GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells.
The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin-Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.
Dipartimento di Biologia e Patologia Cellulare e Molecolare, CEINGE Biotecnologie Avanzate, Università degli Studi di Napoli Federico II, 80131 Napoli, Italy.
Simona
S
Paladino
Thomas
T
Pocard
Maria Agata
MA
Catino
Chiara
C
Zurzolo
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects / Q000502:physiology*; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D021381:Protein Transport / Q000187:drug effects; D020800:Receptor, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013634:Tannins / Q000494:pharmacology; D013696:Temperature; D019108:Tight Junctions / Q000187:drug effects; D014162:Transfection; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology; D021601:trans-Golgi Network / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D020800:Receptor, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D013634:Tannins; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase; D014357:Trypsin