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Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular β-tubulin.

Takemae H, Sugi T, Kobayashi K, Gong H, Ishiwa A, Recuenco FC, Murakoshi F, Iwanaga T, Inomata A, Horimoto T, Akashi H, Kato K - Sci Rep (2013)

Bottom Line: Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion.Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step.Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

View Article: PubMed Central - PubMed

Affiliation: 1] National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan [2] Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

ABSTRACT
Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion. TgRON4 is exposed on the cytosolic side of the host cell during invasion, but its molecular interactions remain unclear. Here, we identified host cellular β-tubulin as a binding partner of TgRON4, but not Plasmodium RON4. Coimmunoprecipitation studies in mammalian cells demonstrated that the C-terminal 15-kDa region of β-tubulin was sufficient for binding to TgRON4, and that a 17-kDa region in the proximal C-terminus of TgRON4 was required for binding to the C-terminal region of β-tubulin. Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step. Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

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Interaction between the C-terminal region of TUBB2C and TgRON4 in T. gondii-infected CHO cells.(a) Upper: Schematic representation of HA-tagged TUBB2C F3 and F2, followed by the IRES2-Venus sequence. Immunoblotting with an anti-HA antibody of the total lysates from CHO cells expressing the lentivirus-mediated HA-tagged TUBB2C F3 (lane 2) and F2 (lane 3). (b) T. gondii RH tachyzoites were allowed to synchronously invade monolayers of CHO cells expressing the indicated HA-TUBB2C for 2 min. The total lysates of infected CHO monolayers were then immunoprecipitated with an anti-HA antibody. Immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-TgRON4 polyclonal antibodies. At the same time, parasite lysates were immunoblotted by using an anti-TgRON4 polyclonal antibody. The arrowhead indicates a 110-kDa band that corresponds to the mature form of TgRON4.
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f5: Interaction between the C-terminal region of TUBB2C and TgRON4 in T. gondii-infected CHO cells.(a) Upper: Schematic representation of HA-tagged TUBB2C F3 and F2, followed by the IRES2-Venus sequence. Immunoblotting with an anti-HA antibody of the total lysates from CHO cells expressing the lentivirus-mediated HA-tagged TUBB2C F3 (lane 2) and F2 (lane 3). (b) T. gondii RH tachyzoites were allowed to synchronously invade monolayers of CHO cells expressing the indicated HA-TUBB2C for 2 min. The total lysates of infected CHO monolayers were then immunoprecipitated with an anti-HA antibody. Immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-TgRON4 polyclonal antibodies. At the same time, parasite lysates were immunoblotted by using an anti-TgRON4 polyclonal antibody. The arrowhead indicates a 110-kDa band that corresponds to the mature form of TgRON4.

Mentions: To better assess the binding of TgRON4 to TUBB2C, we generated stable CHO cell lines expressing HA-tagged TUBB2C by use of lentiviral vector-mediated gene transfer. CHO cells were infected with lentivirus containing HA-tagged TUBB2C consisting of amino acids 315–445 (F3) or 203–334 (F2) followed by the IRES2-Venus sequence. Venus-positive cells that formed colonies were picked up from the diluted cell culture and propagated. The expression of HA-tagged TUBB2C proteins was confirmed by Western blotting using an anti-HA antibody (Fig. 5a). Stable Vero cell lines expressing HA-tagged TUBB2C could not be established due to the low expression of the transgene (data not shown). As a positive control, full-length TUBB2C with an HA tag was transiently expressed in CHO cells. Monolayers of CHO cells expressing HA-tagged TUBB2C were infected with T. gondii RH parasites using a potassium buffer shift30. Parasites were allowed to invade for 2 min and then infected CHO monolayers were lysed. The total lysates were immunoprecipitated with an anti-HA antibody. An anti-TgRON4 polyclonal antibody was produced by immunization of mice with TgRON4 recombinant protein fused with the 3xFLAG tag at the N terminus and an octahistidine tag at the C terminus. Immunoblotting with the anti-TgRON4 polyclonal antibody detected a major band with a molecular mass of approximately 120 kDa in the parasite lysates (Fig. 5b, lane 4). Pulse-chase metabolic labeling and immunoprecipitation with anti-TgRON4 mAb T5 4H1 previously showed that TgRON4 is expressed as a pro-protein of ~ 120 kDa that is cleaved to yield a mature protein of ~ 110 kDa under reducing condition13. In T. gondii–infected CHO cells expressing HA-tagged TUBB2C-Full, TgRON4 coimmunoprecipitated with TUBB2C and was observed at approximately 110 kDa, corresponding to the mature form of TgRON4 (Fig. 5b, lane 5). When stable CHO cell lines expressing HA-tagged truncated TUBB2C were infected with T. gondii RH parasites by means of synchronous invasion, TUBB2C F3 coimmunoprecipitated with TgRON4, but TUBB2C F2 did not (Fig. 5b, lanes 6, 7). These results indicate that the 15-kDa region at the C-terminus of TUBB2C binds to the mature form of TgRON4.


Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular β-tubulin.

