TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.
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.
Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.Show MeSH
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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.
Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.