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Vol. 15, Issue 3, 1407-1416, March 2004

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Specific N-Glycans Direct Apical Delivery of Transmembrane, but Not Soluble or Glycosylphosphatidylinositol-anchored Forms of Endolyn in Madin-Darby Canine Kidney Cells

Beth A. Potter ^*, Gudrun Ihrke , Jennifer R. Bruns ^*, Kelly M. Weixel ^*, and Ora A. Weisz ^* 

^* Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; Cambridge Institute for Medical Research, Department of Clinical Biochemistry, University of Cambridge, Cambridge CB2 2XY, United Kingdom

Submitted August 1, 2003; Revised December 1, 2003; Accepted December 2, 2003
Monitoring Editor: Juan S. Bonifacino

The sialomucin endolyn is a transmembrane protein with a unique trafficking pattern in polarized Madin-Darby canine kidney cells. Despite the presence of a cytoplasmic tyrosine motif that, in isolation, is sufficient to mediate basolateral sorting of a reporter protein, endolyn predominantly traverses the apical surface en route to lysosomes. Apical delivery of endolyn is disrupted in tunicamycin-treated cells, implicating a role for N-glycosylation in apical sorting. Site-directed mutagenesis of endolyn's eight N-glycosylation sites was used to identify two N-glycans that seem to be the major determinants for efficient apical sorting of the protein. In addition, apical delivery of endolyn was disrupted when terminal processing of N-glycans was blocked using glycosidase inhibitors. Missorting of endolyn occurred independently of the presence or absence of the basolateral sorting signal, because apical delivery was also inhibited by tunicamycin when the cytoplasmic tyrosine motif was mutated. However, we found that apical secretion of a soluble mutant of endolyn was N-glycan independent, as was delivery of glycosylphosphatidylinositol-anchored endolyn. Thus, specific N-glycans are only essential for the apical sorting of transmembrane endolyn, suggesting fundamental differences in the mechanisms by which soluble, glycosylphosphatidylinositol-anchored, and transmembrane proteins are sorted.

Abbreviations used: BGN, benzyl 2-acetamido-2-deoxy--D-galactopyranoside; DMJ, deoxymannojirimycin; DNJ, deoxynojirimycin; GPI, glycosylphosphatidylinositol; KIF, kifunensine; mAb, monoclonal antibody; MDCK, Madin-Darby canine kidney; TGN, trans-Golgi network; TM, tunicamycin.

Corresponding author. E-mail address: weisz{at}pitt.edu.

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