SubGolgi localization of GalT1/SialT1/SialT2 multienzyme complex depends on expression levels of SialT2

ULIANA ANDREA S.; CRESPO PILAR M.; MARTINA JOSÉ A; DANIOTTI JOSÉ L; MACCIONI HUGO J.F

Iguazú, Misiones

Congreso; XL Reunión Nacional de la Sociedad Argentina de Investigación en Bioquímica y Biología Celular (SAIB); 2004

Ganglioside glycosyltransferases form multienzyme complexes in the Golgi apparatus. Here we studied whether the proximal Golgi localization of Gal-T1 and Sial-T1 complex is affected when another member of this complex, Sial-T2, is co-expressed.  For this, CHO-K1 cells, which lack endogenous Sial-T2, were stably transfected with full-length Sial-T2. The subGolgi location of these activities was determined by measuring the effect of Brefeldin A on I), the metabolic labeling of glycolipids  from 14C-galactose; II), the subcellular localization of chimeras of Gal-T1 and Sial-T1 and spectral variants of the GFP and by III), isopicnic ultracentrifugation. It was found: in I) that in parental cells BFA imposed a block in the synthesis of glycolipids beyond GM3, while in Sial-T2 expressing cells the block was imposed beyond GlcCer. In II), that in parental cells BFA redistributed Gal-T1-GFP and Sial-T1-GFP to the ER, while in Sial-T2 expressing cells these chimeras mostly remained associated to a distal  Golgi (post BFA) compartment. In III) that in parental cells BFA displaces Gal-T1-GFP and Sial-T1-GFP to fractions enriched in ER membranes while in Sial-T2 expressing cells these chimeras were displaced to fractions floating slightly above Golgi membranes. These results suggest that subGolgi localization of these complexes is dynamic and may change according to relative levels of expression of the participating enzymes.