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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: 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.We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

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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The CD of MIC6 contains sorting signals for the micronemes targeting. (A) IFA analysis by confocal microscopy on monolayers of HFF cells infected with mic6ko mutant expressing stably SAG1TM-CDMIC6. The subcellular distribution of the SAG1TM-CDMIC6 fusion protein was detected by using antibodies raised against the CD of MIC6. The colocalization of SAG1TM-CDMIC6 with MIC2 is illustrated by the yellow color in the merged image and indicated by arrows. In this genetic background, both MIC1 and MIC4 were missorted and accumulated in the vacuolar space, as indicated by arrowheads. (B) SAG1TM-CDMIC6 accumulates precisely in the micronemes as demonstrated by immunoelectron microscopy using anti-SAG1 antibodies. SAG1 is detected by gold particles and, when GPI anchored, was found in its normal location at the parasite surface (arrowheads). In contrast, SAG1TM-CDMIC6 was found in the micronemes (arrow). As micronemes are thinner than the section, some of them were not exposed to the antibody and were not labeled. Bars: (A) 1 μm; (B) 0.2 μm.
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Figure 4: The CD of MIC6 contains sorting signals for the micronemes targeting. (A) IFA analysis by confocal microscopy on monolayers of HFF cells infected with mic6ko mutant expressing stably SAG1TM-CDMIC6. The subcellular distribution of the SAG1TM-CDMIC6 fusion protein was detected by using antibodies raised against the CD of MIC6. The colocalization of SAG1TM-CDMIC6 with MIC2 is illustrated by the yellow color in the merged image and indicated by arrows. In this genetic background, both MIC1 and MIC4 were missorted and accumulated in the vacuolar space, as indicated by arrowheads. (B) SAG1TM-CDMIC6 accumulates precisely in the micronemes as demonstrated by immunoelectron microscopy using anti-SAG1 antibodies. SAG1 is detected by gold particles and, when GPI anchored, was found in its normal location at the parasite surface (arrowheads). In contrast, SAG1TM-CDMIC6 was found in the micronemes (arrow). As micronemes are thinner than the section, some of them were not exposed to the antibody and were not labeled. Bars: (A) 1 μm; (B) 0.2 μm.

Mentions: Mistargeting of MIC1 and MIC4 in absence of MIC6 implies that MIC6 carries sorting signals to the micronemes. A recent study in T. gondii has demonstrated the existence of tyrosine-based sorting signals, the corresponding machinery, and their involvement in the targeting of proteins to the rhoptries (Hoppe et al. 2000). In parallel, a mutagenesis analysis of the CD of MIC2 has revealed that two conserved motifs are both necessary and sufficient for targeting proteins to the micronemes (Di Cristina et al. 2000). One of these signals contains tyrosine residues, whereas the other one is composed of a stretch of acidic residues. Both motifs are also present and strictly conserved in the CD of MIC6. We have replaced the GPI-anchoring signal in the major surface antigen SAG1 with the transmembrane and CDs of MIC6 (TM-CDMIC6) and have expressed stably SAG1TM-CDMIC6 in the mic6ko parasites (Fig. 1 B). Deletion of the GPI anchor signal led to the accumulation of soluble SAG1 in the parasitophorous vacuole (data not shown). In contrast, SAG1TM-CDMIC6 localized essentially to the micronemes, as detected by IFA with antibodies specific for the CD of MIC6 (Fig. 4 A). The expression of SAG1TM-CDMIC6 failed to restore the mic6ko phenotype since both MIC1 and MIC4 still accumulated in the vacuolar space and are absent from micronemes, as evident from the lack of colocalization with MIC2 (Fig. 4 A). Immunoelectron microscopy analysis of parasites expressing SAG1TM-CDMIC6 with anti-SAG1 mAbs demonstrated SAG1 reactivity in micronemes, in addition to its normal location at the parasite surface (Fig. 4 B).


