title: Methods to discriminate the distribution of acidic glycohydrolases between the endosomal-lysosomal systems and the plasma membrane.
abstract: The endosomal-lysosomal system plays important roles in cellular physiology. Beyond the well-known function as terminal degradative compartment, necessary to maintain the health of the cell, lysosomes are critical for many other cellular processes, such as termination of signaling mediated by cell surface receptors and processing of internalized peptides in antigen-presenting cells. Moreover, the intracellular membrane trafficking related to the endosomal-lysosomal system plays a pivotal role in diverse physiological and pathological processes, such as exocytosis, plasma membrane repair, and endocytosis. Increasing evidences suggest that several lysosomal glycohydrolases, together with nonlysosomal glycohydrolases, are associated with cell membranes in their active form, and they are localized into lipid microdomains. The role of these forms in physiological and pathological conditions, such as differentiation and aging, neurodegenerative diseases, and cancer spreading, is under investigation. Here we provide general methods to purify lipid microdomain proteins and to discriminate cell surface lipid microdomains-associated glycohydrolases from those not exposed on cell surface. The methods reported here have been developed to characterize the membrane-associated forms of the acidic glycohydrolases β-hexosaminidase and β-galactosidase, but they may be applied to any other protein of interest.
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, Section of Biochemistry and Molecular Biology, University of Perugia, Perugia, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Magini
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, Section of Biochemistry and Molecular Biology, University of Perugia, Perugia, Italy.
authors.forename: Alice
authors.initials: A
authors.lastname: Polchi
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, Section of Biochemistry and Molecular Biology, University of Perugia, Perugia, Italy.
authors.forename: Brunella
authors.initials: B
authors.lastname: Tancini
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, Section of Biochemistry and Molecular Biology, University of Perugia, Perugia, Italy.
authors.forename: Lorena
authors.initials: L
authors.lastname: Urbanelli
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, Section of Biochemistry and Molecular Biology, University of Perugia, Perugia, Italy.
authors.forename: Manlio
authors.initials: M
authors.lastname: Di Cristina
authors.affiliation: IMAGE Analysis Laboratory, University of Perugia, Perugia, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Mannucci
authors.affiliation: IMAGE Analysis Laboratory, University of Perugia, Perugia, Italy.
authors.forename: Ildo
authors.initials: I
authors.lastname: Nicoletti
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, Section of Biochemistry and Molecular Biology, University of Perugia, Perugia, Italy. Electronic address: emiliani@unipg.it.
authors.forename: Carla
authors.initials: C
authors.lastname: Emiliani
mesh_terms: D019921:Biotinylation; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D011992:Endosomes / Q000737:chemistry* / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008247:Lysosomes / Q000737:chemistry* / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D049452:Green Fluorescent Proteins; D005677:G(M1) Ganglioside; D001616:beta-Galactosidase; D001619:beta-N-Acetylhexosaminidases
keywords: Gangliosides; Glycohydrolases; Lipid microdomains; Lysosomes; Plasma membrane