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.

FIGURE 1.

SM-dependent activity of EqtII-GFP. A, SPR analysis of EqtII-GFP binding to SM:DOPC liposomes (black) and DOPC liposomes (gray) immobilized on the surface of L1 Biacore sensor chip to ∼10000 RU. The concentration of EqtII-GFP was 100 nm. The inset shows the amount of stably bound EqtII-GFP in comparison to the EqtII after 5 min of dissociation, as determined from the sensorgrams. Black columns, SM:DOPC liposomes; gray columns, DOPC liposomes. n = 3–10; average ± S.D. B, insertion of the EqtII-GFP in lipid monolayers composed of DOPC:SM (black) and DOPC (gray). The concentration of EqtII-GFP was 0.2 μm. The inset shows the increase in the surface pressure at the equilibrium. Black columns, SM:DOPC monolayer; gray columns, DOPC monolayer. n = 2; average ± S.D. C, permeabilization of calcein-loaded LUVs by EqtII-GFP. LUVs were composed of DOPC:SM (black) and DOPC (gray). LUVs were at 20 μm lipid concentration. The lipid/toxin ratio used is indicated. D, binding of EqtII-GFP to the MDCK II cells. Cells, seeded on coverslips, were incubated with 0.3 μg of the proteins in PBS for 15 min at 4 °C. After washing, cells were fixed with 3% paraformaldehyde and plasma membrane was stained with R-WGA. E, binding of the EqtII-GFP to MDCK II cells plated on 12-mm clear Transwell filters (Costar). EqtII-GFP (0.5 μg in PBS) was added separately to the apical and to the basolateral side of the membrane. Plasma membrane was stained with R-WGA. Scale bar is 20 μm.

FIGURE 2.

Subcellular distribution of EqtII-GFP and its mutants or homologue protein. MDCK II cells were transfected with DNA constructs encoding EqtII, its mutants or a homologue from zebrafish, Dr1. Proteins used in this experiment are presented schematically in the top row. The structure of EqtII shows the position of the residues that have been changed in mutant proteins. The residues that participate in POC binding are labeled green (17), whereas residues responsible for SM specificity, Trp-112 and Tyr-113 (16), are shown in blue. The position of SM head group is denoted by sticks and surface presentation. Residues at position 8 and 69, which were replaced with cysteines, are labeled pink. The Dr1 region homologous to EqtII is shown by dots. The plasma membrane is stained with R-WGA. Scale bar is 20 μm.

FIGURE 3.

Binding of EqtII-GFP and EqtII to liposomes composed of the inner leaflet mixture. Experimental conditions were as described in Fig. 1A. A, binding of EqtII-GFP to liposomes composed from inner leaflet mixture (DOPC:DPPE:DPPS:CHO:DOPI in the molar ratio 30.5:13.5:19.5:35.5:1) and various % of SM as indicated. B, amount of stably bound protein after 4 min of dissociation. Open circles, EqtII-GFP; solid circles, native EqtII. n = 2–7; average ± S.D.

FIGURE 4.

Subcellular localization of EqtII-GFP. MDCK II cells were transfected with plasmid encoding EqtII-GFP and co-stained with propidium iodide (marker for nucleus) or co-transfected with pDsRed2-ER (marker for endoplasmic reticulum), or pDsRed2-Mito (marker for mitochondria). Scale bar is 20 μm.

FIGURE 5.

Colocalization of EqtII-GFP with BODIPY-TR ceramide. MDCK II cells and NIH 3T3 fibroblasts (FB) transfected with EqtII-GFP or EqtII Y113A-GFP and co-stained with Golgi-specific marker, Bodipy-TR-ceramide (BODIPY). MDCK cells expressing the wild-type EqtII were also treated with 25 μg/ml of D609, while fibroblasts expressing the wild-type EqtII were also treated with BFA at final 50 μg/ml. Scale bar is 20 μm. The colocalization panels show in yellow color the sites where the pixel intensities in both channels are above the calculated threshold values. The framed area is also shown enlarged to reveal details. The BODIPY-GFP scatter-graphs represent the relationship between the intensity of fluorescence in both channels at the same pixel locations in the two channel images. The x axis represents the intensity values 0–255 in the red channel, and the y axis represents the intensity values in the green channel, while the color of the dots represents the frequency of pixels with a given combination of intensities in both channels. The straight white lines represent the automatically calculated threshold values by the method of Costes et al. (25) with the “Colocalisation Threshold” plugin for ImageJ.

FIGURE 6.

Distribution of thresholded Mander's coefficients (tM) of colocalization of EqtII-GFP or EqtII Y113A-GFP and Bodipy-TR-ceramide. The MDCK cells were also analyzed in the presence of 25 μg/ml of D609, while the NIH 3T3 fibroblasts (FB) were also analyzed in the presence of 50 μg/ml BFA. 0 means no colocalization, 1 is perfect colocalization. The average (shown by cross) ± S.D. is also presented. The differences between groups were tested using t test. *, p < 0.05; **, p < 0.01; n.s., not significant.

FIGURE 7.

Subcellular distribution of EqtII-GFP. Cell lysate of NIH 3T3 fibroblasts expressing EqtII-GFP was fractionated by centrifugation on a discontinuous Optiprep gradient and the resulting fractions were immunoblotted and assayed for enzyme activities as indicated. The value in a particular fraction is expressed as percentage to the sum of the enzyme activity in all fractions. Average ± S.D. is shown of two independent fractionations. A, refraction index (solid circles) was measured to check the gradient. Protein content (open circles) was measured by using Bradford reagent. B, markers for plasma membrane (flotillin-2 and K⁺-(p-nitrophenyl phosphatase)). C, markers for ER (calnexin and cytochrome c reductase). D, markers for GA (TGN38, GM130 and mannosidase II). E, Eqt-GFP. The summary of distribution of various markers presented in B–D is shown below schematically.