. 3B), although this was
more accurately discerned by immunoelectron microscopy (see below).
We analyzed the localization of Tgµ1 by qualitative (Fig.
4, A-C) and quantitative
(Fig. 4D) immunolabeling of cryosections. Consistent with
the immunofluorescence localization, endogenous Tgµ1 localized to the
Golgi/TGN, to juxtaposed coated Golgi-associated vesicles and tubules
and endosomes, and to both the rhoptry membrane and membranous lumen.
No significant labeling was observed for dense granules, mitochondria,
or the nucleus, although microneme labeling was also detected.
Altogether, the results suggest that Tgµ1 is localized to multiple
compartments in the secretory and endocytic pathways, including mature
secretory organelles.
Mutation of Residues in Tgµ1 Responsible for Tyrosine Binding
Does Not Alter Tgµ1 Localization--
We next sought to disrupt
Tgµ1 function to assess effects on transport through the secretory
pathway and on organelle biogenesis. We have previously shown that
Tgµ1 binds to the ROP2 cytoplasmic tail in a fashion dependent upon
the YEQL motif in the ROP2 cytoplasmic tail (13). We therefore
introduced mutations in Tgµ1 at residues previously shown in
mammalian adaptins to bind to the YXX
The Tgµ1(D176A)-HA mutant localized to the Golgi/TGN region and to
punctate brefeldin A-resistant clusters (Fig. 3), suggesting that the
protein was incorporated into an adaptin complex. In contrast, the
W415A mutant was diffusely distributed in the cytosol (data not shown),
suggesting that the protein failed to associate with other chains of
the AP-1 complex. Quantification of immunostained cryosections
indicated that Tgµ1(D176A)-HA was still associated predominantly with
the Golgi/TGN, Golgi-associated vesicles and tubules, rhoptries, and
micronemes (Fig. 4D). Overall, the relative association of
Tgµ1(D176A)-HA with organelles was nearly identical to that of
endogenous Tgµ1, further supporting a broad organellar distribution
for AP-1 in T. gondii irrespective of interaction with cargo.
Binding of Tgµ1(D176A)-HA to Tyr-based Motifs Is Inhibited
Compared with That of Tgµ1-HA--
We sought to confirm that
introduction of the D176A and W415A mutations altered binding of Tgµ1
to tyrosine-based motifs. Binding of Tgµ1 to the cytoplasmic tails
containing putative YXX
Tgµ1 bound to the cytoplasmic tail of ROP4 to a greater extent than
to that of ROP2. In contrast, Tgµ1 did not bind to YXX Dominant-negative Tgµ1(D176A) Alters Sorting of Rhoptry
Proteins--
We evaluated the effects of stably overexpressing the
wild-type and D176A constructs on protein sorting and organelle
biogenesis in T. gondii. The most striking phenotype as
shown by immunofluorescence microscopy was an alteration in the
pattern of staining with antibody to ROP2 (Fig.
6). In the majority of parasites
overexpressing Tgµ1-HA, ROP2 labeling was no longer restricted to the
defined apical cluster of mature rhoptries observed in wild-type
parasites (upper panels), but was more fragmented into
vesicles and tubules (middle panels). In parasites
overexpressing dominant-interfering Tgµ1(D176A)-HA, the
labeling of ROP2 decreased drastically in intensity and was detectable
as thin tubules and vesicles extending nearly the length of the cell.
In contrast, there were no detectable differences in the targeting of
glycosylphosphatidylinositol-anchored proteins targeted to the
cell surface (SAG1 (surface antigen
glycoprotein-1)) or of dense granule proteins
(GRA3 and GRA4) to mature dense granules (data not shown). Of note,
there were no differences in the kinetics or extent of processing of
precursor ROP2 to mature ROP2 when native and
Tgµ1(D176A)-HA-expressing parasites were compared (data not shown).
This indicates that ROP2 transport to a post-Golgi processing
compartment is not substantially blocked by the dominant-negative Tgµ1(D176A) construct.
