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: We also determined whether TBC1D9B colocalized to a greater extent with the GFP-tagged Rab11aQ70L mutant. Indeed, we observed higher degree of colocalization between endogenous TBC1D9B and GFP-tagged Rab11a-QL than with GFP-tagged Rab11a-WT (coefficient of colocalization of 0.73 ± 0.03 and 0.61 ± 0.02, respectively; Figure 7, E–G). We also determined whether the localization of Rab11a in MDCK cells was altered by expressing CFP-TBC1D9B-WT or CFP-TBC1D9B-RYQ/AAA. However, we found no difference in the Rab11a distribution (Supplemental Figure S5, A and B; see Figure 9A later in this article), which is consistent with previous reports that the Rab11a-SN mutant remains membrane bound in MDCK cells (Wang et al., 2000b). There was also no change in Rab11a colocalization with these TBC1D9B variants (coefficient of colocalization of 0.72 ± 0.06 and 0.70 ± 0.06 for WT and RYQ/AAA, respectively; Supplemental Figure S5C). In contrast, in HEK cells, there was a significant redistribution of Rab11a from recycling endosomes to the cytosol when TBC1D9B-WT, but not when the inactive mutant TBC1D9B-RYQ/AAA, was expressed (Supplemental Figure S2, A and B). This is the expected result for HEK cells if the GDP-bound form of Rab11a increased as a result of expressing TBC1D9B-WT. Finally, when Rab11a expression was depleted using a specific shRNA, endogenous TBC1D9B's normal accumulation in the apical half of the cell was altered, and its distribution became more dispersed throughout the cell (Supplemental Figure S5D).
Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.