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Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii.

Reiss M, Viebig N, Brecht S, Fourmaux MN, Soete M, Di Cristina M, Dubremetz JF, Soldati D - J. Cell Biol. (2001)

Bottom Line: MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain.MIC4 binds directly to MIC1 and behaves as a passive cargo molecule.MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Biology, University of Heidelberg, Heidelberg D-63120, Germany.

ABSTRACT
The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

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Illustration of the structural domains of the proteins and the recombinant mutants used in this study. (A) Schematic representation of the structural domains on the micronemal proteins MIC1, MIC3, MIC4, MIC6, and the major tachyzoite surface antigen SAG1. The proteolytic cleavages of the micronemal proteins are indicated with two types of symbols distinguishing posttranslational and postexocytosis cleavages. Sequence data for MIC1, MIC4, and MIC6 are available from GenBank/EMBL/DDBJ under accession numbers Z71786, AF143487, and AF110270, respectively. (B) Schematic representation of the constructs used in this study. The epitope tags Ty-1 and myc are represented by a black box. The color code of the diverse domains is as described in A. Schematic drawing of pTMIC6Ty-1, pTMIC6ΔCD, pTMIC6GPI, pTMIC6ΔEGF-1, -2, or -3, pTMIC6ΔAD, pTMIC4Ty-1, pTMIC4ΔA5-6, pTMIC4ΔA3-6, pM2SAG1TM-CD, and pM2MIC1myc.
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Figure 1: Illustration of the structural domains of the proteins and the recombinant mutants used in this study. (A) Schematic representation of the structural domains on the micronemal proteins MIC1, MIC3, MIC4, MIC6, and the major tachyzoite surface antigen SAG1. The proteolytic cleavages of the micronemal proteins are indicated with two types of symbols distinguishing posttranslational and postexocytosis cleavages. Sequence data for MIC1, MIC4, and MIC6 are available from GenBank/EMBL/DDBJ under accession numbers Z71786, AF143487, and AF110270, respectively. (B) Schematic representation of the constructs used in this study. The epitope tags Ty-1 and myc are represented by a black box. The color code of the diverse domains is as described in A. Schematic drawing of pTMIC6Ty-1, pTMIC6ΔCD, pTMIC6GPI, pTMIC6ΔEGF-1, -2, or -3, pTMIC6ΔAD, pTMIC4Ty-1, pTMIC4ΔA5-6, pTMIC4ΔA3-6, pM2SAG1TM-CD, and pM2MIC1myc.

Mentions: The first and essential event in obligate intracellular parasite infection is host cell invasion. In apicomplexan parasites, apical secretory organelles ensure the accumulation and the appropriate release in time and space of adhesins and other invasion factors. An increasing number of micronemal proteins sharing common features have been identified among the Apicomplexa and recent studies illustrated their central role in parasite motility and host cells invasion (for review see Tomley and Soldati 2001). Toxoplasma gondii has developed a remarkable ability to actively penetrate a broad range of cells within the mammalian hosts, whereas the members of the Plasmodium genus exhibit very restricted host range specificities. The molecular bases of host range specificity have not been elucidated yet but might implicate the repertoire of micronemal proteins and their adhesive interactions with host cell receptors (Barnwell and Galinski 1995). The micronemal proteins of the TRAP family have been identified as active players in host cell invasion and gliding motility in the invasive stages of the rodent malaria parasites Plasmodium berghei (Sultan et al. 1997; Dessens et al. 1999; Yuda et al. 1999). Thrombospondin-related adhesive proteins (TRAPs) contain a putative transmembrane spanning domain and a conserved short cytoplasmic tail. MIC2, the homologue of TRAP in T. gondii (Wan et al. 1997), is shed apically on the surface of the parasites and relocalizes toward the posterior pole by a mechanism dependent on the parasite actomyosin system (Sibley et al. 1998). In a recent complementation experiment, the cytoplasmic domain (CD) of MIC2 was shown to functionally replace the corresponding domain in PbTRAP (Kappe et al. 1999), suggesting that the machinery for invasion is conserved among members of the phylum. We have identified a novel family of transmembrane micronemal proteins including MIC6 (Meissner, M., and D. Soldati, unpublished results; sequence data are available from GenBank/EMBL/DDBJ under accession number AF110270). Analysis of the deduced amino acid sequence of MIC6 revealed a secretory signal sequence and three EGF–like domains. The COOH-terminal region exhibits a putative transmembrane spanning domain and a short cytoplasmic tail homologous to MIC2 and to the other members of the TRAP family. Four soluble micronemal proteins, MIC1, MIC3, MIC4, and MIC5 have been characterized so far in T. gondii. These proteins contain multiple thrombospondin-like, EGF-like, or apple domains potentially conferring adhesive properties to these molecules (see Fig. 1 A). Indeed, MIC1 (Fourmaux et al. 1996), MIC3 (Garcia-Réguet et al. 2001), and MIC4 (Brecht et al. 2001) bind to host cells, but it is still unclear how they establish a link between the parasite and the host cell. Similarly, little is known about how in general soluble secretory proteins are sorted to their appropriate organelles. Understanding how T. gondii copes with the sorting of the large variety of secreted proteins in the multiple distinct secretory compartments is an area of intense investigation (Kaasch and Joiner 2000; Ngo et al. 2000). Recent studies have demonstrated that the targeting of transmembrane proteins in specialized organelles is achieved through the use of evolutionary conserved signals and machinery (Hoppe et al. 2000).


Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii.

