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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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Determination of the topology of ROP2 in the PVM. (A) The processing of ROP2 in T. gondii. The gene encoding ROP2 predicts a 64-kD protein including an NH2-terminal signal sequence (SS) (Beckers et al., 1994). Signal sequence cleavage in the parasite ER is followed by an additional processing event (Sadak et al., 1988; Leriche and Dubremetz, 1991). Cleavage at aa 98 predicts a 53.5-kD protein, somewhat smaller than the observed 55 kD for the mature protein (Leriche and Dubremetz, 1991; Sadak et al., 1988), while cleavage at aa 80 would predict a 57.5-kD protein. The protein can be divided into the NH2-terminal domain (aa 98–465) and the transmembrane domain and COOH-terminus (aa 46–561). A chimera of BAP and the COOH-terminal domain of ROP2hc (aa 466–561) was constructed (BAP-ROP2TM/CT). (B) Immunoprecipitation of 35S-Met–labeled in vitro–synthesized NH2-terminal domain (aa 98–465) and BAP-ROP2TM/CT with an antiserum against the R/DG fraction of the parasite. R/DG immunoprecipitates the NH2-terminal domain but not BAP-ROP2TM/CT, indicating that the NH2-terminal domain of ROP2 is exposed to the host cytoplasm.
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fig2: Determination of the topology of ROP2 in the PVM. (A) The processing of ROP2 in T. gondii. The gene encoding ROP2 predicts a 64-kD protein including an NH2-terminal signal sequence (SS) (Beckers et al., 1994). Signal sequence cleavage in the parasite ER is followed by an additional processing event (Sadak et al., 1988; Leriche and Dubremetz, 1991). Cleavage at aa 98 predicts a 53.5-kD protein, somewhat smaller than the observed 55 kD for the mature protein (Leriche and Dubremetz, 1991; Sadak et al., 1988), while cleavage at aa 80 would predict a 57.5-kD protein. The protein can be divided into the NH2-terminal domain (aa 98–465) and the transmembrane domain and COOH-terminus (aa 46–561). A chimera of BAP and the COOH-terminal domain of ROP2hc (aa 466–561) was constructed (BAP-ROP2TM/CT). (B) Immunoprecipitation of 35S-Met–labeled in vitro–synthesized NH2-terminal domain (aa 98–465) and BAP-ROP2TM/CT with an antiserum against the R/DG fraction of the parasite. R/DG immunoprecipitates the NH2-terminal domain but not BAP-ROP2TM/CT, indicating that the NH2-terminal domain of ROP2 is exposed to the host cytoplasm.

Mentions: The ROP2 protein is processed within the secretory pathway of the parasite en route to the rhoptry (Fig. 2 A) (Sadak et al., 1988; Leriche and Dubremetz, 1991). Although the precise processing site is unclear, NH2-terminal sequencing of an ROP2 fragment indicates that aa 98 is at or close to the processing site (Dubremetz, J.-F., personal communication) (Fig. 2 A). Therefore, we divided ROP2 into an NH2-terminal domain (aa 98–465) and a COOH-terminal domain (aa 466–561) around its predicted transmembrane region (Fig. 2 A). These domains were synthesized in vitro as 35S-Met–labeled substrates, and the ability of the R/DG antiserum to immunoprecipitate them was determined. The R/DG antiserum selectively immunoprecipitated the NH2-terminal domain of mature ROP2 (aa 98–465; Fig. 2 B), but not a chimera of bacterial alkaline phosphatase (BAP) with the ROP2 transmembrane domain and COOH terminus (aa 466–561) (Fig. 2 B). This observation indicates that the NH2-terminal domain of PVM-localized ROP2 is exposed to the host cytoplasm (RO2hc; aa 98–465).


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

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

Determination of the topology of ROP2 in the PVM. (A) The processing of ROP2 in T. gondii. The gene encoding ROP2 predicts a 64-kD protein including an NH2-terminal signal sequence (SS) (Beckers et al., 1994). Signal sequence cleavage in the parasite ER is followed by an additional processing event (Sadak et al., 1988; Leriche and Dubremetz, 1991). Cleavage at aa 98 predicts a 53.5-kD protein, somewhat smaller than the observed 55 kD for the mature protein (Leriche and Dubremetz, 1991; Sadak et al., 1988), while cleavage at aa 80 would predict a 57.5-kD protein. The protein can be divided into the NH2-terminal domain (aa 98–465) and the transmembrane domain and COOH-terminus (aa 46–561). A chimera of BAP and the COOH-terminal domain of ROP2hc (aa 466–561) was constructed (BAP-ROP2TM/CT). (B) Immunoprecipitation of 35S-Met–labeled in vitro–synthesized NH2-terminal domain (aa 98–465) and BAP-ROP2TM/CT with an antiserum against the R/DG fraction of the parasite. R/DG immunoprecipitates the NH2-terminal domain but not BAP-ROP2TM/CT, indicating that the NH2-terminal domain of ROP2 is exposed to the host cytoplasm.
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Related In: Results  -  Collection

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fig2: Determination of the topology of ROP2 in the PVM. (A) The processing of ROP2 in T. gondii. The gene encoding ROP2 predicts a 64-kD protein including an NH2-terminal signal sequence (SS) (Beckers et al., 1994). Signal sequence cleavage in the parasite ER is followed by an additional processing event (Sadak et al., 1988; Leriche and Dubremetz, 1991). Cleavage at aa 98 predicts a 53.5-kD protein, somewhat smaller than the observed 55 kD for the mature protein (Leriche and Dubremetz, 1991; Sadak et al., 1988), while cleavage at aa 80 would predict a 57.5-kD protein. The protein can be divided into the NH2-terminal domain (aa 98–465) and the transmembrane domain and COOH-terminus (aa 46–561). A chimera of BAP and the COOH-terminal domain of ROP2hc (aa 466–561) was constructed (BAP-ROP2TM/CT). (B) Immunoprecipitation of 35S-Met–labeled in vitro–synthesized NH2-terminal domain (aa 98–465) and BAP-ROP2TM/CT with an antiserum against the R/DG fraction of the parasite. R/DG immunoprecipitates the NH2-terminal domain but not BAP-ROP2TM/CT, indicating that the NH2-terminal domain of ROP2 is exposed to the host cytoplasm.
Mentions: The ROP2 protein is processed within the secretory pathway of the parasite en route to the rhoptry (Fig. 2 A) (Sadak et al., 1988; Leriche and Dubremetz, 1991). Although the precise processing site is unclear, NH2-terminal sequencing of an ROP2 fragment indicates that aa 98 is at or close to the processing site (Dubremetz, J.-F., personal communication) (Fig. 2 A). Therefore, we divided ROP2 into an NH2-terminal domain (aa 98–465) and a COOH-terminal domain (aa 466–561) around its predicted transmembrane region (Fig. 2 A). These domains were synthesized in vitro as 35S-Met–labeled substrates, and the ability of the R/DG antiserum to immunoprecipitate them was determined. The R/DG antiserum selectively immunoprecipitated the NH2-terminal domain of mature ROP2 (aa 98–465; Fig. 2 B), but not a chimera of bacterial alkaline phosphatase (BAP) with the ROP2 transmembrane domain and COOH terminus (aa 466–561) (Fig. 2 B). This observation indicates that the NH2-terminal domain of PVM-localized ROP2 is exposed to the host cytoplasm (RO2hc; aa 98–465).

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