Apical cargo traverses endosomal compartments on the passage to the cell surface.
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 co-stained 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.
Cramm-Behrens
Catharina I
CI
Department of Cell Biology and Cell Pathology, Philipps-University Marburg, 35033 Marburg, Germany.
Dienst
Martina
M
Jacob
Ralf
R
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004285:Dogs; D011992:Endosomes / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D006056:Golgi Apparatus / Q000378:metabolism; D007700:Kinetics; D010694:Lactase-Phlorizin Hydrolase / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D013394:Sucrase-Isomaltase Complex / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D034741:RNA, Small Interfering; D013394:Sucrase-Isomaltase Complex; D010694:Lactase-Phlorizin Hydrolase; D020691:rab GTP-Binding Proteins