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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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The C-terminal half of TgRON4 binds to host β-tubulin.(a) Schematic representation of TgRON4 and its deletion mutants. (b), (c) Coimmunoprecipitation of β-tubulin with deletion mutants of 3xFLAG-tagged TgRON4. 293 T (b) and Vero (c) cells were transiently transfected with the indicated TgRON4 expression plasmids. Membrane fractions were immunoprecipitated with an anti-β-tubulin antibody. The membrane fractions (3.3% input) and immunoprecipitates (IP) were analyzed by Western blotting with anti-FLAG and anti-β-tubulin antibodies.
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f2: The C-terminal half of TgRON4 binds to host β-tubulin.(a) Schematic representation of TgRON4 and its deletion mutants. (b), (c) Coimmunoprecipitation of β-tubulin with deletion mutants of 3xFLAG-tagged TgRON4. 293 T (b) and Vero (c) cells were transiently transfected with the indicated TgRON4 expression plasmids. Membrane fractions were immunoprecipitated with an anti-β-tubulin antibody. The membrane fractions (3.3% input) and immunoprecipitates (IP) were analyzed by Western blotting with anti-FLAG and anti-β-tubulin antibodies.

Mentions: TUBB5 and TUBB2C are ubiquitously distributed and represent the major isotypes among the seven β-tubulin isotypes in all tissues except for brain29. To validate the binding specificity between TgRON4 and host cellular β-tubulin, we conducted immunoprecipitation assays using mammalian cells expressing TgRON4. We prepared deletion constructs consisting of 3xFLAG-tagged full-length TgRON4 (TgRON4-Full) lacking its putative signal sequence, the N-terminal half of TgRON4 (TgRON4A), and the C-terminal half of TgRON4 (TgRON4B) (Fig. 2a). 293T and Vero cells were transiently transfected with a control or each of these constructs, and the membrane proteins prepared from collected cells were immunoprecipitated with an anti-β-tubulin antibody. Expression of 3xFLAG-tagged TgRON4 protein was observed in the hydrophobic fraction (Fig. 2b and 2c, lanes 2–4), and some smaller bands could also be observed, especially in Vero cells. TgRON4-Full and TgRON4B coimmunoprecipitated with β-tubulin in both 293 T and Vero cells, but RON4A did not (Fig. 2b and 2c, lames 6–8). The ratios of precipitated TgRON4-Full and TgRON4B to TgRON4 in total membrane fractions were 1.7% and 0.82% in 293 T cells, 1.0% and 0.56% in Vero cells, respectively (Fig. 2b and 2c, lames 2, 4, 6, 8). The ratio of precipitated TgRON4-Full is higher than that of TgRON4B. TgRON4A may partially contributed to fully binding to β-tubulin. These results demonstrate that the C-terminus of TgRON4 mainly binds to host cellular β-tubulin.


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)

The C-terminal half of TgRON4 binds to host β-tubulin.(a) Schematic representation of TgRON4 and its deletion mutants. (b), (c) Coimmunoprecipitation of β-tubulin with deletion mutants of 3xFLAG-tagged TgRON4. 293 T (b) and Vero (c) cells were transiently transfected with the indicated TgRON4 expression plasmids. Membrane fractions were immunoprecipitated with an anti-β-tubulin antibody. The membrane fractions (3.3% input) and immunoprecipitates (IP) were analyzed by Western blotting with anti-FLAG and anti-β-tubulin antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: The C-terminal half of TgRON4 binds to host β-tubulin.(a) Schematic representation of TgRON4 and its deletion mutants. (b), (c) Coimmunoprecipitation of β-tubulin with deletion mutants of 3xFLAG-tagged TgRON4. 293 T (b) and Vero (c) cells were transiently transfected with the indicated TgRON4 expression plasmids. Membrane fractions were immunoprecipitated with an anti-β-tubulin antibody. The membrane fractions (3.3% input) and immunoprecipitates (IP) were analyzed by Western blotting with anti-FLAG and anti-β-tubulin antibodies.
Mentions: TUBB5 and TUBB2C are ubiquitously distributed and represent the major isotypes among the seven β-tubulin isotypes in all tissues except for brain29. To validate the binding specificity between TgRON4 and host cellular β-tubulin, we conducted immunoprecipitation assays using mammalian cells expressing TgRON4. We prepared deletion constructs consisting of 3xFLAG-tagged full-length TgRON4 (TgRON4-Full) lacking its putative signal sequence, the N-terminal half of TgRON4 (TgRON4A), and the C-terminal half of TgRON4 (TgRON4B) (Fig. 2a). 293T and Vero cells were transiently transfected with a control or each of these constructs, and the membrane proteins prepared from collected cells were immunoprecipitated with an anti-β-tubulin antibody. Expression of 3xFLAG-tagged TgRON4 protein was observed in the hydrophobic fraction (Fig. 2b and 2c, lanes 2–4), and some smaller bands could also be observed, especially in Vero cells. TgRON4-Full and TgRON4B coimmunoprecipitated with β-tubulin in both 293 T and Vero cells, but RON4A did not (Fig. 2b and 2c, lames 6–8). The ratios of precipitated TgRON4-Full and TgRON4B to TgRON4 in total membrane fractions were 1.7% and 0.82% in 293 T cells, 1.0% and 0.56% in Vero cells, respectively (Fig. 2b and 2c, lames 2, 4, 6, 8). The ratio of precipitated TgRON4-Full is higher than that of TgRON4B. TgRON4A may partially contributed to fully binding to β-tubulin. These results demonstrate that the C-terminus of TgRON4 mainly binds to host cellular β-tubulin.

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