Disruption of Tgµ1 Function Alters Endosome and Rhoptry
Structure--
The above alterations in staining for ROP2 were defined
more precisely and quantitatively by electron microscopy. In comparison with RH, rhoptries in Tgµ1(D176A)-HA parasites were thinner (Fig. 7B), with an overall 2.1-fold
decrease in size (volumetric density of 0.013 for rhoptries in RH
versus 0.006 for those in Tgµ1(D176A)-HA) (Fig.
7A). As shown by immunoelectron microscopy, the localization of ROP2 was substantially altered in the
Tgµ1(D176A)-HA-expressing parasites (Fig.
8). Thin elongated rhoptries extending
nearly the length of the cell were stained. Large tubular and
multivesicular structures consistent with endosomes were most
prominently labeled, in comparison with native parasites. ROP2
accumulated in electron-dense structures resembling the sites to which
ROP2 with mutations in the YXX Targeted Depletion of Tgµ1 Alters Golgi, Endosome, and Rhoptry
Structures as Well as Parasite Segregation--
Finally, we
investigated the consequences of lowering the expression level of
Tgµ1 using the recently described antisense mRNA strategy (29).
Immediately after isolation of Tgµ1 antisense stable clones, up to
90% depletion of Tgµ1 was detected by immunoblotting (Fig.
9A). In contrast, no
alteration in expression of the dense granule protein
nucleoside-triphosphate hydrolase was observed, confirming the
specificity of the antisense effect.
Dramatic effects on parasite morphology were observed. As
shown by differential interference contrast microscopy (Fig.
9B), >50% of the vacuoles contained large irregular cells
suggestive of defective parasite division. This morphologic defect was
shown by electron microscopy instead to reflect parasites that had
completed division, but in which the plasma membranes of neighboring
cells were still closely associated; hence, parasites were aggregated in the vacuole (data not shown). Staining for ROP2 was aberrant, clumped, and irregularly distributed.
Parasites depleted of Tgµ1 had swollen Golgi cisternae
and an accumulation of lucent endocytic vesicles containing internal vesicles and membranes and also of immature rhoptries (Fig.
10A, IR),
containing the honeycombed appearance characteristic of rhoptry luminal
contents. There was a 7-8-fold increase in volumetric density of both
endosome-like structures as well as immature rhoptries (Fig.
10B) in the µ1AS6 clone. There was no significant
alteration in the density of dense granules and micronemes. These
results substantially extend the evidence that Tgµ1 mediates rhoptry
biogenesis from a post-Golgi compartment with characteristics of
multivesicular endosomes and immature rhoptries while also supporting
an additional role for Tgµ1 in transport from the Golgi.
Delivery of ROP2 and ROP4 from the TGN to rhoptries can occur by
three possible routes. The first pathway is direct transport from the
TGN to mature rhoptries. This pathway is unlikely because ROP2 mutated
to remove either tyrosine-based or dileucine motifs is not retained in
the TGN, but rather in a post-Golgi organelle (Ref. 13 and this study).
The second possibility is that rhoptry proteins are delivered first to
the plasma membrane and then internalized to the endosomal/lysosomal
and rhoptry pathway. We do not favor this explanation because neither
native nor mutant ROP2 is observed at the plasma membrane at steady
state as shown by immunofluorescence (13) and immunoelectron (this
study) microscopy localization. The third possibility is that rhoptry
proteins are delivered from the TGN to an endosomal compartment and
subsequently to the mature rhoptry. The data presented in this work
strongly support this notion and directly implicate AP-1 in the
process. The data contained herein (in particular, see Fig. 10) also
support the expected involvement of AP-1 in transport from the Golgi.
Our model for the role of T. gondii AP-1 in protein
transport is illustrated in Fig.
11.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) in the cytoplasmic tail of the
rhoptry protein ROP2, when mutated,
inhibits delivery of the protein to rhoptries (13). The mutant ROP2
protein accumulates in a compartment of unknown origin, but distinct
from the Golgi. Taken together, these results suggest that rhoptries
may be analogous to secretory lysosomes.
