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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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Analysis of mic1ko. MIC1 is necessary for MIC6 and MIC4 to leave the ER and Golgi. (A) Subcellular distribution of MIC4 and MIC6 in mic1ko strain analyzed by IFA. MIC3 was faithfully sorted to the micronemes, whereas MIC4 and MIC6 were retained in the early compartments of the secretory pathway. Two vacuoles stained with anti-MIC4 and three vacuoles stained with anti-MIC6 are presented here to illustrate the various compartments where the two proteins are retained. A double IFA of MIC4 and MIC6 in mic1ko indicated that in all vacuoles, both proteins colocalized perfectly as shown in the merged image. (B) Immunolocalization of MIC6 and MIC4 in mic1ko by electron microscopy revealed various patterns of distribution in the early secretory compartments. (a) MIC4 was detected in the nuclear envelope (arrowheads) and in the successive stacks of the Golgi (arrows). N, nucleus; G, Golgi apparatus. (b) MIC6 stagged in the cis-Golgi. (c) MIC4 stagged in the ER (arrows) and nuclear envelope (arrowheads). (C) The mic1ko mutant expressing MIC1myc showed rescue of the phenoptype by IFA. In presence of MIC1myc, both MIC4 and MIC6 were quantitatively sorted to the micronemes. A slight overexpression of MIC1 led to some leakage to the vacuolar space as detected by anti-MIC1 mAb. (D) Western blot analysis of lysates form RH, mic1ko, and mic1ko complemented with MIC1myc using either anti-MIC1sera or anti-myc mAb. A slight increase in the size of MIC1myc was apparent compared with endogenous MIC1, due to the presence of the myc tag. Bars: (A and C) 1 μm; (B) 0.2 μm.
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Figure 9: Analysis of mic1ko. MIC1 is necessary for MIC6 and MIC4 to leave the ER and Golgi. (A) Subcellular distribution of MIC4 and MIC6 in mic1ko strain analyzed by IFA. MIC3 was faithfully sorted to the micronemes, whereas MIC4 and MIC6 were retained in the early compartments of the secretory pathway. Two vacuoles stained with anti-MIC4 and three vacuoles stained with anti-MIC6 are presented here to illustrate the various compartments where the two proteins are retained. A double IFA of MIC4 and MIC6 in mic1ko indicated that in all vacuoles, both proteins colocalized perfectly as shown in the merged image. (B) Immunolocalization of MIC6 and MIC4 in mic1ko by electron microscopy revealed various patterns of distribution in the early secretory compartments. (a) MIC4 was detected in the nuclear envelope (arrowheads) and in the successive stacks of the Golgi (arrows). N, nucleus; G, Golgi apparatus. (b) MIC6 stagged in the cis-Golgi. (c) MIC4 stagged in the ER (arrows) and nuclear envelope (arrowheads). (C) The mic1ko mutant expressing MIC1myc showed rescue of the phenoptype by IFA. In presence of MIC1myc, both MIC4 and MIC6 were quantitatively sorted to the micronemes. A slight overexpression of MIC1 led to some leakage to the vacuolar space as detected by anti-MIC1 mAb. (D) Western blot analysis of lysates form RH, mic1ko, and mic1ko complemented with MIC1myc using either anti-MIC1sera or anti-myc mAb. A slight increase in the size of MIC1myc was apparent compared with endogenous MIC1, due to the presence of the myc tag. Bars: (A and C) 1 μm; (B) 0.2 μm.

