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A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

Lookup NU author(s): Dr Gavin McHaffie, Dr Andreas Werner, Professor Jeremy LakeyORCiD

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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.

Publication metadata

Author(s): Bakrač B, Kladnik A, Maček P, McHaffie G, Werner A, Lakey JH, Anderluh G

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2010

Volume: 285

Issue: 29

Pages: 22186-22195

Print publication date: 12/05/2010

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology, Inc.

URL: http://dx.doi.org/10.1074/jbc.M110.105122

DOI: 10.1074/jbc.M110.105122

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