Abstract
Ganglioside glycosyltransferases organize as multienzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking CMP-NeuAc: GM3 sialyltransferase (SialT2), the sub-Golgi localization of UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase (GalT1) and CMP-NeuAc:lactosylceramide sialyltransferase (SialT1) complex is affected when SialT2, another member of this complex, is coexpressed. GalT1 and SialT1 sub-Golgi localization was determined by studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT1(1-52)-CFP (cyan fluorescent protein) and SialT1(1-54)-YFP (yellow fluorescent protein) chimeras by single cell fluorescence microscopy and by isopycnic subfractionation. 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. Although BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in WT cells, a fraction of these chimeras remained associated with 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 with Golgi-like membrane fractions separated by isopycnic subfractionation 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 change according to the relative levels of the 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.
Highlights
Glycolipid oligosaccharides are built up in the Golgi apparatus by a complex membrane-bound machinery formed by glycosyltransferases, sugar nucleotide transporters, and ceramidebound acceptors
At least two complexes of ganglioside glycosyltransferases have been described in Chinese hamster ovary (CHO)-K1 cells, one formed by GalT1, SialT1, and SialT2 and another by GalT2 and GalNAcT, of more distal Golgi location (Ref. 11) (Fig. 1C, scheme), raising the question of how changes in the relative mass contribution of partners may affect their topological distribution along the Golgi apparatus
We address this issue in wild type (WT) CHO-K1 cells, which lack the expression of SialT2, and in CHO-K1 cell transfectants that stably express SialT2 (SialT2ϩ cells)
Summary
Glycolipid oligosaccharides are built up in the Golgi apparatus by a complex membrane-bound machinery formed by glycosyltransferases, sugar nucleotide transporters, and ceramidebound acceptors. The nature of the GalT1-CFP and SialT1-YFP containing Golgi elements in the post-BFA compartment of SialT2ϩ cells was investigated by comparing their localization with that of GalNAcT (Fig. 4A), a TGN resident glycosyltransferase (4), and with that of the GTPase Rab11a (Fig. 4B), an established marker of pericentriolar recycling endosomes (28, 29).
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