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

Abstract

Beta 1,4 galactosyl- and alpha 2,6 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 beta-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 structures 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.

Vacuolar ATPase inactivation blocks recycling to the trans‐Golgi network from the plasma membrane

“…ing little if any effect on the gross morphology of that organelle [23]. NRK cells were therefore incubated in the presence of increasing concentrations of chloroquine for one hour at 37°C prior to processing for double label immunofluorescence analysis (see section 2) using a polyclonal antibody to TGN38141 and a monoclonal antibody to marmosidase II (an integral membrane protein of the cis, medial Golgi stacks [20,21]).…”

Human β1,4 galactosyltransferase and α2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum

“…The antibodies against GT or ST, respectively, used for immunoprecipitation were affinity purified on immobilized /3-galactosidase fusion proteins expressed in E. coli (Watzele et al, 1991;Berger et al, 1993). The immunoisolated proteins were analyzed by SDSPAGE and visualized by fluorography.…”

Sequence and overexpression of GPP130/GIMPc: evidence for saturable pH-sensitive targeting of a type II early Golgi membrane protein.

“…Therefore, chloroquine treatment results in a redistribution of TGN proteins from the Golgi to dilated endosomes. In contrast, type II Golgi proteins such as N-acetylglucosaminyltransferase I, mannosidase II, sialyltransferase, and galactosyltransferase remain associated with the Golgi stack in chloroquinetreated cells (Berger et al, 1993;Chapman and Munro, 1994). Because the behavior of these various proteins in response to chloroquine treatment is probably related to their targeting mechanisms, we examined the distribution of GPP130, giantin, and mannose-6-phosphate receptor in COS-7 cells treated with 0.1 mM chloroquine for 3 h. In untreated cells the giantin and GPP130 staining patterns were coincident (Figure 4, A and B).…”