Abstract
We report the first isolation of purified coated vesicles (CVs) from thyroid gland. Bovine thyroid CVs were isolated by differential centrifugaron, including a step through sucrose-D2O, using a modification of the method described by Nandi et al. (1) for bovine brain CVs. The CVs were characterized by electron microscopy, sedimentation properties, and SDS-PAGE of the protein components. Thyroglobulin (Tg) was found to be associated with the purified CVs. When the thyroid CVs were exposed to conditions known to remove the protein coat from brain CVs, such as low ionic strength at pH 8.5, most of the Tg dissociated from the vesicles along with the coat proteins. Moreover, the Tg remaining with the uncoated vesicles (UVs) was trypsin sensitive, and therefore judged to be associated with the external surface of the vesicle. Since ligand-receptor complexes are normally located within CVs and not on their outer surface, no evidence was found for Tg-receptor complexes within thyroid CVs. Thyroid slices were incubated in the presence of [35S] methionine with subsequent isolation of labeled CVs in order to study the incorporation of newly-synthesized proteins into these structures. At0.5 and 2 hours of incubation, the 180K MW subunit of clathrin, as well as other proteins, but not Tg, had become labeled in the purified CVs. Extracellular 19S-[35S] thyroglobulin was isolated from the incubation medium, however, demonstrating release of newly-synthesized Tg (presumably into cut follicles). It is concluded that thyroid CVs do not seem to be involved in the secretion of newly-synthesized Tg from the rough endoplasmic reticulum into the follicular lumen. While a possible role of thyroid CVs in the reabsorption of small quantities Tg by micropinocytosis cannot be completely excluded, the present data do not support a primary role for thyroid CVs in either endocytosis or exocytosis of Tg.
Similar content being viewed by others
References
Nandi P.K., Irace G., Van Jaarsveld P.P., Lippoldt R.E., Edelhoch H. Instability of coated vesicles in concentrated sucrose solutions. Proc. Natl. Acad. Sci. USA 79: 5881, 1982.
Brown M.S., Goldstein J.L. Receptor-mediated endocytosis: Insights from the lipoprotein receptor system. Proc. Natl. Acad. Sci. USA 76: 3330, 1979.
Pilch P.F., Shia M.A., Benson R.J.J., Fine R.E. Coated vesicles participate in the receptor-mediated endocytosis of insulin. J. Cell. Biol. 93:133, 1983.
Steer C.J., Klausner R.D. Clathrin-coated pits and coated vesicles: Functional and structural studies. Hepatology 3: 437, 1983.
Rothman J.E., Fine R.E. Coated vesicles transport newly synthesized membrane glycoproteins from endoplasmic reticulum in two successive stages. Proc. Natl. Acad. Sci. USA 77: 780, 1980.
Herzog V. Transcytosis in thyroid follicle cells. J. Cell Biol. 97:607, 1983.
van den Hove-Vandenbroucke M.F. Secretion of thyroid hormones. In: De Visscher M. (Ed.), The thyroid gland. Raven Press, New York, 1980, p. 61.
Consiglio E., Salvatore G., Rall J.E., Kohn L.D. Thyroglobulin interactions with thyroid plasma membranes. The existence of specific receptors and their potential role. J. Biol. Chem. 254:5065, 1979.
van den Hove M.F., Couvreur M., de Visscher M., Salvatore G. A new mechanism for the reabsorption of thyroid iodoproteins: Selective fluid pinocytosis. Eur. J. Biochem. 122: 415, 1982.
Seljelid R. Endocytosis in thyroid follicle cells II. A microinjection study of the origin of colloid droplets. J. Ultrastructure Res. 17: 401, 1976.
Seljelid R. Endocytosis in thyroid follicle cells. III. An electron microscopic study of the cell surface and related structures. J. Ultrastructure Res. 18:1, 1976.
Zeligs J.D., Wollman S.H. Mitosis in rat thyroid epithelial cells in vivo. J. Ultrastructure Res. 66: 53, 1979.
Irace G., Lippoldt R.E., Edelhoch H., Nandi P.K. Properties of clathrin coat structures. Biochemistry 21: 5764, 1982.
Laemmli U.K. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227: 680, 1970.
Nossal R., Weiss G.H., Nandi P.K., Lippoldt R.E., Edelhoch H. Sizes and mass distributions of clathrin-coated vesicles from bovine brain. Arch. Biochem. Biophys. 226: 593, 1983.
Zaremba S., Keen J.H. Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats. J. Cell. Biol. 97:1339, 1983.
Keen J.H., Willingham M.C., Pastan I. Clathrin coated vesicles: Isolation, dissociation and factor-dependent reassociation of clathrin baskets. Cell 16: 303, 1979.
Pearse B.M.F., Robinson M.S. Purification and properties of 100-Kd proteins from coated vesicles and their reconstitution with clathrin. EMBO J. 3:1951, 1984.
Nandi P.K., Prasad K., Lippoldt R.E., Alfsen A., Edelhoch H. Reversibility of coated vesicle dissociation. Biochemistry 21: 6434, 1982.
Novikoff A.B., Novikoff P.M., Ma M., Shin W., Quintaine N. Cytochemical studies of secretory and other granules associated with the endoplasmic reticulum in rat thyroid epithelial cells. In: Ceccarelli B., Clement F., Meldolesi J. (Eds.), Adv. cytopharmacol., Raven Press, N.Y., vol. 2, 1974, p. 349.
Erickson L.E. Exocytosis and endocytosis in the thyroid follicle cell. Mol. Cell. Endocrinol. 22:1, 1981.
Wollman S.H., Loewenstein J.E. Rates of colloid droplet and apical vesicle production and membrane turnover during thyroglobulin secretion and resorption. Endocrinology 93: 248, 1973.
Rocmans P.A., Kettelbant-Balasse P., Dumont J.E., Neve P. Hormonal secretion by hyperactive thyroid cells is not secondary to apical phagocytosis. Endocrinology 103:1834, 1978.
Pearse B.M.F., Bretscher M.S. Membrane recycling by coated vesicles. Ann. Rev. Biochem. 50: 85, 1981.
Ericson L.E., Engstrom G. Quantitative electron microscopic studies on exocytosis and endocytosis in the thyroid follicle cell. Endocrinology 103: 883, 1978.
Kinnon C., Owen M.J. Clathrin-coated vesicles purified from a human β lymphocyte cell line contain newly synthesized glycoproteins. J. Biol. Chem. 258: 8470, 1983.
Consiglio E., Shrifrin S., Yavin Z., Ambesilmpiombato F.S., Rall J.E., Salvatore G., Kohn L.D. Relationship between receptor recognition of N-acetyl glucosamine residues and the iodine content of thyroglobulin preparations. J. Biol. Chem. 256:10592, 1981.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Pierce, L.R., Zurzolo, C., Salvatore, G. et al. Coated vesicles from the thyroid gland: isolation, characterization, and a search for a possible role in thyroglobulin transport. J Endocrinol Invest 8, 303–312 (1985). https://doi.org/10.1007/BF03348502
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03348502