Intracellular glycosylation of vitellogenin in the liver of estrogen-stimulated Xenopus laevis.
Pulse-chase experiments measuring the rates of incorporation of radiolabeled glucosamine and galactose into intracellular vitellogenin show that glycosylation of this multicomponent protein occurs in a Golgi-enriched fraction isolated from homogenized liver slices. No apparent role of the rough endoplasmic reticulum in this process was demonstrable. Kinetics of the intracellular translocation of glycosylated vitellogenin indicate that the galactosylated intermediate is secreted more rapidly than the glucosamine-labeled precursor. This was corroborated by measuring the rates of accumulation of various pulse-labeled forms of vitellogenin in the chase medium. In addition, a negligible amount of mannose was incorporated into intracellular or secreted vitellogenin. The antibiotic tunicamycin was shown to inhibit [3H] glucosamine incorporation into microsomal vitellogenin by 70%, without any significant effect on the synthesis of the protein backbone. In addition, nonglycosylated vitellogenin showed normal secretion kinetics. After suitable pretreatment with the antibiotic followed by a labeling period in tunicamycin-free medium, mannose was still not incorporated into vitellogenin, whereas glucosamine behaved in a typical manner. In contrast to this finding, gas-liquid chromatography of the alditol acetate derivatives of the neutral hexoses of vitellogenin showed that mannose was indeed a major component of the vitellogenin oligosaccharide side chain. These preliminary results indicate that the oligosaccharide component of vitellogenin in Xenopus laevis is a "complex" type of carbohydrate unit which is linked via an N-glycosidic bond between an asparagine residue and N-acetylglucosamine. With respect to the subcellular localization of glycoprotein assembly in Xenopus liver, there is a significant departure from currently accepted models of glycoprotein synthesis.
D000818:Animals; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D005690:Galactose / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008074:Lipoproteins / Q000096:biosynthesis*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium; D014415:Tunicamycin / Q000494:pharmacology; D014819:Vitellogenins / Q000096:biosynthesis*; D014981:Xenopus
D006023:Glycoproteins; D008074:Lipoproteins; D014819:Vitellogenins; D014316:Tritium; D014415:Tunicamycin; D004958:Estradiol; D007930:Leucine; D005944:Glucosamine; D005690:Galactose