) or dileucine-based (LL)
motifs in the cytoplasmic tail of transmembrane receptors are
recognized by the medium µ or
subunit, respectively, of
heterotetrameric adaptin complexes. In turn, adaptins bind clathrin and
induce the formation of clathrin-coated vesicles (16-18). The six AP
(adaptor protein) complexes (AP-1A/AP-1B, AP-2,
AP-3A/AP-3B, and AP-4) are distributed to different organelles in the
cell, defining their specific function and cargo selection (15). AP-2
mediates endocytosis from the plasma membrane, whereas AP-1A/AP-1B,
AP-3A/AP-3B, and AP-4 sort proteins at the trans-Golgi
network (TGN)1 and/or
endosomes. Although AP-1 was originally suggested to function only in
anterograde transport from the TGN to endosomes, recent evidence
suggests that the complex mediates retrograde transport of mannose
6-phosphate receptors from endosomes to the TGN (19, 20). In
neuroendocrine, endocrine, and mammary epithelial cells, AP-1 is
localized to immature secretory granules, where it functions in the
removal of membranes and nonspecific cargo, presumably via a retrograde
pathway, to facilitate maturation to mature secretory granules (21,
22). AP-3 mediates direct transport of selected cargo from the TGN to
lysosome-like secretory organelles and may also mediate transport from
endosomes to lysosome-like organelles (reviewed in Ref. 12, 15, and
23). In contrast, there is no evidence supporting a role for AP-1 in
anterograde transport from a post-Golgi compartment to another
organelle and especially to a lysosome-like organelle.
-arrestins (reviewed in Ref. 26), cannot participate because they
are absent from the T. gondii genome. This result
substantially expands the role for AP-1 in anterograde transport and
more specifically in transport to and biogenesis of lysosome-related organelles.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
motif previously implicated in rhoptry protein transport. An L529A/L530A mutation was introduced into the cytoplasmic tail of ROP2 using the
hemagglutinin (HA)-tagged ROP2 construct previously described (13).
This construct was transiently transfected into T. gondii
and localized as previously reported (13).
motifs, were PCR-amplified and
cloned in-frame behind the Gal4-binding domain in the pGBT9
vector. Tgµ1, Tgµ1(D176A), and Tgµ1(W415A) were cloned in-frame
with the Gal4 activation domain in the pACT2 vector.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
motif previously shown to be involved in targeting to
mature rhoptries. Because dileucine residues can also mediate endocytic
targeting by binding to adaptin
chains (see Refs. 34 and 35;
reviewed in Ref. 11), these residues were mutated to di-alanine, and
the ROP2 mutant was expressed in T. gondii. As
illustrated in Fig.
1 (A), the
L529A/L530A mutant localized to a compartment distinct from, but
adjacent to, mature rhoptries, similar to the localization of ROP2 with
a deletion of the YXX
motif (ROP2
) (13). This
compartment was distinct from the dense granules, micronemes, and
Golgi/TGN of the parasite (Fig. 1B), as was the ROP2
mutant (13). Importantly, both the ROP2
mutant and the L529A/L530A
mutant substantially co-localized with a multivesicular endosomal
compartment marked by the endosomal marker VPS4 (vacuolar
protein
sorting-4).4
Altogether, these data provide further evidence that rhoptry targeting
occurs along the endocytic pathway and is mediated by adaptins. They
also implicate adaptin machinery in mediating targeting from a
post-Golgi compartment to a mature secretory organelle. We undertook
experiments to further support and understand this observation.
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Fig. 1.
Mutation of dileucine residues in the ROP2
cytoplasmic tail abolishes targeting to mature rhoptries.
A: upper panels, ROP2-HA was expressed in
T. gondii. When localized with anti-HA antibody, ROP2-HA
precisely co-localized with the mature rhoptry compartment, as detected
with anti-ROP2 polyclonal antibody. Lower panels,
ROP2 with an L529A/L530A (LL529-530AA) mutation in the
cytoplasmic tail localized to a compartment (arrow) adjacent
to, but largely distinct from, mature rhoptries. Although a vacuole
showing minimal staining of the L529A/L530A compartment with anti-ROP2
polyclonal antibody is illustrated here for emphasis (and was also
observed previously for ROP2 ) (13), many vacuoles showed the
expected partial staining of the mutant ROP2 compartment with anti-ROP2
polyclonal antibody. B: the L529A/L530A mutant of ROP2 did
not co-localize with dense granules (GRA3), micronemes (MIC2), or the
Golgi/TGN.
sorting motif (16, 18, 36). We accordingly termed the T. gondii homolog Tgµ1.
