Analysis of protein sorting at the trans-Golgi network and the post-TGN compartments of polarized epithelial cells

Abstract

Epithelial cells rely on the generation of surface polarity, which requires initial segregation of apical and basolateral domains on the cell surface and continuous regulated secretion of apical and basolateral domain constituents. For several decades, the trans-Golgi network (TGN) was considered the most distal stop and hence the ultimate protein sorting station for distinct apical and basolateral transport carriers that reach their respective surface domains in the direct trafficking pathway. However, recent reports of apical and basolateral cargoes traversing post-Golgi compartments accessible to endocytic ligands prior to their arrival at the cell surface and the postTGN breakup of large pleomorphic membrane fragments that exit the Golgi region toward the surface, raised the possibility that compartments distal to the TGN mediate or contribute to biosynthetic sorting. Here we describe the development of a novel assay that quantitatively distinguishes different cargo pairs by their degree of colocalization at the TGN and by the evolution of colocalization during their TGN-to-surface transport. Key to the high resolution of our approach are: 1) the conversion of perinuclear organelle clustering into a 2D microsomal spread and 2) the identification of TGN and post-TGN cargo without the need for a TGN marker that universally co-segregates with all cargo. Employing this novel technique with three different cargo pairs, we determined that the two model cargoes, basolateral VSVG and apical NTRp75, undergo iterative sorting in the TGN and in post-TGN compartments, while two differently tagged versions of VSVG or the apical proteins DPPIV and NTRp75 remain colocalized throughout their exocytic itineraries.