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)

The CD of MIC6 contains sorting signals for the micronemes targeting. (A) IFA analysis by confocal microscopy on monolayers of HFF cells infected with mic6ko mutant expressing stably SAG1TM-CDMIC6. The subcellular distribution of the SAG1TM-CDMIC6 fusion protein was detected by using antibodies raised against the CD of MIC6. The colocalization of SAG1TM-CDMIC6 with MIC2 is illustrated by the yellow color in the merged image and indicated by arrows. In this genetic background, both MIC1 and MIC4 were missorted and accumulated in the vacuolar space, as indicated by arrowheads. (B) SAG1TM-CDMIC6 accumulates precisely in the micronemes as demonstrated by immunoelectron microscopy using anti-SAG1 antibodies. SAG1 is detected by gold particles and, when GPI anchored, was found in its normal location at the parasite surface (arrowheads). In contrast, SAG1TM-CDMIC6 was found in the micronemes (arrow). As micronemes are thinner than the section, some of them were not exposed to the antibody and were not labeled. Bars: (A) 1 μm; (B) 0.2 μm.
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Figure 4: The CD of MIC6 contains sorting signals for the micronemes targeting. (A) IFA analysis by confocal microscopy on monolayers of HFF cells infected with mic6ko mutant expressing stably SAG1TM-CDMIC6. The subcellular distribution of the SAG1TM-CDMIC6 fusion protein was detected by using antibodies raised against the CD of MIC6. The colocalization of SAG1TM-CDMIC6 with MIC2 is illustrated by the yellow color in the merged image and indicated by arrows. In this genetic background, both MIC1 and MIC4 were missorted and accumulated in the vacuolar space, as indicated by arrowheads. (B) SAG1TM-CDMIC6 accumulates precisely in the micronemes as demonstrated by immunoelectron microscopy using anti-SAG1 antibodies. SAG1 is detected by gold particles and, when GPI anchored, was found in its normal location at the parasite surface (arrowheads). In contrast, SAG1TM-CDMIC6 was found in the micronemes (arrow). As micronemes are thinner than the section, some of them were not exposed to the antibody and were not labeled. Bars: (A) 1 μm; (B) 0.2 μm.
Mentions: Mistargeting of MIC1 and MIC4 in absence of MIC6 implies that MIC6 carries sorting signals to the micronemes. A recent study in T. gondii has demonstrated the existence of tyrosine-based sorting signals, the corresponding machinery, and their involvement in the targeting of proteins to the rhoptries (Hoppe et al. 2000). In parallel, a mutagenesis analysis of the CD of MIC2 has revealed that two conserved motifs are both necessary and sufficient for targeting proteins to the micronemes (Di Cristina et al. 2000). One of these signals contains tyrosine residues, whereas the other one is composed of a stretch of acidic residues. Both motifs are also present and strictly conserved in the CD of MIC6. We have replaced the GPI-anchoring signal in the major surface antigen SAG1 with the transmembrane and CDs of MIC6 (TM-CDMIC6) and have expressed stably SAG1TM-CDMIC6 in the mic6ko parasites (Fig. 1 B). Deletion of the GPI anchor signal led to the accumulation of soluble SAG1 in the parasitophorous vacuole (data not shown). In contrast, SAG1TM-CDMIC6 localized essentially to the micronemes, as detected by IFA with antibodies specific for the CD of MIC6 (Fig. 4 A). The expression of SAG1TM-CDMIC6 failed to restore the mic6ko phenotype since both MIC1 and MIC4 still accumulated in the vacuolar space and are absent from micronemes, as evident from the lack of colocalization with MIC2 (Fig. 4 A). Immunoelectron microscopy analysis of parasites expressing SAG1TM-CDMIC6 with anti-SAG1 mAbs demonstrated SAG1 reactivity in micronemes, in addition to its normal location at the parasite surface (Fig. 4 B).

Bottom Line: 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.We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

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