title: Iterative sorting of apical and basolateral cargo in Madin-Darby canine kidney cells.
abstract: 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 before their arrival at the cell surface and the post-TGN 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. Keys to the high resolution of our approach are 1) conversion of perinuclear organelle clustering into a two-dimensional microsomal spread and 2) identification of TGN and post-TGN cargo without the need for a TGN marker that universally cosegregates with all cargo. Using our assay, we provide the first evidence that apical NTRp75 and basolateral VSVG in Madin-Darby canine kidney cells still undergo progressive sorting after they exit the TGN toward the cell surface.
authors.affiliation: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Treyer
authors.affiliation: Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, NY 10461 Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.
authors.forename: Mario
authors.initials: M
authors.lastname: Pujato
authors.affiliation: Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: Ximo
authors.initials: X
authors.lastname: Pechuan
authors.affiliation: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461 anne.muesch@einstein.yu.edu.
authors.forename: Anne
authors.initials: A
authors.lastname: Müsch
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology; D004285:Dogs; D006056:Golgi Apparatus / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D021381:Protein Transport; D020800:Receptor, Nerve Growth Factor / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D020800:Receptor, Nerve Growth Factor; D014759:Viral Envelope Proteins