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  • 1
    ISSN: 0730-2312
    Keywords: brefeldin A ; monensin ; nocodazole ; chloroquine ; confocal laser scanning fluorescence microscopy ; fusion protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: β1,4 galactosyl- and α2,6 sialyltransferase (gal-T EC and sialyl-T EC 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.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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