Secretory traffic in the eukaryotic parasite Toxoplasma gondii : less
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《细胞学杂志》
1 Section of Infectious Diseases, Yale University School of Medicine, New Haven, CT 06520
2 Department of Biology, University of Pennsylvania, Philadelphia, PA 19104
Address correspondence to Keith Joiner, Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-8022. Tel.: (203) 785-2115. Fax: (203) 785-3864. E-mail: keith.joiner@yale.edu
Abstract
Name a single-celled eukaryote that boasts a small genome size, is easily cultivated in haploid form, for which a wide variety of molecular genetic tools are available, and that exhibits a simple, polarized secretory apparatus with a well-defined endoplasmic reticulum and Golgi that can serve as a model for understanding secretion. Got it? Now name a cell with all these attributes that contains at least a dozen distinct and morphologically well-defined intracellular organelles, including three distinct types of secretory vesicles and two endosymbiotic organelles. Not so sure anymore?
Key Words: secretory pathway; vesicular trafficking; protozoan cell biology; Apicomplexan parasites; eukaryotic evolution
Toxoplasma gondii is an obligate intracellular protozoan parasite that is a leading cause of focal central nervous system infections in patients with AIDS/HIV (Luft and Remington, 1992). This parasite is a member of the phylum Apicomplexa, which includes Plasmodium (the cause of malaria) and 5,000 additional species, most of which are poorly characterized (Levine, 1988). Among all of the Apicomplexa, T. gondii is one of the easiest to cultivate and the most amenable to genetic manipulation (Boothroyd et al., 1994; Roos et al., 1994). The nuclear genome of T. gondii is 80 Mb in size; numerous ESTs are available (Ajioka et al., 1998), and a genome sequencing project is now underway. The parasite also harbors two organellar genomes associated with its mitochondrion and plastid (of which more below) (Feagin, 1994). The rapidly dividing haploid "tachyzoite" form of T. gondii can be propagated inside of virtually any mammalian host cell, and classical genetic crosses can be performed in cats (the parasite sexual cycle has not yet been established in vitro) (Boothroyd et al., 1994). Available tools for molecular genetic manipulation include a wide variety of selectable markers, integrating and episomal vectors, and high-efficiency transformation systems that permit gene knockouts, insertional mutagenesis, complementation cloning, antisense repression, inducible expression, etc. (Boothroyd et al., 1994; Roos et al., 1994; Black and Boothroyd, 1998; Nakaar et al., 1999; Meissner et al., 2001; Striepen et al., 2002).
A banana-shaped organism 8-μm-long and 2 μm in diameter, T. gondii is substantially smaller than a typical mammalian cell (Fig. 1). The parasite's architecture can be appreciated in a few electron microscopic thin sections, displaying a single nucleus, a single mitochondrion, a single plastid, a single interconnected ER network, a single Golgi apparatus, and an apically clustered complex of secretory organelles (this apical complex gives the phylum Apicomplexa its name). Virtually all of these organelles exhibit a distinctive morphology when labeled with fluorescent protein tags (Fig. 2), permitting quantitative ultrastructural studies and time-lapse analysis in living cells. In sum, T. gondii can be viewed as optimally situated between the morphologically complex mammalian cell and smaller organisms with poor ultrastructural resolution, such as Saccharomyces cerevisiae or Plasmodium sp. (Hager et al., 1999). Although T. gondii parasites are unable to replicate outside of nucleated host cells, tachyzoites remain viable long enough in an extracellular environment to permit standard analyses of secretory processes, and a permeabilized cell secretion system has been established (Chaturvedi et al., 1998). In this mini-review, we describe insights into both unique and conserved features of the T. gondii secretory apparatus, providing comparisons with systems more familiar to mainstream cell biologists.
Figure 1. Intracellular parasitophorous vacuole containing two T. gondii parasites within a human host cell. The ER is distributed throughout the cell, but predominantly in the basal region. The Golgi apparatus is invariably found adjacent to the apical end of the nucleus. Rhoptries and micronemes are found at the apical end of the parasite (terminating in the conoid region), whereas dense granules are distributed throughout the cell. The inner membrane complex is comprised of a series of closed sacs of uncertain origin, underlying the plasma membrane. The micropore (not visible in this micrograph) is the only stable structure bridging the parasite plasma membrane and inner membrane complex. Clathrin-coated vesicles are often observed at the micropore (Nichols et al., 1994), suggesting that endocytosis may occur at this site. Bar, 2 μm. Ap, Apicoplast; Co, conoid; DG, dense granule; ER, endoplasmic reticulum; Go, Golgi; IMC, inner membrane complex; Mn, microneme; Mt, mitochondrion; Nu, nucleus; PM, plasma membrane; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; Rh, rhoptry; HC, host cell cytoplasm; HC-ER, host cell endoplasmic reticulum; HC-Mt; host cell mitochondrion.
Figure 2. Fluorescent protein labeling of subcellular organelles in T. gondii. Fusions between endogenous parasite proteins and GFP, YFP, or other reporters have been expressed in transgenic T. gondii, and localization has been determined by fluorescence microscopy. The central cartoon, showing subcellular structures (also see Fig. 1), illustrates proper targeting of GFP chimeras. Labeling of the conoid and subpellicular microtubules was achieved using a YFP–-tubulin construct (Striepen et al., 2000; Hu et al., 2002; Swedlow et al., 2002), micronemes using MIC3-GFP (Striepen et al., 2001), Golgi using MIC3-GFP (Striepen et al., 2001), mitochondria using HSP60-GFP (Hu et al., 2001), plasma membrane using P30-GFP-GPI (Striepen et al., 2000); rhoptries using ROP1-GFP (Striepen et al., 1998), dense granules using P30-GFP (Striepen et al., 1998), nucleus using PCNA-GFP (Radke et al., 2001), ER using P30-GFP-HDEL (Hager et al., 1999), and inner membrane complex using IMC1-YFP (Hu et al., 2001).
The T. gondii secretory pathway is highly polarized
Considered from the standpoint of an experimental system for secretion, one of the most appealing aspects of T. gondii is the polarized organization of its secretory organelles (Hager et al., 1999)—a consequence of the parasite's mechanism of replication, in which two daughter cells are assembled within the mother (Hu et al., 2001). The nucleus is centrally located, essentially bisecting the organism (Figs. 1 and 2). The endoplasmic reticulum, although distributed throughout the cell, is concentrated posterior to the nucleus, and is so reduced that the nuclear envelope itself provides a substantial fraction of the ER volume. Thinly coated vesicles bud from the anterior end of the nucleus/ER, destined for the closely juxtaposed Golgi stack, which consists of a limited number of cisternae (typically three to five). Reporters containing the COOH-terminal ER retention signal of T. gondii BiP (HDEL) localize most prominently to a cup-like region anterior to the apical end of the nucleus, just below the Golgi (Figs. 1 and 2). The use of the nuclear envelope as an obligatory intermediate between the ER and Golgi is comparable to other small eukaryotic cells, such as Pichia pastoris (Rossanese et al., 1999) but contrasts with mammalian systems, where transitional ER elements are dispersed throughout the cell.
Forward transport from the ER to Golgi takes advantage of acidic/hydrophobic/acidic motifs in the cytoplasmic tails of secretory proteins, along with upstream tyrosines, likely by recruiting COPII coats as observed in mammalian cells and yeast (Hoppe and Joiner, 2000). Both COPII and COPI coat components (including Arf1 and Sar1) are present in T. gondii genome and EST databases (Ajioka et al., 1998), and COPI retrieval motifs have been shown to operate in the parasite (Liendo et al., 2001). Protein transport through the Golgi is inhibited by low temperature treatment, brefeldin A, and microtubule inhibitors (Stokkermans et al., 1996; Soldati et al., 1998). Clathrin-coated vesicles are observed at the lateral margins of the trans-most Golgi stacks (Liendo et al., 2001). The target for these vesicles is likely to be one or more of the multitude of unusual secretory organelles found in T. gondii and other Apicomplexans, as discussed below. In contrast, proteins destined for the parasite surface are delivered via an alternative route (Karsten et al., 1998). Many T. gondii surface antigens are GPI anchored, and the parasite may use the GPI anchor itself as a targeting motif.
Apicomplexan parasites have three secretory organelles: micronemes, rhoptries, and dense granules
The tachyzoite form of T. gondii contains at least three morphologically distinct secretory organelles: micronemes, rhoptries, and dense granules (Figs. 1 and 2). The former two organelles are located in the anterior portion of the cell, whereas dense granules are more broadly distributed. Morphologically, dense granules are essentially indistinguishable from the mature secretory granules found in endocrine, neuroendocrine, or exocrine cells (Table I and Fig. 1). At the other end of the spectrum, rhoptries bear little morphological resemblance to subcellular organelles in any other cell type. These three organelles discharge sequentially: microneme exocytosis occurs upon host cell binding, rhoptry secretion coincides with invasion, and dense granule secretion is most prominent after parasite entry into the host cell (Carruthers and Sibley, 1997). Micronemes and rhoptries are thought to be critical for host cell invasion, a process that is completed within 15–20 s. Dense granule proteins are thought to be required for intracellular replication, including establishment of the parasitophorous vacuole within which parasites reside and divide until lysis of the host cell. The requirements for precise temporal regulation of differential organelle secretion are stringent, distinguishing T. gondii parasites from most secretory cells.
Table I. Comparison of organellar protein trafficking and secretion in Toxoplasma and "higher" eukaryotes
Dense granules are functionally analogous to constitutive secretory vesicles
Soluble recombinant proteins (from various sources) are delivered to dense matrix granules by the bulk flow pathway. Dense granules are quantitatively secreted in a constitutive, calcium-independent fashion (Chaturvedi et al., 1998; Karsten et al., 1998); although there is also likely to be a triggered component to the release process (Dubremetz et al., 1993; Carruthers and Sibley, 1997; Coppens et al., 1999). Even T. gondii proteins from which specific signals for targeting to other organelles have been deleted are routed through the dense granules as soluble proteins (Striepen et al., 1998, 2001; Reiss et al., 2001). It is therefore difficult to invoke the notion that aggregation or retention in dense granules requires specific protein sequence motifs, a low pH/high Ca+2 environment, lipid rafts, or other distinguishing characteristics, in contrast to observations in mammalian secretory cells (Arvan and Castle, 1998; Tooze et al., 2001) (Table I). On balance, dense granules appear to be most similar to the post-Golgi vesicles involved in constitutive secretion (Table I).
Rhoptries are associated with both the endocytic and secretory pathways
The name "rhoptry" is derived from the Greek word meaning "club," reflecting the bulbous shape of this organelle (Fig. 1), thought to contain vesicular/membranous material that is secreted via the long, slender neck. Labeling with DAMP (3--3'amino-N-methyldipropylamine) suggests that rhoptries are the only acidified organelles in T. gondii; the parasite contains no morphological equivalent of secondary lysosomes (Shaw et al., 1998). Unusual organelles designated acidocalciosomes have been reported (Moreno and Zhong, 1996), but these function in the storage of calcium and pyrophosphate (and possibly other materials as well), and do not appear to be directly related to either exocytic or endocytic trafficking. Like multivesicular bodies and late endosomes (Bishop and Woodman, 2000), rhoptries are enriched in cholesterol, but their cholesterol/phospholipid ratio of 1.5:1 (Foussard et al., 1991) is too high for lipid bilayer stability, suggesting that at least some of this cholesterol may be organized in a crystalline array.
Secretion from the rhoptries contributes to the formation of a distinctive parasitophorous vacuole, defining the compartment within the host cell where T. gondii parasites reside. The majority of lipids making up the parasitophorous vacuole at the time of invasion are of host cell (rather than rhoptry) origin (Suss-Toby et al., 1996), but rhoptry proteins are rapidly incorporated into the vacuolar membrane. The parasitophorous vacuole neither acidifies nor fuses with organelles of the host cell endomembrane system, highlighting the unusual nature of this structure (Sibley et al., 1985; Joiner et al., 1990; Mordue et al., 1999). Inhibitors of parasite actin polymerization (Sibley and Andrews, 2000) prevent host cell invasion but not rhoptry discharge, producing small vesicular structures in the host cell. These empty "e-vacuoles" (H?kansson et al., 2001) contain rhoptry markers and associate with host cell mitochondria and ER (without fusing), just as seen for parasite-containing vacuoles in the same cell (Sinai et al., 1997).
The biogenesis of rhoptries is not well understood. Rhoptries of the mother cell disappear as morphologically distinct entities during the early phases of cell division (endodyogeny). Following division of the Golgi apparatus, two distinct rhoptry antigen-positive punctae appear immediately anterior to Golgi; these structures may be regenerated de novo, as precursors to the fully formed rhoptries that will ultimately develop in the two daughter cells. Rhoptry protein processing is thought to occur in these immature rhoptries (Soldati et al., 1998), which therefore exhibit some functional similarity to immature secretory granules (Table I).
Protein targeting to the rhoptries has long been a matter of interest, since the evolutionary origin of these unique secretory structures is unknown. Soluble rhoptry proteins can harbor multiple independent targeting signals (Bradley and Boothroyd, 2001; Striepen et al., 2001). Members of the ROP2 family, which contain a putative transmembrane domain, display both YXX and LL motifs within the predicted cytoplasmic tail (Hoppe et al., 2000). In higher eukaryotes, both of these motifs mediate binding to adaptor subunits and facilitate clathrin-coated vesicle formation from the trans-Golgi (Bonifacino and Dell-Angelica, 1999). Deletion or alteration of the YXX motif (Hoppe et al., 2000) or LL motif (unpublished data) abolishes ROP2 delivery to mature T. gondii rhoptries, providing the first evidence for tyrosine-dependent sorting machinery in protozoan parasites. T. gondii μ1 binds to the cytoplasmic tail of ROP2 family members in a tyrosine-dependent fashion and expression of either dominant–negative T. gondii μ1 or antisense mRNA ablation of T. gondii μ1 expression impairs rhoptry targeting (unpublished data). Alteration of rhoptry targeting motifs leads to protein accumulation in a compartment located just anterior to (but distinct from) the Golgi (the precursor compartment noted in Table I). Combined with the observation that rhoptries are acidic (Shaw et al., 1998), it is tempting to consider the rhoptry a lysosome-like organelle (Dell'Angelica et al., 2000) (see Table I). A model for rhoptry biogenesis consistent with the existing data is provided in Fig. 3.
Figure 3. Post-Golgi protein targeting in the T. gondii secretory pathway. Protein traffic through the ER and Golgi likely depends on both COPI- and COPII-coated vesicles, and is regulated by forward targeting signals, ER retrieval and retention motifs, and Rab proteins. Targeting of soluble proteins from the trans-Golgi network to dense granules is signal independent, whereas targeting of membrane proteins to these organelles depends on transmembrane domain length (unpublished data). T. gondii rab6 mediates retrograde transport from dense granules to the parasite Golgi (unpublished data). Rhoptry proteins appear likely to be transported from the Golgi via a precursor compartment, possibly part of the endosomal pathway (Robibaro et al., 2002). Transmembrane rhoptry proteins are targeted in a tyrosine-, dileucine-, and adaptor-dependent fashion. Targeting of soluble microneme proteins proceeds by association with transmembrane escorters; transmembrane proteins are capable of using adaptor- and tyrosine-dependent signals, although typical endocytic motifs are not apparent in known microneme proteins. Results using dominant–negative adaptors suggest that microneme targeting may exploit the same precursor compartment involved in rhoptry targeting. Nuclear-encoded proteins destined for the apicoplast exhibit a bipartite NH2-terminal domain (Roos et al., 1999; DeRocher et al., 2000; Waller et al., 2000; Yung et al., 2001), mediating transport first into the secretory pathway using a classical secretory signal sequence, and subsequently into the apicoplast using a plastid–transit peptide akin to that found in plants. Whether all secreted proteins transit this organelle after exit from the Golgi remains to be determined, as does the ultimate destination of products produced in the apicoplast (dashed black arrows). A, apicoplast; DG, dense granule; E, endosome; Mn, micronemes; PC, precursor compartment; Rh, rhoptries.
