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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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MIC1, MIC4, and MIC6 physically interact. (A) Immunoprecipitation of a complex containing MIC1, MIC4, and MIC6 from wild-type parasite lysate (RH lysate) by anti-MIC1 mAbs. The immunoprecipitates were shown to contain also MIC6 and MIC4 by Western blot analysis using rabbit polyclonals anti-MIC6 or anti-MIC4. The two processed forms of MIC6 and the two forms of MIC4 are indicated by arrows. Controls included immunoprecipitation in absence of lysate or in absence of the mAb anti-MIC1. (B) Coimmunoprecipitation of MIC4 with anti-MIC1 in cell lysates of mic6ko parasites showed that they interact in absence of MIC6. (C) Coimmunoprecipitation of MIC1 (detected with mAb anti-myc) and MIC4 using the polyclonals anti-CDMIC6 from lysates of mic1ko parasites expressing MIC1myc. The immunoprecipitation was carried out with increasing amount of antibodies (5, 20, and 40 μl). (D) Coimmunoprecipitation of MIC4 with anti-MIC6 from RH lysate. (E) Model. A complex between MIC1, MIC4, and MIC6 forms in the early compartments of the secretory pathway and ensures the proper targeting of MIC1 and MIC4 to the micronemes via the interaction of MIC6 cytoplasmic tail with the sorting machinery. During its transport, MIC6 is processed at the NH2 terminus and looses the first EGF-like domain. Upon contact with host cells, an increase in free intracellular calcium stimulates the discharge of the microneme contents. The complex of MICs is liberated at the surface of the parasites, potentially anchored within the plasma membrane via MIC6.
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Figure 6: MIC1, MIC4, and MIC6 physically interact. (A) Immunoprecipitation of a complex containing MIC1, MIC4, and MIC6 from wild-type parasite lysate (RH lysate) by anti-MIC1 mAbs. The immunoprecipitates were shown to contain also MIC6 and MIC4 by Western blot analysis using rabbit polyclonals anti-MIC6 or anti-MIC4. The two processed forms of MIC6 and the two forms of MIC4 are indicated by arrows. Controls included immunoprecipitation in absence of lysate or in absence of the mAb anti-MIC1. (B) Coimmunoprecipitation of MIC4 with anti-MIC1 in cell lysates of mic6ko parasites showed that they interact in absence of MIC6. (C) Coimmunoprecipitation of MIC1 (detected with mAb anti-myc) and MIC4 using the polyclonals anti-CDMIC6 from lysates of mic1ko parasites expressing MIC1myc. The immunoprecipitation was carried out with increasing amount of antibodies (5, 20, and 40 μl). (D) Coimmunoprecipitation of MIC4 with anti-MIC6 from RH lysate. (E) Model. A complex between MIC1, MIC4, and MIC6 forms in the early compartments of the secretory pathway and ensures the proper targeting of MIC1 and MIC4 to the micronemes via the interaction of MIC6 cytoplasmic tail with the sorting machinery. During its transport, MIC6 is processed at the NH2 terminus and looses the first EGF-like domain. Upon contact with host cells, an increase in free intracellular calcium stimulates the discharge of the microneme contents. The complex of MICs is liberated at the surface of the parasites, potentially anchored within the plasma membrane via MIC6.

Mentions: Evidence for the existence of a complex between MIC1, MIC4, and MIC6 was confirmed and supported biochemically by coimmunoprecipitation experiments. Parasite lysates were prepared under mild conditions by sonification and subjected to immunoprecipitation with mAb anti-MIC1. Western blot analysis revealed that both MIC4 and MIC6 coprecipitated (Fig. 6 A). Interestingly, the two processed forms of MIC6 coimmunoprecipitated with anti-MIC1, strongly suggesting that the complex is present both in the micronemes and at the surface of the parasites, where the COOH-terminal processing of MIC6 takes place. Coimmunoprecipitation experiments using mic6ko parasites clearly indicated that an interaction exists between MIC1 and MIC4 even in the absence of MIC6 (Fig. 6 B). Cell lysates from mic1ko parasites expressing MIC1myc were used to demonstrate that the polyclonal anti-CDMIC6 can coimmunoprecipitate MIC1 and MIC4 as detected on immunoblot using the mAb anti-myc 9E10 and the mAb anti-MIC4 5B2 (Fig. 6 C). Finally, the polyclonal anti-NtMIC6 also coimmunoprecipitated MIC4 in lysates from wild-type parasites (Fig. 6 D). Together, these data provided complementary information with regard to the topology of the complex, which is schematically depicted in Fig. 6 E.


