title: Determining membrane protein topologies in single cells and high-throughput screening applications.
abstract: Correct localization and topology are crucial for a protein's cellular function. To determine topologies of membrane proteins, a new technique, called fluorescence protease protection (FPP) assay, has recently been established. The sole requirements for FPP are the expression of fluorescent-protein fusion proteins and the selective permeabilization of the plasma membrane, permitting a wide range of cell types and organelles to be investigated. Proteins topologies in organelles like endoplasmic reticulum, Golgi apparatus, mitochondria, peroxisomes, and autophagosomes have already been determined by FPP. Here, two different step-by-step protocols of the FPP assay are provided. First, we describe the FPP assay using fluorescence microscopy for single adherent cells, and second, we outline the FPP assay for high-throughput screening applications.
authors.affiliation: National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Wunder
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lippincott-Schwartz
authors.forename: Holger
authors.initials: H
authors.lastname: Lorenz
mesh_terms: D000818:Animals; D002460:Cell Line; D003584:Cytological Techniques; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D010447:Peptide Hydrolases / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis
chemical_list: D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases