Topology of glucosylceramide synthesis in Golgi membranes from porcine submaxillary glands

Coste, Hervé; Martel, M.B.; Got, R.

doi:10.1016/0005-2736(86)90285-3

Cited by 137 publications

(77 citation statements)

References 24 publications

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“…near the N-terminus (amino acids 11-32) of GCS was suggested to be a transmembrane domain [17]. We recently showed that the carboxy tail and a hydrophilic loop near the putative transmembrane domain of GCS are accessible to the cytosol [18], consistent with previous reports by us and others that the active site of GCS is on the cytosolic face of the Golgi membrane [19][20][21]. No studies have been performed, however, which identify the active site or substrate-binding regions of GCS.…”

Section: Nitrobenzo-2-oxa-13-diazole)]-6-aminohexanoyl-d-erythro-sphsupporting

confidence: 89%

Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study

Marks

Watanabe

et al. 1999

Biochemical Journal

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Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor of most higher-order glycosphingolipids. Inhibition of GCS activity has been proposed as a possible target of chemotherapeutic agents for a number of diseases, including cancer. Design of new GCS inhibitors with desirable pharmaceutical properties is hampered by lack of knowledge of the secondary structure or catalytic mechanism of the GCS protein. Thus we cloned the rat homologue of GCS to begin studies to identify its catalytic regions. The histidinemodifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat GCS expressed in bacteria ; this inhibition was rapidly reversible by hydroxylamine and could be diminished by preincubation of GCS with UDP-Glc. These data suggest that DEPC acts on histidine residues within or near the UDP-Glcbinding site of GCS. Mutant proteins were expressed in which

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Section: Nitrobenzo-2-oxa-13-diazole)]-6-aminohexanoyl-d-erythro-sphsupporting

confidence: 89%

Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study

Marks

Watanabe

et al. 1999

Biochemical Journal

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“…Using BFA as a tool to inspect the subcellular localization of SM biosynthesis, we demonstrated that this compound exerts a dual action on ceramide metabolic processing, consisting of a striking increase in Glc-Cer and a marked reduction of SM biosynthesis in both quiescent and bFGF-treated astrocytes. Since BFA disrupts the Golgi apparatus, resulting in the redistribution of the cis,trans-Golgi stacks in the endoplasmic reticulum (32), the increase in Glc-Cer upon BFA treatment is in agreement with the cis,medial-Golgi stack location of the glucosyltransferase involved in its biosynthesis (53)(54)(55). On the other hand, the inhibition of SM biosynthesis by BFA in astrocytes is in contrast with the cis-Golgi as the major location of SM synthase (38 -44).…”

Section: Discussionmentioning

confidence: 78%

Basic Fibroblast Growth Factor-induced Proliferation of Primary Astrocytes

Riboni

Viani

Bassi

et al. 2001

Journal of Biological Chemistry

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We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137-145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from either L-[ 3 H]serine or [ 3 H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.Ceramide, a key sphingolipid metabolite in both the biosynthesis and degradation of complex sphingolipids, is involved in the signal transduction of different extracellular stimuli that lead to cell proliferation, cell differentiation, cell cycle arrest, and apoptotic cell death (reviewed in Refs. 1-3). In all these events, the ceramide concentration, possibly at specific subcellular sites, is crucial. There is evidence that different activators such as cytokines, growth factors, and hormones elicit their biological effect by modulating the activity of sphingomyelinases, ceramide synthase, or neutral ceramidase (reviewed in Refs. 1 and 3-5) and thus affect ceramide levels.A role of ceramide as an intracellular mediator of specific extracellular agents has been recognized also in cells from the central nervous system (reviewed in Refs. 6 -8). In neuronal and glial cells, the administration of differentiating or apoptotic agents results in increased cellular levels of ceramide, which, in turn, participate in the cascade of events producing the final effects (9 -12). In a recent study (13), we demonstrated that ceramide plays a role in the growth control of glial cells by basic fibroblast growth factor (bFGF), 1 a factor stimul...

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“…Our preferred interpretation of this observation is that cytosol is required for intermembrane transport of Cer from the ER to the Golgi compartment for SM synthesis. However, because catalytic sites of SM synthase and GlcCer synthase have been suggested to exist in the lumenal and cytoplasmic sides, respectively, of the Golgi complex (25)(26)(27)(28)(29), there is the alternative interpretation that cytosol is required for transport of Cer across the Golgi membrane but that cytosol-independent transbilayer movement of Cer occurs in the merged organelle formed by BFA treatment. Unfortunately, the effects of BFA treatment on transbilayer movement of natural Cer are unknown.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of ATP- and Cytosol-dependent Transport of de Novo Synthesized Ceramide to the Site of Sphingomyelin Synthesis in Semi-intact Cells

Funakoshi

Yasuda

Fukasawa

et al. 2000

Journal of Biological Chemistry

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Topology of glucosylceramide synthesis in Golgi membranes from porcine submaxillary glands

Cited by 137 publications

References 24 publications

Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study

Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study

Basic Fibroblast Growth Factor-induced Proliferation of Primary Astrocytes

Reconstitution of ATP- and Cytosol-dependent Transport of de Novo Synthesized Ceramide to the Site of Sphingomyelin Synthesis in Semi-intact Cells

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