Functional Organization of the Golgi Apparatus in Glycosphingolipid Biosynthesis

Lannert, H.; Gorgas, Karin; Meißner, Ingrid; Wieland, Felix; Jeckel, D

doi:10.1074/jbc.273.5.2939

Cited by 119 publications

(42 citation statements)

References 80 publications

(92 reference statements)

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“…15 and 38). These findings have been con- firmed to some extent by biochemical and immunohistochemical studies that, for example, localized SAT I to the medial and transGolgi cisternae (50,51).…”

Section: Regulationmentioning

confidence: 95%

Combinatorial Ganglioside Biosynthesis

Kolter¹,

Proia

Sandhoff

2002

Journal of Biological Chemistry

290

212

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Section: Regulationmentioning

confidence: 95%

Combinatorial Ganglioside Biosynthesis

Kolter¹,

Proia

Sandhoff

2002

Journal of Biological Chemistry

290

212

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“…On the other hand, results of in itro labelling of endogenous glycolipid acceptors bound to the Golgi membranes indicate that the activities for synthesis of simple gangliosides extend also to the distal [trans-Golgi network (TGN)] Golgi, and seem to depend on the provision of glucosylceramide (GlcCer) from the proximal Golgi, via vesicular transport [13]. Results of subfractionation of the Golgi complex by centrifugation procedures also point to an enrichment [14,15], or to an almost complete segregation [16], of GalNAc-T to the distal Golgi. However, direct examination of the sub-Golgi location of this enzyme by immunoelectron microscopy in Chinese hamster ovary (CHO) cells transfected with a c-myc-tagged form of GalNAc-T revealed its presence also in the proximal Golgi [17].…”

Section: Introductionmentioning

confidence: 99%

GA2/GM2/GD2 synthase localizes to the trans-Golgi network of CHO-K1 cells

Giraudo

Fritz

Maccioni

1999

Biochemical Journal

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U D P - G a l N A c: l a c t o s y l c e r a m i d e / G M 3 / G D 3 β-1,4-N-a c e t y lgalactosaminyltransferase (GalNAc-T) transforms its acceptors into the gangliosides GA2, GM2 and GD2. It is well established that it is a Golgi-located glycosyltransferase, but its sub-Golgi localization is still unclear. We addressed this question in Chinese hamster ovary K1 cell clones stably transfected with a c-myc-tagged version of GalNAc-T which express the enzyme at different levels of activity. In these cell clones we examined the effect of brefeldin A (BFA) on the synthesis of glycolipids (in metabolic-labelling experiments) and on the sub-Golgi localization of the GalNAc-T (by immunocytochemistry). We found that in cell clones expressing moderate levels of activity, GalNAc-T immunoreactivity behaved as thetrans-Golgi network (TGN) marker mannose-6-P receptor (M6PR) both in BFA-treated and untreated cells, and that BFA completely blocked the synthesis of GM2, GM1 and GD1a. On the other hand, in cell clones expressing high levels of activity and treated with BFA, most GalNAc-T immunoreactivity redistributed to the endoplasmic reticulum, as did the medial-Golgi marker mannosidase II, and the synthesis of GM2, GM1 and GD1a was not completely blocked. These results indicate that GalNAc-T is a TGN-located enzyme and that the mechanism that localizes it to this compartment involves steps that, when saturated, lead to its mislocalization to the cis-, medial- or trans-Golgi. Changes of Golgi membrane properties by modification of local glycolipid composition due to the activity of the expressed enzyme were not the main cause of mislocalization, since it persists when glycolipid synthesis is inhibited with D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl.

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“…The second enzyme, lactosylceramide synthase, the product of which is the precursor for most GSLs, is within the lumen of the Golgi (26,27). Thus a mechanism for the translocation of the substrate, glucosylceramide, for this enzyme from the cytosolic face to the lumen of the Golgi must exist.…”

Section: Surface and Subcellular P-gp And Gbmentioning

confidence: 99%

“…We propose that P-gp acts at this point. The subsequent steps in the globo-series biosynthesis occur in a stepwise fashion via Golgi-located lumenal transferases (27). Earlier studies had shown, in polarized epithelial cells, that P-gp acts as a lipid translocase of limited specificity (8).…”

Section: Surface and Subcellular P-gp And Gbmentioning

confidence: 99%

Retroviral Transfection of Madin-Darby Canine Kidney Cells with Human MDR1 Results in a Major Increase in Globotriaosylceramide and 105- to 106-Fold Increased Cell Sensitivity to Verocytotoxin

Lala¹,

Itô²,

Lingwood³

2000

Journal of Biological Chemistry

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Retroviral infection of the Madin-Darby canine kidney (MDCK) renal cell line with human MDR1 cDNA, encoding the P-glycoprotein (P-gp) multidrug resistance efflux pump, induces a major accumulation of the glycosphingolipid (GSL), globotriaosylceramide (Gal␣1-4Gal␤1-4glucosylceramide-Gb 3 ), the receptor for the E. coli-derived verotoxin (VT), to effect a ϳmillion-fold increase in cell sensitivity to VT. The shorter chain fatty acid isoforms of Gb 3 (primarily C16 and C18) are elevated and VT is internalized to the endoplasmic reticulum/nuclear envelope as we have reported for other hypersensitive cell lines. P-gp (but not MRP) inhibitors, e.g. ketoconazole or cyclosporin A (CsA) prevented the increased Gb 3 and VT sensitivity, concomitant with increased vinblastine sensitivity. Gb 3 synthase was not significantly elevated in MDR1-MDCK cells and was not affected by CsA. In MDR1-MDCK cells, synthesis of fluorescent N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-aminocaproyl (NBD)-lactosylceramide (LacCer) and NBD-Gb 3 via NBD-glucosylceramide (GlcCer) from exogenous NBD-C 6 -ceramide, was prevented by CsA. We therefore propose that P-gp can mediate GlcCer translocation across the bilayer, from the cytosolic face of the Golgi to the lumen, to provide increased substrate for the lumenal synthesis of LacCer and subsequently Gb 3 . These results provide a molecular mechanism for the observed increased sensitivity of multidrug-resistant tumors to VT and emphasize the potential of verotoxin as an antineoplastic. Two strains (I and II) of MDCK cells, which differ in their glycolipid profile, have been described. The original MDR1-MDCK parental cell was not specified, but the MDR1-MDCK GSL phenotype and glycolipid synthase activities indicate MDCK-I cells. However, the partial drug resistance of MDCK-I cells precludes their being the parental cell. We speculate that the retroviral transfection per se, or the subsequent selection for drug resistance, selected a subpopulation of MDCK-I cells in the parental MDCK-II cell culture and that drug resistance in MDR1-MDCK cells is thus a result of both MDR1 expression and a second, previously unrecognized, component, likely the high level of GlcCer synthesis in these cells.

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Functional Organization of the Golgi Apparatus in Glycosphingolipid Biosynthesis

Cited by 119 publications

References 80 publications

Combinatorial Ganglioside Biosynthesis

Combinatorial Ganglioside Biosynthesis

GA2/GM2/GD2 synthase localizes to the trans-Golgi network of CHO-K1 cells

Retroviral Transfection of Madin-Darby Canine Kidney Cells with Human MDR1 Results in a Major Increase in Globotriaosylceramide and 105- to 106-Fold Increased Cell Sensitivity to Verocytotoxin

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