Apical Cargo Traverses Endosomal Compartments on the Passage to the Cell Surface

Abstract

Epithelial polarity is based on intracellular sorting machinery that maintains the asymmetric distribution of lipids and proteins to the cell surface. Dependent on their lipid raft affinity, newly synthesized apical polypeptides are segregated into distinct vesicle populations subsequent to the passage through the Golgi apparatus. Using a combined fluorescence microscopic and biochemical approach, we found that lipid raft-associated sucrase-isomaltase (SI) as well as non-raft-associated lactase-phlorizin hydrolase (LPH) traverse endosomal compartments before entering the apical membrane. Fluorescent fusion proteins of both hydrolases were costained with Rab4-, Rab8-and Rab11-positive endosomes in polarized Madin-Darby canine kidney and non-polarized COS-1 cells. Immunoisolation of post-Golgi vesicles subsequent to different times of TGN release revealed that LPH and SI navigate in chronological order through Rab4-, Rab8-and Rab11-positive endosomes. Thereafter, the two hydrolases are segregated into distinct vesicle populations. In addition, apical membrane traffic could be significantly inhibited by RNA interference-mediated depletion of these guanosine triphosphatases. These results suggest that in epithelial cells, lipid raft-dependent and -independent apical cargo follow a transendosomal route.

Apical trafficking in epithelial cells: signals, clusters and motors

“…Fluorescence and pulse-chase analysis detected the passage of two newly synthesized PM proteins, lactase-phlorizin hydrolase and sucrase-isomaltase, successively through Rab4-, Rab8-and Rab11-positive endosomes. Their transport was inhibited by RNA interference against these Rabs, suggesting that these proteins reach the AEE/ASE first after leaving the TGN (Cramm-Behrens et al, 2008). Route 2 is probably used by rhodopsin, the major component of the outer segments of retinal photoreceptors, which constitute an enlarged version of the primary cilium that is present in most vertebrate cells (Singla and Reiter, 2006).…”

Rab GTPase–Myo5B complexes control membrane recycling and epithelial polarization

“…In polarized epithelial cells, Rab11a and Rab8a subsume roles more specific to trafficking to the apical membrane (16). Therefore, we examined the dependence of Myo5B on interaction with Rab8a or Rab11a in polarized cells using 3D MDCK cyst cultures.…”

Trafficking to the Apical and Basolateral Membranes in Polarized Epithelial Cells

“…In the case of the disaccharidases lactase phlorizin hydrolase and sucrase isomaltase-a raftdependent protein and raft-independent protein, respectively-separation does not occur immediately after release from the TGN, but rather after sequential movement through Rab4-, Rab8-, and Rab11-positive endosomal compartments (or perhaps subcompartments). 100 Mounting evidence demonstrates that, in addition to being separated into different endosomal compartments, cargoes are segregated into different subdomains within endosomes and that this lateral segregation facilitates cargo sorting (reviewed by Miaczynska and Zerial 101 ). Using immuno-electron microscopy, Sonnichsen et al demonstrated separation of resident Rab GTPases into distinct endosomal subdomains.…”

“…99,167 Conversely, lactase phlorizin hydrolase and sucrase isomalatase traffic between the apical early and apical recycling endosomes (6). 100 Proteins internalized from the apical membrane to the AEE can traffic to the ARE for recycling back to the surface, or to the CRE and late endosomes if they are targeted for degradation (unlabeled arrows). LE, late endosome; lys, lysosome; MVB, multivesicular body.…”