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The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane.

Sinai AP, Joiner KA - J. Cell Biol. (2001)

Bottom Line: Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle.Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER.These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases Section, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. sinai@pop.uky.edu

ABSTRACT
Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.

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Mitochondrial association with the T. gondii PVM occurs at the time of invasion. (A) Ultrastructural features of a T. gondii parasitophorous vacuole 20 h after infection of CHO cells, containing four parasites (P) within the vacuole. The major secretory organelles including rhoptries (R), dense granules (DG), and micronemes (m) are indicated. The PV is delimited from the host cell by the PVM, which associates intimately with host mitochondria (M) and ER. (B) Host mitochondria associate with the PVM immediately after infection. PVs containing intracellular T. gondii, with associated mitochondria (arrowheads), were identified on the basis of their phase contrast characteristics (C, F, and I) and the distribution of the dense granule and PVM marker GRA3 (A, D, and G). Host mitochondria were identified using the specific dye MitoTracker (B, E, and H). Host mitochondria associated with the PVM follow the contour of the vacuole (arrowhead). Parasite infection was stopped at 1 (A–C), 5 (D–F), and 10 (G–I) min postinfection, and the extent of release of the dense granule marker GRA3 determined by immunofluorescence (A, D, and G). GRA3 labeling is confined to dense granules and is not detected in either the PV or PVM 1 min postinfection (A). At 5 min postinfection, patchy PVM staining (D, arrowhead) is detected in most vacuoles, which becomes circumferential by 10 min postinfection (G, arrowheads), indicating dense granule exocytosis. In contrast, MitoTracker-labeled PVM-associated mitochondria are readily apparent, surrounding the vacuole at all time points (B, E, and G, arrowhead). Detection of PVM-associated mitochondria (B) before dense granule exocytosis (A) indicates dense granule products are not required. Bars, 5 μm.
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fig1: Mitochondrial association with the T. gondii PVM occurs at the time of invasion. (A) Ultrastructural features of a T. gondii parasitophorous vacuole 20 h after infection of CHO cells, containing four parasites (P) within the vacuole. The major secretory organelles including rhoptries (R), dense granules (DG), and micronemes (m) are indicated. The PV is delimited from the host cell by the PVM, which associates intimately with host mitochondria (M) and ER. (B) Host mitochondria associate with the PVM immediately after infection. PVs containing intracellular T. gondii, with associated mitochondria (arrowheads), were identified on the basis of their phase contrast characteristics (C, F, and I) and the distribution of the dense granule and PVM marker GRA3 (A, D, and G). Host mitochondria were identified using the specific dye MitoTracker (B, E, and H). Host mitochondria associated with the PVM follow the contour of the vacuole (arrowhead). Parasite infection was stopped at 1 (A–C), 5 (D–F), and 10 (G–I) min postinfection, and the extent of release of the dense granule marker GRA3 determined by immunofluorescence (A, D, and G). GRA3 labeling is confined to dense granules and is not detected in either the PV or PVM 1 min postinfection (A). At 5 min postinfection, patchy PVM staining (D, arrowhead) is detected in most vacuoles, which becomes circumferential by 10 min postinfection (G, arrowheads), indicating dense granule exocytosis. In contrast, MitoTracker-labeled PVM-associated mitochondria are readily apparent, surrounding the vacuole at all time points (B, E, and G, arrowhead). Detection of PVM-associated mitochondria (B) before dense granule exocytosis (A) indicates dense granule products are not required. Bars, 5 μm.

Mentions: The T. gondii PVM exhibits a remarkable association with host mitochondria and ER (DeMelo et al., 1992; Sinai et al., 1997) (see Fig. 1 A). We have previously termed this phenomenon PVM–organelle association (Sinai et al., 1997). Association of host organelles with the vacuolar membranes surrounding intracellular pathogens is a feature restricted to organisms that either never enter the endocytic cascade, or that exit it soon after entry (Sinai and Joiner, 1997). Both Legionella pneumophila (Swanson and Isberg, 1995) and Brucella abortus (Pizarro-Cerda et al., 1998) replicate in a compartment associated with the ER. In L. pneumophila–infected cells, a bacterially encoded machinery with homology to bacterial DNA conjugation systems is required for establishment and maintenance of the ER-associated replicative phagosome (Purcell and Shuman, 1998; Vogel et al., 1998). However, the substrates potentially being exported by this machinery remain elusive (Purcell and Shuman, 1998; Vogel et al., 1998). Even less well understood is the molecular basis for mitochondrial association observed with the inclusion (vacuole) membrane housing certain strains of Chlamydia psittaci (Matsumoto et al., 1991; Sinai and Joiner, 1997). Although host cytoplasm–exposed proteins of chlamydial origin have been identified in the inclusion membrane, no link to mitochondrial association has been made (Rockey et al., 1997; Bannantine et al., 1998). What is clear is that both mitochondrial association by C. psittaci (Matsumoto et al., 1991) and ER association by L. pneumophila (Swanson and Isberg, 1995) and B. abortus (Pizarro-Cerda et al., 1998) are important in the establishment of the replication-permissive compartment, and likely involved in nutrient acquisition (Sinai and Joiner, 1997).


The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane.

