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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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Localization of Δ98–127ROP2hcGFP after transient transfection of CHO cells. After expression in CHO cells, Δ98–127ROP2hcGFP exhibited a predominantly juxta- or perinuclear staining pattern (A, D, and G) that radiated in a reticulated pattern (most evident in D and G). This pattern (A and D, green arrowhead) was largely distinct in its distribution from mitochondria (red arrowhead, visualized with anticytochrome c [B] and MitoTracker [E]). This results in a merged image with clearly defined areas of Δ98–127ROP2hcGFP localization (C and F, green arrowheads) and those of mitochondria (C and F, red arrowheads). Some coincidental colocalization of the signals was detected in areas where both ROP2hc and mitochondria were abundant (F, yellow arrowhead) and are likely due to the limits of resolution of the confocal microscope. The possibility that Δ98–127ROP2hcGFP (G, detected using anti-GFP) was localizing to ER was examined using an anti-KDEL antibody (H). The merged image revealed near perfect concordance of the two signals (I, yellow, indicating Δ98–127ROP2hcGFP targets the ER). Bars, 15 μm.
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fig6: Localization of Δ98–127ROP2hcGFP after transient transfection of CHO cells. After expression in CHO cells, Δ98–127ROP2hcGFP exhibited a predominantly juxta- or perinuclear staining pattern (A, D, and G) that radiated in a reticulated pattern (most evident in D and G). This pattern (A and D, green arrowhead) was largely distinct in its distribution from mitochondria (red arrowhead, visualized with anticytochrome c [B] and MitoTracker [E]). This results in a merged image with clearly defined areas of Δ98–127ROP2hcGFP localization (C and F, green arrowheads) and those of mitochondria (C and F, red arrowheads). Some coincidental colocalization of the signals was detected in areas where both ROP2hc and mitochondria were abundant (F, yellow arrowhead) and are likely due to the limits of resolution of the confocal microscope. The possibility that Δ98–127ROP2hcGFP (G, detected using anti-GFP) was localizing to ER was examined using an anti-KDEL antibody (H). The merged image revealed near perfect concordance of the two signals (I, yellow, indicating Δ98–127ROP2hcGFP targets the ER). Bars, 15 μm.

Mentions: Next, we examined whether deletion of the NH2-terminal 30 aa required for in vitro mitochondrial import played a role in ROP2hc localization in vivo. After expression of a Δ98–127ROP2hcGFP chimera, a distinct pattern of localization was observed. Unlike ROP2hc, Δ98–127ROP2hcGFP (Fig. 6, A, C, D, and F , green arrowheads) did not target significantly to mitochondria, as detected with either MitoTracker (Fig. 6, B and C, red arrowhead) or anti-COXI antibody (Fig. 6, E and F, red arrowheads). Some colocalizing was observed (Fig. 6, D–F, bottom cell, yellow arrowhead) and is likely due to the limitations in the resolving power of confocal microscopy. Such patches of apparent colocalization were only found where a high concentration of both signals was present (Fig. 6, D and E). Notably, expression of Δ98–127ROP2hcGFP did not lead to the clumping and other changes in organelle organization (Fig. 6, B, C, E, and F) observed with ROP2hc (Fig. 5, B, C, E, and F).


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

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

Localization of Δ98–127ROP2hcGFP after transient transfection of CHO cells. After expression in CHO cells, Δ98–127ROP2hcGFP exhibited a predominantly juxta- or perinuclear staining pattern (A, D, and G) that radiated in a reticulated pattern (most evident in D and G). This pattern (A and D, green arrowhead) was largely distinct in its distribution from mitochondria (red arrowhead, visualized with anticytochrome c [B] and MitoTracker [E]). This results in a merged image with clearly defined areas of Δ98–127ROP2hcGFP localization (C and F, green arrowheads) and those of mitochondria (C and F, red arrowheads). Some coincidental colocalization of the signals was detected in areas where both ROP2hc and mitochondria were abundant (F, yellow arrowhead) and are likely due to the limits of resolution of the confocal microscope. The possibility that Δ98–127ROP2hcGFP (G, detected using anti-GFP) was localizing to ER was examined using an anti-KDEL antibody (H). The merged image revealed near perfect concordance of the two signals (I, yellow, indicating Δ98–127ROP2hcGFP targets the ER). Bars, 15 μm.
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Related In: Results  -  Collection

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fig6: Localization of Δ98–127ROP2hcGFP after transient transfection of CHO cells. After expression in CHO cells, Δ98–127ROP2hcGFP exhibited a predominantly juxta- or perinuclear staining pattern (A, D, and G) that radiated in a reticulated pattern (most evident in D and G). This pattern (A and D, green arrowhead) was largely distinct in its distribution from mitochondria (red arrowhead, visualized with anticytochrome c [B] and MitoTracker [E]). This results in a merged image with clearly defined areas of Δ98–127ROP2hcGFP localization (C and F, green arrowheads) and those of mitochondria (C and F, red arrowheads). Some coincidental colocalization of the signals was detected in areas where both ROP2hc and mitochondria were abundant (F, yellow arrowhead) and are likely due to the limits of resolution of the confocal microscope. The possibility that Δ98–127ROP2hcGFP (G, detected using anti-GFP) was localizing to ER was examined using an anti-KDEL antibody (H). The merged image revealed near perfect concordance of the two signals (I, yellow, indicating Δ98–127ROP2hcGFP targets the ER). Bars, 15 μm.
Mentions: Next, we examined whether deletion of the NH2-terminal 30 aa required for in vitro mitochondrial import played a role in ROP2hc localization in vivo. After expression of a Δ98–127ROP2hcGFP chimera, a distinct pattern of localization was observed. Unlike ROP2hc, Δ98–127ROP2hcGFP (Fig. 6, A, C, D, and F , green arrowheads) did not target significantly to mitochondria, as detected with either MitoTracker (Fig. 6, B and C, red arrowhead) or anti-COXI antibody (Fig. 6, E and F, red arrowheads). Some colocalizing was observed (Fig. 6, D–F, bottom cell, yellow arrowhead) and is likely due to the limitations in the resolving power of confocal microscopy. Such patches of apparent colocalization were only found where a high concentration of both signals was present (Fig. 6, D and E). Notably, expression of Δ98–127ROP2hcGFP did not lead to the clumping and other changes in organelle organization (Fig. 6, B, C, E, and F) observed with ROP2hc (Fig. 5, B, C, E, and F).

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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