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Fig. 2.
A medium adaptin in Toxoplasma
is a µ1. Shown is a sequence
alignment of Tgµ1 with C. elegans (Celeg),
A. thaliana (Arab), mouse (Mou),
Drosophila (Dros), and human (Hum)
adaptin µ1 chains. Percentages of amino acid identity
(%I) and similarity (%S) to Tgµ1 are
indicated. Asterisks highlight underlined amino
acids known to interact with the YXX motif (18,
23).
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Fig. 3.
Tgµ1 is predominantly
localized to the Golgi/TGN as indicated by immunofluorescence
microscopy. A, endogenous Tgµ1 was
detected by an affinity-purified antiserum against
Tgµ1, whereas overexpressed wild-type (Tgµ1-HA)
and dominant-negative (Tgµ1(D176A)-HA) Tgµ1
were detected with anti-HA monoclonal antibody. Staining of the
putative Golgi (arrows) was partially disrupted by a 10-min
treatment with 5 µM brefeldin A (BFA).
B, Tgµ1-HA (detected by anti-HA monoclonal antibody)
partially co-localized with the Golgi/TGN as visualized in Tgµ1-HA
parasites transiently expressing the Golgi/TGN marker bacterial
alkaline phosphatase-low density lipoprotein receptor (LDLR)
(13) or by staining for dynamin (polyclonal antibody MC63, provided by
Mark McNiven, Mayo Clinic, Rochester, MD) or ADP-ribosylation factor-1
(ARF-1) (38) in Tgµ1-HA parasites. Tgµ1 also partially
co-localized with structures labeled by anti-ROP2 polyclonal antibody.
In any given microscopic field, Tgµ1-HA was concentrated to different
extents in the Golgi relative to other structures in the organism,
probably reflecting different levels of protein expression.
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Fig. 4.
Localization of Tgµ1
by cryoimmunoelectron microscopy. Shown are electron microscopic
images of Tgµ1-HA parasites demonstrating the gold localization of
Tgµ1 (arrows) predominantly to the trans-most
cisterna of the Golgi (G) and vesicles and tubules extending
from or juxtaposed to the TGN (GVT) (panels A and
B) and to membranes of microneme (M) and
endosomes (E) and also to the membrane and lumen of aberrant
rhoptries (R) (panel C). Endosomes are defined as
vesicles 200-300 nm in diameter that are clear (panel C,
lower inset) or that contain membrane whorls or dense
deposits and that are known to contain the early endosomal marker
T. gondii Rab5 (44) or VPS4 (see Footnote 5). Similar
localization was observed for endogenous Tgµ1 in RH, as detected by
affinity-purified anti-Tgµ1 polyclonal antibody, and for
Tgµ1(D176A)-HA in the stable transgenic parasites (data not shown).
The organellar density of gold labeling (gold
particles/µM2) for endogenous Tgµ1
in RH and Tgµ1(D176A)-HA was determined from a random sample of
20-30 cell profiles (panel D). Density was calculated by
estimating the number of gold particles divided by the
estimated volume of organelles using a double-square test system. The
density was expressed as a percentage of the sum of all organelles and
cytoplasm estimates. Only well defined Golgi stacks were categorized as
Golgi cisternae, whereas vesicles and tubules adjacent to the TGN were
categorized as Golgi-associated vesicles and tubules. N,
nucleus. Scale bars = 200 nm.
motif, specifically
D176A and W415A (18, 30), and stably expressed these mutants in
T. gondii.
motifs of dense granule (GRA4,
YAEL; and GRA7, YRHF) and rhoptry (ROP2, YEQL; and ROP4, YREM) proteins
was tested by yeast two-hybrid analysis.
motifs within the putative cytoplasmic tails of the dense granule proteins GRA4 and GRA7 (Fig.
5A). The avidity of Tgµ1
binding to the ROP2 tail was increased when a triplicated YEQL motif
was added to the truncated tail (ROP2.3Y) (Fig. 5, A and
B), as previously reported (13). Importantly, both D176A and
W415A mutations impaired binding to ROP2.3Y and ROP4 (Fig.