Mentions: MIC1 localizes to micronemes and has been shown to bind to host cells (Fourmaux et al. 1996). MIC1 gene encodes a polypeptide of 456 amino acids containing two domains bearing some homology to the thrombospondin domains present in the TRAP family (Naitza et al. 1998). The absence of MIC1 gene is not essential for the survival of the parasites in culture, and, like in mic4ko, the targeting of other micronemal proteins was investigated in the mic1ko strain. Unlike MIC4, the absence of MIC1 has a drastic and specific effect on the sorting of the two other members of the complex. In absence of MIC1, both MIC2 and MIC3 are properly localized in the micronemes, whereas MIC4 and MIC6 are retained in the early compartments of the secretory pathway. A selective accumulation of these proteins in the perinuclear region, ER, Golgi, and possibly TGN was observed. Within each single vacuole, all the parasites showed a homogeneous distribution in a particular compartment of the secretory pathway, whereas the distribution varied between vacuoles. The heterogeneity of staining observed within the population of vacuoles is suggestive of a cell cycle–dependent effect. Fig. 9 A shows retention of MIC4 and MIC6 in various compartments of the secretory pathway. Analysis by immunoelectron microscopy confirmed the localization of MIC4 in the Golgi stacks and ER (Fig. 9 B). In all circumstances, MIC4 and MIC6 perfectly colocalized, and we failed to identify a single vacuole with a MIC4 or MIC6 localized to the micronemes. A complete rescue of this phenotype was observed by IFA when mic1ko were stably transformed with the vector pM2MIC1myc (Fig. 9 C). Expression of MIC1myc fully restored the transport of MIC4 and MIC6 to the micronemes. In this mutant, MIC1 did not only accumulate in the micronemes, but a small proportion of it also transited via the dense granules and reached the parasitophorous vacuole. This result suggests that MIC6 is present in limiting amount, and when this escorter becomes saturated, both MIC1 and MIC4 are missorted. Western blot analysis of the mic1ko rescue showed that the reintroduced MIC1myc was slightly overexpresssed compared with MIC1 in RH (Fig. 9 D). MIC6 in mic1ko indicated that retention of MIC6 in the early compartments of the secretory pathway correlated with the accumulation of the unprocessed form of the protein (Fig. 2 B). In the rescued mutant expressing MIC1myc, the ratio between full-length MIC6 and the NH2-terminal processed form is restored as in wild type (data not shown).


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)

Analysis of mic1ko. MIC1 is necessary for MIC6 and MIC4 to leave the ER and Golgi. (A) Subcellular distribution of MIC4 and MIC6 in mic1ko strain analyzed by IFA. MIC3 was faithfully sorted to the micronemes, whereas MIC4 and MIC6 were retained in the early compartments of the secretory pathway. Two vacuoles stained with anti-MIC4 and three vacuoles stained with anti-MIC6 are presented here to illustrate the various compartments where the two proteins are retained. A double IFA of MIC4 and MIC6 in mic1ko indicated that in all vacuoles, both proteins colocalized perfectly as shown in the merged image. (B) Immunolocalization of MIC6 and MIC4 in mic1ko by electron microscopy revealed various patterns of distribution in the early secretory compartments. (a) MIC4 was detected in the nuclear envelope (arrowheads) and in the successive stacks of the Golgi (arrows). N, nucleus; G, Golgi apparatus. (b) MIC6 stagged in the cis-Golgi. (c) MIC4 stagged in the ER (arrows) and nuclear envelope (arrowheads). (C) The mic1ko mutant expressing MIC1myc showed rescue of the phenoptype by IFA. In presence of MIC1myc, both MIC4 and MIC6 were quantitatively sorted to the micronemes. A slight overexpression of MIC1 led to some leakage to the vacuolar space as detected by anti-MIC1 mAb. (D) Western blot analysis of lysates form RH, mic1ko, and mic1ko complemented with MIC1myc using either anti-MIC1sera or anti-myc mAb. A slight increase in the size of MIC1myc was apparent compared with endogenous MIC1, due to the presence of the myc tag. Bars: (A and C) 1 μm; (B) 0.2 μm.