Microneme targeting involves membrane escorts for soluble proteins
Microneme proteins typically exhibit one or more of a variety of adhesive domains, and are thought to be involved in host cell adhesion (Carruthers et al., 2000; Garcia-Reguet et al., 2000; Brecht et al., 2001). Chimeras containing the cytoplasmic tail of a mammalian lysosomal membrane protein are targeted to micronemes in a tyrosine-dependent fashion (Hoppe et al., 2000). No endogenous T. gondii microneme proteins bearing a transmembrane domain and YXX or LL motifs in the cytoplasmic tail have yet been identified, however, and microneme proteins can possess multiple independent targeting domains (Striepen et al., 2001). The transmembrane protein MIC6 forms a trimeric complex with the soluble microneme proteins MIC1 and MIC4, and deletion of MIC6 prevents targeting of these molecules (Reiss et al., 2001), suggesting that MIC6 functions an escort protein. A similar escort role has been described for MIC8 in targeting MIC3 (Meissner et al., 2002), and for rhoptry proteins in P. falciparum (Baldi et al., 2000).
MIC2 is probably the most intensively studied microneme protein in T. gondii. T. gondii MIC2 is predicted to contain a transmembrane domain with cytoplasmic tail tyrosine motifs (SYHYY, EIEYE) that play a role in sorting (Di Cristina et al., 2000), and MIC2 has been shown to associate with another protein (MIC2AP) during transport to and storage in micronemes (Rabenau et al., 2001). Genetic deletion of the cytoplasmic domain of the MIC2 orthologue TRAP in P. berghei does not completely abolish microneme targeting (Kappe et al., 1999), however, suggesting functional redundancy in organellar targeting pathways.
The T. gondii plastid resides within the secretory pathway
Perhaps the most unusual subcellular organelle in T. gondii (and other Apicomplexan parasites) is a relict plastid, acquired by secondary endosymbiosis of a eukaryotic alga and retention of the algal plastid (K?hler et al., 1997). The Apicomplexan plastid—or "apicoplast"—is essential for parasite survival (Fichera and Roos, 1997; He et al., 2001). This organelle is known to play a role in lipid metabolism (Waller et al., 1998; Jomaa et al., 1999; Jelenska et al., 2001) and possibly other metabolic functions as well. Although the apicoplast has a its own genome (Wilson et al., 1996), this 35-kb circular element encodes only a limited protein repertoire; the majority of apicoplast proteins are synthesized on cytoplasmic ribosomes and posttranslationally imported (Waller et al., 1998; Roos et al., 1999).
As previously noted in other systems containing complex plastids, nuclear-encoded proteins destined for the apicoplast exhibit a bipartite NH2-terminal domain. Molecular genetic manipulation in T. gondii and P. falciparum demonstrates that the extreme NH2 terminus functions as a secretory signal sequence, whereas the subterminal domain (presumed to be exposed after cleavage of the secretory signal) functions as a plastid-targeting signal, directing the cargo protein from the secretory pathway into the apicoplast lumen (Roos et al., 1999; DeRocher et al., 2000; Waller et al., 2000; Yung et al., 2001). Remarkably, this entire process can be reconstituted from heterologous components. Thus, the combination of two normally distinct targeting processes—cotranslational translocation into the endoplasmic reticulum and posttranslational translocation into chloroplasts—combine to provide an elegant mechanism for targeting across the four membranes that surround the apicoplast. Based on the characteristics of the apicoplast targeting signal, a large number of candidate apicoplast proteins have been identified in the T. gondii EST and P. falciparum genome databases (Ajioka et al., 1998; Bahl et al., 2002), including plastid import machinery of the tic and toc family (McFadden, G.I., personal communication).
Although the molecular details of protein targeting to the apicoplast are now clear, precisely how—in morphological terms—these proteins traffic from the secretory pathway to the apicoplast remains a mystery, as vesicles are never observed fusing with (or budding from) the organelle (compare Fig. 1). Moreover, treatment with brefeldin A or appending an ER retention signal to nuclear-encoded apicoplast proteins fails to inhibit trafficking to the organelle. These observations raise the possibility that the apicoplast lies at a proximal position within the secretory pathway—perhaps within the ER itself—and that all secreted proteins bearing an NH2-terminal signal sequence wash over the apicoplast! As noted above, the function of the apicoplast is also uncertain, but circumstantial evidence suggests that it may play an important role in establishing the parasitophorous vacuole during host cell invasion (Fichera and Roos, 1997).
Conclusions
Protein targeting in T. gondii and related parasites utilizes a combination of conserved and unusual motifs and transport machinery (Ng? et al., 2000). The simplified, polarized, and morphologically distinctive organization of this cell readily permits comparison with mammalian cells and yeast, as detailed in Table I. Where mechanisms are conserved, T. gondii provides an excellent model for eukaryotes in general. For example, studies on the use of COPI and COPII coats in ER–Golgi transport, or the process of Golgi division, should be fruitful areas for study. Where mechanisms are different—as in the secretion of rhoptry lipids, the targeting across four membranes surrounding the apicoplast, and the trafficking of proteins destined for association with membrane compartments that lie beyond the plasma membrane—studies on these parasites are likely to reveal the diversity of eukaryotic evolution and highlight potential targets for antiparasitic drug development.
Acknowledgments
We wish to thank Lewis G. Tilney for Fig. 1, Cynthia Y. He for Fig. 2, and all members of the Joiner and Roos laboratories for helpful comments and stimulating discussions.
