Abstract
Previous studies provide evidence for an endocytic mechanism in mammalian cells that is distinct from both clathrin-coated pits and caveolae1,2,3,4,5, and is not inhibited by overexpression of GTPase-null dynamin mutants1,2,3,4,6. 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)7,8. 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 subunit4,9. 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 caveolae10. 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.
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Acknowledgements
H. Pelham and S. Munro provided criticism of the manuscript. S.-Y. Peak-Chew and F. Begum carried out protein identification by mass spectrometry. D. Bowser and B. Khakh set up the TIR microscope and assisted with its use. J. Skepper carried out preparation of samples for electron microscopy and assisted in their analysis.
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Glebov, O., Bright, N. & Nichols, B. Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells. Nat Cell Biol 8, 46–54 (2006). https://doi.org/10.1038/ncb1342
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DOI: https://doi.org/10.1038/ncb1342
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