title: Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus.
abstract: Glycolipid glycosyltransferases catalyze the stepwise transfer of monosaccharides from sugar nucleotides to proper glycolipid acceptors. They are Golgi resident proteins that colocalize functionally in the organelle, but their intimate relationships are not known. Here, we show that the sequentially acting UDP-GalNAc:lactosylceramide/GM3/GD3 beta-1,4-N-acetyl-galactosaminyltransferase and the UDP-Gal:GA2/GM2/GD2 beta-1,3-galactosyltransferase associate physically in the distal Golgi. Immunoprecipitation of the respective epitope-tagged versions expressed in transfected CHO-K1 cells resulted in their mutual coimmunoprecipitation. The immunocomplexes efficiently catalyze the two transfer steps leading to the synthesis of GM1 from exogenous GM3 in the presence of UDP-GalNAc and UDP-Gal. The N-terminal domains (cytosolic tail, transmembrane domain, and few amino acids of the stem region) of both enzymes are involved in the interaction because (i) they reproduce the coimmunoprecipitation behavior of the full-length enzymes, (ii) they compete with the full-length counterpart in both coimmunoprecipitation and GM1 synthesis experiments, and (iii) fused to the cyan and yellow fluorescent proteins, they localize these proteins to the Golgi membranes in an association close enough as to allow fluorescence resonance energy transfer between them. We suggest that these associations may improve the efficiency of glycolipid synthesis by channeling the intermediates from the position of product to the position of acceptor along the transfer steps.
authors.affiliation: Centro de Investigaciones en Quimica Biológica de Córdoba, Departamento de Quimica Biológica, Facultad de Ciencias Quimicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina.
authors.forename: C G
authors.initials: CG
authors.lastname: Giraudo
authors.forename: J L
authors.initials: JL
authors.lastname: Daniotti
authors.forename: H J
authors.initials: HJ
authors.lastname: Maccioni
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D005677:G(M1) Ganglioside / Q000378:metabolism; D005679:G(M3) Ganglioside / Q000378:metabolism; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D051147:Ganglioside Galactosyltransferase; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005679:G(M3) Ganglioside; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005677:G(M1) Ganglioside; C108454:GD2 beta1,3-galactosyltransferase; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; D051147:Ganglioside Galactosyltransferase; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase