Interaction of nSMase2 with APLs. A, schematic diagram of nSMase2-GFP in pEGFP-N2 and nSMase2-FLAG in pYES2 expression vectors. Mouse nSMase2 was fused to GFP or FLAG tag at the C terminus. B, HEK293 cells were transfected with pEGFP vector or an expression construct of nSMase2-GFP. The immunoblot analysis of nSMase2-GFP and GFP was performed with anti-GFP antibody as described under “Experimental Procedures.” C, lipid-protein overlay assay showed nSMase2 binding to immobilized lipids. Equimolar amounts of lipids were immobilized on nitrocellulose membranes and probed with HEK293 cell lysates. The lipid-protein binding was identified by immunostaining with an anti-GFP monoclonal antibody. The following dioleoyl-lipids were immobilized on the membrane as indicated: CL, PA, PC, PE, PG, PI, PS, D, the immunoblot analysis of nSMase2-FLAG expressed in yeast was performed with anti-FLAG antibody. nSMase2-FLAG was expressed in ISC1Δ S. cerevisiae and detected as described under “Experimental Procedures”. E, lipid-protein overlay assay showed nSMase2 expressed in yeast binding to immobilized lipids. F, interactions between APLs and immunoprecipitated nSMase2-FLAG. The nSMase-FLAG expressed in the yeast was immunoprecipitated as described under “Experimental Procedures.” The same percentage of the supernatant of immunoprecipitation (lane 1) or eluted nSMase2 (lane 2) was analyzed by Western blot analysis (upper panel). Lipid-protein overlay assays were performed using eluted nSMase2-FLAG (lower panel). G, competition overlay assays were performed. 50 μm PA (middle panel) or (right panel) PS was incubated with lysates overexpressing nSMase2-FLAG for 1 h prior to performing the overlay assays. The lysate together with PA or PS was then added to the blot for lipid-protein overlay assay. The lipid binding signals were detected using an antibody against FLAG. The results are representative experiments of at least two independent experiments.

nSMase2 N-terminal domain is essential for nSMase interaction with APLs. A, schematic diagram of the four mutants of nSMase2. The nSMase2 mutants were fused to GFP at the C terminus. B, immunoblot analysis of deletion mutants of nSMase2 with anti-GFP antibody. Lane 1, GFP; lane 2, nSMase2 (aa1–123)-GFP; lane 3, nSMase2(Δ12–123)-GFP. C, lipid-protein overlay assay showing the binding capabilities of various nSMase2 constructs to APLs. D, immunoblot analysis of deletion mutants of nSMase2 with anti-GFP antibody. Lane 1, nSMase2 (aa 1–52)-GFP; lane 2, nSMase2 (aa 1–123/Δ12–59)-GFP; lane 3, nSMase2 (aa 1–123)-GFP. E, lipid-protein overlay assay showing the binding capabilities of various nSMase2 constructs to APLs. The results are representative experiments of at least two independent experiments.

Identification of the first APL binding determinants of N terminus mutants of nSMase. A, a series of GFP-tagged deletion or point mutants were made from mutants within aa 1–59 in nSMase2 tagged by GFP at C terminus. Schematic diagram of deletion mutants is shown in the left panel. The APL binding signal on the fat blot was examined by incubation with HEK293 lysates expressing the truncated or point mutants of nSMase2 (aa1–59)-GFP. B, immunoblot analysis of expression of the deletion mutants of nSMase2 with anti-GFP antibody. C, densitometry analyses were performed using ImageQuant TL software (GE Healthcare). The relative value of each lipid-protein interaction signal was calculated by subtracting the background and normalized to its western signals from the upper band (the correct sized band). Similar results were obtained in two separate experiments.

Effects of PS and PA on nSMase2 enzymatic activity. FLAG-tagged nSMase2 was expressed in isc1Δ S. cerevisiae and analyzed as described under “Experimental Procedures.” A, N-SMase activity of nSMase2 was measured at various concentrations of PS. B, N-SMase activity of nSMase2 was measured at various concentrations of PA. C-D, PS effects on kinetics of nSMase2 activity. Under three various PS concentrations (1.6, 3.2, and 6.5 mol%), enzymatic kinetics for N-SMase activity were analyzed using 0.8–25.8 mol % SM as substrate. E-F, PA effects on kinetics of nSMase2 activity. Under different PA concentrations (0.2, 0.4, and 0.8 mol%), enzymatic kinetics for N-SMase activity were analyzed. The data are the averages of duplicates. The values are expressed as the mean ± S.D. The data are representative of at least two independent experiments.

Effects of mutations of the APL binding domains on lipid binding capability of full-length nSMase2. A, several mutants of full-length nSMase2 were generated, including nSMase2(R33A/Δ45–48)-FLAG (nSM2-APL1 mutant), nSMase2(R92A/R93A)-FLAG (nSM2-APL2 mutant) and nSMase2(R33A/R92A/R93A/Δ45–48)-FLAG (combined mutant). The wild type and mutated nSMase2 were transformed and expressed in isc1Δ S. cerevisiae cells. The APL binding signal on the fat blot was examined by incubation with cell lysates expressing nSMase2-FLAG and its mutants. B. immunoblot analysis of nSMase2 and its mutants were performed using anti-FLAG antibody. The data are representative of at least two independent experiments.

Effects of the mutations in APL binding domains of full-length nSMase2 on enzymatic activity. The wild type and mutated nSMase2 were transformed and expressed in isc1Δ cells, and the cell lysates were subjected to N-SMase activity assay as described in “Experimental Procedures.” A, effects of mutations in APL binding domains on PS activation of nSMase2. B, effects of mutations in APL binding domains on PS activation of nSMase2. The data are the averages of duplicates. The values are expressed as the mean ± S.D. The data are representative of at least two independent experiments.

APL binding domains are important for nSMase2 to localize at the PM. HEK293 cells were transfected with nSMase-V5 and mutants (R33A/Δ45–48, R92A/R93A and R33A/Δ45–48/R92A/R93A) expression vectors. After 24 h, the cells were fixed and co-stained with an antibody against calreticulin (red, upper panel) and antibody against V5 (green, middle panel) for nSMase2 signal and then subjected to confocal microscopy evaluation. The colocalization signals were observed as yellow or orange (lower panel). The data are representative of at least two independent experiments.

APL binding domain mutants fail to correct the defect of isc1Δ S. cerevisiae cells. nSMase2 APL binding domain mutant of nSMase2 (R33A/Δ45–48/R92A/R93A), nSMase2, ISC1, or its nonfunctional mutant ISC1-K168A was expressed in isc1Δ S. cerevisiae cells, respectively. Exponentially growing cells were first diluted to an A₆₀₀ of 0.3, and then serially diluted and spotted onto agar Raf/Galactose uracil minus plates with or without 10 mg/ml hydroxyurea (HU). The plates were incubated for 3–4 days at 30 °C. This experiment was performed two times with similar results.