Double immunofluorescent staining of α2,6 sialyltransferase and β1,4 galactosyltransferase in monensin‐treated cells: Evidence for different Golgi compartments?

β1,4 galactosyl‐ and α2,6 sialyltransferase (gal‐T EC 2.4.1.22 and sialyl‐T EC 2.4.99.1) sequentially elongate and terminate complex N‐glycan chains of glycoproteins. Both enzymes reside in trans Golgi cisternae; their ultrastructural relationship, however, is unknown. To delineate their respective Golgi compartment(s) we conducted a double label immunofluorescent study by conventional and confocal laser scanning microscopy in HepG2, HeLa, and other cells in presence of Golgi‐disturbing agents. Polyclonal, peptide‐specific antibodies to human sialyl‐T expressed as a β‐galactosidase‐sialyl‐T fusion protein in E. coli were developed and applied together with mABs to human milk gal‐T. In untreated HepG2 and HeLa cells Golgi morphology identified by immunofluorescent labeling of sialyl‐T and gal‐T, respectively, was nearly identical. Treatment of cells with brefeldin A (BFA) led to rapid and coordinated disappearance of immunostaining of both enzymes; after BFA washout, vesicular structures reappeared which first stained for gal‐T followed by sialyl‐T; in the reassembled Golgi apparatus sialyl‐T and gal‐T were co‐localized again. In contrast, monensin treatment produced a reversible swelling and scattering of gal‐T positive Golgi elements while sialyl‐T positive structures showed little change. Treatment with nocodazole led to dispersal of Golgi elements in which gal‐T and sialyl‐T remained co‐localized. Treatment with chloroquine affected Golgi structure less than monensin and led to condensation of gal‐T positive and to slight enlargement of sialyl‐T positive structures. Sequential recovery from BFA of gal‐T and sialyl‐T and their segregation by monensin suggest that these enzymes are targeted to different Golgi subcompartments.