MODULATION OF GalT1 AND SialT1 SUB-GOLGI LOCALIZATION BY

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

JOURNAL OF BIOLOGICAL CHEMISTRY

Año: 2006

0021-9258

Ganglioside glycosyltransferases organize as multi-enzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking SialT2, the sub-Golgi localization of GalT1 and SialT1 complex is affected when SialT2, another member of this complex, is co-expressed. GalT1 and SialT1 sub-Golgi localization was determined studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT11-52-CFP and SialT11-54-YFP chimeras by single cell fluorescence microscopy and by isopicnic sub-fractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (wt) cells, but beyond GlcCer in SialT2+ cells. While BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in wt cells, a fraction of these chimeras remained associated to a distal Golgi compartment, enriched in trans Golgi network and recycling endosome markers in SialT2+ cells. In BFA treated cells the percentage of GalT1-CFP and SialT1-YFP associated to Golgi like membrane fractions separated by isopicnic sub-fractionation was higher in SialT2+ cells than in wt cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may changeaccording to the relative levels of expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.1-52-CFP and SialT11-54-YFP chimeras by single cell fluorescence microscopy and by isopicnic sub-fractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (wt) cells, but beyond GlcCer in SialT2+ cells. While BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in wt cells, a fraction of these chimeras remained associated to a distal Golgi compartment, enriched in trans Golgi network and recycling endosome markers in SialT2+ cells. In BFA treated cells the percentage of GalT1-CFP and SialT1-YFP associated to Golgi like membrane fractions separated by isopicnic sub-fractionation was higher in SialT2+ cells than in wt cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may changeaccording to the relative levels of expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.