Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway.
Epithelial cell polarity depends on mechanisms for targeting proteins to different plasma membrane domains. Here, we dissect the pathway for apical delivery of several raft-associated, glycosyl phosphatidylinositol (GPI)-anchored proteins in polarized MDCK cells using live-cell imaging and selective inhibition of apical or basolateral exocytosis. Rather than trafficking directly from the trans-Golgi network (TGN) to the apical plasma membrane as previously thought, the GPI-anchored proteins followed an indirect, transcytotic route. They first exited the TGN in membrane-bound carriers that also contained basolateral cargo, although the two cargoes were laterally segregated. The carriers were then targeted to and fused with a zone of lateral plasma membrane adjacent to tight junctions that is known to contain the exocyst. Thereafter, the GPI-anchored proteins, but not basolateral cargo, were rapidly internalized, together with endocytic tracer, into clathrin-free transport intermediates that transcytosed to the apical plasma membrane. Thus, apical sorting of these GPI-anchored proteins occurs at the plasma membrane, rather than at the TGN.
Polishchuk
Roman
R
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Di Pentima
Alessio
A
Lippincott-Schwartz
Jennifer
J
D000818:Animals; D015415:Biomarkers; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology*; D006224:Cricetinae; D004285:Dogs; D004705:Endocytosis / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D047348:Hydrolyzable Tannins / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism / Q000648:ultrastructure; D008561:Membrane Fusion / Q000502:physiology; D021962:Membrane Microdomains / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D021601:trans-Golgi Network / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
D015415:Biomarkers; D017261:Glycosylphosphatidylinositols; D047348:Hydrolyzable Tannins; D011993:Recombinant Fusion Proteins