Double immunofluorescent staining of alpha 2,6 sialyltransferase and beta 1,4 galactosyltransferase in monensin-treated cells: evidence for different Golgi compartments?
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.
Berger
E G
EG
Institute of Physiology, University of Zurich, Switzerland.
Grimm
K
K
Bächi
T
T
Bosshart
H
H
Kleene
R
R
Watzele
M
M
D000911:Antibodies, Monoclonal / Q000032:analysis; D001483:Base Sequence; D020126:Brefeldin A; D002451:Cell Compartmentation / Q000502:physiology*; D002738:Chloroquine / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology*; D009237:N-Acetyllactosamine Synthase / Q000032:analysis / Q000187:drug effects*; D015739:Nocodazole / Q000494:pharmacology; D012799:Sialyltransferases / Q000032:analysis / Q000187:drug effects*; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D000911:Antibodies, Monoclonal; D003517:Cyclopentanes; D020126:Brefeldin A; D002738:Chloroquine; D008985:Monensin; D009237:N-Acetyllactosamine Synthase; D012799:Sialyltransferases; D015739:Nocodazole; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase