A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin |
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| Authors: | Biserka Bakra? Ale? Kladnik Peter Ma?ek Gavin McHaffie Andreas Werner Jeremy H Lakey Gregor Anderluh |
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| Institution: | From the ‡Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna Pot 111, 1000 Ljubljana, Slovenia.;the §Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom, and ;the ¶Nottingham Renal Unit, Nottingham University Hospitals NHS Trust, Nottingham NG5 1PB, United Kingdom |
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| Abstract: | Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles. |
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| Keywords: | Golgi Lipid Membrane Bilayer Membrane Lipids Plasma Membrane |
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