title: Biochemical analysis of secretory trafficking in mammalian cells.
abstract: Protein trafficking within the secretory pathway of mammalian cells is amenable to analysis by biochemical methods. This can be achieved by monitoring posttranslational modifications that occur naturally within the secretory pathway, or by measuring the delivery of cargo to the cell surface or extracellular medium. These approaches can be combined with additional manipulations such as specific temperature blocks that permit analysis of distinct trafficking steps. Biochemical analysis is advantageous in that it permits both a sensitive and quantitative measure of trafficking along the pathway. The methods discussed in this chapter permit the analysis of trafficking of both endogenous cargo proteins and ectopically expressed model cargos, which can be followed using either Western blotting or metabolic pulse-chase approaches. These methods are relatively straightforward and suitable for use in most modern cell biology laboratories. In addition to the well-established methods that we describe here in detail, we also refer to the development of more recent tailored approaches that add further to the arsenal of tools that can be used to assess trafficking in the secretory pathway.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Peristera
authors.initials: P
authors.lastname: Roboti
authors.forename: Tomasz M
authors.initials: TM
authors.lastname: Witkos
authors.forename: Martin
authors.initials: M
authors.lastname: Lowe
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D055571:Secretory Pathway; D013194:Staining and Labeling; D014161:Transduction, Genetic; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins
keywords: Brefeldin A; Cycloheximide chase; Endoglycosidase H; Golgi apparatus; Immunoprecipitation; Protein sorting; Pulse-chase; Surface biotinylation; Temperature block; Temperature-sensitive ts045 mutant of vesicular stomatitis virus G protein