The authors greatfully acknowledge the National Institutes of Health and the Burroughs Wellcome Fund for support of this work.Revised: 15 February 2002References
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2 Department of Biology, University of Pennsylvania, Philadelphia, PA 19104
Address correspondence to Keith Joiner, Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-8022. Tel.: (203) 785-2115. Fax: (203) 785-3864. E-mail: keith.joiner@yale.edu
Abstract
Name a single-celled eukaryote that boasts a small genome size, is easily cultivated in haploid form, for which a wide variety of molecular genetic tools are available, and that exhibits a simple, polarized secretory apparatus with a well-defined endoplasmic reticulum and Golgi that can serve as a model for understanding secretion. Got it? Now name a cell with all these attributes that contains at least a dozen distinct and morphologically well-defined intracellular organelles, including three distinct types of secretory vesicles and two endosymbiotic organelles. Not so sure anymore?
Key Words: secretory pathway; vesicular trafficking; protozoan cell biology; Apicomplexan parasites; eukaryotic evolution
Toxoplasma gondii is an obligate intracellular protozoan parasite that is a leading cause of focal central nervous system infections in patients with AIDS/HIV (Luft and Remington, 1992). This parasite is a member of the phylum Apicomplexa, which includes Plasmodium (the cause of malaria) and 5,000 additional species, most of which are poorly characterized (Levine, 1988). Among all of the Apicomplexa, T. gondii is one of the easiest to cultivate and the most amenable to genetic manipulation (Boothroyd et al., 1994; Roos et al., 1994). The nuclear genome of T. gondii is 80 Mb in size; numerous ESTs are available (Ajioka et al., 1998), and a genome sequencing project is now underway. The parasite also harbors two organellar genomes associated with its mitochondrion and plastid (of which more below) (Feagin, 1994). The rapidly dividing haploid "tachyzoite" form of T. gondii can be propagated inside of virtually any mammalian host cell, and classical genetic crosses can be performed in cats (the parasite sexual cycle has not yet been established in vitro) (Boothroyd et al., 1994). Available tools for molecular genetic manipulation include a wide variety of selectable markers, integrating and episomal vectors, and high-efficiency transformation systems that permit gene knockouts, insertional mutagenesis, complementation cloning, antisense repression, inducible expression, etc. (Boothroyd et al., 1994; Roos et al., 1994; Black and Boothroyd, 1998; Nakaar et al., 1999; Meissner et al., 2001; Striepen et al., 2002).
A banana-shaped organism 8-μm-long and 2 μm in diameter, T. gondii is substantially smaller than a typical mammalian cell (Fig. 1). The parasite's architecture can be appreciated in a few electron microscopic thin sections, displaying a single nucleus, a single mitochondrion, a single plastid, a single interconnected ER network, a single Golgi apparatus, and an apically clustered complex of secretory organelles (this apical complex gives the phylum Apicomplexa its name). Virtually all of these organelles exhibit a distinctive morphology when labeled with fluorescent protein tags (Fig. 2), permitting quantitative ultrastructural studies and time-lapse analysis in living cells. In sum, T. gondii can be viewed as optimally situated between the morphologically complex mammalian cell and smaller organisms with poor ultrastructural resolution, such as Saccharomyces cerevisiae or Plasmodium sp. (Hager et al., 1999). Although T. gondii parasites are unable to replicate outside of nucleated host cells, tachyzoites remain viable long enough in an extracellular environment to permit standard analyses of secretory processes, and a permeabilized cell secretion system has been established (Chaturvedi et al., 1998). In this mini-review, we describe insights into both unique and conserved features of the T. gondii secretory apparatus, providing comparisons with systems more familiar to mainstream cell biologists.
Figure 1. Intracellular parasitophorous vacuole containing two T. gondii parasites within a human host cell. The ER is distributed throughout the cell, but predominantly in the basal region. The Golgi apparatus is invariably found adjacent to the apical end of the nucleus. Rhoptries and micronemes are found at the apical end of the parasite (terminating in the conoid region), whereas dense granules are distributed throughout the cell. The inner membrane complex is comprised of a series of closed sacs of uncertain origin, underlying the plasma membrane. The micropore (not visible in this micrograph) is the only stable structure bridging the parasite plasma membrane and inner membrane complex. Clathrin-coated vesicles are often observed at the micropore (Nichols et al., 1994), suggesting that endocytosis may occur at this site. Bar, 2 μm. Ap, Apicoplast; Co, conoid; DG, dense granule; ER, endoplasmic reticulum; Go, Golgi; IMC, inner membrane complex; Mn, microneme; Mt, mitochondrion; Nu, nucleus; PM, plasma membrane; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; Rh, rhoptry; HC, host cell cytoplasm; HC-ER, host cell endoplasmic reticulum; HC-Mt; host cell mitochondrion.
Figure 2. Fluorescent protein labeling of subcellular organelles in T. gondii. Fusions between endogenous parasite proteins and GFP, YFP, or other reporters have been expressed in transgenic T. gondii, and localization has been determined by fluorescence microscopy. The central cartoon, showing subcellular structures (also see Fig. 1), illustrates proper targeting of GFP chimeras. Labeling of the conoid and subpellicular microtubules was achieved using a YFP–-tubulin construct (Striepen et al., 2000; Hu et al., 2002; Swedlow et al., 2002), micronemes using MIC3-GFP (Striepen et al., 2001), Golgi using MIC3-GFP (Striepen et al., 2001), mitochondria using HSP60-GFP (Hu et al., 2001), plasma membrane using P30-GFP-GPI (Striepen et al., 2000); rhoptries using ROP1-GFP (Striepen et al., 1998), dense granules using P30-GFP (Striepen et al., 1998), nucleus using PCNA-GFP (Radke et al., 2001), ER using P30-GFP-HDEL (Hager et al., 1999), and inner membrane complex using IMC1-YFP (Hu et al., 2001).
