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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 EGF-3 domain of MIC6 is necessary for accurate targeting of MIC1 and MIC4 to the micronemes. (A) Western blot analysis of cell lysates from RH and recombinant parasites expressing MIC6ΔEGF1, MIC6ΔEGF2, and MIC6ΔEGF3 with anti-CDMIC6 antibodies (raised against the CD of MIC6). MIC6ΔEGF1 and MIC6ΔEGF3 were still subjected to NH2-terminal cleavage, whereas a single form of MIC6ΔEGF2 was detectable, suggesting that the cleavage site has been deleted by removing the EGF-2 domain. (B) IFA analysis by confocal microscopy. MIC6ΔEGF-3 expressed in mic6ko was accurately targeted to the micronemes, as detected with anti-CDMIC6, and colocalized with MIC2 (arrows). In this mutant, MIC4 was only partially sorted to the micronemes (arrowhead), with a significant amount of protein still accumulating in the dense granules. MIC1 and MIC4 colocalized (arrows). Bar, 1 μm.
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Figure 7: The EGF-3 domain of MIC6 is necessary for accurate targeting of MIC1 and MIC4 to the micronemes. (A) Western blot analysis of cell lysates from RH and recombinant parasites expressing MIC6ΔEGF1, MIC6ΔEGF2, and MIC6ΔEGF3 with anti-CDMIC6 antibodies (raised against the CD of MIC6). MIC6ΔEGF1 and MIC6ΔEGF3 were still subjected to NH2-terminal cleavage, whereas a single form of MIC6ΔEGF2 was detectable, suggesting that the cleavage site has been deleted by removing the EGF-2 domain. (B) IFA analysis by confocal microscopy. MIC6ΔEGF-3 expressed in mic6ko was accurately targeted to the micronemes, as detected with anti-CDMIC6, and colocalized with MIC2 (arrows). In this mutant, MIC4 was only partially sorted to the micronemes (arrowhead), with a significant amount of protein still accumulating in the dense granules. MIC1 and MIC4 colocalized (arrows). Bar, 1 μm.

Mentions: MIC6 mutants with various deletions in their lumenal domains were generated and stably expressed in the mic6ko strain. Phenotypic analysis of these mutants by IFA allowed identifying the domain on MIC6, interacting with MIC1 and MIC4. MIC6 mutants lacking either the three EGF domains or the acidic domain were poorly targeted to the micronemes. These large deletions are likely to cause significant alteration of the folding properties of the truncated proteins. However, more subtle deletions of each individual EGF-like domain produced truncated proteins that were perfectly targeted to the organelle. Western blot analysis of the deletion mutants using the anti-MIC6 tail demonstrated that the MIC6 mutants were expressed at comparable levels (Fig. 7 A). The migration behavior of the various mutants and the absence of processing on MIC6ΔEGF-2 indicated that the NH2-terminal cleavage of MIC6 occurred within the second EGF domain. The subcellular distribution of MIC1 and MIC4 in these mutants was analyzed by IFA. In summary, MIC6ΔEGF-1 and MIC6ΔEGF-2 were able to fully rescue the missorting phenotype, with both MIC1 and MIC4 faithfully targeted to the micronemes (data not shown). In contrast, in parasites expressing MIC6ΔEGF-3, neither MIC1 nor MIC4 were quantitatively sorted to the micronemes, although MIC6ΔEGF-3 localized to these organelles as shown by colocalization with MIC2 (Fig. 7 B). These results suggest that the EGF-3 domain is necessary for an efficient interaction of MIC6 with MIC1 and MIC4 individually or with one of the two, whereas MIC1 and MIC4 are directly associated. A possible contribution of the adjacent acidic domain can not be excluded by this analysis.


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 EGF-3 domain of MIC6 is necessary for accurate targeting of MIC1 and MIC4 to the micronemes. (A) Western blot analysis of cell lysates from RH and recombinant parasites expressing MIC6ΔEGF1, MIC6ΔEGF2, and MIC6ΔEGF3 with anti-CDMIC6 antibodies (raised against the CD of MIC6). MIC6ΔEGF1 and MIC6ΔEGF3 were still subjected to NH2-terminal cleavage, whereas a single form of MIC6ΔEGF2 was detectable, suggesting that the cleavage site has been deleted by removing the EGF-2 domain. (B) IFA analysis by confocal microscopy. MIC6ΔEGF-3 expressed in mic6ko was accurately targeted to the micronemes, as detected with anti-CDMIC6, and colocalized with MIC2 (arrows). In this mutant, MIC4 was only partially sorted to the micronemes (arrowhead), with a significant amount of protein still accumulating in the dense granules. MIC1 and MIC4 colocalized (arrows). Bar, 1 μm.
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Related In: Results  -  Collection

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Figure 7: The EGF-3 domain of MIC6 is necessary for accurate targeting of MIC1 and MIC4 to the micronemes. (A) Western blot analysis of cell lysates from RH and recombinant parasites expressing MIC6ΔEGF1, MIC6ΔEGF2, and MIC6ΔEGF3 with anti-CDMIC6 antibodies (raised against the CD of MIC6). MIC6ΔEGF1 and MIC6ΔEGF3 were still subjected to NH2-terminal cleavage, whereas a single form of MIC6ΔEGF2 was detectable, suggesting that the cleavage site has been deleted by removing the EGF-2 domain. (B) IFA analysis by confocal microscopy. MIC6ΔEGF-3 expressed in mic6ko was accurately targeted to the micronemes, as detected with anti-CDMIC6, and colocalized with MIC2 (arrows). In this mutant, MIC4 was only partially sorted to the micronemes (arrowhead), with a significant amount of protein still accumulating in the dense granules. MIC1 and MIC4 colocalized (arrows). Bar, 1 μm.
Mentions: MIC6 mutants with various deletions in their lumenal domains were generated and stably expressed in the mic6ko strain. Phenotypic analysis of these mutants by IFA allowed identifying the domain on MIC6, interacting with MIC1 and MIC4. MIC6 mutants lacking either the three EGF domains or the acidic domain were poorly targeted to the micronemes. These large deletions are likely to cause significant alteration of the folding properties of the truncated proteins. However, more subtle deletions of each individual EGF-like domain produced truncated proteins that were perfectly targeted to the organelle. Western blot analysis of the deletion mutants using the anti-MIC6 tail demonstrated that the MIC6 mutants were expressed at comparable levels (Fig. 7 A). The migration behavior of the various mutants and the absence of processing on MIC6ΔEGF-2 indicated that the NH2-terminal cleavage of MIC6 occurred within the second EGF domain. The subcellular distribution of MIC1 and MIC4 in these mutants was analyzed by IFA. In summary, MIC6ΔEGF-1 and MIC6ΔEGF-2 were able to fully rescue the missorting phenotype, with both MIC1 and MIC4 faithfully targeted to the micronemes (data not shown). In contrast, in parasites expressing MIC6ΔEGF-3, neither MIC1 nor MIC4 were quantitatively sorted to the micronemes, although MIC6ΔEGF-3 localized to these organelles as shown by colocalization with MIC2 (Fig. 7 B). These results suggest that the EGF-3 domain is necessary for an efficient interaction of MIC6 with MIC1 and MIC4 individually or with one of the two, whereas MIC1 and MIC4 are directly associated. A possible contribution of the adjacent acidic domain can not be excluded by this analysis.

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