Reiss M, Viebig N, Brecht S, Fourmaux MN, Soete M, Di Cristina M, Dubremetz JF, Soldati D - J. Cell Biol. (2001)

Illustration of the structural domains of the proteins and the recombinant mutants used in this study. (A) Schematic representation of the structural domains on the micronemal proteins MIC1, MIC3, MIC4, MIC6, and the major tachyzoite surface antigen SAG1. The proteolytic cleavages of the micronemal proteins are indicated with two types of symbols distinguishing posttranslational and postexocytosis cleavages. Sequence data for MIC1, MIC4, and MIC6 are available from GenBank/EMBL/DDBJ under accession numbers Z71786, AF143487, and AF110270, respectively. (B) Schematic representation of the constructs used in this study. The epitope tags Ty-1 and myc are represented by a black box. The color code of the diverse domains is as described in A. Schematic drawing of pTMIC6Ty-1, pTMIC6ΔCD, pTMIC6GPI, pTMIC6ΔEGF-1, -2, or -3, pTMIC6ΔAD, pTMIC4Ty-1, pTMIC4ΔA5-6, pTMIC4ΔA3-6, pM2SAG1TM-CD, and pM2MIC1myc.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196004&req=5

Figure 1: Illustration of the structural domains of the proteins and the recombinant mutants used in this study. (A) Schematic representation of the structural domains on the micronemal proteins MIC1, MIC3, MIC4, MIC6, and the major tachyzoite surface antigen SAG1. The proteolytic cleavages of the micronemal proteins are indicated with two types of symbols distinguishing posttranslational and postexocytosis cleavages. Sequence data for MIC1, MIC4, and MIC6 are available from GenBank/EMBL/DDBJ under accession numbers Z71786, AF143487, and AF110270, respectively. (B) Schematic representation of the constructs used in this study. The epitope tags Ty-1 and myc are represented by a black box. The color code of the diverse domains is as described in A. Schematic drawing of pTMIC6Ty-1, pTMIC6ΔCD, pTMIC6GPI, pTMIC6ΔEGF-1, -2, or -3, pTMIC6ΔAD, pTMIC4Ty-1, pTMIC4ΔA5-6, pTMIC4ΔA3-6, pM2SAG1TM-CD, and pM2MIC1myc.
Mentions: The first and essential event in obligate intracellular parasite infection is host cell invasion. In apicomplexan parasites, apical secretory organelles ensure the accumulation and the appropriate release in time and space of adhesins and other invasion factors. An increasing number of micronemal proteins sharing common features have been identified among the Apicomplexa and recent studies illustrated their central role in parasite motility and host cells invasion (for review see Tomley and Soldati 2001). Toxoplasma gondii has developed a remarkable ability to actively penetrate a broad range of cells within the mammalian hosts, whereas the members of the Plasmodium genus exhibit very restricted host range specificities. The molecular bases of host range specificity have not been elucidated yet but might implicate the repertoire of micronemal proteins and their adhesive interactions with host cell receptors (Barnwell and Galinski 1995). The micronemal proteins of the TRAP family have been identified as active players in host cell invasion and gliding motility in the invasive stages of the rodent malaria parasites Plasmodium berghei (Sultan et al. 1997; Dessens et al. 1999; Yuda et al. 1999). Thrombospondin-related adhesive proteins (TRAPs) contain a putative transmembrane spanning domain and a conserved short cytoplasmic tail. MIC2, the homologue of TRAP in T. gondii (Wan et al. 1997), is shed apically on the surface of the parasites and relocalizes toward the posterior pole by a mechanism dependent on the parasite actomyosin system (Sibley et al. 1998). In a recent complementation experiment, the cytoplasmic domain (CD) of MIC2 was shown to functionally replace the corresponding domain in PbTRAP (Kappe et al. 1999), suggesting that the machinery for invasion is conserved among members of the phylum. We have identified a novel family of transmembrane micronemal proteins including MIC6 (Meissner, M., and D. Soldati, unpublished results; sequence data are available from GenBank/EMBL/DDBJ under accession number AF110270). Analysis of the deduced amino acid sequence of MIC6 revealed a secretory signal sequence and three EGF–like domains. The COOH-terminal region exhibits a putative transmembrane spanning domain and a short cytoplasmic tail homologous to MIC2 and to the other members of the TRAP family. Four soluble micronemal proteins, MIC1, MIC3, MIC4, and MIC5 have been characterized so far in T. gondii. These proteins contain multiple thrombospondin-like, EGF-like, or apple domains potentially conferring adhesive properties to these molecules (see Fig. 1 A). Indeed, MIC1 (Fourmaux et al. 1996), MIC3 (Garcia-Réguet et al. 2001), and MIC4 (Brecht et al. 2001) bind to host cells, but it is still unclear how they establish a link between the parasite and the host cell. Similarly, little is known about how in general soluble secretory proteins are sorted to their appropriate organelles. Understanding how T. gondii copes with the sorting of the large variety of secreted proteins in the multiple distinct secretory compartments is an area of intense investigation (Kaasch and Joiner 2000; Ngo et al. 2000). Recent studies have demonstrated that the targeting of transmembrane proteins in specialized organelles is achieved through the use of evolutionary conserved signals and machinery (Hoppe et al. 2000).

Bottom Line: MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain.MIC4 binds directly to MIC1 and behaves as a passive cargo molecule.MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Biology, University of Heidelberg, Heidelberg D-63120, Germany.

ABSTRACT
The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

Show MeSH
Related in: MedlinePlus