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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 TUBB2C-binding region in TgRON4.(a) Schematic representation of TgRON4B and its deletion mutants. (b), (c) The indicated 3xFLAG-tagged RON4 proteins were coexpressed with HA-tagged TUBB2C F3R in Vero and CHO cells. The cell lysates were immunoprecipitated with an anti-FLAG antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies.
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f4: Identification of the TUBB2C-binding region in TgRON4.(a) Schematic representation of TgRON4B and its deletion mutants. (b), (c) The indicated 3xFLAG-tagged RON4 proteins were coexpressed with HA-tagged TUBB2C F3R in Vero and CHO cells. The cell lysates were immunoprecipitated with an anti-FLAG antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies.

Mentions: HA-tagged TUBB2C consisting of amino acids 315–445 (TUBB2C F3R) was sufficient to bind 3xFLAG-tagged TgRON4B (Fig. 3a, lane 20). To determine the TUBB2C-binding region of TgRON4, we made 3xFLAG-tagged expression constructs containing various deletions in the C-terminal half of TgRON4 (Fig. 4a). These deletion mutants were coexpressed with HA-tagged TUBB2C F3R in Vero and CHO cells and immunoprecipitated with an anti-FLAG antibody. Of the N-terminal truncated deletions, the 3xFLAG-tagged TgRON4B mutants (F1R and F2R) consisting of amino acids 594–984 and 727–984 coimmunoprecipitated with HA-tagged TUBB2C F3R in both Vero and CHO cells, indicating that they bound to the HA-tagged TUBB2C F3R (Fig. 4blanes 21, 22 and 4c lanes 18, 19), whereas the 3xFLAG-tagged TgRON4B mutant (B4) consisting of amino acids 861–984 did not (Fig. 4blane 23 and 4c lane 20). In Vero cells, the 3xFLAG-tagged TgRON4B mutant (B3) consisting of amino acids 727–880 bound to HA-tagged TUBB2C F3R (Fig. 4b, lane 24). Because the transient expression of TgRON4B B3 was limited in CHO cells, cotransfection was scaled up 4-fold. The 3xFLAG-tagged TgRON4B B3 mutant coimmunoprecipitated with HA-tagged TUBB2C F3R in both Vero and CHO cells (Supplemental Fig. 1lanes 14, 16). Together, these results suggest that the TUBB2C-binding region is localized within amino acids 727–860 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)

Identification of the TUBB2C-binding region in TgRON4.(a) Schematic representation of TgRON4B and its deletion mutants. (b), (c) The indicated 3xFLAG-tagged RON4 proteins were coexpressed with HA-tagged TUBB2C F3R in Vero and CHO cells. The cell lysates were immunoprecipitated with an anti-FLAG antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Identification of the TUBB2C-binding region in TgRON4.(a) Schematic representation of TgRON4B and its deletion mutants. (b), (c) The indicated 3xFLAG-tagged RON4 proteins were coexpressed with HA-tagged TUBB2C F3R in Vero and CHO cells. The cell lysates were immunoprecipitated with an anti-FLAG antibody. The cell lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-FLAG antibodies.
Mentions: HA-tagged TUBB2C consisting of amino acids 315–445 (TUBB2C F3R) was sufficient to bind 3xFLAG-tagged TgRON4B (Fig. 3a, lane 20). To determine the TUBB2C-binding region of TgRON4, we made 3xFLAG-tagged expression constructs containing various deletions in the C-terminal half of TgRON4 (Fig. 4a). These deletion mutants were coexpressed with HA-tagged TUBB2C F3R in Vero and CHO cells and immunoprecipitated with an anti-FLAG antibody. Of the N-terminal truncated deletions, the 3xFLAG-tagged TgRON4B mutants (F1R and F2R) consisting of amino acids 594–984 and 727–984 coimmunoprecipitated with HA-tagged TUBB2C F3R in both Vero and CHO cells, indicating that they bound to the HA-tagged TUBB2C F3R (Fig. 4blanes 21, 22 and 4c lanes 18, 19), whereas the 3xFLAG-tagged TgRON4B mutant (B4) consisting of amino acids 861–984 did not (Fig. 4blane 23 and 4c lane 20). In Vero cells, the 3xFLAG-tagged TgRON4B mutant (B3) consisting of amino acids 727–880 bound to HA-tagged TUBB2C F3R (Fig. 4b, lane 24). Because the transient expression of TgRON4B B3 was limited in CHO cells, cotransfection was scaled up 4-fold. The 3xFLAG-tagged TgRON4B B3 mutant coimmunoprecipitated with HA-tagged TUBB2C F3R in both Vero and CHO cells (Supplemental Fig. 1lanes 14, 16). Together, these results suggest that the TUBB2C-binding region is localized within amino acids 727–860 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