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TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.

Gallo LI, Liao Y, Ruiz WG, Clayton DR, Li M, Liu YJ, Jiang Y, Fukuda M, Apodaca G, Yin XM - Mol. Biol. Cell (2014)

Bottom Line: In contrast, TBC1D9B had no effect on two Rab11a-independent pathways--basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor.Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A.We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.

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TBC1D9B interacts with Rab GTPases. (A) Flag-tagged TBC1D9B, along with the indicated GFP-Rabs containing an activating Q-to-L mutation, were coexpressed in HEK cells. Flag-TBC1D9B was recovered using an anti-flag tag antibody and coimmunopreciptated GFP-tagged Rab-QL detected using an anti-GFP antibody. IgG was used as a nonspecific control. (B) Quantification of the percentage GFP-Rab-QL coimmunoprecipitated with TBC1D9B normalized to the total amount of GFP-Rab-QL in the lysate. Data were obtained from three independent experiments, and the mean ± SEM is shown. Values significantly different from the group means, assessed by ANOVA, are indicated (*p < 0.05).
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Figure 2: TBC1D9B interacts with Rab GTPases. (A) Flag-tagged TBC1D9B, along with the indicated GFP-Rabs containing an activating Q-to-L mutation, were coexpressed in HEK cells. Flag-TBC1D9B was recovered using an anti-flag tag antibody and coimmunopreciptated GFP-tagged Rab-QL detected using an anti-GFP antibody. IgG was used as a nonspecific control. (B) Quantification of the percentage GFP-Rab-QL coimmunoprecipitated with TBC1D9B normalized to the total amount of GFP-Rab-QL in the lysate. Data were obtained from three independent experiments, and the mean ± SEM is shown. Values significantly different from the group means, assessed by ANOVA, are indicated (*p < 0.05).

Mentions: As an additional method to screen for TBC1D9B interactions, we determined whether flag-TBC1D9B could coimmunoprecipitate a broader array of GFP-tagged, GTP-locked mutants of Rab proteins previously implicated in endocytic and exocytic membrane traffic. Of the Rabs tested, we observed that Rab11a-QL, Rab11b-QL, and Rab4a-QL formed significant interactions with flag-TBC1D9B (Figure 2). In contrast, little interaction was observed with nominally GTP-locked mutants of Rab3a, Rab5a, Rab6a, Rab7, Rab8a, Rab10, Rab13, Rab25, Rab27a, or Rab33a (Figure 2). Taken together, these results indicate that TBC1D9B forms interactions with a selective number of Rab GTPases, including Rab11a, Rab11b, and Rab4a.


TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.

Gallo LI, Liao Y, Ruiz WG, Clayton DR, Li M, Liu YJ, Jiang Y, Fukuda M, Apodaca G, Yin XM - Mol. Biol. Cell (2014)

TBC1D9B interacts with Rab GTPases. (A) Flag-tagged TBC1D9B, along with the indicated GFP-Rabs containing an activating Q-to-L mutation, were coexpressed in HEK cells. Flag-TBC1D9B was recovered using an anti-flag tag antibody and coimmunopreciptated GFP-tagged Rab-QL detected using an anti-GFP antibody. IgG was used as a nonspecific control. (B) Quantification of the percentage GFP-Rab-QL coimmunoprecipitated with TBC1D9B normalized to the total amount of GFP-Rab-QL in the lysate. Data were obtained from three independent experiments, and the mean ± SEM is shown. Values significantly different from the group means, assessed by ANOVA, are indicated (*p < 0.05).
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Figure 2: TBC1D9B interacts with Rab GTPases. (A) Flag-tagged TBC1D9B, along with the indicated GFP-Rabs containing an activating Q-to-L mutation, were coexpressed in HEK cells. Flag-TBC1D9B was recovered using an anti-flag tag antibody and coimmunopreciptated GFP-tagged Rab-QL detected using an anti-GFP antibody. IgG was used as a nonspecific control. (B) Quantification of the percentage GFP-Rab-QL coimmunoprecipitated with TBC1D9B normalized to the total amount of GFP-Rab-QL in the lysate. Data were obtained from three independent experiments, and the mean ± SEM is shown. Values significantly different from the group means, assessed by ANOVA, are indicated (*p < 0.05).
Mentions: As an additional method to screen for TBC1D9B interactions, we determined whether flag-TBC1D9B could coimmunoprecipitate a broader array of GFP-tagged, GTP-locked mutants of Rab proteins previously implicated in endocytic and exocytic membrane traffic. Of the Rabs tested, we observed that Rab11a-QL, Rab11b-QL, and Rab4a-QL formed significant interactions with flag-TBC1D9B (Figure 2). In contrast, little interaction was observed with nominally GTP-locked mutants of Rab3a, Rab5a, Rab6a, Rab7, Rab8a, Rab10, Rab13, Rab25, Rab27a, or Rab33a (Figure 2). Taken together, these results indicate that TBC1D9B forms interactions with a selective number of Rab GTPases, including Rab11a, Rab11b, and Rab4a.

Bottom Line: In contrast, TBC1D9B had no effect on two Rab11a-independent pathways--basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor.Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A.We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.

Show MeSH
Related in: MedlinePlus