The segregation into microsomal vesicles and core-glycosylation in vitro of a 300-kDa rat thyroglobulin subunit.

Translation of rat thyroid mRNA in a cell-free protein synthesis system derived from rabbit reticulocytes results in synthesis of a 300-kDa thyroglobulin polypeptide [C.G. Alvino et al. (1982) FEBS Lett. 137, 307-313]. In the presence of dog pancreas microsomal membranes this polypeptide is segregated into the microsomal vesicles and core-glycosylated, as shown by increased protection against proteolytic treatment and binding to concanavalin-A-Sepharose. The segregation process appears to be strictly coupled to glycosylation, i.e. no detectable amounts of unglycosylated chains are found in the vesicles, and both processes only occur cotranslationally. The glycosylated protein exhibits lower electrophoretic mobility with respect to its non-glycosylated form and comigrates with a glycosylated 330-kDa polypeptide found in rat thyroid epithelial cells cultured in vitro [F.S. Ambesi-Impiombato et al. (1980) Proc. Natl Acad. Sci. USA, 77, 3455-3459]. Both glycosylated and non-glycosylated forms are immunoprecipitated by antibodies to 19-S rat thyroglobulin. Some features of the tyroglobulin polypeptide segregation and glycosylation have been studied by synchronized translation of rat thyroid mRNA: (a) the signal sequence seems to be located at the amino-terminal portion of the nascent polypeptide chain; (b) the distribution of the carbohydrate units is mostly clustered in the amino-terminal half of the protein as monitored by posttranslational analysis of the polypeptides synthesized in the presence of pancreatic membranes during competition experiments carried out with the detergent Nonidet P40.