title: Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.
abstract: Previous studies provide evidence for an endocytic mechanism in mammalian cells that is distinct from both clathrin-coated pits and caveolae, and is not inhibited by overexpression of GTPase-null dynamin mutants. This mechanism, however, has been defined largely in these negative terms. We applied a ferro-fluid-based purification of endosomes to identify endosomal proteins. One of the proteins identified in this way was flotillin-1 (also called reggie-2). Here, we show that flotillin-1 resides in punctate structures within the plasma membrane and in a specific population of endocytic intermediates. These intermediates accumulate both glycosylphosphatidylinositol (GPI)-linked proteins and cholera toxin B subunit. Endocytosis in flotillin-1-containing intermediates is clathrin-independent. Total internal reflection microscopy and immuno-electron microscopy revealed that flotillin-1-containing regions of the plasma membrane seem to bud into the cell, and are distinct from clathrin-coated pits and caveolin-1-positive caveolae. Flotillin-1 small interfering RNA (siRNA) inhibited both clathrin-independent uptake of cholera toxin and endocytosis of a GPI-linked protein. We propose that flotillin-1 is one determinant of a clathrin-independent endocytic pathway in mammalian cells.
authors.affiliation: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
authors.forename: Oleg O
authors.initials: OO
authors.lastname: Glebov
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Bright
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Nichols
mesh_terms: D000818:Animals; D019556:COS Cells; D022461:Caveolins / Q000378:metabolism; D002522:Chlorocebus aethiops; D002772:Cholera Toxin / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D034285:Dynamin II / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007295:Inositol Phosphates / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D021381:Protein Transport; D034622:RNA Interference; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
chemical_list: D022461:Caveolins; D002966:Clathrin; D007295:Inositol Phosphates; D008565:Membrane Proteins; D014168:Transferrin; C106354:flotillins; C014575:glycerylphosphoinositol; D002772:Cholera Toxin; D034285:Dynamin II