Takemae H, Sugi T, Kobayashi K, Gong H, Ishiwa A, Recuenco FC, Murakoshi F, Iwanaga T, Inomata A, Horimoto T, Akashi H, Kato K - Sci Rep (2013)

Interaction between the C-terminal region of TUBB2C and TgRON4 in T. gondii-infected CHO cells.(a) Upper: Schematic representation of HA-tagged TUBB2C F3 and F2, followed by the IRES2-Venus sequence. Immunoblotting with an anti-HA antibody of the total lysates from CHO cells expressing the lentivirus-mediated HA-tagged TUBB2C F3 (lane 2) and F2 (lane 3). (b) T. gondii RH tachyzoites were allowed to synchronously invade monolayers of CHO cells expressing the indicated HA-TUBB2C for 2 min. The total lysates of infected CHO monolayers were then immunoprecipitated with an anti-HA antibody. Immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-TgRON4 polyclonal antibodies. At the same time, parasite lysates were immunoblotted by using an anti-TgRON4 polyclonal antibody. The arrowhead indicates a 110-kDa band that corresponds to the mature form of TgRON4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: Interaction between the C-terminal region of TUBB2C and TgRON4 in T. gondii-infected CHO cells.(a) Upper: Schematic representation of HA-tagged TUBB2C F3 and F2, followed by the IRES2-Venus sequence. Immunoblotting with an anti-HA antibody of the total lysates from CHO cells expressing the lentivirus-mediated HA-tagged TUBB2C F3 (lane 2) and F2 (lane 3). (b) T. gondii RH tachyzoites were allowed to synchronously invade monolayers of CHO cells expressing the indicated HA-TUBB2C for 2 min. The total lysates of infected CHO monolayers were then immunoprecipitated with an anti-HA antibody. Immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-TgRON4 polyclonal antibodies. At the same time, parasite lysates were immunoblotted by using an anti-TgRON4 polyclonal antibody. The arrowhead indicates a 110-kDa band that corresponds to the mature form of TgRON4.
Mentions: To better assess the binding of TgRON4 to TUBB2C, we generated stable CHO cell lines expressing HA-tagged TUBB2C by use of lentiviral vector-mediated gene transfer. CHO cells were infected with lentivirus containing HA-tagged TUBB2C consisting of amino acids 315–445 (F3) or 203–334 (F2) followed by the IRES2-Venus sequence. Venus-positive cells that formed colonies were picked up from the diluted cell culture and propagated. The expression of HA-tagged TUBB2C proteins was confirmed by Western blotting using an anti-HA antibody (Fig. 5a). Stable Vero cell lines expressing HA-tagged TUBB2C could not be established due to the low expression of the transgene (data not shown). As a positive control, full-length TUBB2C with an HA tag was transiently expressed in CHO cells. Monolayers of CHO cells expressing HA-tagged TUBB2C were infected with T. gondii RH parasites using a potassium buffer shift30. Parasites were allowed to invade for 2 min and then infected CHO monolayers were lysed. The total lysates were immunoprecipitated with an anti-HA antibody. An anti-TgRON4 polyclonal antibody was produced by immunization of mice with TgRON4 recombinant protein fused with the 3xFLAG tag at the N terminus and an octahistidine tag at the C terminus. Immunoblotting with the anti-TgRON4 polyclonal antibody detected a major band with a molecular mass of approximately 120 kDa in the parasite lysates (Fig. 5b, lane 4). Pulse-chase metabolic labeling and immunoprecipitation with anti-TgRON4 mAb T5 4H1 previously showed that TgRON4 is expressed as a pro-protein of ~ 120 kDa that is cleaved to yield a mature protein of ~ 110 kDa under reducing condition13. In T. gondii–infected CHO cells expressing HA-tagged TUBB2C-Full, TgRON4 coimmunoprecipitated with TUBB2C and was observed at approximately 110 kDa, corresponding to the mature form of TgRON4 (Fig. 5b, lane 5). When stable CHO cell lines expressing HA-tagged truncated TUBB2C were infected with T. gondii RH parasites by means of synchronous invasion, TUBB2C F3 coimmunoprecipitated with TgRON4, but TUBB2C F2 did not (Fig. 5b, lanes 6, 7). These results indicate that the 15-kDa region at the C-terminus of TUBB2C binds to the mature form of TgRON4.

Bottom Line: Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion.Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step.Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

View Article: PubMed Central - PubMed

Affiliation: 1] National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan [2] Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

ABSTRACT
Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion. TgRON4 is exposed on the cytosolic side of the host cell during invasion, but its molecular interactions remain unclear. Here, we identified host cellular β-tubulin as a binding partner of TgRON4, but not Plasmodium RON4. Coimmunoprecipitation studies in mammalian cells demonstrated that the C-terminal 15-kDa region of β-tubulin was sufficient for binding to TgRON4, and that a 17-kDa region in the proximal C-terminus of TgRON4 was required for binding to the C-terminal region of β-tubulin. Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step. Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

Show MeSH
Related in: MedlinePlus