Vectorial apical delivery and slow endocytosis of a glycolipid-anchored fusion protein in transfected MDCK cells.
To characterize the mechanisms that determine the apical polarity of proteins anchored by glycosylphosphatidylinositol (GPI), we studied the targeting of a GPI-anchored form of a herpes simplex glycoprotein, gD-1, in transfected MDCK cells. Using a biotin-based targeting assay, we found that GPI-anchored gD-1 was sorted intracellularly and delivered directly to the apical surface. Endocytosis of GPI-anchored gD-1 occurred slowly and preferentially from the apical domain, while transcytosis of the basolateral fraction did not occur at a significant rate (incompatible with being a precursor to the apical pool). Prevention of tight junction formation by incubation in medium with micromolar Ca2+ resulted in expression of GPI-anchored gD-1 on the free surface, but not on the attached surface of the cell. Our results indicate that the apical polarity of a GPI-anchored protein is generated by vectorial delivery to the apical membrane, where its distribution is maintained by slow endocytosis and by a retention system not necessarily involving the tight junction.
Lisanti
M P
MP
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021.
Caras
I W
IW
Gilbert
T
T
Hanzel
D
D
Rodriguez-Boulan
E
E
D000818:Animals; D002460:Cell Line; D004285:Dogs; D004705:Endocytosis; D006017:Glycolipids / Q000502:physiology*; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols; D007668:Kidney; D007700:Kinetics; D008563:Membrane Lipids / Q000502:physiology; D008856:Microscopy, Fluorescence; D010716:Phosphatidylinositols / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008563:Membrane Lipids; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C488986:glycoprotein D, Feline herpesvirus 1; C039447:glycoprotein D, Human herpesvirus 1