The T. gondii secretory pathway is highly polarized
Considered from the standpoint of an experimental system for secretion, one of the most appealing aspects of T. gondii is the polarized organization of its secretory organelles (Hager et al., 1999)—a consequence of the parasite's mechanism of replication, in which two daughter cells are assembled within the mother (Hu et al., 2001). The nucleus is centrally located, essentially bisecting the organism (Figs. 1 and 2). The endoplasmic reticulum, although distributed throughout the cell, is concentrated posterior to the nucleus, and is so reduced that the nuclear envelope itself provides a substantial fraction of the ER volume. Thinly coated vesicles bud from the anterior end of the nucleus/ER, destined for the closely juxtaposed Golgi stack, which consists of a limited number of cisternae (typically three to five). Reporters containing the COOH-terminal ER retention signal of T. gondii BiP (HDEL) localize most prominently to a cup-like region anterior to the apical end of the nucleus, just below the Golgi (Figs. 1 and 2). The use of the nuclear envelope as an obligatory intermediate between the ER and Golgi is comparable to other small eukaryotic cells, such as Pichia pastoris (Rossanese et al., 1999) but contrasts with mammalian systems, where transitional ER elements are dispersed throughout the cell.
Forward transport from the ER to Golgi takes advantage of acidic/hydrophobic/acidic motifs in the cytoplasmic tails of secretory proteins, along with upstream tyrosines, likely by recruiting COPII coats as observed in mammalian cells and yeast (Hoppe and Joiner, 2000). Both COPII and COPI coat components (including Arf1 and Sar1) are present in T. gondii genome and EST databases (Ajioka et al., 1998), and COPI retrieval motifs have been shown to operate in the parasite (Liendo et al., 2001). Protein transport through the Golgi is inhibited by low temperature treatment, brefeldin A, and microtubule inhibitors (Stokkermans et al., 1996; Soldati et al., 1998). Clathrin-coated vesicles are observed at the lateral margins of the trans-most Golgi stacks (Liendo et al., 2001). The target for these vesicles is likely to be one or more of the multitude of unusual secretory organelles found in T. gondii and other Apicomplexans, as discussed below. In contrast, proteins destined for the parasite surface are delivered via an alternative route (Karsten et al., 1998). Many T. gondii surface antigens are GPI anchored, and the parasite may use the GPI anchor itself as a targeting motif.
Apicomplexan parasites have three secretory organelles: micronemes, rhoptries, and dense granules
The tachyzoite form of T. gondii contains at least three morphologically distinct secretory organelles: micronemes, rhoptries, and dense granules (Figs. 1 and 2). The former two organelles are located in the anterior portion of the cell, whereas dense granules are more broadly distributed. Morphologically, dense granules are essentially indistinguishable from the mature secretory granules found in endocrine, neuroendocrine, or exocrine cells (Table I and Fig. 1). At the other end of the spectrum, rhoptries bear little morphological resemblance to subcellular organelles in any other cell type. These three organelles discharge sequentially: microneme exocytosis occurs upon host cell binding, rhoptry secretion coincides with invasion, and dense granule secretion is most prominent after parasite entry into the host cell (Carruthers and Sibley, 1997). Micronemes and rhoptries are thought to be critical for host cell invasion, a process that is completed within 15–20 s. Dense granule proteins are thought to be required for intracellular replication, including establishment of the parasitophorous vacuole within which parasites reside and divide until lysis of the host cell. The requirements for precise temporal regulation of differential organelle secretion are stringent, distinguishing T. gondii parasites from most secretory cells.
Table I. Comparison of organellar protein trafficking and secretion in Toxoplasma and "higher" eukaryotes
Dense granules are functionally analogous to constitutive secretory vesicles
Soluble recombinant proteins (from various sources) are delivered to dense matrix granules by the bulk flow pathway. Dense granules are quantitatively secreted in a constitutive, calcium-independent fashion (Chaturvedi et al., 1998; Karsten et al., 1998); although there is also likely to be a triggered component to the release process (Dubremetz et al., 1993; Carruthers and Sibley, 1997; Coppens et al., 1999). Even T. gondii proteins from which specific signals for targeting to other organelles have been deleted are routed through the dense granules as soluble proteins (Striepen et al., 1998, 2001; Reiss et al., 2001). It is therefore difficult to invoke the notion that aggregation or retention in dense granules requires specific protein sequence motifs, a low pH/high Ca+2 environment, lipid rafts, or other distinguishing characteristics, in contrast to observations in mammalian secretory cells (Arvan and Castle, 1998; Tooze et al., 2001) (Table I). On balance, dense granules appear to be most similar to the post-Golgi vesicles involved in constitutive secretion (Table I).
