A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

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.

Transbilayer lipid distribution in nano scale

“…The molecular mechanism of the distribution of SM in the inner leaflet of the plasma membranes from HSF and neutrophils is obscure. However, recent results using equinatoxin-EGFP as a SM probe suggest the exposure of SM to the cytoplasmic side of cis-Golgi compartment in fibroblasts (Bakrac et al, 2010). These results suggest that the exposure of SM to the inner leaflet of the plasma membranes in HSF is dependent on the active de novo biosynthesis of SM, which is defective in RBC.…”

Molecular recognition of a single sphingolipid species by a protein’s transmembrane domain

“…6b). In addition, in vivo analyses employing the SM-binding toxin Equinatoxin II point to an occurrence of SM in the cytoplasmic leaflet of the Golgi membrane 14 .…”

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

“…Cell lysates were prepared by resuspending the pellet in binding buffer consisting of 20 mM sodium phosphate buffer (pH 7.4) containing 500 mM NaCl, 20 mM imidazole, and protease inhibitor cocktail set I (Calbiochem), followed by domains ( 15 ). Several nontoxic mutants of these toxins that specifi cally bind either SM (16)(17)(18) or Chol ( 19 ) have been employed to study the distribution, dynamics, and function of SM/Chol domains. Because both sphingolipids and Chol are required to assemble lipid rafts ( 6 ), a protein that binds to the SM/Chol complex would be a very useful tool to study lipid microdomain organization in cell membranes.…”