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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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Identification of the TgRON4-binding region in TUBB2C.(a), (b) Upper: Schematic representation of TUBB2C and its N- and C-truncated mutants. Lower: The indicated HA-tagged TUBB2C proteins were coexpressed with 3xFLAG-tagged TgRON4B. The cell lysates were immunoprecipitated with an anti-HA antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. Owing to low expression, transient transfection of TUBB2C FR3 was scaled up (see Methods).
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f3: Identification of the TgRON4-binding region in TUBB2C.(a), (b) Upper: Schematic representation of TUBB2C and its N- and C-truncated mutants. Lower: The indicated HA-tagged TUBB2C proteins were coexpressed with 3xFLAG-tagged TgRON4B. The cell lysates were immunoprecipitated with an anti-HA antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. Owing to low expression, transient transfection of TUBB2C FR3 was scaled up (see Methods).

Mentions: To identify the TgRON4-binding region of host cellular β-tubulin, we generated expression vectors encoding various fragments of HA-tagged TUBB2C (Fig. 3a and 3b, upper). We used the coding sequence of TUBB2C to prepare the deletion constructs, because of the slightly higher transient expression level of TUBB2C (data not shown). HA-tagged TUBB2C mutants were coexpressed with 3xFLAG-tagged TgRON4B in Vero cells and immunoprecipitated with an anti-HA antibody. All N-terminal truncated deletions of TUBB2C did not affect the binding to TgRON4B (Fig. 3a, 17–20). To determine whether the C-terminal region of TUBB2C is important for the binding to TgRON4B, we generated a C-terminal truncated mutant of HA-tagged TUBB2C. The TUBB2C mutant, consisting of amino acids 1–334, did not coprecipitate with 3xFLAG-tagged TgRON4B (Fig. 3b, lane 12), suggesting that the TgRON4-binding region is contained within amino acids 335–445.


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)

Identification of the TgRON4-binding region in TUBB2C.(a), (b) Upper: Schematic representation of TUBB2C and its N- and C-truncated mutants. Lower: The indicated HA-tagged TUBB2C proteins were coexpressed with 3xFLAG-tagged TgRON4B. The cell lysates were immunoprecipitated with an anti-HA antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. Owing to low expression, transient transfection of TUBB2C FR3 was scaled up (see Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824165&req=5

f3: Identification of the TgRON4-binding region in TUBB2C.(a), (b) Upper: Schematic representation of TUBB2C and its N- and C-truncated mutants. Lower: The indicated HA-tagged TUBB2C proteins were coexpressed with 3xFLAG-tagged TgRON4B. The cell lysates were immunoprecipitated with an anti-HA antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. Owing to low expression, transient transfection of TUBB2C FR3 was scaled up (see Methods).
Mentions: To identify the TgRON4-binding region of host cellular β-tubulin, we generated expression vectors encoding various fragments of HA-tagged TUBB2C (Fig. 3a and 3b, upper). We used the coding sequence of TUBB2C to prepare the deletion constructs, because of the slightly higher transient expression level of TUBB2C (data not shown). HA-tagged TUBB2C mutants were coexpressed with 3xFLAG-tagged TgRON4B in Vero cells and immunoprecipitated with an anti-HA antibody. All N-terminal truncated deletions of TUBB2C did not affect the binding to TgRON4B (Fig. 3a, 17–20). To determine whether the C-terminal region of TUBB2C is important for the binding to TgRON4B, we generated a C-terminal truncated mutant of HA-tagged TUBB2C. The TUBB2C mutant, consisting of amino acids 1–334, did not coprecipitate with 3xFLAG-tagged TgRON4B (Fig. 3b, lane 12), suggesting that the TgRON4-binding region is contained within amino acids 335–445.

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