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. 2019 Feb 5;8:e43230. doi: 10.7554/eLife.43230

Figure 3. caCers are light-sensitive substrates of sphingomyelin synthase SMS2.

(a) Blue, UV-A or dark-adapted caCers were incubated with lysates of control or SMS2-expressing yeast cells for 30 min at 37°C and their metabolic conversion to SM was determined by TLC analysis of total lipid extracts click-reacted with Alexa-647. (b) Lysates of yeast cells transfected with empty vector (EV) or V5-tagged SMS2 were analyzed by immunoblotting with an anti-V5 antibody. (c) Lysates of control (EV) and SMS2-expressing yeast cells were incubated with caCers or cCer as outlined in (a). Reaction samples were subjected to lipid extraction, click-reacted with Alexa-647 and analyzed by TLC. (d) SMS2 was produced cell-free in the dark at 26°C in the presence of caCer-containing liposomes and then incubated at 37°C in the dark or upon illumination with blue or UV-A light. Reaction samples were subjected to lipid extraction, click-reacted with Alexa-647 and analyzed by TLC. (e) Cell-free translation reactions with or without SMS2-V5 mRNA were analyzed by immunoblotting with an anti-V5 antibody. (f) SMS2 was produced cell-free in the presence of caCer-1 or cCer-containing liposomes and then incubated for 0 or 60 min at 37°C as outlined in (d). Reaction samples were subjected to lipid extraction, click-reacted with Alexa-647 and analyzed by TLC.

Figure 3.

Figure 3—figure supplement 1. Monitoring SMS2-mediated metabolic conversion of caCers and cCer using 3-azido-7-hydroxycoumarin as click reagent.

Figure 3—figure supplement 1.

(a) Blue, UV-A or dark-adapted caCers and cCer were incubated with lysates of control (EV) or SMS2-expressing yeast cells for 30 min at 37°C and their metabolic conversion to SM was determined by TLC analysis of lipid extracts click-reacted with 3-azido-7-hydroxycoumarin. Imaging of fluorescent lipids with UV-trans-illumination indicated that the azobenzene moiety in caCers causes a distance-dependent intramolecular quenching of the coumarin fluorescence. (b) Chemical structures of coumarin-derivatized caCers and cCer, with the azobenzene and coumarin moieties marked in red and blue, respectively.
Figure 3—figure supplement 2. Monitoring SMS2-mediated metabolic conversion of caCers independently of fluorescent click-reagents.

Figure 3—figure supplement 2.

Blue, UV-A or dark-adapted caCers were incubated with lysates of control (EV) or SMS2-expressing yeast cells for 30 min at 37°C and their metabolic conversion to SM was determined by TLC analysis. Azobenzene-containing lipids were visualized by UV-trans-illumination, taking advantage of the UV-A absorbing properties of the azobenzene group.