Multiple Biosynthetic Trafficking Routes for Apically Secreted Proteins in MDCK Cells

Abstract

Many newly synthesized membrane proteins traverse endocytic intermediates en route to the surface in polarized epithelial cells; however, the biosynthetic route of secreted proteins has not been elucidated. We monitored the trafficking route of two secreted proteins with different apical sorting signals: the N-glycan dependent cargo glycosylated growth hormone (gGH) and Ensol, a soluble version of endolyn whose apical sorting is independent of N-glycans. Both proteins were observed to colocalize in part with apical recycling endosome (ARE) markers. Cargo that lacks an apical targeting signal and is secreted in a nonpolarized manner did not localize to the ARE. Expression of dominant-negative mutant of Myosin Vb, which disrupts ARE export of glycan dependent membrane proteins, selectively inhibited apical release of gGH but not Ensol. FRAP measurements revealed that gGH in the ARE was less mobile than Ensol, consistent with tethering to a sorting receptor. However, knockdown of galectin-3 or galectin-4, lectins implicated in apical sorting, had no effect on the rate or polarity of gGH secretion. Together, our results suggest that apically-secreted cargo selectively access the ARE and are exported via differentially regulated pathways.

Polarized sorting and trafficking in epithelial cells

“…Proteins secreted apically have been shown to depend on N-glycans to be sorted correctly [125]. Galectin-3 seems not to be involved in this pathway [126]. Whether other galectins such as galectin-9 in MDCK cells plays a role in transporting secretory proteins to the apical side of the epithelium remains to be analyzed.…”

Role of membrane traffic in the generation of epithelial cell asymmetry

“…Depletion of galectin-3 in MDCK cells leads to mistargeting of several lipid-raft-independent apical membrane proteins to the basolateral cell surface, a phenotype also observed in the enterocytes of galectin-3-knockout mice 70,71 . Galectin-3 function may be specific to trafficking of membrane proteins, as depletion of galectin-3 (and galectin-4) has no effect on transport of glycosylated apical secretory cargoes 72 . Other galectins implicated in apical targeting include galectin-4, which associates with sulphatides, and galectin-9, which binds Forssman glycosphingolipids 73–75 .…”

Galectin-4-mediated transcytosis of transferrin receptor

“…Canine galectin-3 siRNAs were: 59-ACCCAAACCCUCAAGGAUGdTdT-39 (Delacour et al, 2006) and 59-AUACCAAGCUGGAUAAUAUdTdT-39. Canine galectin-4 siRNA was 59-GGG-ACAAGGUGGUGUUCAAUU-39 (Mattila et al, 2012). Sus scrofa galectin-4 siRNAs (designed using Dharmacon algorithm) were: siRNA 1 , 59-CAGUAAAGGCCCUCAUCCAUU-39; siRNA 2 , 59-CUGGAAAGC-ACAACCAACAUU-39; siRNA 3 , 59-GGACAAAGUGUAUGAACAU-UU-39.…”