Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide

Phosphoinositides mediate a large number of signaling processes in mammalian cells. Here, we report that phophatidylinositol-4-phosphate (Ptdlns(4)P) downregulates the cellular glucosylceramide (GlcCer) level by inhibiting the interaction between GlcCer synthase (UGCG) and UDP-glucose in the Golgi apparatus. In this study, we used two PH domain probes to bind phosphoinositides; one derived from FAPP1 for targeting to the Golgi Ptdlns(4)P and the other from PLC delta for targeting to the plasma membrane Ptdlns(4,5)P-2. The levels of GlcCer and lactosylceramide, but not of sphingomyelin (SM), were increased following expression of the FAPP1 PH domain in cells, accompanied by an increase in UGCG activity. However, no elevated GIcCer level was observed after expression of the PLC delta PH domain. Ptdlns(4)P inhibited UGCG activity, but not SMS activity, in a concentration-dependent manner, and UGCG activity was restored by the addition of UDP-glucose in the reaction mixture. These results indicate that Ptdlns(4)P inhibits UGCG activity by competing with UDP-glucose. We conclude that the increase in UGCG activity due to the expression of the FAPP1 PH domain was caused by an attenuation of the inhibitory effect of Ptdlns(4)P on UGCG. This study provides new insights into the regulation of GlcCer synthesis by Ptdlns(4)P in the Golgi apparatus. (C) 2018 Elsevier Inc. All rights reserved.