Sinai AP, Joiner KA - J. Cell Biol. (2001)

Mitochondrial association with the T. gondii PVM occurs at the time of invasion. (A) Ultrastructural features of a T. gondii parasitophorous vacuole 20 h after infection of CHO cells, containing four parasites (P) within the vacuole. The major secretory organelles including rhoptries (R), dense granules (DG), and micronemes (m) are indicated. The PV is delimited from the host cell by the PVM, which associates intimately with host mitochondria (M) and ER. (B) Host mitochondria associate with the PVM immediately after infection. PVs containing intracellular T. gondii, with associated mitochondria (arrowheads), were identified on the basis of their phase contrast characteristics (C, F, and I) and the distribution of the dense granule and PVM marker GRA3 (A, D, and G). Host mitochondria were identified using the specific dye MitoTracker (B, E, and H). Host mitochondria associated with the PVM follow the contour of the vacuole (arrowhead). Parasite infection was stopped at 1 (A–C), 5 (D–F), and 10 (G–I) min postinfection, and the extent of release of the dense granule marker GRA3 determined by immunofluorescence (A, D, and G). GRA3 labeling is confined to dense granules and is not detected in either the PV or PVM 1 min postinfection (A). At 5 min postinfection, patchy PVM staining (D, arrowhead) is detected in most vacuoles, which becomes circumferential by 10 min postinfection (G, arrowheads), indicating dense granule exocytosis. In contrast, MitoTracker-labeled PVM-associated mitochondria are readily apparent, surrounding the vacuole at all time points (B, E, and G, arrowhead). Detection of PVM-associated mitochondria (B) before dense granule exocytosis (A) indicates dense granule products are not required. Bars, 5 μm.
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Related In: Results  -  Collection

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fig1: Mitochondrial association with the T. gondii PVM occurs at the time of invasion. (A) Ultrastructural features of a T. gondii parasitophorous vacuole 20 h after infection of CHO cells, containing four parasites (P) within the vacuole. The major secretory organelles including rhoptries (R), dense granules (DG), and micronemes (m) are indicated. The PV is delimited from the host cell by the PVM, which associates intimately with host mitochondria (M) and ER. (B) Host mitochondria associate with the PVM immediately after infection. PVs containing intracellular T. gondii, with associated mitochondria (arrowheads), were identified on the basis of their phase contrast characteristics (C, F, and I) and the distribution of the dense granule and PVM marker GRA3 (A, D, and G). Host mitochondria were identified using the specific dye MitoTracker (B, E, and H). Host mitochondria associated with the PVM follow the contour of the vacuole (arrowhead). Parasite infection was stopped at 1 (A–C), 5 (D–F), and 10 (G–I) min postinfection, and the extent of release of the dense granule marker GRA3 determined by immunofluorescence (A, D, and G). GRA3 labeling is confined to dense granules and is not detected in either the PV or PVM 1 min postinfection (A). At 5 min postinfection, patchy PVM staining (D, arrowhead) is detected in most vacuoles, which becomes circumferential by 10 min postinfection (G, arrowheads), indicating dense granule exocytosis. In contrast, MitoTracker-labeled PVM-associated mitochondria are readily apparent, surrounding the vacuole at all time points (B, E, and G, arrowhead). Detection of PVM-associated mitochondria (B) before dense granule exocytosis (A) indicates dense granule products are not required. Bars, 5 μm.
Mentions: The T. gondii PVM exhibits a remarkable association with host mitochondria and ER (DeMelo et al., 1992; Sinai et al., 1997) (see Fig. 1 A). We have previously termed this phenomenon PVM–organelle association (Sinai et al., 1997). Association of host organelles with the vacuolar membranes surrounding intracellular pathogens is a feature restricted to organisms that either never enter the endocytic cascade, or that exit it soon after entry (Sinai and Joiner, 1997). Both Legionella pneumophila (Swanson and Isberg, 1995) and Brucella abortus (Pizarro-Cerda et al., 1998) replicate in a compartment associated with the ER. In L. pneumophila–infected cells, a bacterially encoded machinery with homology to bacterial DNA conjugation systems is required for establishment and maintenance of the ER-associated replicative phagosome (Purcell and Shuman, 1998; Vogel et al., 1998). However, the substrates potentially being exported by this machinery remain elusive (Purcell and Shuman, 1998; Vogel et al., 1998). Even less well understood is the molecular basis for mitochondrial association observed with the inclusion (vacuole) membrane housing certain strains of Chlamydia psittaci (Matsumoto et al., 1991; Sinai and Joiner, 1997). Although host cytoplasm–exposed proteins of chlamydial origin have been identified in the inclusion membrane, no link to mitochondrial association has been made (Rockey et al., 1997; Bannantine et al., 1998). What is clear is that both mitochondrial association by C. psittaci (Matsumoto et al., 1991) and ER association by L. pneumophila (Swanson and Isberg, 1995) and B. abortus (Pizarro-Cerda et al., 1998) are important in the establishment of the replication-permissive compartment, and likely involved in nutrient acquisition (Sinai and Joiner, 1997).

Bottom Line: Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle.Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER.These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases Section, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. sinai@pop.uky.edu

ABSTRACT
Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.

Show MeSH
Related in: MedlinePlus