Figure 6. caCer-1 is a light-sensitive substrate of glucosylceramide synthase GCS.

(a) Lysates of yeast cells transfected with V5-tagged GCS or empty vector (EV; control) were analyzed by immunoblotting using anti-V5 and anti-Vti1p antibodies. (b) UV-A pretreated caCer-1 (25 μM) was incubated with lysates of control or GCS-expressing yeast cells for 30 min at 37°C in the presence or absence of 1 mM UDP-glucose. Reactions were subjected to lipid extraction, click-reacted with Alexa-647 and then analyzed by TLC. (c) UV-A, blue or dark pretreated caCer-1 and cCer (25 μM, each) were incubated in the dark for 30 min at 37°C with lysates of GCS-expressing yeast in the presence of 1 mM UDP-glucose and analyzed as in (b). (d) Quantitative analysis of GlcCer formed from caCer-1 or cCer in lysates treated as in (c). (e) Dark-adapted caCer-1 or cCer (25 μM, each) and UDP-glucose (1 mM) were incubated with lysates of GCS-expressing yeast in the dark at 37°C. After 15 min, lysates were flash-illuminated by blue light followed by UV-A or vice versa and then incubated for another 20 min. Lysates kept in the dark for the entire incubation period served as control. GlcCer formation was determined by TLC analysis of total lipid extracts click-reacted with Alexa-647. (f) Cell-free translation reactions with or without GCS-V5 mRNA were carried out overnight in the dark at 26°C. GCS was expressed in the presence of liposomes containing caCer-1 or cCer and then analyzed by immunoblotting using an anti-V5 antibody. (g) GCS produced cell-free as in (f) was flash-illuminated with UV-A or kept in the dark and then incubated with 1 mM UDP-glucose for 30 min at 37°C in the dark. GlcCer formation was monitored by TLC as in (e). Data shown are average values ± s.d. (cCer, n = 2; caCer-1, n = 3).