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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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Expression of MIC4 deletion mutants in mic4ko. The NH2-terminal region of MIC4 containing the first two apple domains was correctly sorted to the micronemes. (A) Western blot analysis of cell lysates from RH and recombinant parasites expressing MIC4ΔA5-6 or MIC4ΔA3-6. The migration of MIC4 and the truncated forms on SDS-PAGE were slower than their predicted molecular weight. (B) IFA analysis by confocal microscopy of parasites expressing MIC4ΔA5-6 and MIC4ΔA3-6. Proper targeting to the micronemes was confirmed by colocalization with MIC3. Bar, 1 μm.
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Figure 8: Expression of MIC4 deletion mutants in mic4ko. The NH2-terminal region of MIC4 containing the first two apple domains was correctly sorted to the micronemes. (A) Western blot analysis of cell lysates from RH and recombinant parasites expressing MIC4ΔA5-6 or MIC4ΔA3-6. The migration of MIC4 and the truncated forms on SDS-PAGE were slower than their predicted molecular weight. (B) IFA analysis by confocal microscopy of parasites expressing MIC4ΔA5-6 and MIC4ΔA3-6. Proper targeting to the micronemes was confirmed by colocalization with MIC3. Bar, 1 μm.

Mentions: To assess the contribution of each partner in the complex, we have abrogated selectively their expression. MIC4 cDNA has a predicted ORF of 580 amino acids that contains a single NH2-terminal hydrophobic region. The protein contains six apple domains comprising six cysteine residues separated by variable spacing that are predicted to form a structure resembling an apple (McMullen et al. 1991a,McMullen et al. 1991b). Homologues of this protein have been identified in Spironucleus muris and in Eimeria species (Brown et al. 2000). The protein is an adhesin, which is proteolytically cleaved both at the NH2 and COOH terminus at the surface of the parasite after release from the micronemes (Brecht et al. 2001). The level of expression and subcellular distribution of the other micronemal proteins was carefully examined in the mic4ko. We concluded that all micronemal proteins described in T. gondii so far were appropriately expressed and sorted in this mutant (data not shown). The correct targeting of MIC1 in absence of MIC4 testified for a direct association of MIC1 with MIC6, excluding an association via MIC4. Two deletion mutants of MIC4 lacking the last two, MIC4ΔA5-6, or the last four apple domains, MIC4ΔA3-6 (Fig. 1 B) were generated and expressed stably in the mic4ko recipient strain. These clones were analyzed by Western blotting and showed products of the expected sizes (Fig. 8 A). IFA analysis of these mutants confirmed that the truncated proteins lacking the NH2-terminal two or four apple domains were still localized to the micronemes and have consequently retained the ability to associate with MIC1 (Fig. 8 B). The MIC4ΔA5-6 mutant was substantially overexpressed compared with MIC4 in wild type, causing a significant spill over of the truncated protein into the default pathway leading to an accumulation of the protein into the dense granules, as perceptible on IFA.


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)

Expression of MIC4 deletion mutants in mic4ko. The NH2-terminal region of MIC4 containing the first two apple domains was correctly sorted to the micronemes. (A) Western blot analysis of cell lysates from RH and recombinant parasites expressing MIC4ΔA5-6 or MIC4ΔA3-6. The migration of MIC4 and the truncated forms on SDS-PAGE were slower than their predicted molecular weight. (B) IFA analysis by confocal microscopy of parasites expressing MIC4ΔA5-6 and MIC4ΔA3-6. Proper targeting to the micronemes was confirmed by colocalization with MIC3. Bar, 1 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196004&req=5

Figure 8: Expression of MIC4 deletion mutants in mic4ko. The NH2-terminal region of MIC4 containing the first two apple domains was correctly sorted to the micronemes. (A) Western blot analysis of cell lysates from RH and recombinant parasites expressing MIC4ΔA5-6 or MIC4ΔA3-6. The migration of MIC4 and the truncated forms on SDS-PAGE were slower than their predicted molecular weight. (B) IFA analysis by confocal microscopy of parasites expressing MIC4ΔA5-6 and MIC4ΔA3-6. Proper targeting to the micronemes was confirmed by colocalization with MIC3. Bar, 1 μm.
Mentions: To assess the contribution of each partner in the complex, we have abrogated selectively their expression. MIC4 cDNA has a predicted ORF of 580 amino acids that contains a single NH2-terminal hydrophobic region. The protein contains six apple domains comprising six cysteine residues separated by variable spacing that are predicted to form a structure resembling an apple (McMullen et al. 1991a,McMullen et al. 1991b). Homologues of this protein have been identified in Spironucleus muris and in Eimeria species (Brown et al. 2000). The protein is an adhesin, which is proteolytically cleaved both at the NH2 and COOH terminus at the surface of the parasite after release from the micronemes (Brecht et al. 2001). The level of expression and subcellular distribution of the other micronemal proteins was carefully examined in the mic4ko. We concluded that all micronemal proteins described in T. gondii so far were appropriately expressed and sorted in this mutant (data not shown). The correct targeting of MIC1 in absence of MIC4 testified for a direct association of MIC1 with MIC6, excluding an association via MIC4. Two deletion mutants of MIC4 lacking the last two, MIC4ΔA5-6, or the last four apple domains, MIC4ΔA3-6 (Fig. 1 B) were generated and expressed stably in the mic4ko recipient strain. These clones were analyzed by Western blotting and showed products of the expected sizes (Fig. 8 A). IFA analysis of these mutants confirmed that the truncated proteins lacking the NH2-terminal two or four apple domains were still localized to the micronemes and have consequently retained the ability to associate with MIC1 (Fig. 8 B). The MIC4ΔA5-6 mutant was substantially overexpressed compared with MIC4 in wild type, causing a significant spill over of the truncated protein into the default pathway leading to an accumulation of the protein into the dense granules, as perceptible on IFA.

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