title: Raft-mediated trafficking of apical resident proteins occurs in both direct and transcytotic pathways in polarized hepatic cells: role of distinct lipid microdomains.
abstract: In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains ("rafts") are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway.
authors.affiliation: Department of Membrane Cell Biology, University of Groningen, The Netherlands.
authors.forename: Tounsia Aït
authors.initials: TA
authors.lastname: Slimane
authors.forename: Germain
authors.initials: G
authors.lastname: Trugnan
authors.forename: Sven C D
authors.initials: SC
authors.lastname: Van IJzendoorn
authors.forename: Dick
authors.initials: D
authors.lastname: Hoekstra
mesh_terms: D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D015638:Cytochalasin D / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D006801:Humans; D050356:Lipid Metabolism; D008099:Liver / Q000166:cytology*; D008164:Luminescent Proteins / Q000378:metabolism; D021962:Membrane Microdomains; D018613:Microscopy, Confocal; D017830:Octoxynol / Q000494:pharmacology; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013997:Time Factors; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D003902:Detergents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D017830:Octoxynol; D002784:Cholesterol