A toxin-based probe reveals cytoplasmic exposure of Golgi sphingomyelin.
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.
Bakrac
Biserka
B
Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna Pot 111, 1000 Ljubljana, Slovenia.
Kladnik
Ales
A
Macek
Peter
P
McHaffie
Gavin
G
Werner
Andreas
A
Lakey
Jeremy H
JH
Anderluh
Gregor
G
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism; D003064:Cnidarian Venoms / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004285:Dogs; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D051379:Mice; D015335:Molecular Probes / Q000378:metabolism*; D041681:NIH 3T3 Cells; D011162:Porphobilinogen / Q000031:analogs & derivatives / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
C538930:3-anilino difluoroboron dipyrromethene; D002518:Ceramides; D003064:Cnidarian Venoms; D008081:Liposomes; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D013109:Sphingomyelins; D049452:Green Fluorescent Proteins; C012307:equinatoxin; D011162:Porphobilinogen
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