Abstract
Polarized epithelial cells maintain the polarized distribution of basolateral and apical membrane proteins through a process of receptor-mediated endocytosis, sorting, and then recycling to the appropriate membrane domain. We have previously shown that the small GTP-binding proteins, Rab11a and Rab25, are associated with the apical recycling system of Madin-Darby canine kidney cells. Here we have utilized inducible expression of wild-type, dominant negative, and constitutively active mutants to directly compare the functions of Rab25 and Rab11a in postendocytic vesicular transport. We found that a Rab11a mutant deficient in GTP binding, Rab11aS25N, potently inhibited both transcytosis and apical recycling yet failed to inhibit transferrin recycling. Similarly, expression of either wild type Rab25 or the active mutant Rab25S21V inhibited both apical recycling and transcytosis of IgA by greater than 50% but had no effect on basolateral recycling of transferrin. Interestingly, the GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling. These results indicate that neither Rab11a nor Rab25 function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Thus, transferrin receptors must be recycled to the plasma membrane prior to sorting of apically directed cargoes into Rab11a/Rab25-positive apical recycling endosomes.
Highlights
Eukaryotic cells continually take up fluid, solutes, and macromolecules through endocytosis
Electron microscopy studies indicate that transferrin internalized from the basolateral side of the Caco-2 cell is found in an apical endosomal compartment that is accessible to apical endocytic markers [8, 9]
In polarized MDCK cells, polymeric IgA receptor internalized from the basolateral surface is first delivered to basolateral sorting endosomes and transported to apical tubular endosomal compartments before being delivered to the apical plasma membrane, and these endosomes are accessible to apically internalized IgA (10 –13). These results suggest the existence of apical recycling endosomes that receive cargoes from both apical and basolateral early endosomes
Summary
Vol 275, No 37, Issue of September 15, pp. 29138 –29146, 2000 Printed in U.S.A. Regulation of Vesicle Trafficking in Madin-Darby Canine Kidney Cells by Rab11a and Rab25*. The GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling These results indicate that neither Rab11a nor Rab function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Wild type Rab and GTPase-deficient Rab behaved with both potently inhibiting apical recycling and transcytosis, without affecting transferrin recycling These studies demonstrate that both Rab11a and Rab function at a late stage in the transcytotic and apical recycling pathways, beyond the point at which transferrin has been sorted away from IgA. This pattern of sorting is clearly different from the situation in nonpolarized cells in which Rab11a is present on transferrin-containing compartments and regulates transferrin recycling
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