Drosophila Glucosylceramide Synthase

Abstract

Glucosylceramide synthase (GlcT-1) catalyzes the formation of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs), from ceramide and UDP-glucose. Ceramide and its metabolites, such as sphingosine-1-phosphate, are now known to be important mediators of apoptosis and cell survival. Recently, we have shown that GlcT-1 functions to regulate intracellular ceramide levels via glycosylation of ceramide. In this study, we employ the fruit fly Drosophila melanogaster as a model system for understanding the in vivo roles of GlcT-1. We isolated and characterized a GlcT-1 homologue (DGlcT-1) from Drosophila. When DGlcT-1 was expressed in GM-95 cells deficient in GSLs (because of the absence of GlcT-1 activity), these cells regained the ability to synthesize GSLs. Northern blot and in situ hybridization analyses revealed that the expression of DGlcT-1 mRNA was ubiquitous throughout development, suggesting that DGlcT-1 is important for development and differentiation. Indeed, RNA interference experiments demonstrated that the loss of GlcT-1 function enhances apoptotic cell death. Conversely, targeted expression of GlcT-1 partially rescued cell death caused by the proapoptotic factors Reaper and Grim, suggesting that ceramide generation might be one signal pathway that executes the cell death program. We also found that GlcT-1 localized not only in the Golgi apparatus but also in the perinuclear endoplasmic reticulum, providing the first visual evidence of GlcT-1 in membranes. These results indicate that GlcT-1 might down-regulate ceramide generated in these membranes.Ceramide and its metabolites, such as sphingosine-1-phosphate, have emerged as novel second messengers for intracellular signaling pathways that are involved in regulation of diverse cellular responses to exogenous stimuli (1-3).

c-Fos Activates Glucosylceramide Synthase and Glycolipid Synthesis in PC12 Cells

“…6A, lower row) and with the Golgi marker (Fig. 6A, upper row) as already shown (26). Endogenous c-Fos was found clearly colocalizing with GlcCerS (Fig.…”

“…A key step in glycolipid synthesis is the formation of GlcCer, the first glycosylated intermediate. This synthesis is catalyzed by GlcCerS, an ER and Golgi (26,33,34) or Golgi membraneconcentrated enzyme (35,36). Inhibition of GlcCerS with the specific inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol results in glycosphingolipid depletion and reduced growth of neuroblastoma (37) and PC12 cells (38) and axonal growth of hippocampus (39) and cerebral cortical neurons (40) in culture.…”

Recycling of Sphingosine Is Regulated by the Concerted Actions of Sphingosine-1-phosphate Phosphohydrolase 1 and Sphingosine Kinase 2

“…A certain pool of ceramide molecules might be selectively translocated to a Golgi sub-fraction for glucosylceramide synthesis by a vesicular and non-CERT pathway (41). Another possibility is that ceramide is converted to hexosylceramides in the ER without inter-organelle transfer, as galactosylceramide synthase is located in the ER, and it has been suggested that de novo synthesis of glucosylceramide takes place not only in the cis-Golgi but also in a sub-region of the ER (42)(43)(44). Interestingly, it has been shown previously that glycosphingolipids are synthesized predominantly from sphingosine salvaged from the lysosomal pathway in slowly dividing cells, whereas in rapidly dividing cells, the need for increased synthesis is met by up-regulation of the de novo pathway (45).…”

Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis

“…A first clue has come from the finding that the synthesis of SM but not GlcCer depended on ceramide transport to the trans-Golgi by ceramide transport protein (CERT; Hanada et al, 2003), a pathway that is regulated via phosphoinositides, sterols, and CERT phosphorylation (Perry and Ridgway, 2006). However, both SMS1 and GCS have been localized biochemically to the cis-medial Golgi, whereas GCS has also been assigned to pre-Golgi and trans-Golgi membranes (Futerman et al, 1990; Jeckel et al, 1990, 1992; Futerman and Pagano, 1991; Kohyama-Koganeya et al, 2004). The current agreement is that GlcCer is synthesized on a cytosolic surface and translocates across the Golgi membrane for higher GSL synthesis in the late Golgi (Lannert et al, 1994, 1998).…”