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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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Trafficking of MIC6GPI and SAG1TM-CDMIC6 in mic1ko. Pools of parasites expressing MIC6TyGPI and SAG1Ty1TM-CDMIC6 were analyzed by IFA. (A) MIC6TyGPI localized predominantly to the perinuclear region as detected by the anti–Ty-1 antibodies (arrowhead). The endogenous MIC4 was retained in the early compartments of the secretory pathway (arrow). (B) SAG1TM-CDMIC6 was accurately sorted to the micronemes as shown by the extensive colocalization with the microneme marker MIC7 (rabbit polyclonal anti-MIC7 raised against the EGF-like domains; Meissne, M., and D. Soldati, unpublished results). The vacuole on the left was not transformed with SAG1TM-CDMIC6 and showed the absence of background with the mAb anti-Ty-1. The pool of transformants was analyzed with the anti-CDMIC6 revealing that the endogenous MIC6 (green, arrows) is retained in the ER and Golgi. Bar, 1 μm.
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Figure 10: Trafficking of MIC6GPI and SAG1TM-CDMIC6 in mic1ko. Pools of parasites expressing MIC6TyGPI and SAG1Ty1TM-CDMIC6 were analyzed by IFA. (A) MIC6TyGPI localized predominantly to the perinuclear region as detected by the anti–Ty-1 antibodies (arrowhead). The endogenous MIC4 was retained in the early compartments of the secretory pathway (arrow). (B) SAG1TM-CDMIC6 was accurately sorted to the micronemes as shown by the extensive colocalization with the microneme marker MIC7 (rabbit polyclonal anti-MIC7 raised against the EGF-like domains; Meissne, M., and D. Soldati, unpublished results). The vacuole on the left was not transformed with SAG1TM-CDMIC6 and showed the absence of background with the mAb anti-Ty-1. The pool of transformants was analyzed with the anti-CDMIC6 revealing that the endogenous MIC6 (green, arrows) is retained in the ER and Golgi. Bar, 1 μm.

Mentions: The absence of MIC1 is expected to interfere with the trafficking of the lumenal part of MIC6, whereas the CD of MIC6 should not be affected. To test this hypothesis, we have generated constructs, the MIC6TyGPI and SAG1TyTM-CDMIC6, to follow their expression in mic1ko strain. The presence of a Ty-1 tag allowed us to unambiguously distinguish the transgenes from the endogenous MIC6 and SAG1. These constructs were introduced stably in mic1ko by cotransfection with a plasmid expressing CAT gene as selectable marker, and pools of transformants were analyzed by IFA. As opposed to the plasma membrane localization of MIC6GPI in wild-type parasites (data not shown) and in mic6ko (Fig. 5 D), MIC6TyGPI expressed in mic1ko was retained predominantly in the ER and to a lesser extent in the Golgi compartments (Fig. 10 A). In this genetic background, endogenous MIC4 is still retained in the early compartments of the secretory pathway and MIC6 (data not shown). In contrast, the trafficking of SAG1TyTM-CDMIC6 (Fig. 1 B) in mic1ko was not affected, confirming the MIC1 acts on the lumenal part of MIC6. As expected, SAG1TyTM-CDMIC6 colocalized perfectly with another micronemal marker, MIC7 (Meissner et al., unpublished results) (Fig. 10 B). Detection of the SAG1TyTM-CDMIC6 in mic1ko using the polyclonal antibody anti-CDMIC6 confirmed that the endogenous MIC6 is still retained in the early secretory pathway in this mutant.


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)

Trafficking of MIC6GPI and SAG1TM-CDMIC6 in mic1ko. Pools of parasites expressing MIC6TyGPI and SAG1Ty1TM-CDMIC6 were analyzed by IFA. (A) MIC6TyGPI localized predominantly to the perinuclear region as detected by the anti–Ty-1 antibodies (arrowhead). The endogenous MIC4 was retained in the early compartments of the secretory pathway (arrow). (B) SAG1TM-CDMIC6 was accurately sorted to the micronemes as shown by the extensive colocalization with the microneme marker MIC7 (rabbit polyclonal anti-MIC7 raised against the EGF-like domains; Meissne, M., and D. Soldati, unpublished results). The vacuole on the left was not transformed with SAG1TM-CDMIC6 and showed the absence of background with the mAb anti-Ty-1. The pool of transformants was analyzed with the anti-CDMIC6 revealing that the endogenous MIC6 (green, arrows) is retained in the ER and Golgi. Bar, 1 μm.
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Related In: Results  -  Collection

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Figure 10: Trafficking of MIC6GPI and SAG1TM-CDMIC6 in mic1ko. Pools of parasites expressing MIC6TyGPI and SAG1Ty1TM-CDMIC6 were analyzed by IFA. (A) MIC6TyGPI localized predominantly to the perinuclear region as detected by the anti–Ty-1 antibodies (arrowhead). The endogenous MIC4 was retained in the early compartments of the secretory pathway (arrow). (B) SAG1TM-CDMIC6 was accurately sorted to the micronemes as shown by the extensive colocalization with the microneme marker MIC7 (rabbit polyclonal anti-MIC7 raised against the EGF-like domains; Meissne, M., and D. Soldati, unpublished results). The vacuole on the left was not transformed with SAG1TM-CDMIC6 and showed the absence of background with the mAb anti-Ty-1. The pool of transformants was analyzed with the anti-CDMIC6 revealing that the endogenous MIC6 (green, arrows) is retained in the ER and Golgi. Bar, 1 μm.
Mentions: The absence of MIC1 is expected to interfere with the trafficking of the lumenal part of MIC6, whereas the CD of MIC6 should not be affected. To test this hypothesis, we have generated constructs, the MIC6TyGPI and SAG1TyTM-CDMIC6, to follow their expression in mic1ko strain. The presence of a Ty-1 tag allowed us to unambiguously distinguish the transgenes from the endogenous MIC6 and SAG1. These constructs were introduced stably in mic1ko by cotransfection with a plasmid expressing CAT gene as selectable marker, and pools of transformants were analyzed by IFA. As opposed to the plasma membrane localization of MIC6GPI in wild-type parasites (data not shown) and in mic6ko (Fig. 5 D), MIC6TyGPI expressed in mic1ko was retained predominantly in the ER and to a lesser extent in the Golgi compartments (Fig. 10 A). In this genetic background, endogenous MIC4 is still retained in the early compartments of the secretory pathway and MIC6 (data not shown). In contrast, the trafficking of SAG1TyTM-CDMIC6 (Fig. 1 B) in mic1ko was not affected, confirming the MIC1 acts on the lumenal part of MIC6. As expected, SAG1TyTM-CDMIC6 colocalized perfectly with another micronemal marker, MIC7 (Meissner et al., unpublished results) (Fig. 10 B). Detection of the SAG1TyTM-CDMIC6 in mic1ko using the polyclonal antibody anti-CDMIC6 confirmed that the endogenous MIC6 is still retained in the early secretory pathway in this mutant.

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