Rhoptries are associated with both the endocytic and secretory pathways
The name "rhoptry" is derived from the Greek word meaning "club," reflecting the bulbous shape of this organelle (Fig. 1), thought to contain vesicular/membranous material that is secreted via the long, slender neck. Labeling with DAMP (3--3'amino-N-methyldipropylamine) suggests that rhoptries are the only acidified organelles in T. gondii; the parasite contains no morphological equivalent of secondary lysosomes (Shaw et al., 1998). Unusual organelles designated acidocalciosomes have been reported (Moreno and Zhong, 1996), but these function in the storage of calcium and pyrophosphate (and possibly other materials as well), and do not appear to be directly related to either exocytic or endocytic trafficking. Like multivesicular bodies and late endosomes (Bishop and Woodman, 2000), rhoptries are enriched in cholesterol, but their cholesterol/phospholipid ratio of 1.5:1 (Foussard et al., 1991) is too high for lipid bilayer stability, suggesting that at least some of this cholesterol may be organized in a crystalline array.
Secretion from the rhoptries contributes to the formation of a distinctive parasitophorous vacuole, defining the compartment within the host cell where T. gondii parasites reside. The majority of lipids making up the parasitophorous vacuole at the time of invasion are of host cell (rather than rhoptry) origin (Suss-Toby et al., 1996), but rhoptry proteins are rapidly incorporated into the vacuolar membrane. The parasitophorous vacuole neither acidifies nor fuses with organelles of the host cell endomembrane system, highlighting the unusual nature of this structure (Sibley et al., 1985; Joiner et al., 1990; Mordue et al., 1999). Inhibitors of parasite actin polymerization (Sibley and Andrews, 2000) prevent host cell invasion but not rhoptry discharge, producing small vesicular structures in the host cell. These empty "e-vacuoles" (H?kansson et al., 2001) contain rhoptry markers and associate with host cell mitochondria and ER (without fusing), just as seen for parasite-containing vacuoles in the same cell (Sinai et al., 1997).
The biogenesis of rhoptries is not well understood. Rhoptries of the mother cell disappear as morphologically distinct entities during the early phases of cell division (endodyogeny). Following division of the Golgi apparatus, two distinct rhoptry antigen-positive punctae appear immediately anterior to Golgi; these structures may be regenerated de novo, as precursors to the fully formed rhoptries that will ultimately develop in the two daughter cells. Rhoptry protein processing is thought to occur in these immature rhoptries (Soldati et al., 1998), which therefore exhibit some functional similarity to immature secretory granules (Table I).
Protein targeting to the rhoptries has long been a matter of interest, since the evolutionary origin of these unique secretory structures is unknown. Soluble rhoptry proteins can harbor multiple independent targeting signals (Bradley and Boothroyd, 2001; Striepen et al., 2001). Members of the ROP2 family, which contain a putative transmembrane domain, display both YXX and LL motifs within the predicted cytoplasmic tail (Hoppe et al., 2000). In higher eukaryotes, both of these motifs mediate binding to adaptor subunits and facilitate clathrin-coated vesicle formation from the trans-Golgi (Bonifacino and Dell-Angelica, 1999). Deletion or alteration of the YXX motif (Hoppe et al., 2000) or LL motif (unpublished data) abolishes ROP2 delivery to mature T. gondii rhoptries, providing the first evidence for tyrosine-dependent sorting machinery in protozoan parasites. T. gondii μ1 binds to the cytoplasmic tail of ROP2 family members in a tyrosine-dependent fashion and expression of either dominant–negative T. gondii μ1 or antisense mRNA ablation of T. gondii μ1 expression impairs rhoptry targeting (unpublished data). Alteration of rhoptry targeting motifs leads to protein accumulation in a compartment located just anterior to (but distinct from) the Golgi (the precursor compartment noted in Table I). Combined with the observation that rhoptries are acidic (Shaw et al., 1998), it is tempting to consider the rhoptry a lysosome-like organelle (Dell'Angelica et al., 2000) (see Table I). A model for rhoptry biogenesis consistent with the existing data is provided in Fig. 3.
Figure 3. Post-Golgi protein targeting in the T. gondii secretory pathway. Protein traffic through the ER and Golgi likely depends on both COPI- and COPII-coated vesicles, and is regulated by forward targeting signals, ER retrieval and retention motifs, and Rab proteins. Targeting of soluble proteins from the trans-Golgi network to dense granules is signal independent, whereas targeting of membrane proteins to these organelles depends on transmembrane domain length (unpublished data). T. gondii rab6 mediates retrograde transport from dense granules to the parasite Golgi (unpublished data). Rhoptry proteins appear likely to be transported from the Golgi via a precursor compartment, possibly part of the endosomal pathway (Robibaro et al., 2002). Transmembrane rhoptry proteins are targeted in a tyrosine-, dileucine-, and adaptor-dependent fashion. Targeting of soluble microneme proteins proceeds by association with transmembrane escorters; transmembrane proteins are capable of using adaptor- and tyrosine-dependent signals, although typical endocytic motifs are not apparent in known microneme proteins. Results using dominant–negative adaptors suggest that microneme targeting may exploit the same precursor compartment involved in rhoptry targeting. Nuclear-encoded proteins destined for the apicoplast exhibit a bipartite NH2-terminal domain (Roos et al., 1999; DeRocher et al., 2000; Waller et al., 2000; Yung et al., 2001), mediating transport first into the secretory pathway using a classical secretory signal sequence, and subsequently into the apicoplast using a plastid–transit peptide akin to that found in plants. Whether all secreted proteins transit this organelle after exit from the Golgi remains to be determined, as does the ultimate destination of products produced in the apicoplast (dashed black arrows). A, apicoplast; DG, dense granule; E, endosome; Mn, micronemes; PC, precursor compartment; Rh, rhoptries.
