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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 aa 98–127GFP after transient transfection into CHO cells. After expression in CHO cells, aa 98–127GFP localized primarily to rod-shaped and punctate structures (A, yellow arrowhead) that colocalized to a significant extent with mitochondria decorated with an antibody against COXI (B, yellow arrowhead). The merged image indicates a high degree of colocalization (C, yellow arrowhead) in the transfected cell. Increasing levels of expression result in the accumulation of aa 98–127GFP on distended and clumped structures (D and F, yellow arrowhead) revealed to be mitochondria using MitoTracker (E and F, yellow arrowhead) and COXI (unpublished data). In addition, regions of aa 98–127GFP not colocalizing with mitochondria (D and F, green arrowhead) as well as mitochondria not colocalizing with aa 98-127GFP (E and F, red arrowhead) are apparent. Localization of aa 98–127GFP (G) to the ER was examined using anti-KDEL (H). Limited colocalization was only observed in areas with a high concentration of both signals (G–I, yellow arrowhead). However, most of the signal failed to colocalize with aa 98–127GFP (G–I, green arrowhead) and ER (G–I, red arrowhead) being found in distinct regions of the cell. Bars, 15 μm.
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fig7: Localization of aa 98–127GFP after transient transfection into CHO cells. After expression in CHO cells, aa 98–127GFP localized primarily to rod-shaped and punctate structures (A, yellow arrowhead) that colocalized to a significant extent with mitochondria decorated with an antibody against COXI (B, yellow arrowhead). The merged image indicates a high degree of colocalization (C, yellow arrowhead) in the transfected cell. Increasing levels of expression result in the accumulation of aa 98–127GFP on distended and clumped structures (D and F, yellow arrowhead) revealed to be mitochondria using MitoTracker (E and F, yellow arrowhead) and COXI (unpublished data). In addition, regions of aa 98–127GFP not colocalizing with mitochondria (D and F, green arrowhead) as well as mitochondria not colocalizing with aa 98-127GFP (E and F, red arrowhead) are apparent. Localization of aa 98–127GFP (G) to the ER was examined using anti-KDEL (H). Limited colocalization was only observed in areas with a high concentration of both signals (G–I, yellow arrowhead). However, most of the signal failed to colocalize with aa 98–127GFP (G–I, green arrowhead) and ER (G–I, red arrowhead) being found in distinct regions of the cell. Bars, 15 μm.

Mentions: The contribution of the 30-aa putative mitochondrial targeting signal (aa 98–127) to the in vivo targeting was examined directly using the GFP chimera aa 98–127GFP chimera. With relatively low levels of expression, the aa 98–127GFP construct (Fig. 7 A, yellow arrowhead) localized primarily to mitochondria visualized using an anti-COXI antibody (Fig. 7 B, yellow arrowhead). This is best visualized as yellow staining in the merged image (Fig. 7 C, yellow arrowhead). Like ROP2hc (Fig. 5, A, D, and G), increased accumulation of aa 98–127GFP exhibited, in addition to colocalization with mitochondria (Fig. 7, D–F, yellow arrowhead), evidence of clumping (Fig. 7 D) and localization to nonmitochondrial sites (Fig. 7, D–F, green arrowhead). In addition, a subpopulation of mitochondria did not label with aa 98–127GFP (Fig. 7, D–F, red arrowhead).


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

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

Localization of aa 98–127GFP after transient transfection into CHO cells. After expression in CHO cells, aa 98–127GFP localized primarily to rod-shaped and punctate structures (A, yellow arrowhead) that colocalized to a significant extent with mitochondria decorated with an antibody against COXI (B, yellow arrowhead). The merged image indicates a high degree of colocalization (C, yellow arrowhead) in the transfected cell. Increasing levels of expression result in the accumulation of aa 98–127GFP on distended and clumped structures (D and F, yellow arrowhead) revealed to be mitochondria using MitoTracker (E and F, yellow arrowhead) and COXI (unpublished data). In addition, regions of aa 98–127GFP not colocalizing with mitochondria (D and F, green arrowhead) as well as mitochondria not colocalizing with aa 98-127GFP (E and F, red arrowhead) are apparent. Localization of aa 98–127GFP (G) to the ER was examined using anti-KDEL (H). Limited colocalization was only observed in areas with a high concentration of both signals (G–I, yellow arrowhead). However, most of the signal failed to colocalize with aa 98–127GFP (G–I, green arrowhead) and ER (G–I, red arrowhead) being found in distinct regions of the cell. Bars, 15 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196872&req=5

fig7: Localization of aa 98–127GFP after transient transfection into CHO cells. After expression in CHO cells, aa 98–127GFP localized primarily to rod-shaped and punctate structures (A, yellow arrowhead) that colocalized to a significant extent with mitochondria decorated with an antibody against COXI (B, yellow arrowhead). The merged image indicates a high degree of colocalization (C, yellow arrowhead) in the transfected cell. Increasing levels of expression result in the accumulation of aa 98–127GFP on distended and clumped structures (D and F, yellow arrowhead) revealed to be mitochondria using MitoTracker (E and F, yellow arrowhead) and COXI (unpublished data). In addition, regions of aa 98–127GFP not colocalizing with mitochondria (D and F, green arrowhead) as well as mitochondria not colocalizing with aa 98-127GFP (E and F, red arrowhead) are apparent. Localization of aa 98–127GFP (G) to the ER was examined using anti-KDEL (H). Limited colocalization was only observed in areas with a high concentration of both signals (G–I, yellow arrowhead). However, most of the signal failed to colocalize with aa 98–127GFP (G–I, green arrowhead) and ER (G–I, red arrowhead) being found in distinct regions of the cell. Bars, 15 μm.
Mentions: The contribution of the 30-aa putative mitochondrial targeting signal (aa 98–127) to the in vivo targeting was examined directly using the GFP chimera aa 98–127GFP chimera. With relatively low levels of expression, the aa 98–127GFP construct (Fig. 7 A, yellow arrowhead) localized primarily to mitochondria visualized using an anti-COXI antibody (Fig. 7 B, yellow arrowhead). This is best visualized as yellow staining in the merged image (Fig. 7 C, yellow arrowhead). Like ROP2hc (Fig. 5, A, D, and G), increased accumulation of aa 98–127GFP exhibited, in addition to colocalization with mitochondria (Fig. 7, D–F, yellow arrowhead), evidence of clumping (Fig. 7 D) and localization to nonmitochondrial sites (Fig. 7, D–F, green arrowhead). In addition, a subpopulation of mitochondria did not label with aa 98–127GFP (Fig. 7, D–F, red arrowhead).

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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