title: Image-based and biochemical assays to investigate endosomal protein sorting.
abstract: The sorting of membrane proteins within the endosomal system occurs through a panoply of highly dynamic sequential molecular interactions that together govern many physiologically important processes. A key component of the endosomal protein sorting machinery is the retromer complex. Through two distinct subcomplexes, retromer operates to select cargo for endosome-to-Golgi retrieval and also drives membrane tubule formation. Many accessory proteins associate with retromer to facilitate protein sorting and/or tubule formation. The experience we have gained from studying retromer-mediated endosomal protein sorting and the assays developed and applied in the course of these studies can provide a template for researchers interested in related endosomal trafficking pathways. Herein we describe image-based assays that can be applied to study endosomal protein sorting through the use of antibody-uptake assays in low-, medium-, and high-throughput formats. We additionally detail simple but effective native immunoprecipitation methods that can be employed to identify novel proteins that may interact transiently with a protein of interest within the endosomal pathway.
authors.affiliation: Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom. Electronic address: syab2@cam.ac.uk.
authors.forename: Sophia Y
authors.initials: SY
authors.lastname: Breusegem
authors.affiliation: Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom. Electronic address: mnjs100@cam.ac.uk.
authors.forename: Matthew N J
authors.initials: MN
authors.lastname: Seaman
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D016827:CD8 Antigens / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D017527:Receptor, IGF Type 2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D058305:Sorting Nexins / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D016827:CD8 Antigens; C429024:GOLPH3 protein, human; D008565:Membrane Proteins; D017527:Receptor, IGF Type 2; C513978:SNX1 protein, human; D058305:Sorting Nexins; C498296:VPS26A protein, human; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins
keywords: Antibody-uptake assay; Endosome; Native immunoprecipitation; Quantitative imaging; Retromer