Microneme targeting involves membrane escorts for soluble proteins
Microneme proteins typically exhibit one or more of a variety of adhesive domains, and are thought to be involved in host cell adhesion (Carruthers et al., 2000; Garcia-Reguet et al., 2000; Brecht et al., 2001). Chimeras containing the cytoplasmic tail of a mammalian lysosomal membrane protein are targeted to micronemes in a tyrosine-dependent fashion (Hoppe et al., 2000). No endogenous T. gondii microneme proteins bearing a transmembrane domain and YXX or LL motifs in the cytoplasmic tail have yet been identified, however, and microneme proteins can possess multiple independent targeting domains (Striepen et al., 2001). The transmembrane protein MIC6 forms a trimeric complex with the soluble microneme proteins MIC1 and MIC4, and deletion of MIC6 prevents targeting of these molecules (Reiss et al., 2001), suggesting that MIC6 functions an escort protein. A similar escort role has been described for MIC8 in targeting MIC3 (Meissner et al., 2002), and for rhoptry proteins in P. falciparum (Baldi et al., 2000).
MIC2 is probably the most intensively studied microneme protein in T. gondii. T. gondii MIC2 is predicted to contain a transmembrane domain with cytoplasmic tail tyrosine motifs (SYHYY, EIEYE) that play a role in sorting (Di Cristina et al., 2000), and MIC2 has been shown to associate with another protein (MIC2AP) during transport to and storage in micronemes (Rabenau et al., 2001). Genetic deletion of the cytoplasmic domain of the MIC2 orthologue TRAP in P. berghei does not completely abolish microneme targeting (Kappe et al., 1999), however, suggesting functional redundancy in organellar targeting pathways.
The T. gondii plastid resides within the secretory pathway
Perhaps the most unusual subcellular organelle in T. gondii (and other Apicomplexan parasites) is a relict plastid, acquired by secondary endosymbiosis of a eukaryotic alga and retention of the algal plastid (K?hler et al., 1997). The Apicomplexan plastid—or "apicoplast"—is essential for parasite survival (Fichera and Roos, 1997; He et al., 2001). This organelle is known to play a role in lipid metabolism (Waller et al., 1998; Jomaa et al., 1999; Jelenska et al., 2001) and possibly other metabolic functions as well. Although the apicoplast has a its own genome (Wilson et al., 1996), this 35-kb circular element encodes only a limited protein repertoire; the majority of apicoplast proteins are synthesized on cytoplasmic ribosomes and posttranslationally imported (Waller et al., 1998; Roos et al., 1999).
As previously noted in other systems containing complex plastids, nuclear-encoded proteins destined for the apicoplast exhibit a bipartite NH2-terminal domain. Molecular genetic manipulation in T. gondii and P. falciparum demonstrates that the extreme NH2 terminus functions as a secretory signal sequence, whereas the subterminal domain (presumed to be exposed after cleavage of the secretory signal) functions as a plastid-targeting signal, directing the cargo protein from the secretory pathway into the apicoplast lumen (Roos et al., 1999; DeRocher et al., 2000; Waller et al., 2000; Yung et al., 2001). Remarkably, this entire process can be reconstituted from heterologous components. Thus, the combination of two normally distinct targeting processes—cotranslational translocation into the endoplasmic reticulum and posttranslational translocation into chloroplasts—combine to provide an elegant mechanism for targeting across the four membranes that surround the apicoplast. Based on the characteristics of the apicoplast targeting signal, a large number of candidate apicoplast proteins have been identified in the T. gondii EST and P. falciparum genome databases (Ajioka et al., 1998; Bahl et al., 2002), including plastid import machinery of the tic and toc family (McFadden, G.I., personal communication).
Although the molecular details of protein targeting to the apicoplast are now clear, precisely how—in morphological terms—these proteins traffic from the secretory pathway to the apicoplast remains a mystery, as vesicles are never observed fusing with (or budding from) the organelle (compare Fig. 1). Moreover, treatment with brefeldin A or appending an ER retention signal to nuclear-encoded apicoplast proteins fails to inhibit trafficking to the organelle. These observations raise the possibility that the apicoplast lies at a proximal position within the secretory pathway—perhaps within the ER itself—and that all secreted proteins bearing an NH2-terminal signal sequence wash over the apicoplast! As noted above, the function of the apicoplast is also uncertain, but circumstantial evidence suggests that it may play an important role in establishing the parasitophorous vacuole during host cell invasion (Fichera and Roos, 1997).
Conclusions
Protein targeting in T. gondii and related parasites utilizes a combination of conserved and unusual motifs and transport machinery (Ng? et al., 2000). The simplified, polarized, and morphologically distinctive organization of this cell readily permits comparison with mammalian cells and yeast, as detailed in Table I. Where mechanisms are conserved, T. gondii provides an excellent model for eukaryotes in general. For example, studies on the use of COPI and COPII coats in ER–Golgi transport, or the process of Golgi division, should be fruitful areas for study. Where mechanisms are different—as in the secretion of rhoptry lipids, the targeting across four membranes surrounding the apicoplast, and the trafficking of proteins destined for association with membrane compartments that lie beyond the plasma membrane—studies on these parasites are likely to reveal the diversity of eukaryotic evolution and highlight potential targets for antiparasitic drug development.
Acknowledgments
We wish to thank Lewis G. Tilney for Fig. 1, Cynthia Y. He for Fig. 2, and all members of the Joiner and Roos laboratories for helpful comments and stimulating discussions.
The authors greatfully acknowledge the National Institutes of Health and the Burroughs Wellcome Fund for support of this work.Revised: 15 February 2002References
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