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Figure 9: Analysis of mic1ko. MIC1 is necessary for MIC6 and MIC4 to leave the ER and Golgi. (A) Subcellular distribution of MIC4 and MIC6 in mic1ko strain analyzed by IFA. MIC3 was faithfully sorted to the micronemes, whereas MIC4 and MIC6 were retained in the early compartments of the secretory pathway. Two vacuoles stained with anti-MIC4 and three vacuoles stained with anti-MIC6 are presented here to illustrate the various compartments where the two proteins are retained. A double IFA of MIC4 and MIC6 in mic1ko indicated that in all vacuoles, both proteins colocalized perfectly as shown in the merged image. (B) Immunolocalization of MIC6 and MIC4 in mic1ko by electron microscopy revealed various patterns of distribution in the early secretory compartments. (a) MIC4 was detected in the nuclear envelope (arrowheads) and in the successive stacks of the Golgi (arrows). N, nucleus; G, Golgi apparatus. (b) MIC6 stagged in the cis-Golgi. (c) MIC4 stagged in the ER (arrows) and nuclear envelope (arrowheads). (C) The mic1ko mutant expressing MIC1myc showed rescue of the phenoptype by IFA. In presence of MIC1myc, both MIC4 and MIC6 were quantitatively sorted to the micronemes. A slight overexpression of MIC1 led to some leakage to the vacuolar space as detected by anti-MIC1 mAb. (D) Western blot analysis of lysates form RH, mic1ko, and mic1ko complemented with MIC1myc using either anti-MIC1sera or anti-myc mAb. A slight increase in the size of MIC1myc was apparent compared with endogenous MIC1, due to the presence of the myc tag. Bars: (A and C) 1 μm; (B) 0.2 μm.
Mentions: MIC1 localizes to micronemes and has been shown to bind to host cells (Fourmaux et al. 1996). MIC1 gene encodes a polypeptide of 456 amino acids containing two domains bearing some homology to the thrombospondin domains present in the TRAP family (Naitza et al. 1998). The absence of MIC1 gene is not essential for the survival of the parasites in culture, and, like in mic4ko, the targeting of other micronemal proteins was investigated in the mic1ko strain. Unlike MIC4, the absence of MIC1 has a drastic and specific effect on the sorting of the two other members of the complex. In absence of MIC1, both MIC2 and MIC3 are properly localized in the micronemes, whereas MIC4 and MIC6 are retained in the early compartments of the secretory pathway. A selective accumulation of these proteins in the perinuclear region, ER, Golgi, and possibly TGN was observed. Within each single vacuole, all the parasites showed a homogeneous distribution in a particular compartment of the secretory pathway, whereas the distribution varied between vacuoles. The heterogeneity of staining observed within the population of vacuoles is suggestive of a cell cycle–dependent effect. Fig. 9 A shows retention of MIC4 and MIC6 in various compartments of the secretory pathway. Analysis by immunoelectron microscopy confirmed the localization of MIC4 in the Golgi stacks and ER (Fig. 9 B). In all circumstances, MIC4 and MIC6 perfectly colocalized, and we failed to identify a single vacuole with a MIC4 or MIC6 localized to the micronemes. A complete rescue of this phenotype was observed by IFA when mic1ko were stably transformed with the vector pM2MIC1myc (Fig. 9 C). Expression of MIC1myc fully restored the transport of MIC4 and MIC6 to the micronemes. In this mutant, MIC1 did not only accumulate in the micronemes, but a small proportion of it also transited via the dense granules and reached the parasitophorous vacuole. This result suggests that MIC6 is present in limiting amount, and when this escorter becomes saturated, both MIC1 and MIC4 are missorted. Western blot analysis of the mic1ko rescue showed that the reintroduced MIC1myc was slightly overexpresssed compared with MIC1 in RH (Fig. 9 D). MIC6 in mic1ko indicated that retention of MIC6 in the early compartments of the secretory pathway correlated with the accumulation of the unprocessed form of the protein (Fig. 2 B). In the rescued mutant expressing MIC1myc, the ratio between full-length MIC6 and the NH2-terminal processed form is restored as in wild type (data not shown).

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