Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol‐anchored proteins in biosynthetic and recycling routes in Madin‐Darby canine kidney 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 mistargeting of endogenous glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) and soluble secretory proteins in Madin-Darby canine kidney (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 in 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.

Clustering in the Golgi apparatus governs sorting and function of GPI‐APs in polarized epithelial cells

“…Galectin-4, which binds glycosphingolipids [44], could be a good candidate as it could crosslink raft-associated proteins into lipid platforms from which vesicle could bud. Furthermore, the new findings showing the involvement of clathrin and AP1 in the Golgi-to-PM transport of GPI-APs [72] point out a putative role of these proteins in promoting GPI-AP vesicle formation.…”

“…Interestingly a recent study has shown that clathrin and AP1 are required for apical sorting of GPI‐APs . The knockdown of either clathrin or AP1 leads to the basolateral missorting of the GPI‐AP CD59 in MDCK cells, while it does not affect the apical and basolateral transport of transmembrane proteins . However, whether the reduced expression of these two proteins affects the GPI‐AP clustering remains to be checked.…”

Clathrin adaptor AP-1 and Stratum act in parallel pathways to control Notch activation in Drosophila Sensory Organ Precursor Cells

“…However the role of Rab8is not restricted to the basolateral transport as the loss of one or the other has also been previously associated to defects in apical targeting and secretion (Castillon et al, 2018;Gillard et al, 2015;Holloway et al, 2013;Nakajo et al, 2016;Norgate et al, 2006;Sato et al, 2014;Sato et al, 2007;Zhang et al, 2012). In the same way, the role of AP-1 is not restricted to the basolateral transport.…”

Plasma membrane to vacuole traffic induced by glucose starvation requires Gga2‐dependent sorting at the trans‐Golgi network

“…Several lines of evidence suggest AP-1 might participate in secretory traffic in other organisms. In epithelia of mammals, worms and flies, AP-1 contributes to both apical and basolateral sorting, and in budding yeast, an AP-1 binding partner is implicated in the formation of a subclass of secretory vesicles (Gall et al, 2002;Liu et al, 2008;Kametaka et al, Sikorski and Hieter (1989) 2012; Folsch, 2015;Gillard et al, 2015;Castillon et al, 2018;Caceres et al, 2019;Gravotta et al, 2019). Moreover, AP-1 appears to be important for secretory traffic in the filamentous yeast Aspergillus nidulans; therefore, it might be expected that in budding yeast AP-1 would participate in secretory traffic (Martzoukou et al, 2018).…”