Limits...
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.

Show MeSH
ROP2hc localizes to both mitochondria and ER upon transient expression in CHO cells. Expression of ROP2hc results in the protein targeting to mitochondrial as well as nonmitochondrial locations. Colocalization of ROP2hc (A and E) with mitochondria (visualized using an anti-COXII antibody [B] or MitoTracker [F]) is detected as a yellow signal in the merged image (C, D, and G, yellow arrowhead). ROP2hc targeting to nonmitochondrial sites is detected as either a red (A and D, red arrowhead) or green (D and G, green arrowhead) in the merged image (D and G). Nonmitochondrial targeting of ROP2hc was particularly apparent with high levels of expression that resulted in the clumping of the ROP2hc signal (A, compare top and bottom cells). The possibility that the nonmitochondrial localization of ROP2hc corresponded to ER was examined using T34A7 (H) and anticalnexin (I) antibodies. The merged image reveals perinuclear colocalization (J, yellow signal in the merged image) with distinct non-ER labeling (J, red signal in the merged image). This signal likely represents mitochondria. Note the clumping of the ROP2hc signal in (H). Bars, 15 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196872&req=5

fig5: ROP2hc localizes to both mitochondria and ER upon transient expression in CHO cells. Expression of ROP2hc results in the protein targeting to mitochondrial as well as nonmitochondrial locations. Colocalization of ROP2hc (A and E) with mitochondria (visualized using an anti-COXII antibody [B] or MitoTracker [F]) is detected as a yellow signal in the merged image (C, D, and G, yellow arrowhead). ROP2hc targeting to nonmitochondrial sites is detected as either a red (A and D, red arrowhead) or green (D and G, green arrowhead) in the merged image (D and G). Nonmitochondrial targeting of ROP2hc was particularly apparent with high levels of expression that resulted in the clumping of the ROP2hc signal (A, compare top and bottom cells). The possibility that the nonmitochondrial localization of ROP2hc corresponded to ER was examined using T34A7 (H) and anticalnexin (I) antibodies. The merged image reveals perinuclear colocalization (J, yellow signal in the merged image) with distinct non-ER labeling (J, red signal in the merged image). This signal likely represents mitochondria. Note the clumping of the ROP2hc signal in (H). Bars, 15 μm.

Mentions: Next, we tested the targeting of ROP2hc in vivo. The level of expression of ROP2hc and ROP2hc80 (unpublished data) had a significant effect on the localization of the protein. Relatively low levels of ROP2hc resulted in localization to rod-shaped and punctate structures reminiscent of mitochondria (Fig. 5, A and C , yellow arrowheads). These structures were confirmed to be mitochondria based on colocalization with the mitochondrial marker cytochrome c oxidase subunit III (COXIII) (Fig. 5, B and C, yellow arrowheads). Colocalization with mitochondria was confirmed with antibody to cytochrome c oxidase subunit 1 (COXI) (unpublished data), hamster MOM (unpublished data), and the mitochondrion-specific, membrane potential–sensitive dye MitoTracker (Molecular Probes) (Fig. 5, E–G).


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

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

ROP2hc localizes to both mitochondria and ER upon transient expression in CHO cells. Expression of ROP2hc results in the protein targeting to mitochondrial as well as nonmitochondrial locations. Colocalization of ROP2hc (A and E) with mitochondria (visualized using an anti-COXII antibody [B] or MitoTracker [F]) is detected as a yellow signal in the merged image (C, D, and G, yellow arrowhead). ROP2hc targeting to nonmitochondrial sites is detected as either a red (A and D, red arrowhead) or green (D and G, green arrowhead) in the merged image (D and G). Nonmitochondrial targeting of ROP2hc was particularly apparent with high levels of expression that resulted in the clumping of the ROP2hc signal (A, compare top and bottom cells). The possibility that the nonmitochondrial localization of ROP2hc corresponded to ER was examined using T34A7 (H) and anticalnexin (I) antibodies. The merged image reveals perinuclear colocalization (J, yellow signal in the merged image) with distinct non-ER labeling (J, red signal in the merged image). This signal likely represents mitochondria. Note the clumping of the ROP2hc signal in (H). Bars, 15 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196872&req=5

fig5: ROP2hc localizes to both mitochondria and ER upon transient expression in CHO cells. Expression of ROP2hc results in the protein targeting to mitochondrial as well as nonmitochondrial locations. Colocalization of ROP2hc (A and E) with mitochondria (visualized using an anti-COXII antibody [B] or MitoTracker [F]) is detected as a yellow signal in the merged image (C, D, and G, yellow arrowhead). ROP2hc targeting to nonmitochondrial sites is detected as either a red (A and D, red arrowhead) or green (D and G, green arrowhead) in the merged image (D and G). Nonmitochondrial targeting of ROP2hc was particularly apparent with high levels of expression that resulted in the clumping of the ROP2hc signal (A, compare top and bottom cells). The possibility that the nonmitochondrial localization of ROP2hc corresponded to ER was examined using T34A7 (H) and anticalnexin (I) antibodies. The merged image reveals perinuclear colocalization (J, yellow signal in the merged image) with distinct non-ER labeling (J, red signal in the merged image). This signal likely represents mitochondria. Note the clumping of the ROP2hc signal in (H). Bars, 15 μm.
Mentions: Next, we tested the targeting of ROP2hc in vivo. The level of expression of ROP2hc and ROP2hc80 (unpublished data) had a significant effect on the localization of the protein. Relatively low levels of ROP2hc resulted in localization to rod-shaped and punctate structures reminiscent of mitochondria (Fig. 5, A and C , yellow arrowheads). These structures were confirmed to be mitochondria based on colocalization with the mitochondrial marker cytochrome c oxidase subunit III (COXIII) (Fig. 5, B and C, yellow arrowheads). Colocalization with mitochondria was confirmed with antibody to cytochrome c oxidase subunit 1 (COXI) (unpublished data), hamster MOM (unpublished data), and the mitochondrion-specific, membrane potential–sensitive dye MitoTracker (Molecular Probes) (Fig. 5, E–G).

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