Clathrin and AP1 are required for apical sorting of GPI ‐APs in biosynthetic and recycling routes in MDCK cells

Abstract
Recently studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mis‐targeting of endogenous GPI‐APs and soluble secretory proteins in MDCK cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI‐APs in biosynthetic pathway but also for their apical recycling and basal to apical transcytosis routes. The apical distribution of the t‐SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical destined cargo proteins both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI‐APs in polarized MDCK cells.

Knock downs of Clathrin and AP1 subunit affect the t‐SNARE syntaxin3 distribution from apical to basolateral in polarized MDCK cells. As a result, Glycosyl Phosphatidyl Inositol anchored proteins (GPI‐AP) containing secretory vesicles are targeted to basolateral membranes. Additionally, in AP1 knockdown cells GPI‐AP containing recycling endosomes are rerouted to the basolateral surface. All together our data dem...

Source: Traffic - January 1, 2018 Category: Research Authors: Guillaume Alain Castillon, Patricia Burriat ‐Couleru, Daniel Abegg, Nina Criado Santos, Reika Watanabe Tags: ORIGINAL ARTICLE Source Type: research