5B), confirming the functional roles of the conserved
residues within Tgµ1 and the utility of the mutants as
dominant-negative inhibitors of endogenous µ1 function. Because the
W415A mutant was not incorporated into an AP-1 complex, the remainder
of the experiments were conducted with the D176A construct.
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Fig. 5.
Disruption of the
YXX -binding pocket of
Tgµ1 abolishes its specific interactions with
rhoptry proteins in a yeast two-hybrid growth assay. A,
the T. gondii µ1 homolog was fused to the Gal4 activation
domain (AD), whereas the cytoplasmic tails of GRA4, GRA7,
ROP2, and ROP4 were fused to the Gal4 binding domain (BD) as
described under "Experimental Procedures." The strength of
interaction between the activation and binding domains co-transformed
into S. cerevisiae was measured by growth
(A600 nm) in 5 mM
3-amino-1,2,4-triazole, a His3 inhibitor. Interactions between
Tgµ1 and GRA4 and GRA7 were comparable to the negative control (yeast
co-transformed with pGBT9 and pACT2, expressing the binding and
activation domains alone). Interaction between Tgµ1 and ROP4 was
3-fold that between Tgµ1 and ROP2 and similar to that between a
truncated ROP2 tail and a triplicated YEQL motif at the C terminus
(ROP2.3Y) (13). B, either the ROP4 cytoplasmic tail or
ROP2.3Y was fused to the activation domain, whereas wild-type Tgµ1
and dominant-negative Tgµ1 (described under "Results")
were fused to the binding domain. Mutations in tyrosine-binding
residues within Tgµ1 blocked binding to ROP4 and ROP2.3Y. Results
shown are from a single experiment, representative of two experiments
performed.
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Fig. 6.
Overexpressing dominant-negative
Tgµ1-HA alters the distribution of ROP2.
Shown are the results from immunofluorescence microscopy of RH and
parasites stably overexpressing Tgµ1-HA or dominant-negative
Tgµ1(D176A)-HA. Parasites were stained with polyclonal antibody to
the rhoptry protein ROP2. It is likely that the intermediate phenotype
of the Tgµ1-HA parasites results from a dominant-negative effect of
overexpressing wild-type protein, potentially by saturating
membrane-binding sites for Tgµ1 (45).
motif localized (Fig. 8,
ROP2
panel). On the other hand, minimal accumulation of
ROP2 label was observed in the T. gondii Golgi, largely
excluding the notion that AP-1 mediates direct transport from the Golgi
to rhoptries. Overall, the results indicate that the AP-1
adaptin mediates transport of ROP2 from a post-Golgi endosomal
compartment to the mature rhoptry organelle.
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Fig. 7.
Expression of dominant-negative
Tgµ1 alters rhoptry morphology. Rhoptries
in parasites expressing Tgµ1(D176A)-HA were smaller in comparison
with those in RH as determined by quantitative (A) and
qualitative (B) electron microscopy. Volumetric density
analysis of a random sample of cells (RH, n = 46; and
Tgµ1(D176A)-HA, n = 43) indicated a 2.1-fold decrease
in the size of rhoptries in parasites expressing dominant-negative
Tgµ1. Volumetric density for unit rhoptry (R) was
determined by dividing total volumetric density (volume of
organelle/volume of cell) into organelle density (organelle
number/cell). The arrow indicates either an endosome or an
acidocalcisome. Scale bars = 200 nm.
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Fig. 8.
ROP2 accumulates in endosomes and immature
rhoptries in parasites expressing dominant-negative
Tgµ1 or when the YXX
sorting signal is deleted. Shown are the results form
immunoelectron microscopy of cryofixed RH, Tgµ1(D176A)-HA, and
ROP2
-HA parasite strains. ROP2 was localized predominantly in
mature rhoptries (R) in RH (left column,
arrows) as indicated by labeling by anti-ROP2/3/4 monoclonal
antibody. In parasites expressing Tgµ1(D176A)-HA, ROP2/3/4 was found
in abnormally elongated and thin rhoptries (middle column,
arrow) as well as in multivesicular organelles and immature
rhoptries (right column, upper two panels,
arrows). The ROP2
-HA mutant, which is deleted of the YEQL
motif (13), was localized by anti-HA monoclonal antibody to endosomes
and multivesicular organelles and to the electron-dense structures
shown (right column, lower panel,
arrow). Scale bar = 200 nm.
