title: Ganglioside glycosyltransferases organize in distinct multienzyme complexes in CHO-K1 cells.
abstract: The synthesis of gangliosides is compartmentalized in the Golgi complex. In most cells, glycosylation of LacCer, GM3, and GD3 to form higher order species (GA2, GM2, GD2, GM1, GD1b) is displaced toward the most distal aspects of the Golgi and the trans-Golgi network, where the involved transferases (GalNAcT and GalT2) form physical and functional associations. Glycosylation of the simple species LacCer, GM3, and GD3, on the other hand, is displaced toward more proximal Golgi compartments, and we investigate here whether the involved transferases (GalT1, SialT1, and SialT2) share the property of forming physical associations. Co-immunoprecipitation experiments from membranes of CHO-K1 cells expressing epitope-tagged versions of these enzymes indicate that GalT1, SialT1, and SialT2 associate physically in a SialT1-dependent manner and that their N-terminal domains participate in these interactions. Microscopic fluorescence resonance energy transfer and fluorescence recovery after photobleaching in living cells confirmed the interactions, and in addition to showing a Golgi apparatus localization of the complexes, mapped their formation to the endoplasmic reticulum. Neither co-immunoprecipitation nor fluorescence resonance energy transfer detected interactions between either GalT2 or GalNAcT and GalT1 or SialT1 or SialT2. These results, and triple color imaging of Golgi-derived microvesicles in nocodazole-treated cells, suggest that ganglioside synthesis is organized in distinct units each formed by associations of particular glycosyltransferases, which concentrate in different sub-Golgi compartments.
authors.affiliation: CIQUIBIC, Universidad Nacional de Cordoba-Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Argentina.
authors.forename: Claudio G
authors.initials: CG
authors.lastname: Giraudo
authors.forename: Hugo J F
authors.initials: HJ
authors.lastname: Maccioni
mesh_terms: D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005732:Gangliosides / Q000096:biosynthesis* / Q000737:chemistry; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D005732:Gangliosides; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D016695:Glycosyltransferases; D005700:Galactosyltransferases; C039090:UDPgalactose-glucosylceramide galactosyltransferase; C023235:glucosylceramide beta-1-4-galactosyltransferase; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase; C021343:haematoside synthetase