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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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Subcellular localization of TBC1D9B in MDCK cells. Filter-grown MDCK cells were immunolabeled to show the distribution of the following endogenous proteins. (A) TBC1D9B and the tight junction–associated protein ZO-1 (xy-section, left), the apical membrane protein gp-135 (xz-section, top right), or actin (xz-section, bottom right). (B) Coefficient of colocalizations for endogenous TBC1D9B and the following markers: (C) TBC1D9B, actin, and EEA1 (an early endosome marker); (D) TBC1D9B, actin, and Lamp2 (a late endosome/lysosome marker); (E) TBC1D9B, actin, and giantin (a Golgi marker); (F) TBC1D9B, ZO-1, and TfR (a marker of the common recycling endosome and basolateral early endosomes); and (G) TBC1D9B, actin, and Rab11a (which is associated with the apical recycling endosomes in these cells). In A and C–G, single optical sections from the region just under the apical membrane (subapical), just above the nucleus (supranuclear), or at the level of the nucleus along the lateral membranes (medial) are shown. Boxed regions are magnified in the images below the word ZOOM. Scale bar, 5 μm. In B, data are mean ± SEM from three experiments (n > 100 cells).
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Figure 6: Subcellular localization of TBC1D9B in MDCK cells. Filter-grown MDCK cells were immunolabeled to show the distribution of the following endogenous proteins. (A) TBC1D9B and the tight junction–associated protein ZO-1 (xy-section, left), the apical membrane protein gp-135 (xz-section, top right), or actin (xz-section, bottom right). (B) Coefficient of colocalizations for endogenous TBC1D9B and the following markers: (C) TBC1D9B, actin, and EEA1 (an early endosome marker); (D) TBC1D9B, actin, and Lamp2 (a late endosome/lysosome marker); (E) TBC1D9B, actin, and giantin (a Golgi marker); (F) TBC1D9B, ZO-1, and TfR (a marker of the common recycling endosome and basolateral early endosomes); and (G) TBC1D9B, actin, and Rab11a (which is associated with the apical recycling endosomes in these cells). In A and C–G, single optical sections from the region just under the apical membrane (subapical), just above the nucleus (supranuclear), or at the level of the nucleus along the lateral membranes (medial) are shown. Boxed regions are magnified in the images below the word ZOOM. Scale bar, 5 μm. In B, data are mean ± SEM from three experiments (n > 100 cells).

Mentions: To better understand the site of TBC1D9B function in the cell, we studied its subcellular localization in polarized epithelial MDCK cells. In these cells, endogenous TBC1D9B was vesicular in appearance and accumulated in the apical cytoplasm of the cell up to the level of the apical membrane (defined by GP-135 or actin staining in Figure 6A). TBC1D9B did not localize to EEA1-positive early endosomes or to LAMP2-labeled late endosomes/lysosomes (Figure 6, B–D; Manders coefficient of colocalization of 0.031 ± 0.005 and 0.035 ± 0.005, respectively). TBC1D9B also partially localized with the giantin-labeled Golgi (coefficient of colocalization of 0.31 ± 0.04; Figure 6, B and E), which is consistent with previous observations that Rab11a is localized in part to this organelle (Ullrich et al., 1996; Chen et al., 1998). Small amounts of TBC1D9B colocalized with the transferrin receptor (TfR; coefficient of colocalization of 0.20 ± 0.05; Figure 6, B and F), which shows only modest colocalization with Rab11a in MDCK cells (Brown et al., 2000; Leung et al., 2000). However, as expected for a protein that interacts with Rab11a, a relatively large fraction of TBC1D9B was localized to Rab11a-positive vesicles (coefficient of colocalization of 0.48 ± 0.1; Figure 6, B and G.


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)

Subcellular localization of TBC1D9B in MDCK cells. Filter-grown MDCK cells were immunolabeled to show the distribution of the following endogenous proteins. (A) TBC1D9B and the tight junction–associated protein ZO-1 (xy-section, left), the apical membrane protein gp-135 (xz-section, top right), or actin (xz-section, bottom right). (B) Coefficient of colocalizations for endogenous TBC1D9B and the following markers: (C) TBC1D9B, actin, and EEA1 (an early endosome marker); (D) TBC1D9B, actin, and Lamp2 (a late endosome/lysosome marker); (E) TBC1D9B, actin, and giantin (a Golgi marker); (F) TBC1D9B, ZO-1, and TfR (a marker of the common recycling endosome and basolateral early endosomes); and (G) TBC1D9B, actin, and Rab11a (which is associated with the apical recycling endosomes in these cells). In A and C–G, single optical sections from the region just under the apical membrane (subapical), just above the nucleus (supranuclear), or at the level of the nucleus along the lateral membranes (medial) are shown. Boxed regions are magnified in the images below the word ZOOM. Scale bar, 5 μm. In B, data are mean ± SEM from three experiments (n > 100 cells).
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Figure 6: Subcellular localization of TBC1D9B in MDCK cells. Filter-grown MDCK cells were immunolabeled to show the distribution of the following endogenous proteins. (A) TBC1D9B and the tight junction–associated protein ZO-1 (xy-section, left), the apical membrane protein gp-135 (xz-section, top right), or actin (xz-section, bottom right). (B) Coefficient of colocalizations for endogenous TBC1D9B and the following markers: (C) TBC1D9B, actin, and EEA1 (an early endosome marker); (D) TBC1D9B, actin, and Lamp2 (a late endosome/lysosome marker); (E) TBC1D9B, actin, and giantin (a Golgi marker); (F) TBC1D9B, ZO-1, and TfR (a marker of the common recycling endosome and basolateral early endosomes); and (G) TBC1D9B, actin, and Rab11a (which is associated with the apical recycling endosomes in these cells). In A and C–G, single optical sections from the region just under the apical membrane (subapical), just above the nucleus (supranuclear), or at the level of the nucleus along the lateral membranes (medial) are shown. Boxed regions are magnified in the images below the word ZOOM. Scale bar, 5 μm. In B, data are mean ± SEM from three experiments (n > 100 cells).
Mentions: To better understand the site of TBC1D9B function in the cell, we studied its subcellular localization in polarized epithelial MDCK cells. In these cells, endogenous TBC1D9B was vesicular in appearance and accumulated in the apical cytoplasm of the cell up to the level of the apical membrane (defined by GP-135 or actin staining in Figure 6A). TBC1D9B did not localize to EEA1-positive early endosomes or to LAMP2-labeled late endosomes/lysosomes (Figure 6, B–D; Manders coefficient of colocalization of 0.031 ± 0.005 and 0.035 ± 0.005, respectively). TBC1D9B also partially localized with the giantin-labeled Golgi (coefficient of colocalization of 0.31 ± 0.04; Figure 6, B and E), which is consistent with previous observations that Rab11a is localized in part to this organelle (Ullrich et al., 1996; Chen et al., 1998). Small amounts of TBC1D9B colocalized with the transferrin receptor (TfR; coefficient of colocalization of 0.20 ± 0.05; Figure 6, B and F), which shows only modest colocalization with Rab11a in MDCK cells (Brown et al., 2000; Leung et al., 2000). However, as expected for a protein that interacts with Rab11a, a relatively large fraction of TBC1D9B was localized to Rab11a-positive vesicles (coefficient of colocalization of 0.48 ± 0.1; Figure 6, B and G.

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