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)

MIC1, MIC4, and MIC6 physically interact. (A) Immunoprecipitation of a complex containing MIC1, MIC4, and MIC6 from wild-type parasite lysate (RH lysate) by anti-MIC1 mAbs. The immunoprecipitates were shown to contain also MIC6 and MIC4 by Western blot analysis using rabbit polyclonals anti-MIC6 or anti-MIC4. The two processed forms of MIC6 and the two forms of MIC4 are indicated by arrows. Controls included immunoprecipitation in absence of lysate or in absence of the mAb anti-MIC1. (B) Coimmunoprecipitation of MIC4 with anti-MIC1 in cell lysates of mic6ko parasites showed that they interact in absence of MIC6. (C) Coimmunoprecipitation of MIC1 (detected with mAb anti-myc) and MIC4 using the polyclonals anti-CDMIC6 from lysates of mic1ko parasites expressing MIC1myc. The immunoprecipitation was carried out with increasing amount of antibodies (5, 20, and 40 μl). (D) Coimmunoprecipitation of MIC4 with anti-MIC6 from RH lysate. (E) Model. A complex between MIC1, MIC4, and MIC6 forms in the early compartments of the secretory pathway and ensures the proper targeting of MIC1 and MIC4 to the micronemes via the interaction of MIC6 cytoplasmic tail with the sorting machinery. During its transport, MIC6 is processed at the NH2 terminus and looses the first EGF-like domain. Upon contact with host cells, an increase in free intracellular calcium stimulates the discharge of the microneme contents. The complex of MICs is liberated at the surface of the parasites, potentially anchored within the plasma membrane via MIC6.
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Related In: Results  -  Collection

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Figure 6: MIC1, MIC4, and MIC6 physically interact. (A) Immunoprecipitation of a complex containing MIC1, MIC4, and MIC6 from wild-type parasite lysate (RH lysate) by anti-MIC1 mAbs. The immunoprecipitates were shown to contain also MIC6 and MIC4 by Western blot analysis using rabbit polyclonals anti-MIC6 or anti-MIC4. The two processed forms of MIC6 and the two forms of MIC4 are indicated by arrows. Controls included immunoprecipitation in absence of lysate or in absence of the mAb anti-MIC1. (B) Coimmunoprecipitation of MIC4 with anti-MIC1 in cell lysates of mic6ko parasites showed that they interact in absence of MIC6. (C) Coimmunoprecipitation of MIC1 (detected with mAb anti-myc) and MIC4 using the polyclonals anti-CDMIC6 from lysates of mic1ko parasites expressing MIC1myc. The immunoprecipitation was carried out with increasing amount of antibodies (5, 20, and 40 μl). (D) Coimmunoprecipitation of MIC4 with anti-MIC6 from RH lysate. (E) Model. A complex between MIC1, MIC4, and MIC6 forms in the early compartments of the secretory pathway and ensures the proper targeting of MIC1 and MIC4 to the micronemes via the interaction of MIC6 cytoplasmic tail with the sorting machinery. During its transport, MIC6 is processed at the NH2 terminus and looses the first EGF-like domain. Upon contact with host cells, an increase in free intracellular calcium stimulates the discharge of the microneme contents. The complex of MICs is liberated at the surface of the parasites, potentially anchored within the plasma membrane via MIC6.
Mentions: Evidence for the existence of a complex between MIC1, MIC4, and MIC6 was confirmed and supported biochemically by coimmunoprecipitation experiments. Parasite lysates were prepared under mild conditions by sonification and subjected to immunoprecipitation with mAb anti-MIC1. Western blot analysis revealed that both MIC4 and MIC6 coprecipitated (Fig. 6 A). Interestingly, the two processed forms of MIC6 coimmunoprecipitated with anti-MIC1, strongly suggesting that the complex is present both in the micronemes and at the surface of the parasites, where the COOH-terminal processing of MIC6 takes place. Coimmunoprecipitation experiments using mic6ko parasites clearly indicated that an interaction exists between MIC1 and MIC4 even in the absence of MIC6 (Fig. 6 B). Cell lysates from mic1ko parasites expressing MIC1myc were used to demonstrate that the polyclonal anti-CDMIC6 can coimmunoprecipitate MIC1 and MIC4 as detected on immunoblot using the mAb anti-myc 9E10 and the mAb anti-MIC4 5B2 (Fig. 6 C). Finally, the polyclonal anti-NtMIC6 also coimmunoprecipitated MIC4 in lysates from wild-type parasites (Fig. 6 D). Together, these data provided complementary information with regard to the topology of the complex, which is schematically depicted in Fig. 6 E.

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