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Fig. 9.
Targeted depletion of
Tgµ1 by antisense RNA alters ROP2/3/4
localization and impairs parasite growth. Clones depleted of
Tgµ1 by antisense RNA (µ1AS) isolated from two different
experiments were immunoprecipitated from 106 RH and µ1AS
parasites using anti-Tgµ1 and anti-nucleoside-triphosphate hydrolase
(NTPase) polyclonal antibodies, separated by SDS-PAGE, and
blotted with the same antibodies (A). Up to 90% of Tgµ1,
but not nucleoside-triphosphate hydrolase, was depleted in µ1AS
clones. Normal vacuoles in RH and distorted vacuoles commonly observed
in µ1AS clones were labeled by anti-ROP2/3/4 monoclonal antibody
(B). Increased Tgµ1 depletion as quantitated by
densitometry correlated directly with an increase in the percentage of
vacuoles with distorted morphology (data not shown).
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Fig. 10.
Targeted depletion of
Tgµ1 by antisense RNA generates swollen Golgi,
multivesicular endosomes, and immature rhoptries. A,
shown are the results from transmission electron microscopy of µ1AS
parasites showing dilated Golgi cisternae (G) adjacent to
the nucleus (N) and a profound accumulation of
multivesicular endosomes (E) rhoptries (R) and
immature rhoptries (IR). B, quantitative
analyses of volumetric density revealed a drastic accumulation of
lucent and multivesicular endosomes and immature rhoptries in parasite
clone µ1AS6 (n = 23) in comparison with RH
(n = 47). Scale bar = 200 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 11.
Model for the role of T. gondii AP-1 in protein transport. Based on the
data obtained during this study, previous publications, and submitted
work, we present a model for the role of T. gondii AP-1 in
protein transport. AP-1 mediates transport from the T. gondii Golgi to a post-Golgi endocytic compartment, to which
the T. gondii early endocytic marker Rab5
(TgRab5) also localizes. Transport from the post-Golgi
Rab5-positive compartment may then occur to a second endosomal
compartment, bearing functional similarity to multivesicular endosomes
(see Footnote 5). Experiments focusing on transport of ROP2
specifically (by mutating motifs in the protein necessary for
interaction with adaptors) indicate that delivery of ROP2 from a
post-Golgi compartment to the mature rhoptry is a key function for
T. gondii AP-1. This does not obviate a predominate
role for T. gondii AP-1 in transport of other cargo (that
accumulate in the Golgi when AP-1-interacting motifs are mutated) from
the Golgi to a post-Golgi compartment. IR, immature
rhoptries; MVB, multivesicular body.
We had previously suggested (24, 39) that rhoptries are lysosome-like
organelles, analogous to melanosomes and platelet-dense granules (11,
12). This raised the possibility that ROP2 was sorted by AP-3,
especially because of the acidic "AP-3-like" YXX binding motif (EYEQL) (23) within the ROP2 cytoplasmic tail. Three
lines of evidence argue against a role for AP-3 in rhoptry protein
transport. First and by far the most important, no µ3 or
3 chains
are present in the T. gondii genome (of interest, the same
is true in the closely related Plasmodium falciparum). Second, as shown by two-hybrid analysis, ROP2 (13) and
ROP4 5 bind substantially
less well to human µ3 than to human µ1. Finally, a chimera between
the ROP2 ectodomain/transmembrane domain and the cytoplasmic tail of
human tyrosinase (targeted to melanosomes in an
AP-3-dependent fashion) (40) did not localize to rhoptries when expressed in T. gondii.6 Taken together,
these results implicate AP-1 rather than AP-3 in the transport of
rhoptry proteins from a post-Golgi endosomal compartment to mature
rhoptry organelles. The absence from the T. gondii genome of
other adaptors that mediate endocytic transport, including GGA
proteins, stonins, and
-arrestins (26), lends further credence to
the argument that Tgµ1 is responsible for protein transport to
the T. gondii rhoptry.
The crystal structure of a rat µ2 fragment (residue
158-435) bound to tyrosine-containing peptides resolved the critical
residues that interact with the aromatic ring (Phe174 and
Trp421) and phenolic hydroxyl group (Asp176,
Lys203, and Arg423) of the critical tyrosine
(18). An independent mutational analysis of dominant-interfering µ2
confirmed in vitro that Asp176 and
Trp421 are critical for the internalization of TGN38 and
the epidermal growth factor receptor (30), albeit no changes in the
cell morphology or protein localization were detected. We adopted this
mutational strategy to analyze the role of the adaptin µ1
chain in mediating protein sorting in T. gondii.
The partial phenotype of the dominant-interfering D176A mutant in
T. gondii is almost certainly due to the presence of
endogenous adaptin µ1 chain and also to the capacity of other residues (Phe174, Lys203, and
Arg417 in T. gondii) within Tgµ1
to interact with the YXX motif. Although this partial
phenotype might not have been apparent in other eukaryotic cells, the
unique structure and polarized distribution of rhoptries allowed us to
uncover the sorting defect.
It is now well established that the antisense RNA strategy inherently does not generate complete inhibition or complete loss of function of a gene and therefore is not suitable for the creation of a null mutant (41-43). Targeted depletion of the T. gondii adaptin µ1 chain by antisense RNA allowed the stable clone to survive through at least three passages of the host cell prior to our isolation and analyses of single clones. At this stage, the mutants grew at an irregular and slow rate, and depletion of Tgµ1 eventually resulted in the substantial abortion of parasite vacuoles in the host cell. On the other hand, when maintained in continuous passage, µ1 levels returned to those in wild-type parasites, and normal growth and morphology were restored, similar to what we observed in clones depleted of ROP2 by antisense mRNA.6 Protein recovery that is accompanied by loss of phenotypes further supports the specificity of defects due to Tgµ1 depletion.
We have now identified two separate circumstances in which blocking
transport of ROP2 to the mature rhoptry alters rhoptry morphology (this
work).7 These results are
most consistent with a model in which transmembrane ROP2 (and perhaps
other associated proteins) is required for establishing and maintaining
structural integrity of the rhoptry. Whether components analogous to
ROP2 exist in other lysosome-like organelles and whether their
transport is AP-1-dependent remain to be determined.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kim Murphy-Zichichi (Yale Center for Cellular and Molecular Imaging) for technical assistance. Genomic data were provided by the Institute for Genomic Research and by the Sanger Center (Wellcome Trust). Expressed sequence tags were generated by Washington University.
![]() |
FOOTNOTES |
---|
* This work was supported by National Research Service Awards 5T32AI07404 and 5F32AI10044 (to H. M. N.) and Grant RO1 AI30060 (to K. A. J.) from the National Institutes of Health and by a scholar award in molecular parasitology from the Burroughs Wellcome Fund (to K. A. J.). Work performed at the Institute for Genomic Research and Washington University was supported by National Institutes of Health Grants AI05093 and 1R01AI045806-01A1, respectively.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) AY117037.
§ Both authors contributed equally to this work.
Present address: Dept. of Medical Microbiology, University of
Cape Town Medical School, Observatory 7925, Cape Town, South Africa.
** To whom correspondence should be addressed: Dept. of Internal Medicine, Yale University School of Medicine, LCI 808, 333 Cedar St., New Haven, CT 06520-8022. Tel.: 203-785-2115; Fax: 203-785-3864; E-mail: keith.joiner@yale.edu.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M208291200
2 Available at www.toxodb.org.
3 Available at www.tigr.org/tdb/t_gondii/.
4 M. Yang, I. Coppens, S. Wormsley, H. C. Hoppe, and K. A. Joiner, manuscript in preparation.
5 M. Yang, I. Coppens, S. Wormsley, H. C. Hoppe, and K. A. Joiner, unpublished data.
6 K. Paprotka and K. A. Joiner, unpublished data.
7 V. Nakaar, H. M. Ngô, E. Aaronson, I. Coppens, T. Stedman, and K. A. Joiner, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TGN, trans-Golgi network; Tgµ1, T. gondii µ1 chain; HA, hemagglutinin.
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